SciAm Online 2003-09 Germ Wars PDF

COPYRIGHT 2003 SCIENTIFIC AMERICAN, INC.
GERM WARS ScientificAmerican.com

exclusive online issue no. 9
The human body has an impressive arsenal of defenses against pathogens. But bacteria and viruses are
wily opponents, and tackling the most dangerous ones has become a battle of witsone in which scien-
tists have had both stunning successes and frustrating defeats. They must remain vigilant: germs have
plagued our species since its inception and they are here to stay.
Scientific American has long covered developments in the war on germs. In this exclusive online edition,
prominent researchers and journalists discuss the new weapons of this war, such as virus-fighting drugs,
edible vaccines and novel antibiotics; emerging enemies, such as anthrax and chronic wasting disease;
and the all-too familiar foes HIV and hepatitis C. The Editors
TABLE OF CONTENTS
2 Beyond Chicken Soup
BY WILLIAM A. HASELTINE; SCIENTIFIC AMERICAN, NOVEMBER 2001
The antiviral era is upon us, with an array of virus-fighting drugs on the market and in development. Research into viral
genomes is fueling much of this progress
9 Behind Enemy Lines

BY K.C. NICOLAOU AND CHRISTOPHER N.C. BODDY; SCIENTIFIC AMERICAN, MAY 2001
A close look at the inner workings of microbes in the era of escalating antibiotic resistance is offering new strategies for
designing drugs
14 Edible Vaccines
BY WILLIAM H.R. LANGRIDGE, SIDEBAR BY RICKI RUSTING; SCIENTIFIC AMERICAN, SEPTEMBER 2000
One day children may get immunized by munching on foods instead of enduring shots. More important, food vaccines
might save millions who now die for lack of access to traditional inoculants
20 The Unmet Challenges of Hepatitis C

BY BY ADRIAN M. DI BISCEGLIE AND BRUCE R. BACON; SCIENTIFIC AMERICAN, OCTOBER 1999
Some 1.8 percent of the U.S. adult population are infected with the hepatitis C virus, most without knowing it
26 Attacking Anthrax
BY JOHN. A. T. YOUNG AND R. JOHN COLLIER; SCIENTIFIC AMERICAN, MARCH 2002
Recent discoveries are suggesting much-needed strategies for improving prevention and treatment. High on the list:
ways to neutralize the anthrax bacteriums fiendish toxin
33 Shoot This Deer

BY PHILIP YAM; SCIENTIFIC AMERICAN, JUNE 2003
Chronic wasting disease, a cousin of mad cow disease, is spreading among wild deer in parts of the U.S. Left unchecked,
the fatal sickness could threaten North American deer populationsand maybe livestock and humans.
37 Hope in a Vial
BY CAROL EZZELL; SCIENTIFIC AMERICAN, JUNE 2002
Will there be an AIDS vaccine anytime soon?
1 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

Originally published in November 2001
BEYOND By William A. Haseltine
CHICKEN
SOUP
The antiviral era is upon us, with an array of virus-fighting drugs on the market and
in development. Research into viral genomes is fueling much of this progress
Back in the mid-1980s, when scientists first learned that a tis will surely remain a main focus of investigation for some
virus caused a relentless new disease named AIDS, pharmacy time; together they cause more than 250,000 cases of disease
shelves were loaded with drugs able to treat bacterial infections. in the U.S. every year and millions in other countries. Biologists,
For viral diseases, though, medicine had little to offer beyond however, are working aggressively to combat other viral ill-
chicken soup and a cluster of vaccines. The story is dramati- nesses as well. I cannot begin to describe all the classes of an-
cally different today. Dozens of antiviral therapies, including tivirals on the market and under study, but I do hope this arti-
several new vaccines, are available, and hundreds more are in cle will offer a sense of the extraordinary advances that ge-
development. If the 1950s were the golden age of antibiotics, nomics and other sophisticated technologies have made
we are now in the early years of the golden age of antivirals. possible in recent years.
This richness springs from various sources. Pharmaceutical
companies would certainly point to the advent in the past 15 Drug-Search Strategies
years of sophisticated techniques for discovering all manner of THE EARLIEST ANTIVIRALS (mainly against herpes) were
drugs. At the same time, frantic efforts to find lifesaving thera- introduced in the 1960s and emerged from traditional drug-dis-
pies for HIV, the cause of AIDS, have suggested creative ways covery methods. Viruses are structurally simple, essentially con-
to fight not only HIV but other viruses, too. sisting of genes and perhaps some enzymes (biological catalysts)
A little-recognized but more important force has also been encased in a protein capsule and sometimes also in a lipid enve-
at work: viral genomics, which deciphers the sequence of let- lope. Because this design requires viruses to replicate inside cells,
ters, or nucleic acids, in a viruss genetic text. This sequence investigators infected cells, grew them in culture and exposed
includes the letters in all the viruss genes, which form the blue- the cultures to chemicals that might plausibly inhibit viral ac-
prints for viral proteins; these proteins, in turn, serve as the struc- tivities known at the time. Chemicals that reduced the amount
tural elements and the working parts of the virus and thus con- of virus in the culture were considered for in-depth investigation.
trol its behavior. With a full or even a partial genome sequence Beyond being a rather hit-or-miss process, such screening left sci-
in hand, scientists can quickly learn many details of how a virus entists with few clues to other viral activities worth attacking.
causes disease and which stages of the process might be par- This handicap hampered efforts to develop drugs that were more
ticularly vulnerable to attack. In 2001 the full genome of any effective or had fewer side effects.
virus can be sequenced within days, making it possible to spot Genomics has been a springboard for discovering fresh tar-
that viruss weaknesses with unprecedented speed. gets for attack and has thus opened the way to development of
The majority of antivirals on sale these days take aim at whole new classes of antiviral drugs. Most viral targets select-
QUADE PAUL
HIV, herpesviruses (responsible for a range of ills, from cold ed since the 1980s have been identified with the help of ge-
sores to encephalitis), and hepatitis B and C viruses (both of nomics, even though the term itself was only coined in the late
which can cause liver cancer). HIV and these forms of hepati- 1980s, well after some of the currently available antiviral drugs

were developed. gies. In general, though, the stages of viral replication include
After investigators decipher the sequence of code letters in attachment to the cells of a host, release of viral genes into the
a given virus, they can enlist computers to compare that se- cells interiors, replication of all viral genes and proteins (with
quence with those already identified in other organisms, in- help from the cells own protein-making machinery), joining of
cluding other viruses, and thereby learn how the sequence is seg- the components into hordes of viral particles, and escape of
mented into genes. Strings of code letters that closely resemble those particles to begin the cycle again in other cells.
known genes in other organisms are likely to constitute genes in The ideal time to ambush a virus is in the earliest stage of
the virus as well and to give rise to proteins that have similar an infection, before it has had time to spread throughout the
structures. Having located a viruss genes, scientists can study body and cause symptoms. Vaccines prove their worth at that
the functions of the corresponding proteins and thus build a point, because they prime a persons immune system to specifi-
comprehensive picture of the molecular steps by which the virus cally destroy a chosen disease-causing agent, or pathogen, al-
of interest gains a foothold and thrives in the body. most as soon as it enters the body. Historically vaccines have
That picture, in turn, can highlight the proteins and the achieved this priming by exposing a person to a killed or weak-
domains within those proteins that would be good to disable. ened version of the infectious agent that cannot make enough
In general, investigators favor targets whose disruption would copies of itself to cause disease. So-called subunit vaccines are
impair viral activity most. They also like to focus on protein do- the most common alternative to these. They contain mere frag-
mains that bear little resemblance to those in humans, to avoid ments of a pathogen; fragments alone have no way to produce
harming healthy cells and causing intolerable side effects. They an infection but, if selected carefully, can evoke a protective im-
take aim, too, at protein domains that are basically identical mune response.
in all major strains of the virus, so that the drug will be useful An early subunit vaccine, for hepatitis B, was made by iso-
against the broadest possible range of viral variants. lating the virus from the plasma (the fluid component of blood)
After researchers identify a viral target, they can enlist var- of people who were infected and then purifying the desired pro-
ious techniques to find drugs that are able to perturb it. Drug teins. Today a subunit hepatitis B vaccine is made by genetic en-
sleuths can, for example, take advantage of standard genetic gineering. Scientists use the gene for a specific hepatitis B pro-
engineering (introduced in the 1970s) to produce pure copies tein to manufacture pure copies of the protein. Additional vac-
of a selected protein for use in drug development. They insert cines developed with the help of genomics are in development
the corresponding gene into bacteria or other types of cells, for other important viral diseases, among them dengue fever,
which synthesize endless copies of the encoded protein. The re- genital herpes and the often fatal hemorrhagic fever caused by
sulting protein molecules can then form the basis of rapid the Ebola virus.
screening tests: only substances that bind to them are pursued Several vaccines are being investigated for preventing or
further. treating HIV. But HIVs genes mutate rapidly, giving rise to
Alternatively, investigators might analyze the three- di- many viral strains; hence, a vaccine that induces a reaction
mensional structure of a protein domain and then design drugs against certain strains might have no effect against others. By
that bind tightly to that region. For instance, they might con- comparing the genomes of the various HIV strains, researchers
struct a compound that inhibits the active site of an enzyme cru- can find sequences that are present in most of them and then
cial to viral reproduction. Drugmakers can also combine old- use those sequences to produce purified viral protein fragments.
fashioned screening methods with the newer methods based on These can be tested for their ability to induce immune protec-
structures. tion against strains found worldwide. Or vaccines might be tai-
Advanced approaches to drug discovery have generated lored to the HIV variants prominent in particular regions.
ideas for thwarting viruses at all stages of their life cycles. Vi-
ral species vary in the fine details of their reproductive strate- Bar Entry
T R E A T M E N T S B E C O M E important when a vaccine is not
Overview/Antiviral Drugs available or not effective. Antiviral treatments effect cures

for some patients, but so far most of them tend to reduce the
Deciphering the genetic sequences, or genomes, of humans and severity or duration of a viral infection. One group of ther-
of a variety of viruses has enabled scientists to devise drugs for apies limits viral activity by interfering with entry into a fa-
diseases such AIDS, hepatitis and influenza. vored cell type.
After decoding the genetic sequence of a virus, researchers can The term entry actually covers a few steps, beginning
use computers to compare its sequence with those of other with the binding of the virus to some docking site, or recep-
viruses a process known loosely as genomics. The comparison tor, on a host cell and ending with uncoating inside the
allows drugmakers to identify genes in the new virus that encode cell; during uncoating, the protein capsule (capsid) breaks
molecules worth targeting. up, releasing the viruss genes. Entry for enveloped viruses
Viruses have complex life cycles but are vulnerable to attack by requires an extra step. Before uncoating can occur, these mi-
pharmaceuticals at nearly every stage. croorganisms must fuse their envelope with the cell mem-
brane or with the membrane of a vesicle that draws the virus

A VIRUS IN ACTION
HIV LIFE CYCLE, deciphered with the help of genomic opting the cells machinery and raw materials), and pack
analyses, is unusually complex in its details, but all virus- the fresh copies into new viral particles able to spread to
es undergo the same basic steps to infect cells and re- and infect other cells. The viral components involved in
produce. They enter a cell (bind to it and inject their genes any of these steps can serve as targets for drugs, as the
into the interior), copy their genes and proteins (by co- table on page 7 demonstrates.
1 BINDING
Virus attaches to a cell
Envelope
protein
HIV 2 FUSION
Viral and cell membranes fuse
New viral
particle
Envelope
CCR5 receptor CD4 receptor

for HIV for HIV
Cell membrane Capsid 9 VIRUS ASSEMBLY

AND SPREAD
New viral particles
bud from cell and
3 UNCOATING move on to infect
Capsid, or coat, breaks other cells
up, freeing viral genes
and enzymes
Reverse transcriptase
Protease
HIVs RNA genome 6 GENOME REPLICATION
Integrase Cell uses the viral DNA as a
template for reproducing
HELPER T CELL the HIV RNA genome
Viral
proteins
4 REVERSE TRANSCRIPTION
HIV reverse transcriptase
copies viral RNA to DNA Copies of HIVs 8 PROTEIN CLEAVAGE
Viral DNA RNA genome Protease enzyme
cuts long protein
chain into individual
7 PROTEIN SYNTHESIS proteins
Cell uses HIV RNA as a template
for synthesizing viral proteins Protease
Integrase
Nascent
Cellular DNA Integrated protein
viral DNA chain
5 GENOME INTEGRATION
Viral integrase splices
QUADE PAUL
viral DNA into cellular DNA Cellular protein

Nucleus making machinery

into the cells interior. caused by varicella zoster and inflammation of the retina caused
Several entry-inhibiting drugs in development attempt to by cytomegalovirus.
block HIV from penetrating cells. Close examination of the The first drug approved for use against HIV, zidovudine
way HIV interacts with its favorite hosts (white blood cells (AZT), is a nucleoside analogue as well. Initially developed as
called helper T cells) has indicated that it docks with molecules an anticancer drug, it was shown to interfere with the activity
on those cells called CD4 and CCR5. Although blocking CD4 of reverse transcriptase, an enzyme that HIV uses to copy its
has failed to prevent HIV from entering cells, blocking CCR5 RNA genome into DNA. If this copying step is successful, oth-
may yet do so. er HIV enzymes splice the DNA into the chromosomes of an
Amantidine and rimantidine, the first two (of four) influen- invaded cell, where the integrated DNA directs viral reproduc-
za drugs to be introduced, interrupt other parts of the entry pro- tion.
cess. Drugmakers found the compounds by screening likely AZT can cause severe side effects, such as anemia. But stud-
chemicals for their overall ability to interfere with viral replica- ies of reverse transcriptase, informed by knowledge of the en-
tion, but they have since learned more specifically that the com- zymes gene sequence, have enabled drug developers to intro-
pounds probably act by inhibiting fusion and uncoating. Fusion duce less toxic nucleoside analogues. One of these, lamivudine,
inhibitors discovered with the aid of genomic information are has also been approved for hepatitis B, which uses reverse tran-
also being pursued against respiratory syncytial virus (a cause scriptase to convert RNA copies of its DNA genome back into
of lung disease in infants born prematurely), hepatitis B and C, DNA. Intense analyses of HIV reverse transcriptase have led as
and HIV. well to improved versions of a class of reverse transcriptase in-
Many colds could soon be controlled by another entry hibitors that do not resemble nucleosides.
blocker, pleconaril, which is reportedly close to receiving fed- Genomics has uncovered additional targets that could be hit
eral approval. Genomic and structural comparisons have to interrupt replication of the HIV genome. Among these is
shown that a pocket on the surface of rhinoviruses (responsi- RNase H, a part of reverse transcriptase that separates freshly
ble for most colds) is similar in most variants. Pleconaril binds minted HIV DNA from RNA. Another is the active site of inte-
to this pocket in a way that inhibits the uncoating of the virus. grase, an enzyme that splices DNA into the chromosomal DNA
The drug also appears to be active against enteroviruses, which of the infected cell. An integrase inhibitor is now being tested
can cause diarrhea, meningitis, conjunctivitis and encephali- in HIV-infected volunteers.
tis.
Impede Protein Production
Jam the Copier A L L V I R U S E S M U S T at some point in their life cycle tran-
A NUMBER OF ANTIVIRALS on sale and under study oper- scribe genes into mobile strands of messenger RNA, which the
ate after uncoating, when the viral genome, which can take the host cell then translates, or uses as a guide for making the en-
form of DNA or RNA, is freed for copying and directing the coded proteins. Several drugs in development interfere with the
production of viral proteins. Several of the agents that inhibit transcription stage by preventing proteins known as transcrip-
genome replication are nucleoside or nucleotide analogues, tion factors from attaching to viral DNA and switching on the
which resemble the building blocks of genes. The enzymes that production of messenger RNA.
copy viral DNA or RNA incorporate these mimics into the Genomics helped to identify the targets for many of these
nascent strands. Then the mimics prevent the enzyme from agents. It also made possible a novel kind of drug: the antisense
adding any further building blocks, effectively aborting viral molecule. If genomic research shows that a particular protein
replication. is needed by a virus, workers can halt the proteins production
Acyclovir, the earliest antiviral proved to be both effective by masking part of the corresponding RNA template with a cus-
and relatively nontoxic, is a nucleoside analogue that was dis- tom-designed DNA fragment able to bind firmly to the selected
covered by screening selected compounds for their ability to in- RNA sequence. An antisense drug, fomivirsen, is already used
terfere with the replication of herpes simplex virus. It is pre- to treat eye infections caused by cytomegalovirus in AIDS pa-
scribed mainly for genital herpes, but chemical relatives have tients. And antisense agents are in development for other viral
value against other herpesvirus infections, such as shingles diseases; one of them blocks production of the HIV protein Tat,
which is needed for the transcription of other HIV genes.
Drugmakers have also used their knowledge of viral ge-
THE AUTHOR
WILLIAM A. HASELTINE, who has a doctorate in biophysics from Harvard

University, is the chairman of the board of directors and chief executive nomes to identify sites in viral RNA that are susceptible to cut-
officer of Human Genome Sciences; he is also editor in chief of a new pub- ting by ribozymesenzymatic forms of RNA. A ribozyme is be-
lication, the Journal of Regenerative Medicine, and serves on the editor- ing tested in patients with hepatitis C, and ribozymes for HIV
ial boards of several other scientific journals. He was a professor at the are in earlier stages of development. Some such projects employ
Dana-Farber Cancer Institute, an affiliate of Harvard Medical School, and gene therapy: specially designed genes are introduced into cells,
at the Harvard School of Public Health from 1988 to 1995. His laborato- which then produce the needed ribozymes. Other types of HIV
ry was the first to assemble the sequence of the AIDS virus genome. Since gene therapy under study give rise to specialized antibodies that
1981 he has helped found more than 20 biotechnology companies. seek targets inside infected cells or to other proteins that latch

Antiviral Drugs Today
Sampling of antiviral drugs on the market appears below. Many owe their existence, at least in part, to viral genomics.
About 30 other viral drugs based on an understanding of viral genomics are in human tests.
DRUG NAMES SPECIFIC ROLES MAIN VIRAL DISEASES TARGETED
DISRUPTORS OF GENOME
abacavir, didanosine, stavudine, Nucleoside analogue inhibitors of reverse HIV infection
zalcitabine, zidovudine transcriptase
acyclovir, ganciclovir, penciclovir Nucleoside analogue inhibitors of the Herpes infections; retinal inflammation
enzyme that duplicates viral DNA caused by cytomegalovirus
cidofovir Nucleotide analogue inhibitor of the Retinal inflammation caused by

enzyme that duplicates viral DNA cytomegalovirus
delavardine, efavirenz Nonnucleoside, nonnucleotide inhibitors of HIV infection

reverse transcriptase
lamivudine Nucleoside analogue inhibitor of reverse HIV, hepatitis B infections

transcriptase
ribavirin Synthetic nucleoside that induces mutations Hepatitis C infection

in viral genes
DISRUPTORS OF PROTEIN SYNTHESIS
amprenavir, indinavir, lopinavir, Inhibitors of HIV protease HIV infection
nelfinavir, ritonavir, saquinavir
fomivirsen Antisense molecule that blocks translation Retinal inflammation caused by

of viral RNA cytomegalovirus
interferon alpha Activator of intracellular immune defenses Hepatitis B and C infections

that block viral protein synthesis
BLOCKERS OF VIRAL SPREAD FROM CELL TO CELL
oseltamivir, zanamivir Inhibitors of viral release Influenza
palivizumab Humanized monoclonal antibody that Respiratory syncytial infection

marks virus for destruction
onto certain viral gene sequences within those cells. been known to play a role in helping viral particles escape from
Some viruses produce a protein chain in a cell that must be the cells that produced them. Genomic comparisons revealed
spliced to yield functional proteins. HIV is among them, and that the active site of neuraminidase is similar among various
an enzyme known as a protease performs this cutting. When influenza strains, and structural studies enabled researchers to
analyses of the HIV genome pinpointed this activity, scientists design compounds able to plug that site. The other flu drugs
began to consider the protease a drug target. With enormous act only against type A.
help from computer-assisted structure-based research, potent Drugs can prevent the cell-to-cell spread of viruses in a dif-
protease inhibitors became available in the 1990s, and more ferent way by augmenting a patients immune responses.
are in development. The inhibitors that are available so far can Some of these responses are nonspecific: the drugs may restrain
cause disturbing side effects, such as the accumulation of fat in the spread through the body of various kinds of invaders rather
unusual places, but they nonetheless prolong overall health and than homing in on a particular pathogen. Molecules called in-
life in many people when taken in combination with other HIV terferons take part in this type of immunity, inhibiting protein
antivirals. A new generation of protease inhibitors is in the re- synthesis and other aspects of viral replication in infected cells.
search pipeline. For that reason, one form of human interferon, interferon al-
pha, has been a mainstay of therapy for hepatitis B and C. (For
Stop Traffic hepatitis C, it is used with an older drug, ribavirin.) Other in-
E V E N I F V I R A L G E N O M E S and proteins are reproduced in terferons are under study, too.
a cell, they will be harmless unless they form new viral parti- More specific immune responses include the production of
cles able to escape from the cell and migrate to other cells. The standard antibodies, which recognize some fragment of a pro-
most recent influenza drugs, zanamivir and oseltamivir, act at tein on the surface of a viral invader, bind to that protein and
this stage. A molecule called neuraminidase, which is found on mark the virus for destruction by other parts of the immune
the surface of both major types of influenza (A and B), has long system. Once researchers have the gene sequence encoding a

viral surface protein, they can generate pure, or monoclonal,
antibodies to selected regions of the protein. One monoclonal Deciphered Viruses
is on the market for preventing respiratory syncytial virus in ba- Some medically important viruses whose genomes have been
bies at risk for this infection; another is being tested in patients sequenced are listed below. Frederick Sanger of the University of
suffering from hepatitis B. Cambridge and his colleagues determined the DNA sequence of
Comparisons of viral and human genomes have suggested the first viral genome from a virus that infects bacteria in 1977.
yet another antiviral strategy. A number of viruses, it turns out,
YEAR
produce proteins that resemble molecules involved in the im- VIRUS DISEASE SEQUENCED
mune response. Moreover, certain of those viral mimics disrupt
Human poliovirus Poliomyelitis 1981
the immune onslaught and thus help the virus to evade destruc-
tion. Drugs able to intercept such evasion-enabling proteins may Influenza A virus Influenza 1981
preserve full immune responses and speed the organisms re- Hepatitis B virus Hepatitis B 1984
covery from numerous viral diseases. The hunt for such agents
Human rhinovirus type 14 Common cold 1984
is under way.
HIV-1 AIDS 1985
The Resistance Demon Human papillomavirus type 16 Cervical cancer 1985
THE PACE OF ANTIVIRAL drug discovery is nothing short
Dengue virus type 1 Dengue fever 1987
of breathtaking, but at the same time, drugmakers have to con-
front a hard reality: viruses are very likely to develop resistance, Hepatitis A virus Hepatitis A 1987
or insensitivity, to many drugs. Resistance is especially proba- Herpes simplex virus type 1 Cold sores 1988
ble when the compounds are used for long periods, as they are
Hepatitis C virus Hepatitis C 1990
in such chronic diseases as HIV and in quite a few cases of he-
patitis B and C. Indeed, for every HIV drug in the present ar- Cytomegalovirus Retinal infections 1991
in HIV-infected people
senal, some viral strain exists that is resistant to it and, often,
to additional drugs. This resistance stems from the tendency of Variola virus Smallpox 1992
viruses especially RNA viruses and most especially HIV to Ebola virus Ebola hemorrhagic fever 1993
mutate rapidly. When a mutation enables a viral strain to over-
Respiratory syncytial virus Childhood respiratory 1996
come some obstacle to reproduction (such as a drug), that infections
strain will thrive in the face of the obstacle.
Human parainfluenzavirus 3 Childhood respiratory 1998
To keep the resistance demon at bay until effective vaccines infections
are found, pharmaceutical companies will have to develop more
drugs. When mutants resistant to a particular drug arise, read-
ing their genetic text can indicate where the mutation lies in the genome is available in draft form, drug designers will identify a
viral genome and suggest how that mutation might alter the in- number of previously undiscovered proteins that stimulate the
teraction between the affected viral protein and the drug. Armed production of antiviral antibodies or that energize other parts
with that information, researchers can begin structure-based or of the immune system against viruses. I fully expect these dis-
other studies designed to keep the drug working despite the mu- coveries to translate into yet more antivirals. The insights
tation. gleaned from the human genome, viral genomes and other ad-
Pharmaceutical developers are also selecting novel drugs vanced drug-discovery methods are sure to provide a flood of
based on their ability to combat viral strains that are resistant needed antivirals within the next 10 to 20 years. SA
to other drugs. Recently, for instance, DuPont Pharmaceuticals

chose a new HIV nonnucleoside reverse transcriptase inhibitor,
DPC 083, for development precisely because of its ability to MORE TO E XPLORE
overcome viral resistance to such inhibitors. The companys re- Viral Strategies of Immune Evasion. Hidde L. Ploegh in Science, Vol.
searchers first examined the mutations in the reverse tran- 280, No. 5361, pages 248253; April 10, 1998.
scriptase gene that conferred resistance. Next they turned to Strategies for Antiviral Drug Discovery. Philip S. Jones in Antiviral
computer modeling to find drug designs likely to inhibit the re- Chemistry and Chemotherapy, Vol. 9, No. 4, pages 283302; July 1998.
verse transcriptase enzyme in spite of those mutations. Then, New Technologies for Making Vaccines. Ronald W. Ellis in Vaccine, Vol.
using genetic engineering, they created viruses that produced 17, No. 13-14, pages 15961604; March 26, 1999.
the mutant enzymes and selected the compound best able to Protein Design of an HIV-1 Entry Inhibitor. Michael J. Root, Michael S.
limit reproduction by those viruses. The drug is now being eval- Kay and Peter S. Kim in Science, Vol. 291, No. 5505, pages 884888;
February 2, 2001.
uated in HIV-infected patients.
Antiviral Chemotherapy: General Overview. Jack M. Bernstein, Wright
It may be some time before virtually all serious viral infec- State University School of Medicine, Division of Infectious Diseases,
tions are either preventable by vaccines or treatable by some ef- 2000.
fective drug therapy. But now that the sequence of the human Available at www.med.wright.edu/im/AntiviralChemotherapy.html

Originally published in May 2001
BEHIND
ENEMY
A close look at the inner workings of microbes in this era of escalating
LINES
ANTIBIOTIC RESISTANCE is offering new strategies for designing drugs
by K. C. Nicolaou and Christopher N. C. Boddy
In the celebrated movie Crouching Tiger, Hidden Dragon, One of the foundations of the modern medical system is be-
two warriors face each other in a closed courtyard whose walls ing similarly overcome. Health care workers are increasingly
are lined with a fantastic array of martial-arts weaponry, in- finding that nearly every weapon in their arsenal of more than
cluding iron rods, knives, spears and swords. 150 antibiotics is becoming useless. Bacteria that have survived
The older, more experienced warrior grabs one instrument attack by antibiotics have learned from the enemy and have
after another from the arsenal and battles energetically and flu- grown stronger; some that have not had skirmishes themselves
idly with them. But one after another, the weapons prove use- have learned from others that have. The result is a rising num-
less. Each, in turn, is broken or thrown aside, the shards of an ber of antibiotic-resistant strains. Infections including tuber-
era that can hold little contest against a young, triumphant, up- culosis, meningitis and pneumoniathat would once have been
start warrior who has learned not only the old ways but some easily treated with an antibiotic are no longer so readily thwart-
that are new. ed. More and more bacterial infections are proving deadly.
Bacteria are wily warriors, but even so, we have given
THE AUTHORS
K. C. NICOLAOU and CHRISTOPHER N. C. BODDY have worked together themand continue to give themexactly what they need for
at the Scripps Research Institute in La Jolla, Calif., where Nicolaou is their stunning success. By misusing and overusing antibiotics,
chairman of the department of chemistry and Boddy recently received
his Ph.D. Nicolaou holds the Darlene Shiley Chair in Chemistry, the Aline W. we have encouraged super-races of bacteria to evolve. We dont
and L. S. Skaggs Professorship in Chemical Biology and a professorship finish a course of antibiotics. Or we use them for viral and oth-
at the University of California, San Diego. His work in chemistry, biology er inappropriate infectionsin fact, researchers estimate that
and medicine has been described in more than 500 publications and 50 one third to one half of all antibiotic prescriptions are unnec-
patents. Boddys research has focused on the synthesis of vancomycin. essary. We put 70 percent of the antibiotics we produce in the
He will soon be moving to Stanford University, where as a postdoctoral
fellow he will continue work on antibiotics and anticancer agents. The U.S. each year into our livestock. We add antibiotics to our
authors are indebted to Nicolas Winssinger and Joshua Gruber for dishwashing liquid and our hand soap. In all these ways, we en-
valuable discussions and assistance in preparing this article. courage the weak to die and the strong to become stronger [see

The Challenge of Antibiotic Resistance, by Stuart B. Levy; This selective pressure is the force driving the development of
Scientific American, March 1998]. antibiotics in nature.
Yet even absent the massive societal and medical misuse of Our window onto this biological arms race first opened with
these medications, the unavoidable destiny of any antibiotic is the discovery of penicillin in 1928. Alexander Fleming of St.
obsolescence. Bacteria which grow quickly through many cell Marys Hospital Medical School at London University noticed
divisions a day will always learn something new; some of the that the mold Penicillium notatum was able to kill nearby
strongest will always survive and thrive. So we have had to be- Staphylococcus bacteria growing in agar in a petri dish. Thus
come ever more wily ourselves. was the field of antibiotics born. By randomly testing com-
In the past 10 years, long-standing complacency about van- pounds, such as other molds, to see if they could kill bacteria
quishing infection has been replaced by a dramatic increase in or retard their growth, later researchers were able to identify a
antibacterial research in academic, government and industrial whole suite of antibiotics.
laboratories. Scientists the world over are finding imaginative One of the most successful of these has been vancomycin,
strategies to attack bacteria. Although they will have a limited first identified by Eli Lilly and Company in 1956. Understand-
life span, new antibiotics are being developed using information ing how it works a feat that has taken three decades to ac-
gleaned from genome and protein studies. This exciting research complish has allowed us insight into the mechanism behind a
and drug development is no panacea, but if combined with the class of antibiotics called the glycopeptides, one of the seven or
responsible use of antibiotics, it can offer some hope. Indeed, so major kinds of antibiotics. This insight is proving important
in April 2000 the Food and Drug Administration approved the because vancomycin has become the antibiotic of last resort, the
first new kind of clinical antibiotic in 35 years linezolid and only remaining drug effective against the most deadly of all hos-
several agents are already in the pharmaceutical pipeline. pital-acquired infections: methicillin-resistant Staphylococcus
aureus. And yet vancomycins power like that of the great, ex-
Dismantling the Wall perienced warrior is itself in jeopardy.
almost all the antibiotics that have been developed so Vancomycin works by targeting the bacterial cell wall,
far have come from nature. Scientists have identified them and which surrounds the cell and its membrane, imparting struc-
improved on them, but they certainly did not invent them. Since ture and support. Because human and other mammalian cells
the beginning of life on this planet, organisms have fought over lack such a wall (instead their cells are held up by an internal
limited resources. These battles resulted in the evolution of an- structure called a cytoskeleton), vancomycin and related drugs
tibiotics. The ability to produce such powerful compounds gives are not dangerous to them. This bacterial wall is composed
an organism a fungus or plant or even another species of bac- mostly of peptidoglycan, a material that contains both peptides
teria an advantage over bacteria susceptible to the antibiotic. and sugars (hence its name). As the cell assembles this materi-
RISING RESISTANCE
MANY ANTIBIOTICS are STAPHYLOCOCCUS AUREUS ENTEROCOCCUS FAECIUM STREPTOCOCCUS PNEUMONIAE
VS. PENICILLIN VS. CIPROFLOXACIN (CIPRO) VS. TETRACYCLINE
no longer effective against
certain strains of bacteria,
as these examples
collected from different
hospitals in the late
1990s show. One strain 98%
RESISTANT
70% 10%
of Staphylococcus aureus
found in Korea, for
STAPHYLOCOCCUS AUREUS ENTEROCOCCUS FAECIUM STREPTOCOCCUS PNEUMONIAE
instance, is 98 percent VS. METHICILLIN VS. AMPICILLIN VS. PENICILLIN
resistant to penicillin (top
left); another, found in
the U.S., is 32 percent
resistant to methicillin 32% 37%
(bottom left). All these
70%
SLIM FILMS
strains are not resistant to

vancomycin, for now.

ala constant process, because old peptidoglycan needs to be rowed this idea and attached hydrophobic chains to vancomycin,
replaced as it breaks downsugar units are linked together by creating an analogue called LY333328. The drug connects to the
an enzyme called transglycosidase to form a structural core. cell membrane in high concentrations, allowing it more purchase
Every other sugar unit along this core has a short peptide chain and, as a consequence, more power against peptidoglycan. This
attached to it. Each peptide chain is composed of five amino analogue is effective against VRE and is now in clinical trials.
acids, the last three being an L-lysine and two D-alanines. An Other glycopeptide antibiotics use a different strategy: dimer-
enzyme called transpeptidase then hooks these peptide chains ization. This process occurs when two molecules bind to each
together, removing the final D-alanine and attaching the penul- other to form a single complex. By creating couples, or dimers,
timate D-alanine to an L-lysine from a different sugar chain. As of vancomycin, researchers can enhance the drugs strength. One
a result, the sugar chains are crocheted together through their vancomycin binds to peptidoglycan, bringing the other half of
peptide chains. All this linking and cross-linking creates a thick- the pair the other molecule of vancomycin into proximity as
ly woven material essential for the cells survival: without it, the well. The drug is more effective because more of it is present.
cell would burst from its own internal pressure. One of the aims of our laboratory is to alter vancomycin so it
Vancomycin meddles in the formation of this essential ma- pairs up more readily, and we have recently developed a num-
terial. The antibiotic is perfectly suited to bind to the peptide ber of dimeric vancomycin molecules with exceptional activity
chains before they are linked to one another by transpeptidase. against VRE.
The drug fastens onto the terminal D-alanines, preventing the Even so, the good news may be short-lived. A second mech-
enzyme from doing its work. Without the thicket of cross-link- anism by which VRE foils vancomycin has recently been dis-
ing connections, peptidoglycan becomes weak, like an ill-woven covered. Rather than substituting an atom in the final D-ala-
fabric. The cell wall rends, and cell death rapidly occurs. nine, the bacterium adds an amino acid that is much larger than
D-alanine to the very end of the peptide chain. Like a muscular
Resisting Resistance bouncer blocking a doorway, the amino acid prevents van-
vancomycins lovely fit at the end of the peptide chain comycin from reaching its destination.
is the key to its effectiveness as an antibiotic. Unfortunately, its One method by which the deadly S. aureus gains resistance
peptide connection is also the key to resistance on the part of is becoming clear as well. The bacterium thickens the peptido-
bacteria. In 1998 vancomycin-resistant S. aureus emerged in glycan layer but simultaneously reduces the linking between the
three geographic locations. Physicians and hospital workers are peptide fragments. So it makes no difference if vancomycin
increasingly worried that these strains will become widespread, binds to D-alanine: thickness has replaced interweaving as the
leaving them with no treatment for lethal staph infections. source of the peptidoglycans strength. Vancomycins meddling
Understanding resistance offers the possibility of overcom- has no effect.
ing it, and so scientists have focused on another bacterium that
has been known to be resistant to the powerful drug since the The Cutting Edge
late 1980s: vancomycin-resistant enterococci (VRE). In most en- as the story of vancomycin shows, tiny molecular alterations
terococci bacteria, vancomycin does what it does best: it binds can make all the difference, and bacteria find myriad strategies
to the terminal two D-alanines. At a molecular level, this bind- to outwit drugs. Obviously, the need for new, improved or even
ing entails five hydrogen bonds think of them as five fingers revived antibiotics is enormous. Historically, the drug discovery
clasping a ball. But in VRE, the peptide chain is slightly differ- process identified candidates using whole-cell screening, in
ent. Its final D-alanine is altered by a simple substitution: an oxy- which molecules of interest were applied to living bacterial cells.
gen replaces a pair of atoms consisting of a nitrogen bonded to This approach has been very successful and underlies the dis-
a hydrogen. In molecular terms, this one substitution means that covery of many drugs, including vancomycin. Its advantage lies
vancomycin can bind to the peptide chain with only four hy- in its simplicity and in the fact that every possible drug target in
drogen bonds. The loss of that one bond makes all the differ- the cell is screened. But screening such a large number of tar-
ence. With only four fingers grasping the ball, the drug cannot gets also has a drawback. Various targets are shared by both
hold on as well, and enzymes pry it off, allowing the peptide bacteria and humans; compounds that act against those are tox-
chains to link up and the peptidoglycan to become tightly wo- ic to people. Furthermore, researchers gain no information
ven once again. One atomic substitution reduces the drugs ac- about the mechanism of action: chemists know that an agent
tivity by a factor of 1,000. worked, but they have no information about how. Without this
Researchers have turned to other members of the glycopep- critical information it is virtually impossible to bring a new drug
tide class of antibiotics to see if some have a strategy that van- all the way to the clinic.
comycin could adopt against VRE. It turns out that some mem- Molecular-level assays provide a powerful alternative. This
bers of the group have long, hydrophobic that is, oily chains form of screen identifies only those compounds that have a
attached to them that have proved useful. These chains prefer to specified mechanism of action. For instance, one such screen
be surrounded by other hydrophobic molecules, such as those would look specifically for inhibitors of the transpeptidase en-
that make up the cell membrane, which is hidden behind the pro- zyme. Although these assays are difficult to design, they yield
tective peptidoglycan shield. Researchers at Eli Lilly have bor- potential drugs with known modes of action. The trouble is that

only one enzyme is usually investigated at a time. It would be the laboratory. Now a technique called in vivo expression tech-
a vast improvement in the drug discovery process if researchers nology (IVET) can insert a unique sequence of DNA, a form
could review more than one target simultaneously, as they do of tag that deactivates a gene, into each bacterial gene. Tagged
in the whole-cell process, but also retain the implicit knowledge bacteria are then used to infect an organism. The bacteria are
of the way the drug works. Scientists have accomplished this later recovered and the tags identified. The disappearance of
feat by figuring out how to assemble the many-enzyme path- any tags means that the genes they were attached to were es-
way of a certain bacterium in a test tube. Using this system, they sential for the bacterias survival so essential that the bacteria
can identify molecules that either strongly disrupt one of the en- could not survive in the host without the use of those genes.
zymes or subtly disrupt many of them. Investigators have long hoped that by identifying and in-
Automation and miniaturization have also significantly im- hibiting these virulence factors, they can allow the bodys im-
proved the rate at which compounds can be screened. Robot- mune system to combat pathogenic bacteria before they gain a
ics in so-called high-throughput machines allow scientists to re- foothold. And it seems that the hypothesis is bearing fruit. In a
view thousands of compounds per week. At the same time, recent study, an experimental molecule that inhibited a virulence
miniaturization has cut the cost of the process by using ever factor of the dangerous S. aureus permitted infected mice to re-
smaller amounts of reagents. In the new ultrahigh-throughput sist and overcome infection.
screening systems, hundreds of thousands of compounds can In addition to identifying essential genes and virulence fac-
be looked at cost-effectively in a single day. Accordingly, tors, researchers are discovering which genes confer antibiotic
chemists have to work hard to keep up with the demand for mol- resistance. Targeting them provides a method to rejuvenate pre-
ecules. Their work is made possible by new methods in combi- viously ineffective antibiotics. This is an approach used with -
natorial chemistry, which allows them to design huge libraries lactam antibiotics such as penicillin. The most common mech-
of compounds quickly [see Combinatorial Chemistry and New anism of resistance to -lactam antibiotics is the bacterial
Drugs, by Matthew J. Plunkett and Jonathan A. Ellman; Sci- production of an enzyme called -lactamase, which breaks one
entific American, April 1997]. In the future, some of these of the antibiotics chemical bonds, changing its structure and
new molecules will most likely come from bacteria themselves. preventing it from inhibiting the enzyme transpeptidase. If -lac-
By understanding the way these organisms produce antibiotics, tamase is silenced, the antibiotics remain useful. A -lactamase
scientists can genetically engineer them to produce new related inhibitor called clavulanic acid does just that and is mixed with
molecules. amoxicillin to create an antibiotic marketed as Augmentin.
In the near future, with improvements in the field of DNA
The Genomic Advantage transcriptional profiling, it will become routine to identify re-
the methodology of drug design and screening has benefit- sistance determinants, such as -lactamase, and virulence fac-
ed tremendously from recent developments in genomics. Infor- tors. Such profiling allows scientists to identify all the genes that
mation about genes and the synthesis of their proteins has al- are in use under different growth conditions in the cell. Virulence
lowed geneticists and chemists to go behind enemy lines and use genes can be determined by identifying bacterial genes whose
inside information against the organism itself. This microbial expression increases on infecting a host. Genes that code for an-
counterintelligence is taking place on several fronts, from sab- tibiotic resistance can be determined by comparing expression
otaging centrally important genes to putting a wrench in the pro- levels in bacteria treated with the antibiotic and those not treat-
duction of a single protein and disrupting a bacteriums ability ed. Though still in its infancy, this technique monitored tiny
to infect an organism or to develop resistance. changes in the number of transcription events. With DNA tran-
Studies have revealed that many of the known targets of an- scriptional profiling, researchers should also be able to deter-
tibiotics are essential genes, genes that cause cell death if they are mine whether certain drugs have entirely new mechanisms of
not functioning smoothly. New genetic techniques are making action or cellular targets that could open up new fields of an-
the identification of these essential genes much faster. For in- tibiotic research.
stance, researchers are systematically analyzing all 6,000 or so
genes of the yeast Saccharomyces cerevisiae for essential genes. Killing the Messenger
Every gene can be experimentally disrupted and its effect on another interesting line of genomic research entails inter-
yeast determined. This effort will ultimately catalogue all the es- fering with bacterial RNA. Most RNA is ribosomal RNA
sential genes and will also provide insight into the action of oth- (rRNA), which forms a major structural component of ribo-
er genes that could serve as targets for new antibiotics. somes, the cellular factories where proteins are assembled. Ri-
The proteins encoded by essential genes are not the only bosomal RNA is vulnerable because it has various places where
molecular-level targets that can lead to antibiotics. Genes that drugs can attach and because it lacks the ability to repair itself.
encode for virulence factors are also important. Virulence fac- In 1987 scientists determined that antibiotics in the aminogly-
tors circumvent the hosts immune response, allowing bacte- coside group which includes streptomycin bind to rRNA,
ria to colonize. In the past, it has been quite hard to identify causing the ribosome to misread the genetic code for protein as-
these genes because they are turned on, or transcribed, by sembly. Many of these antibiotics, however, are toxic and have
events in the hosts tissue that are very difficult to reproduce in only limited usefulness. Recently scientists at the Scripps Re-

search Institute in La Jolla, Calif., have reported a new synthetic vent it from interacting with a required protein activator, thus
aminoglycoside dimer that may have less toxicity. inhibiting the replication of HIV. This proof-of-principle ex-
Investigators can also interfere with messenger RNA (mRNA), periment should help pave the way for further studies of mRNA
which directs the assembly of proteins and travels between the as a drug development target.
genetic code and the ribosome. Messenger RNA is created by Scientific interest has been intense in another approach,
reading one strand of the DNA, using the same nucleic acid, or called antisense therapy. By generating sequences of nucleotides
base pair, interactions that hold the double helix together. The that bind perfectly with a specific mRNA sequence, investiga-
mRNA molecule then carries its message to the ribosome, where tors can essentially straitjacket the mRNA. It cannot free itself
a protein is assembled through the process of translation. Because from the drug, which either destroys it or inhibits it from acting.
each mRNA codes for a specific protein and is distinct from oth- Although the FDA has recently approved the first antisense drug
er mRNAs, researchers have the opportunity to create interac- to treat human cytomegalovirus infections, antisense for bacte-
tions between small organic moleculesthat is, not proteins rial infections has not succeeded yet for several reasons, includ-
and specific mRNAs. Parke-Davis chemists have been able to ing toxicity and the challenge of getting enough of the drug to
use such an approach to combat HIV infection. They identified the right spot. Nevertheless, the approach holds promise.
molecules that bind to a part of an mRNA sequence and pre- As is clear, all these genomic insights are making it possi-
ble to identify and evaluate a range of new biological targets
against which chemists can direct their small, bulletlike mole-
cules. A number of antibiotics developed in the past century
ANTIBIOTICS cannot be used, because they harm us. But by comparing a po-
tential targets genetic sequence with the genes found in hu-
mans, researchers can identify genes that are unique to bacte-
AT WORK ria and can focus on those. Similarly, by comparing a targets

genetic sequence to those of other bacteria, they are able to eval-
uate the selectivity of a drug that would be generated from it.
EXISTING ANTIBIOTICS fight infections by A target sequence that appears in all bacteria would very like-
preventing bacteria from making essential ly generate an antibiotic active against many different bacteria:
substances. Vancomycin and -lactam a broad-spectrum antibiotic. In contrast, a target sequence that
appears in only a few bacterial genomes would generate a nar-
antibiotics interfere with synthesis of the cell
row-spectrum antibiotic.
wall (1). Erythromycin and tetracycline disrupt If physicians can identify early on which strain is causing an
ribosomes that make proteins (2). Quinolone infection, they can hone their prescription to a narrow-spectrum
antibiotics inhibit enzymes involved in antibiotic. Because this drug would affect only a subset of the
replicating DNA (3), and sulfonamide antibiotics bacterial population, selective pressure for the development of
also interfere with DNA synthesis (not shown). resistance would be reduced. Advances in the high-speed repli-
cation of DNA and transcriptional profiling may soon make
identification of bacterial strains a routine medical procedure.
CELL WALL 1 Although the picture looks brighter than it has for several
decades, it is crucial that we recognize that the biological arms
PROTEIN ANTIBIOTIC
race is an ancient one. For every creative counterattack we make,
bacteria will respond in kind changing perhaps one atom in
one amino acid. There will always be young warriors to chal-
lenge the old ones. The hope is that we exercise restraint and that
we use our ever expanding arsenal of weapons responsibly, not
relegating them so quickly to obsolescence.
2 3 MORE TO E XPLORE
The Coming Plague: Newly Emerging Diseases in a World out of Balance. Laurie
Garrett. Penguin USA, 1995.
RIBOSOME The Chemistry, Biology, and Medicine of the Glycopeptide Antibiotics. K. C.

ENZYME Nicolaou, Christopher N. C. Boddy, Stefan Brse and Nicolas Winssinger in
Angewandte Chemie International Edition, Vol. 38, No. 15, pages 20962152;
JEFF JOHNSON
August 2, 1999.
Genome Prospecting. Barbara R. Jasny and Pamela J. Hines in Science, Vol. 286,
pages 443491; October 15, 1999.

EDIBLE
Originally published in
September 2000
VACCINES
One day children may
get immunized by
munching on foods
instead of enduring
shots. More important,
food vaccines might
save millions who now
die for lack of access to
traditional inoculants
by William H. R. Langridge
JOHNSON & FANCHER
FOODS UNDER STUDY as alternatives to injectable vac-

cines include bananas, potatoes and tomatoes, as well as
lettuce, rice, wheat, soybeans and corn.

V accines have accomplished near miracles in
the fight against infectious disease. They have
consigned smallpox to history and should soon do
the same for polio. By the late 1990s an international cam-
paign to immunize all the worlds children against six devas-
tating diseases was reportedly reaching 80 percent of infants
toimmunityin which the bodys defenses mistakenly attack
normal, uninfected tissues. Among the autoimmune disor-
ders that might be prevented or eased are type I diabetes (the
kind that commonly arises during childhood), multiple scle-
rosis and rheumatoid arthritis.
(up from about 5 percent in the mid-1970s) and was reduc- By Any Other Name
ing the annual death toll from those infections by roughly
three million.
Yet these victories mask tragic gaps in delivery. The 20 per-
cent of infants still missed by the six vaccines against diph-
R egardless of how vaccines for infectious diseases are de-
livered, they all have the same aim: priming the immune
system to swiftly destroy specific disease-causing agents, or
theria, pertussis (whooping cough), polio, measles, tetanus pathogens, before the agents can multiply enough to cause
and tuberculosis account for about two million unnecessary symptoms. Classically, this priming has been achieved by pre-
deaths each year, especially in the most remote and impover- senting the immune system with whole viruses or bacteria
ished parts of the globe. Upheavals in many developing na- that have been killed or made too weak to proliferate much.
tions now threaten to erode the advances of the recent past, On detecting the presence of a foreign organism in a vac-
and millions still die from infectious diseases for which immu- cine, the immune system behaves as if the body were under
nizations are nonexistent, unreliable or too costly. attack by a fully potent antagonist. It mobilizes its various
This situation is worrisome not only for the places that forces to root out and destroy the apparent invader target-
lack health care but for the entire world. Regions harboring ing the campaign to specific antigens (proteins recognized as
infections that have faded from other areas are like bombs foreign). The acute response soon abates, but it leaves be-
ready to explode. When environmental or social disasters hind sentries, known as memory cells, that remain on
undermine sanitation systems or displace communities alert, ready to unleash whole armies of defenders if the real
bringing people with little immunity into contact with carri- pathogen ever finds its way into the body. Some vaccines
ersinfections that have been long gone from a population provide lifelong protection; others (such as those for cholera
can come roaring back. Further, as international travel and and tetanus) must be readministered periodically.
trade make the earth a smaller place, diseases that arise in Classic vaccines pose a small but troubling risk that the
one locale are increasingly popping up continents away. Un- vaccine microorganisms will somehow spring back to life,
til everyone has routine access to vaccines, no one will be en- causing the diseases they were meant to forestall. For that
tirely safe. reason, vaccine makers today favor so-called subunit prepa-
In the early 1990s Charles J. Arntzen, then at Texas A&M rations, composed primarily of antigenic proteins divorced
University, conceived of a way to solve many of the prob- from a pathogens genes. On their own, the proteins have no
lems that bar vaccines from reaching all too many children way of establishing an infection. Subunit vaccines, however,
in developing nations. Soon after learning of a World Health are expensive, in part because they are produced in cultures
Organization call for inexpensive, oral vaccines that needed of bacteria or animal cells and have to be purified out; they
no refrigeration, Arntzen visited Bangkok, where he saw a also need to be refrigerated.
mother soothe a crying baby by offering a piece of banana. Food vaccines are like subunit preparations in that they are
Plant biologists had already devised ways of introducing se- engineered to contain antigens but bear no genes that would
lected genes (the blueprints for proteins) into plants and in- enable whole pathogens to form. Ten years ago Arntzen un-
ducing the altered, or transgenic, plants to manufacture derstood that edible vaccines would therefore be as safe as
the encoded proteins. Perhaps, he mused, food could be ge- subunit preparations while sidestepping their costs and de-
netically engineered to produce vaccines in their edible parts, mands for purification and refrigeration. But before he and
which could then be eaten when inoculations were needed. others could study the effects of food vaccines in people, they
The advantages would be enormous. The plants could be had to obtain positive answers to a number of questions.
grown locally, and cheaply, using the standard growing Would plants engineered to carry antigen genes produce
methods of a given region. Because many food plants can be functional copies of the specified proteins? When the food
regenerated readily, the crops could potentially be produced plants were fed to test animals, would the antigens be de-
indefinitely without the growers having to purchase more graded in the stomach before having a chance to act? (Typi-
seeds or plants year after year. Homegrown vaccines would cal subunit vaccines have to be delivered by injection precise-
also avoid the logistical and economic problems posed by ly because of such degradation.) If the antigens did survive,
having to transport traditional preparations over long dis- would they, in fact, attract the immune systems attention?
tances, keeping them cold en route and at their destination. And would the response be strong enough to defend the ani-
And, being edible, the vaccines would require no syringes mals against infection?
which, aside from costing something, can lead to infections Additionally, researchers wanted to know whether edible
if they become contaminated. vaccines would elicit what is known as mucosal immunity.
Efforts to make Arntzens inspired vision a reality are still Many pathogens enter the body through the nose, mouth or
quite preliminary. Yet studies carried out in animals over the other openings. Hence, the first defenses they encounter are
past 10 years, and small tests in people, encourage hope that those in the mucous membranes that line the airways, the di-
edible vaccines can work. The research has also fueled spec- gestive tract and the reproductive tract; these membranes
ulation that certain food vaccines might help suppress au- constitute the biggest pathogen-deterring surface in the body.

HOW TO MAKE AN EDIBLE VACCINE
One way of generating edible vaccines relies on the antigens proteins that elicit a targeted immune
bacterium Agrobacterium tumefaciens to deliver into response in the recipient.The diagram illustrates the
plant cells the genetic blueprints for viral or bacterial production of vaccine potatoes.
BACTERIAL CELL PLANT CELL

ANTIGEN GENE
DNA
PLASMID
ANTIBIOTIC-
RESISTANCE GENE TRANSFER
BACTERIAL
GENE SUSPENSION DEAD CELL CALLUS
VACCINE
POTATOES
ANTIBIOTIC MEDIUM
4 Allow callus to sprout

1 Cut leaf. 2 Expose leaf to bacte- 3 Expose leaf to an anti-
JARED SCHNEIDMAN DESIGN

shoots and roots.
ria carrying an antigen biotic to kill cells that lack
gene and an antibiotic- the new genes. Wait for 5 Put in soil. Within three
resistance gene. Allow surviving (gene-altered) months, the plantlets will
bacteria to deliver the cells to multiply and form grow into plants bearing
genes into leaf cells. a clump (callus). antigen-laden vaccine potatoes.
When the mucosal immune response is deadly when rehydration therapy is not ly or partly protect animals from subse-
effective, it generates molecules known an option. No vaccine practical for quent exposure to the real pathogens or,
as secretory antibodies that dash into wide distribution in the developing na- in the case of V. cholerae and enterotox-
the cavities of those passageways, neu- tions is yet available to prevent these ills. igenic E. coli, to microbial toxins. Edi-
tralizing any pathogens they find. An ef- By 1995 researchers attempting to an- ble vaccines have also provided laborato-
fective reaction also activates a systemic swer the many questions before them ry animals with some protection against
response, in which circulating cells of the had established that plants could indeed challenge by the rabies virus, Helicobac-
immune system help to destroy invaders manufacture foreign antigens in their ter pylori (a bacterial cause of ulcers)
at distant sites. proper conformations. For instance, and the mink enteric virus (which does
Injected vaccines initially bypass mu- Arntzen and his colleagues had intro- not affect humans).
cous membranes and typically do a poor duced into tobacco plants the gene for It is not entirely surprising that anti-
job of stimulating mucosal immune re- a protein derived from the hepatitis B gens delivered in plant foods survive the
sponses. But edible vaccines come into virus and had gotten the plants to syn- trip through the stomach well enough
contact with the lining of the digestive thesize the protein. When they injected to reach and activate the immune sys-
tract. In theory, then, they would activate the antigen into mice, it activated the tem. The tough outer wall of plant cells
both mucosal and systemic immunity. same immune system components that apparently serves as temporary armor
That dual effect should, in turn, help im- are activated by the virus itself. (Hep- for the antigens, keeping them relatively
prove protection against many danger- atitis B can damage the liver and con- safe from gastric secretions. When the
ous microorganisms, including, impor- tribute to liver cancer.) wall finally begins to break up in the in-
tantly, the kinds that cause diarrhea. testines, the cells gradually release their
Those of us attempting to develop Green Lights on Many Fronts antigenic cargo.
food vaccines place a high priority on Of course, the key question is whether
combating diarrhea. Together the main
causes the Norwalk virus, rotavirus,
Vibrio cholerae (the cause of cholera)
B ut injection is not the aim; feeding
is. In the past five years experiments
conducted by Arntzen (who moved to
food vaccines can be useful in people.
The era of clinical trials for this technol-
ogy is just beginning. Nevertheless, Arnt-
and enterotoxigenic Escherichia coli (a the Boyce Thompson Institute for Plant zen and his collaborators obtained reas-
toxin-producing source of travelers Research at Cornell University in 1995) suring results in the first published hu-
diarrhea) account for some three and his collaborators and by my group man trial, involving about a dozen
million infant deaths a year, mainly in at Loma Linda University have demon- subjects. In 1997 volunteers who ate
developing nations. These pathogens strated that tomato or potato plants pieces of peeled, raw potatoes contain-
disrupt cells of the small intestine in can synthesize antigens from the Nor- ing a benign segment of the E. coli toxin
ways that cause water to flow from the walk virus, enterotoxigenic E. coli, V. (the part called the B subunit) displayed
blood and tissues into the intestine. The cholerae and the hepatitis B virus. More- both mucosal and systemic immune re-
resulting dehydration may be combated over, feeding antigen-laced tubers or sponses. Since then, the group has also
by delivering an intravenous or oral so- fruits to test animals can evoke mucosal seen immune reactivity in 19 of 20 peo-
lution of electrolytes, but it often turns and systemic immune responses that ful- ple who ate a potato vaccine aimed at

the Norwalk virus. Similarly, after Hil- tors (adjuvants) and better targeting to mune responses to the added antigens.
ary Koprowski of Thomas Jefferson the immune system might compensate in The B subunit also tends to associate
University fed transgenic lettuce carrying part for low antigen production. with copies of itself, forming a dough-
a hepatitis B antigen to three volunteers, One targeting strategy involves linking nut-shaped, five-membered ring with a
two of the subjects displayed a good sys- antigens to molecules that bind well to hole in the middle. These features raise
temic response. Whether edible vaccines immune system components known as the prospect of producing a vaccine
can actually protect against human dis- M cells in the intestinal lining. M cells that brings several different antigens to
ease remains to be determined, however. take in samples of materials that have M cells at oncethus potentially ful-
entered the small intestine (including fulling an urgent need for a single vac-
Still to Be Accomplished pathogens) and pass them to other cells cine that can protect against multiple
of the immune system, such as antigen- diseases simultaneously.
I n short, the studies completed so far

in animals and people have provided
a proof of principle; they indicate that
presenting cells. Macrophages and oth-
er antigen-presenting cells chop up their
acquisitions and display the resulting
Researchers are also grappling with
the reality that plants sometimes grow
poorly when they start producing large
the strategy is feasible. Yet many issues protein fragments on the cell surface. If amounts of a foreign protein. One solu-
must still be addressed. For one, the white blood cells called helper T lym- tion would be to equip plants with reg-
amount of vaccine made by a plant is phocytes recognize the fragments as ulatory elements that cause antigen genes
low. Production can be increased in dif- foreign, they may induce B lympho- to turn onthat is, give rise to the encod-
ferent waysfor instance, by linking cytes (B cells) to secrete neutralizing an- ed antigensonly at selected times (such
antigen genes with regulatory elements tibodies and may also help initiate a as after a plant is nearly fully grown or
known to help switch on the genes more broader attack on the perceived enemy. is exposed to some outside activator mol-
readily. As researchers solve that chal- It turns out that an innocuous seg- ecule) or only in its edible regions. This
lenge, they will also have to ensure that ment of the V. cholerae toxin the B work is progressing.
any given amount of a vaccine food pro- subunit binds readily to a molecule on Further, each type of plant poses its
vides a predictable dose of antigen. M cells that ushers foreign material into own challenges. Potatoes are ideal in
Additionally, workers could try to en- those cells. By fusing antigens from oth- many ways because they can be propa-
hance the odds that antigens will activate er pathogens to this subunit, it should gated from eyes and can be stored for
the immune system instead of passing be possible to improve the uptake of long periods without refrigeration. But
out of the body unused. General stimula- antigens by M cells and to enhance im- potatoes usually have to be cooked to be
VACCINE
POTATO
HOW EDIBLE VACCINES PROVIDE PROTECTION
An antigen in a food vaccine gets taken up by M infectious agent, not just part of one. That re-
cells in the intestine (below, left) and passed to sponse leaves long-lasting memory cells able
various immune-system cells,which then launch to promptly neutralize the real infectious agent
a defensive attack as if the antigen were a true if it attempts an invasion (right).
ANTIGEN FROM ARRIVING

VACCINE VIRUS
3 Antibodies
quickly
INFECTED neutralize
M CELL ANTIBODY CELL the invader
B CELL
1 M cells pass 4 Activated MEMORY
the antigen to B cells make B CELL
macrophages and release
and B cells
STIMULATORY antibodies 2 Memory
SECRETIONS helper T cells
able to
MACROPHAGE 3 T cells neutralize CYTOTOXIC swiftly stimulate
stimulate the antigen T CELL antibody secretion
2 Macrophages B cells and
display HELPER seek out 1 Memory MEMORY
pieces of T CELL antigens at helper T cells HELPER
the antigen distant sites prod cytotoxic T CELL

to helper T cells to attack
T cells infected cells
INITIAL RESPONSE WHEN A DISEASE AGENT APPEARS

palatable, and heating can denature pro- than most of us expected. sideration are lettuce, carrots, peanuts,
teins. Indeed, as is true of tobacco plants, Bananas need no cooking and are rice, wheat, corn and soybeans.
potatoes were not initially intended to be grown widely in developing nations, but In another concern, scientists need to
used as vaccine vehicles; they were stud- banana trees take a few years to mature, be sure that vaccines meant to enhance
ied because they were easy to manipu- and the fruit spoils fairly rapidly after immune responses do not backfire and
late. Surprisingly, though, some kinds of ripening. Tomatoes grow more quickly suppress immunity instead. Research
potatoes are actually eaten raw in South and are cultivated broadly, but they too into a phenomenon called oral tolerance
America. Also, contrary to expectations, may rot readily. Inexpensive methods has shown that ingesting certain pro-
cooking of potatoes does not always de- of preserving these foodssuch as dry- teins can at times cause the body to shut
stroy the full complement of antigen. So ingmight overcome the spoilage prob- down its responses to those proteins. To
potatoes may have more practical merit lem. Among the other foods under con- determine safe, effective doses and feed-
ing schedules for edible vaccines, manu-
facturers will need to gain a better han-
dle on the manipulations that influence
Moving against Malnutrition whether an orally delivered antigen will

stimulate or depress immunity.
A final issue worth studying is whether
food vaccines ingested by mothers can
A s research into edible vaccines is progressing,so too are efforts to make foods
more nutritious. A much publicized example,golden rice,takes aim at vi-
tamin A deficiency, rampant in many parts of Asia, Africa and Latin America.This
indirectly vaccinate their babies. In the-
ory, a mother could eat a banana or two
and thus trigger production of antibod-
condition can lead to blindness and to immune impairment,which contributes to ies that would travel to her fetus via the
the death of more than a million children each year. placenta or to her infant via breast milk.
Nonscientific challenges accompany
Rice would be a convenient way to deliver the needed vitamin, because the
the technical ones. Not many pharma-
grain is a daily staple for a third or more of all people on the earth. But natural va- ceutical manufacturers are eager to sup-
rieties do not supply vitamin A. Golden rice, though, has been genetically altered port research for products targeted pri-
to make beta-carotene, a pigment the body converts to vitamin A. marily to markets outside the lucrative
A team led by Ingo Potrykus of the Swiss Federal Institute of Technology and West. International aid organizations and
Peter Beyer of the University of Freiburg in Germany formally reported its creation some national governments and philan-
this past January in Science. In May an agribusinessZenecabought the rights thropies are striving to fill the gap, but
the effort to develop edible vaccines re-
and agreed to allow the rice to be donated to facilities that will cross the beta-
mains underfunded.
carotene trait into rice species popular in impoverished areas and will distribute In addition, edible vaccines fall under
the resulting products to farmers at no charge. (Zeneca is hoping to make its the increasingly unpopular rubric of ge-
money from sales of the improved rice in richer countries, where beta-carotenes netically modified plants. Recently a
antioxidant properties are likely to have appeal.) British company (Axis Genetics) that
Golden rice is not yet ready to be commercialized, however. Much testing still was supporting studies of edible vaccines
lies ahead, including analyses of whether the human body can efficiently absorb failed; one of its leaders lays at least
part of the blame on investor worry
the beta-carotene in the rice.Testing is expected to last at least until 2003.
about companies involved with geneti-
Meanwhile scientists are trying to enrich rice with still more beta-carotene,with cally engineered foods. I hope, however,
other vitamins and with minerals. At a conference last year Potrykus announced that these vaccines will avoid serious
success with iron; more than two billion people worldwide are iron deficient. controversy, because they are intended
Investigators are attempting to enhance other foods as well. In June, for in- to save lives and would probably be
stance, a group of British and Japanese investigators reported the creation of a planted over much less acreage than oth-
tomato containing a gene able to supply three times the usual amount of beta- er food plants (if they are raised outside
carotene.Conventional breeding methods are being used,too,such as in an inter- of greenhouses at all). Also, as drugs,
they would be subjected to closer scruti-
national project focused on increasing the vitamin and mineral content of rice
ny by regulatory bodies.
and four other staples wheat, corn, beans and cassava.
Not everyone is thrilled by the recent genetic coups. Genetically modified (GM) Fighting Autoimmunity
foods in general remain controversial.Some opponents contend that malnutrition
can be combated right now in other wayssay,by constructing supply roads.And
they fear that companies will tout the benefits of the new foods to deflect attention C onsideration of one of the chal-
lenges detailed herethe risk of in-
ducing oral tolerancehas recently led
from worries over other GM crops,most of which (such as plants designed to resist
my group and others to pursue edible
damage from pesticides) offer fewer clear advantages for consumers. High on the
vaccines as tools for quashing autoim-
list of concerns are risk to the environment and to people.Supporters of the nutri- munity. Although oral delivery of anti-
tionally improved foods hope,however,that the rice wont be thrown out with the gens derived from infectious agents of-
rinse water. Ricki Rusting,staff writer ten stimulates the immune system, oral
delivery of autoantigens (proteins de-
rived from uninfected tissue in a treated

individual) can sometimes
suppress immune activity
a phenomenon seen fre-
STOPPING AUTOIMMUNITY
INTESTINAL
quently in test animals. The autoimmune reaction responsible for type I diabetes CAVITY
No one fully understands arises when the immune system mistakes proteins that are
the reasons for this differ- AUTO-
made by pancreatic beta cells (the insulin producers) for for- ANTIGEN
ence. eign invaders.The resulting attack, targeted to the offending M CELL DELIVERED
Some of the evidence proteins, or autoantigens, destroys the beta cells (below, IN FOOD
that ingesting autoanti- left). Eating small amounts of autoantigens quiets the pro- SUPPRESSOR
gens, or self-antigens, cess in diabetic mice, for unclear reasons. The autoantigens T CELL
might suppress autoimmu- might act in part by switching on suppressor cells of the
nity comes from studies immune system (inset), which then block the destructive ac-
of type I diabetes, which tivities of their cousins (below,right). Activated
results from autoimmune HELPER
suppressor cells
STIMULATORY go to pancreas
destruction of the insulin- SECRETIONS T CELL
producing cells (beta cells) CYTOTOXIC
of the pancreas. This de- T CELL
struction progresses si-
lently for a time. Eventu-
ally, though, the loss of AUTOANTIGEN
beta cells leads to a dras-
B CELL
tic shortage of insulin, a NATURAL
hormone needed to help KILLER SUPPRESSIVE
CELL SECRETIONS
cells take up sugar from
the blood for energy. The ANTIBODY
loss results in high blood DAMAGED
AREA PRESERVED
sugar levels. Insulin injec-

MACROPHAGE BETA CELL
BETA CELL
tions help to control dia- UNDER ATTACK DESTRUCTIVE
betes, but they are by no IN PANCREAS SECRETIONS
means a cure; diabetics
face an elevated risk of se- BEFORE TREATMENT AFTER TREATMENT
vere complications.
In the past 15 years, in-
vestigators have identified several beta plant-based diabetes vaccines, such as diseases. But, as is true for infectious dis-
cell proteins that can elicit autoimmuni- potatoes containing insulin or GAD eases, investigators are exploring a num-
ty in people predisposed to type I dia- linked to the innocuous B subunit of the ber of promising schemes to overcome
betes. The main culprits, however, are V. cholerae toxin (to enhance uptake of that and other challenges.
insulin and a protein called GAD (glu- the antigens by M cells). Feeding of the Edible vaccines for combating au-
tamic acid decarboxylase). Researchers vaccines to a mouse strain that becomes toimmunity and infectious diseases have
have also made progress in detecting diabetic helped to suppress the immune a long way to go before they will be
when diabetes is brewing. The next attack and to prevent or delay the onset ready for large-scale testing in people.
position, policy, decision or endorsement of the federal government or of the National Medical Technology Testbed, Inc.
step, then, is to find ways of stopping of high blood sugar. The technical obstacles, though, all seem
The views, opinions and/or findings contained in this report are those of the author and should not be construed as a
the underground process before any Transgenic plants cannot yet produce surmountable. Nothing would be more
symptoms arise. the amounts of self-antigens that would satisfying than to protect the health of
To that end, my colleagues and I, as be needed for a viable vaccine against many millions of now defenseless chil-
well as other groups, have developed human diabetes or other autoimmune dren around the globe. SA
The Author Further Information

WILLIAM H. R. LANGRIDGE, a leader in the ef- Oral Immunization with a Recombinant Bacterial Antigen Produced in
fort to develop edible vaccines for infectious and au- Transgenic Plants. Charles J. Arntzen in Science, Vol. 268, No. 5211, pages
toimmune diseases, is professor in the department of 714716; May 5, 1995.
biochemistry and at the Center for Molecular Biolo- Immunogenicity in Humans of a Recombinant Bacterial Antigen Deliv-
gy and Gene Therapy at the Loma Linda University ered in a Transgenic Potato. C. O. Tacket et al. in Nature Medicine, Vol. 4,
School of Medicine. After receiving his Ph.D. in bio- No. 5, pages 607609; May 1998.
chemistry from the University of Massachusetts at A Plant-Based Cholera Toxin B Subunit-Insulin Fusion Protein Protects
Amherst in 1973, he conducted genetic research on against the Development of Autoimmune Diabetes. Takeshi Arakawa, Jie
insect viruses and plants at the Boyce Thompson In- Yu, D. K. Chong, John Hough, Paul C. Engen and William H. R. Langridge in
stitute for Plant Research at Cornell University. In Nature Biotechnology, Vol. 16, No. 10, pages 934938; October 1998.
1987 he moved to the Plant Biotechnology Center of Plant-Based Vaccines for Protection against Infectious and Autoim-
the University of Alberta in Edmonton, and he mune Diseases. James E. Carter and William H. R. Langridge in Critical Re-
joined Loma Linda in 1993. views in Plant Sciences (in press).

Originally published in October 1999
The Unmet Challenges of

Hepatitis C
Some 1.8 percent of the U.S. adult pop-
ulation are infected with the hepatitis C
virus, most without knowing it
by Adrian M. Di Bisceglie and Bruce R. Bacon
A s recently as the late 1980s few

people other than physicians
had heard of hepatitis C, a slowly
progressing viral infection that over a couple
of decades can lead to liver failure or liver can-
virus is a major public health problem
throughout the world.
Physicians, historians and military leaders
have long recognized hepatitis inflammation
of the liver as a cause of jaundice. This yel-
cer. Today the condition is widely recognized low discoloration of the whites of the eyes and
as a huge public health concern. Some 1.8 per- skin occurs when the liver fails to excrete a
cent of the U.S. adult population, almost four pigment called bilirubin, which then accumu-
million people, are infected with the hepatitis lates in the body. In recent decades, however,
C virus, most of them without knowing it. the diagnosis of hepatitis has progressively im-
The virus is one of the major causes of chronic proved, and physicians can now distinguish
liver disease, probably accounting for even several distinct forms. At least five different
more cases than excessive alcohol use, and is viruses can cause the condition, as can drugs
the most common reason for liver transplants. and toxins such as alcohol.
Some 9,000 people die each year in the U.S. Researchers first studied viral hepatitis in the
from complications of the infection, a number 1930s and 1940s in settings where jaundice
that is expected to triple by 2010. Information was common, such as prisons and mental insti-
about the incidence of hepatitis C in other tutions. They identified two distinct forms with
countries is less reliable, but it is clear that the different patterns of transmission. One was

transmitted by contact with feces of in- from infected chimpanzees provided by authorities to screen all blood donated
fected individuals and was called infec- Daniel W. Bradley of the Centers for to blood banks for signs of infection.
tious hepatitis, or hepatitis A. The other Disease Control and Prevention. Hepati- The antibody test soon showed hepati-
appeared to be passed only through tis C accounts for most cases of viral hep- tis C to be a much bigger threat to public
blood and was termed serum hepatitis, atitis that are not types A or B, although health than had generally been recog-
or hepatitis B. a few result from other, rarer viruses. nized. A remarkable feature one that
An important development occurred sets it apart from most other viruses is
in the 1950s, when researchers devised The Needle in an RNA Haystack its propensity to cause chronic disease.
tests for liver injury based on certain en- Most other viruses are self-limited: infec-
zymes in blood serum. When liver cells
known as hepatocytes die, they release
these enzymes into the circulation, where
H epatitis C virus proved difficult to
identify because it cannot be reli-
ably grown in cell cultures, and chim-
tion with hepatitis A, for example, usu-
ally lasts for only a few weeks. In con-
trast, nearly 90 percent of people with
their concentrations can be easily mea- panzees and tamarins appear to be the hepatitis C have it for years or decades.
sured. Elevated serum levels of alanine only nonhuman animals that can be in- Few patients know the source of their
aminotransferase (ALT) and, especially, fected. Because both species are very virus, but on direct questioning many re-
aspartate aminotransferase (AST) be- expensive to use in research, only small call having a blood transfusion, an epi-
came recognized as more reliable signs numbers of animals can be employed. sode of injection drug use or an injury
of liver trouble than jaundice. (In addi- These obstacles, which still impede the from a hypodermic needle containing
tion to hepatitis, some uncommon inher- study of the virus, explain why it was blood from an infected individual.
ited metabolic diseases can cause elevat- the first infectious agent discovered en- About 40 percent of patients have none
ed liver enzymes.) tirely by cloning nucleic acid. of these clear risk factors but fall into
There things stood until Baruch Blum- The Chiron researchers first extracted one of several categories identified in epi-
berg, working at the National Institutes RNA from serum samples strongly sus- demiologic studies. These include having
of Health, made a breakthrough in the pected to contain the unknown viral had sexual contact with someone with
mid-1960s. Blumberg identified the sig- agent. A chemical variant of DNA, RNA hepatitis, having had more than one sex-
nature of a viral agent, now known as is used by many viruses as their genetic ual partner in the past year, and being of
hepatitis B virus, in the blood of patients material. RNA is also found in healthy low socioeconomic status.
with that disease. Blumbergs discovery cells, so the problem was to identify the Whether hepatitis C is sexually trans-
won him a Nobel Prize and allowed re- tiny fraction corresponding to the un- mitted is controversial. Instances of
searchers to develop reliable blood tests known viral genome. transmission between partners in stable,
for the virus. A decade later Stephen M. The Chiron workers used an enzyme monogamous relationships are rarely
Feinstone, a researcher at the same insti- to copy multiple fragments of DNA from identified, and the rate of infection in
tution, identified a different viral agent the RNA, so that each carried some part promiscuous gay men is no higher than
in the stool of patients with hepatitis A. of its genetic sequence. Next, they insert- in the population in general. These ob-
This work led quickly to the developed this complementary DNA into servations suggest that sexual transmis-
ment of tests that accurately detect anti- viruslike entities that infect Escherichia sion is uncommon, but they are hard to
bodies to hepatitis A virus in the blood coli bacteria, which induced some bacte- reconcile with the epidemiologic find-
of those infected. ria to manufacture protein fragments ings. The paradox has not been resolved.
Hepatitis had long been a significant that the DNA encoded. The researchers Some patients who deny injection drug
risk for recipients of blood transfusions grew the bacteria to form colonies, or use may be unwilling or unable to recall
and blood products. As many as 30 per- clones, that were then tested for their it. Others might have been infected from
cent of patients receiving a blood trans- ability to cause a visible reaction with unsterile razors or tattooing instruments.
fusion in the 1960s developed elevated serum from chimpanzees and a human Shared straws put into the nose and used
levels of ALT and AST, or even jaundice, with non-A, non-B hepatitis. to snort street drugs might also transmit
some weeks later. Workers had suspect- The hope was that antibodies in the the virus via minute amounts of blood.
ed an infectious agent was responsible. serum would bind to any clones produc-
When the new tests for hepatitis A and B ing protein from the infectious agent. Slow Progress
became available in the 1970s, re- Out of a million bacterial clones tested,
searchers soon found that a substantial
proportion of cases of post-transfusion
hepatitis were caused by neither of these
just one was found that reacted with
serum from chimpanzees with the dis-
ease but not with serum from the same
T he discovery of hepatitis C virus and
the development of an accurate test
for it mark an important victory for pub-
two viruses. The new disease was labeled chimpanzees before they had been infect- lic health. The formerly substantial risk
non-A, non-B hepatitis. ed. The result indicated that this clone of infection from a blood transfusion has
Most investigators expected that the contained genetic sequences of the dis- been virtually eliminated. Moreover, the
agent responsible for these cases would ease agent. Using the clone as a toehold, rate of infection appears to be dropping
soon be discovered. In reality, it took investigators subsequently characterized among injection drug users, although this
nearly 15 years before Michael Hough- the remainder of the viruss genetic ma- may be because anti-AIDS campaigns
ton and his colleagues at Chiron Corpo- terial and developed the first diagnostic have discouraged sharing of needles. Yet
ration, a biotechnology company in assay, a test that detects antibodies to hepatitis C still presents numerous chal-
Emeryville, Calif., finally identified the hepatitis C in blood. Since 1990 that test lenges, and the prospects for eradicating
hepatitis C virus, using samples of serum and subsequent versions have allowed the virus altogether appear dismal. At-

How the Hepatitis C CHIMPANZEE WITH
NON-A, NON-B RNA EXTRACTED DNA COPY MADE DNA INCORPORATED
Virus Was Discovered HEPATITIS FROM CELLS FROM RNA INTO BACTERIOPHAGES
R esearchers identified the hepati-

tis C virus by making DNA
copies of RNA from the cells of in-
fected chimpanzees.They cloned the
DNA by using bacteriophages to car-
ry it into bacteria.Colonies were then
tested with serum from infected
chimps. One produced an immune
LAURIE GRACE
reaction,indicating it carried viral ge-
netic sequences. A.M.D.and B.R.B.
tempts to develop a vaccine have been genome responsible for the manufacture found in other organs, such as fibro-
hampered because even animals that suc- of proteins on the outside of the virus to blasts in the skin and mesangial cells in
cessfully clear the virus from their bodies which antibodies might bind. Two such the kidney. They store vitamin A as well
acquire no immunity to subsequent in- hypervariable regions have been identi- as produce the livers extracellular ma-
fection. Moreover, millions of people fied within the so-called envelope regions trix, or framework. It is likely that
who are chronically infected are at risk of the genome. As many as six distinct many of the processes that initiate the
of developing severe liver disease. genotypes and many more subtypes of fibrotic response in the liver occur in
The mechanism of damage is known the virus have been identified; numerous these other tissues as well.
in outline. Viral infections can cause in- variants exist even within a single patient. If fibrosis progresses far enough, it re-
jury either because the virus kills cells di- In contrast to the humoral arm, the sults in cirrhosis, which is characterized
rectly or because the immune system at- cellular arm of the immune system, by bands of fibrosis enclosing nodules of
tacks infected cells. Hepatitis C virus which specializes in viral infections, regenerating hepatocytes. Progression is
causes disease through the second mech- mounts a vigorous defense against hep- faster in people over age 50 at the time
anism. The immune system has two op- atitis C. It appears to be responsible for of infection, in those who consume more
erating divisions. The humoral arm, most of the liver injury. Cytotoxic T than 50 grams of alcohol a day, and in
which is responsible for producing anti- lymphocytes primed to recognize hepati- men, but cirrhosis can result even in pa-
bodies, appears to be largely ineffective tis C proteins are found in the circula- tients who never drink alcohol. Fibrosis
against hepatitis C virus. Although it tion and in the liver of chronically infect- and cirrhosis are generally considered ir-
produces antibodies to various viral ed individuals and are thought to kill reversible, although recent findings cast
components, the antibodies fail to neu- hepatocytes that display viral proteins. some doubt on that conclusion.
tralize the invader, and their presence Fortunately, liver tissue can regenerate About 20 percent of patients develop
does not indicate immunity, as is the case well, but that from hepatitis patients of- cirrhosis over the first 20 years of infec-
with hepatitis B. ten contains numerous dead or dying tion. Thereafter some individuals may
It seems likely that hepatitis C virus hepatocytes, as well as chronic inflam- reach a state of equilibrium without fur-
evades this defense through its high mu- matory cells such as lymphocytes and ther liver damage, whereas others may
tation rate, particularly in regions of its monocytes. continue to experience very slow but
progressive fibrosis. End-stage liver dis-
Long-Term Consequences ease often manifests itself as jaundice, as-
cites (accumulation of fluid within the
I f hepatitis persists for long enough

typically some years the condition
escalates, and normally quiescent cells
abdomen), bleeding from varicose veins
within the esophagus, and confusion.
Hepatitis C infection has also come to be
adjacent to hepatocytes, called hepatic recognized as a major indirect cause of
stellate cells, become abnormally acti- primary liver cancer. The virus itself
FIBROUS
TISSUE vated. These cells then secrete collagen seems not to put people at increased risk,
and other proteins, which disrupt the but cirrhosis induced by the virus does.
FIBROUS TISSUE fine-scale structure of the liver and slow- Cirrhosis is responsible for almost all
SURROUNDING BILE DUCT CHRONIC ly impair its ability to process materials. the illness caused by the hepatitis C
AND BLOOD VESSELS INFLAMMATORY
CELLS This pathology is known as fibrosis. virus. Although a small proportion of
Stellate cells are similar in origin and patients recollect an episode of jaundice
ELIZABETH M. BRUNT Saint Louis University
function to the fibrosis-producing cells when they probably acquired their in-
LIVER TISSUE from patients with hepatitis C often shows fibrosis excess collagen (here
stained blue).The top image shows typical mild fibrosis.The bottom image shows cirrhosis,
a more serious condition in which fibrotic tissue surrounds regenerating nodules of hepa-
tocytes; chronic inflammatory cells are also visible.
BILE DUCT CIRRHOTIC NODULES

BACTERIOPHAGES INFECT BACTERIAL COLONIES SEPARATED
E. COLI BACTERIA
SERUM FROM CHIMPANZEE

WITH NON-A, NON-B HEPATITIS
IS ADDED; COLONY CONTAINING
VIRAL SEQUENCES FURTHER
PRODUCES VISIBLE REACTION STUDIES
fection, chronic hepatitis C is often to enhance interferons effects on the im- been classified with them as a member
asymptomatic. When symptoms do oc- mune system. Interferon and ribavirin of the family Flaviviridae. Enzymes in
cur, they are nonspecific: patients some- given together for six to 12 months can an infected cell use the viral RNA as a
times complain of vague feelings of fa- expunge the virus in about 40 percent of template to produce a single large pro-
tigue, nausea or general unwellness. patients, and clinical workers are now tein called a polyprotein, which then
The insidious nature of the condition is studying how to maximize the benefits cleaves to yield a variety of separate pro-
probably another reason why hepatitis from these two agents. Long-acting teins with different functions. Some are
C remained undiscovered for as long as it forms of interferon that require admin- structural proteins that go to form new
did. The disease plays out over decades. istration only once a week are one focus viral particles; others are enzymes that
An aspect confounding investigators is of interest. replicate the original infecting RNA. At
that not all infected individuals react in A new drug is now being tested in either end of the genome are short
the same way. Some may carry the virus small numbers of patients. Vertex Phar- stretches of RNA that are not translated
for decades without significant injury; maceuticals in Cambridge, Mass., is in- into protein. One of these terminal re-
others experience serious damage with- vestigating a compound that inhibits a gions seems to prompt infected cells to
in only a few years. human enzyme called ionosine mono- manufacture the viral polyprotein; it is
Liver transplantation can save some phosphate dehydrogenase. The hepati- an important target for diagnostic as-
end-stage patients, but the supply of hu- tis C virus relies on this enzyme to gen- says. The other appears to play a role in
man livers available for transplant is erate constituents of RNA. No results initiating the replication of viral RNA.
woefully inadequate. Researchers are from these trials are yet available. The structural proteins include the
therefore working intensively to develop In the absence of medications capable core protein, which encloses the RNA in
treatments that will eradicate the virus of dependably eliminating the virus, the a viral particle within a structure known
in patients. NIH recently embarked on a study to as the nucleocapsid, and two envelope
The first therapeutic agent shown to determine whether long-term adminis- proteins that coat the nucleocapsid. The
be effective was alpha interferon, a pro- tration of alpha interferon can slow liv- nonstructural proteins include a viral
tein that occurs naturally in the body. In- er damage in patients who fail to clear protease responsible for cleaving the
terferon appears to have a nonspecific the virus. And we and other researchers polyprotein, as well as other enzymes re-
antiviral action and may also enhance are studying the simple expedient of sponsible for chemically readying the
immune system activity. The drug is gen- taking a pint of blood from patients on components of viral RNA (triphospha-
erally given by subcutaneous injection a regular basis. This treatment reduces tase), for copying the RNA (polymerase)
three times a week for 12 months. Only the amount of iron in the body, a ma- and for unwinding the newly manufac-
15 to 20 percent of patients, however, nipulation that can reduce serum ALT tured copy (helicase).
exhibit a sustained response, as defined and AST levels. Whether it slows liver The protease and helicase enzymes
by the return of ALT and AST to nor- damage is still uncertain. have been well characterized and their
mal levels and the absence of detectable detailed three-dimensional structure
hepatitis C RNA in serum for at least Targeting the Virus elucidated through x-ray crystallogra-
six months after stopping treatment. phy, necessary first steps for designing
Why treatment fails in most patients is
essentially unknown, although some vi-
ral genotypes seem to be more suscepti-
T he best prospects for future treat-
ment for hepatitis C appear to be
agents targeted specifically against the
drugs to inhibit an enzyme. Several
drug companies, including Schering-
Plough, Agouron Pharmaceuticals, and
ble to interferon than others. virus, just as successful treatments for Eli Lilly and Vertex Pharmaceuticals,
Last year the Food and Drug Admin- HIV target that agent. With that goal in are now studying potential hepatitis C
istration approved another drug, rib- mind, researchers have elucidated the protease or helicase inhibitors. Clinical
avirin, to treat hepatitis C in conjunction structure of the hepatitis C virus in de- trials are probably only a few years
with interferon. Ribavirin, which can be tail. Its genetic material, or genome, con- away. Another viral enzyme, the poly-
swallowed in pill form, inhibits many sists of a single strand of RNA. In size merase, is also a possible target. Whether
viruses. Interestingly, though, it appears and organization the genome is similar the virus will evolve resistance to such
to have no effect against the hepatitis C to that of yellow fever and dengue fever agents remains to be seen.
virus by itself and is thought somehow viruses; hepatitis C virus has therefore Developing antihepatitis C therapies

How the Hepatitis C Virus
Reproduces Itself
H epatitis C infection starts when vi-

ral particles in the circulation find
their way to susceptible cells,particularly
CELL MEMBRANE
hepatocytes.A viral protein called E2 ap-

pears to facilitate entry by latching onto
a specific receptor.On entering, the virus
loses its lipid coat and its protein enve-
lope, freeing the RNA cargo. Enzymes in
1 Virus binds
the cell then use this RNA as a template to receptor,
to make a large viral protein,the polypro- enters cell
inclusion
tein. It is cleaved into a variety of small
proteins that go on to form new viral
particles and help to copy the viral RNA.
The original RNA is copied to yield a
negative-stranded RNA that carries
the inverse, or complement, of the origi-
KEITH KASNOT; SOURCE: CHARLES M. RICE Washington University School of Medicine
nal sequence. This serves as a template

to make multiple copies of the original
RNA, which are incorporated into new
viral particles, along with structural pro- 3 RNA directs
cell to make
teins, at a body called the Golgi com- polyprotein
plex.Complete viral particles are eventu-
ally released from the infected cell, after
HUMAN LIVER CELL 2 Viral RNA reaches
acquiring a lipid surface layer. Recent cell interior
studies suggest that a patient produces
as many as 1,000 billion copies of hep-
atitis C virus a day, most of them from
the liver. A.M.D. and B.R.B.
may be about to get easier. Three months being investigated is disruption of the Some workers are trying to develop
ago Ralf Bartenschlager and his col- process that activates hepatic stellate therapeutics aimed at the short terminal
leagues at Johannes-Gutenberg Universi- cells and causes them to instigate fibro- regions of the viruss genome. One idea,
ty in Mainz, Germany, published details sis. This mechanism is known to involve being pursued by Ribozyme Pharmaceu-
of an RNA genetic construct that in- cytokines, or signaling chemicals, that ticals, is to develop therapeutic molecules
cludes the regions coding for the viruss cells in the liver called Kupffer cells re- that can cut specific constant sequences
enzymes and reproduces itself in liver lease when they are stimulated by lym- there. Ribozymes, short lengths of RNA
cancer cell lines. This construct may phocytes. Turning this process off once it or a chemical close relative, can accom-
prove valuable for testing drugs targeted has started should prevent most of the plish this feat. The main challenge
at these enzymes. untoward consequences of hepatitis C may be getting enough ribozymes into
Another possible therapeutic avenue infection. infected cells. Delivering adequate quan-
tities of a therapeutic agent is also a
HEPATITIS C VIRUS GENOME consists of a single RNA gene plus two terminal regions. problem for some other innovative
The gene encodes a polyprotein,which subsequently cleaves to form a variety of small- treatment concepts, such as gene thera-
er proteins. Some of these are used to make new virus particles; others are enzymes py to make liver cells resistant to infec-
that help to replicate the viral RNA for inclusion into new viruses. tion, antisense RNA that can inhibit
Core Enve- Envelope Non- Nonstructural Non- Nonstructural

Terminal lope 2 struc- 3 structural 5 Terminal
GENE region tural 4 region
1 2
LAURIE GRACE
PROTEIN Core Envelope Protease/ RNA

PRODUCTS protein proteins helicase polymerase

8 New viral particles
with lipid outer layer
are released
4 Polyprotein
cleaves
5 Viral enzymes copy GOLGI COMPLEX

negative-stranded RNA
from original
7 RNAs gain
protein coat
as viral precursors
6 Enzymes make multiple

copies of original RNA from
negative-stranded RNA
NUCLEUS
specified genes, and engineered proteins C are hampered by a serious shortage of pies, and possibly a vaccine, will in time
that activate a cells self-destruct mecha- funds for research. The amount of feder- be available. An expanded research pro-
nism when they are cleaved by the hep- al support, considering the threat to mil- gram could ensure that these develop-
atitis C protease. lions of patients, is relatively small. We ments come soon enough to help patients
All these attempts to counter hepatitis are confident that much improved thera- and those at risk. SA
The Authors Further Reading

ADRIAN M. DI BISCEGLIE and BRUCE R. BA- The Crystal Structure of Hepatitis C Virus NS3 Proteinase Reveals a
CON are physicians specializing in hepatitis C. Di Bis- Trypsin-like Fold and a Structural Zinc Binding Site. Robert A. Love
ceglie received his medical training at the University of et al. in Cell, Vol. 87, No. 2, pages 331342; October 18, 1996.
the Witwatersrand in South Africa. Before joining Saint Management of Hepatitis C. National Institutes of Health Consensus Develop-
Louis University School of Medicine as associate chair- ment Conference Panel Statement. In Hepatology, Vol. 26, Supplement No. 1,
man of internal medicine, he was head of the liver dis- pages 2S10S; 1997.
eases section at the National Institutes of Health. His Interferon Alfa-2b Alone or in Combination with Ribavirin as Initial
research interests include viral hepatitis and primary Treatment for Chronic Hepatitis C. John G. McHutchison et al. in New
liver cancer. Bacon is director of the division of gas- England Journal of Medicine, Vol. 339, No. 21, pages 14851492; Novem-
troenterology and hepatology at Saint Louis University ber 19, 1998.
School of Medicine. He completed his medical training Molecular Characterization of Hepatitis C Virus. Second edition. Karen
at Cleveland Metropolitan General Hospital. His re- E. Reed and Charles M. Rice in Hepatitis C Virus. Edited by H. W. Reesink.
search has focused on iron metabolism in the liver. Both Karger, Basel, 1998.
Di Bisceglie and Bacon are associated with the Ameri- Replication of Subgenomic Hepatitis C Virus RNAs in a Hepatoma
can Liver Foundation: Di Bisceglie as medical director Cell Line. V. Lohmann, F. Krner, J.-O. Koch, U. Herian, L. Theilmann and
and Bacon as a member of the board of directors. R. Bartenschlager in Science, Vol. 285, pages 110113; July 2, 1999.

March 2002
ANTHRAX TTACKING
Recent discoveries are suggesting much-needed strategies
for improving prevention and treatment. High on the list: ways
to neutralize the anthrax bacteriums fiendish toxin
by John A. T. Young and R. John Collier
The need for new anthrax therapies became all too clear last fall
when five people died of inhalation anthrax, victims of the first pur-
poseful release of anthrax spores in the U.S. Within days of showing ini-
tially unalarming symptoms, the patients were gone, despite intensive
treatment with antibiotics. Six others became seriously ill as well before
pulling through.

Fortunately, our laboratories and bing buildup of fluid around the lungs.) and Arthur M. Friedlander, while at the
others began studying the causative bac- Extensive replication in the blood is U.S. Army Medical Research Institute of
terium, Bacillus anthracis, and seeking generally what kills patients who suc- Infectious Diseases, initiated that effort
antidotes long before fall 2001. Recent cumb to anthrax. B. anthraciss ability to with their colleagues in the 1980s; the two
findings are now pointing the way to expand so successfully derives from its of us and others took up the task some-
novel medicines and improved vaccines. secretion of two substances, known as what later.
Indeed, in the past year alone, the two of virulence factors, that can profoundly de- The toxin turns out to consist of three
us and our collaborators have reported rail the immune defenses meant to keep proteins: protective antigen, edema fac-
on three promising drug prototypes. bacterial growth in check. One of these tor and lethal factor. These proteins co-
factors encases the vegetative cells in a operate but are not always joined to-
An Elusive Killer polymer capsule that inhibits ingestion gether physically. They are harmless in-
THE NEW IDEAS for fighting anthrax by the immune systems macrophages dividually until they attach to and enter
have emerged from ongoing research into and neutrophilsthe scavenger cells that cells, which they accomplish in a highly
how B. anthracis causes disease and death. normally degrade disease-causing bacte- orchestrated fashion.
Anthrax does not spread from individual ria. The capsules partner in crime is an First, protective antigen binds to the
to individual. A person (or animal) gets extraordinary toxin that works its way surface of a cell, where an enzyme trims
sick only after incredibly hardy spores en- into those scavenger cells, or phagocytes, off its outermost tip. Next, seven of those
ter the body through a cut in the skin, and interferes with their usual bacteria- trimmed molecules combine to form a
through contaminated food or through killing actions. ring-shaped structure, or heptamer, that
spore-laden air. Inside the body the The anthrax toxin, which also enters captures the two factors and is transported
spores molt into vegetative, or active- other cells, is thought to contribute to to an internal membrane-bound compart-
ly dividing, cells. mortal illness not only by dampening im- ment called an endosome. Mild acidity in
Anthrax bacteria that colonize the mune responses but also by playing a di- this compartment causes the heptamer to
skin or digestive tract initially do damage rect role. Evidence for this view includes change shape in a way that leads to the
locally and may cause self-limited ail- the observation that the toxin alone, in transport of edema factor and lethal fac-
ments: black sores and swelling in the the absence of bacteria, can kill animals. tor across the endosomal membrane into
first instance; possibly vomiting and ab- Conversely, inducing the immune system the cytosol (the internal matrix of cells),
dominal pain and bleeding in the second. to neutralize the toxin prevents B. an- where they do their mischief. In essence,
If bacterial growth persists unchecked in thracis from causing disease. the heptamer is like a syringe loaded with
the skin or gastrointestinal tract, howev- edema factor and lethal factor, and the
er, the microbes may eventually invade A Terrible Toxin slight acidity of the endosome causes the
the bloodstream and thereby cause sys- HARRY SMITH and his co-workers at syringe to pierce the membrane of the en-
temic disease. the Microbiological Research Establish- dosome and inject the toxic factors into
Inhaled spores that reach deep into the ment in Wiltshire, England, discovered the cytosol.
lungs tend to waste little time where they the toxin in the 1950s. Aware of its cen- Edema factor and lethal factor cat-
land. They typically convert to the vege- tral part in anthraxs lethality, many re- alyze different molecular reactions in cells.
tative form and travel quickly to lymph searchers have since focused on learning Edema factor upsets the controls on ion
nodes in the middle of the chest, where how the substance intoxicates cells and water flow across cell membranes and
many of the cells find ready access to the gets into them and disrupts their activi- thereby promotes the swelling of tissues. In
blood. (Meanwhile bacteria that remain ties. Such details offer essential clues to phagocytes, it also saps energy that would
in the chest set the stage for a breath-rob- blocking its effects. Stephen H. Leppla otherwise be used to engulf bacteria.
The precise behavior of lethal factor,
Overview/Anthrax which could be more important in caus-
ing patient deaths, is less clear. Scientists
A three-part toxin produced by the anthrax bacterium, Bacillus anthracis, do know that it is a protease (a protein-
contributes profoundly to the symptoms and lethality of anthrax. cutting enzyme) and that it cleaves en-
The toxin causes trouble only when it gets into the cytosol of cells, the material zymes in a family known as MAPKKs.
that bathes the cells internal compartments. Now they are trying to tease out the mo-
Drugs that prevented the toxin from reaching the cytosol would probably go lecular events that follow such cleavage
a long way toward limiting illness and saving the lives of people infected by the and to uncover the factors specific con-
anthrax bacterium. tributions to disease and death.
Analyses of how the toxin enters cells have recently led to the discovery
of several potential antitoxins. Therapeutic Tactics
CERTAINLY DRUGS able to neutralize

the anthrax toxin would help the immune which is a soluble form of the receptor inject edema and lethal factors into the cy-
system fight bacterial multiplication and domain that binds to protective antigen. tosol. Remarkably, some of these mutants
would probably reduce a patients risk of When sATR molecules are mixed into were so disruptive that a single copy in a
dying. At the moment, antibiotics given the medium surrounding cells, they serve heptamer completely prevented injection.
to victims of inhalation anthrax may con- as effective decoys, tricking protective In a study reported last April, these
trol microbial expansion but leave the antigen into binding to them instead of to mutants known as dominant negative
toxin free to wreak havoc. its true receptor on cells. inhibitors, or DNIs proved to be potent
In principle, toxin activity could be We are now trying to produce sATR blockers of the anthrax toxin in cell cul-
halted by interfering with any of the steps in the amounts needed for evaluating its tures and in rats. Relatively small amounts
in the intoxication process. An attractive ability to combat anthrax in rodents and of selected DNIs neutralized an amount
approach would stop the sequence al- nonhuman primates experiments that of protective antigen and lethal factor that
most before it starts, by preventing pro- must be done before any new drug can be would otherwise kill a rat in 90 minutes.
tective antigen from attaching to cells. considered for fighting anthrax in people. These findings suggest that each mutant
Scientists realized almost 10 years ago Other groups are examining whether copy of protective antigen is capable of in-
that this protein initiated toxin entry by carefully engineered antibodies (highly activating six normal copies in the blood-
binding to some specific protein on the specific molecules of the immune system) stream and that it would probably reduce
surface of cells; when cells were treated might bind tightly to protective antigen toxin activity in patients dramatically.
with enzymes that removed all their sur- in ways that will keep it from coupling Of course, as more and more ques-
face proteins, protective antigen found no with its receptor. tions about the toxin are answered, sci-
footing. Until very recently, though, no entists should discover further treatment
one knew which of the countless proteins More Targets ideas. Now that the receptor for protective
on cells served as the crucial receptor. S C I E N T I S T S A R E A L S O seeking ways antigen has been identified, researchers
The two of us, with our colleagues to forestall later steps in the intoxication can use it as a target in screening tests
Kenneth Bradley, Jeremy Mogridge and pathway. For example, a team from Har- aimed at finding drugs able to bar the re-
Michael Mourez, found the receptor last vard has constructed a drug able to clog ceptor from binding to protective antigen.
summer. Detailed analysis of this molecule the regions of the heptamer that grasp And understanding of the receptors
(now named ATR, for anthrax toxin re- edema and lethal factors. The group three-dimensional structure would reveal
ceptor) then revealed that it spans the cell from the laboratories of one of us (Collier) the precise contact points between pro-
membrane and protrudes from it. The and George M. Whitesides reasoned tective antigen and the receptor, enabling
protruding part contains an area resem- that a plugged heptamer would be unable drugmakers to custom-design receptor
bling a region that serves in other recep- to draw the factors into cells. blocking agents.
tors as an attachment site for particular We began by screening randomly con- Scientists would also like to uncover
proteins. This discovery suggested that the structed peptides (short chains of amino the molecular interactions that enable
area was the place where protective anti- acids) to see if any of them bound to the protective antigen heptamers to move
gen latched onto ATR, and indeed it is. heptamer. One did, so we examined its from the cell surface into endosomes in-
We have not yet learned the normal ability to block toxin activity. It worked, side the cell. Impeding that migration
function of the receptor, which surely did but weakly. Assuming that fitting many should be very useful. And what happens
not evolve specifically to allow the an- plugs into the heptamers binding do- after lethal factor cleaves MAPKK en-
thrax toxin into cells. Nevertheless, mains for edema and lethal factor would zymes? How do those subsequent events
knowledge of the molecules makeup is be more effective, we took advantage of affect cells? Although the latter question
enabling us to begin testing inhibitors of chemical procedures devised by White- remains a vexing challenge, recent study
its activity. We have had success, for in- sidess group and linked an average of 22 of lethal factor has brightened the pros-
stance, with a compound called sATR, copies of the peptide to a flexible polymer. pects for finding drugs able to inactivate
That construction showed itself to be a it. Last November, Robert C. Liddington
JOHN A. T. YOUNG and R. JOHN COLLIER strong inhibitor of toxin action more of the Burnham Institute in La Jolla,
THE AUTHORS
have collaborated for several years on than 7,000 times better than the free pep- Calif., and his colleagues in several labo-
investigating the anthrax toxin. Young is tide both in cell cultures and in rats. ratories published the three-dimensional
Howard M. Temin Professor of Cancer Re- Another exciting agent, and the one structure of the part of lethal factor that
search in the McArdle Laboratory for probably closest to human testing, would acts on MAPKK molecules. That site can
Cancer Research at the University of alter the heptamer itself. This compound now become a target for drug screening
WisconsinMadison. Collier, who has was discovered after Bret R. Sellman in or design.
studied anthrax for more than 14 years, Colliers group noted that when certain New leads for drugs should also
is Maude and Lillian Presley Professor of mutant forms of protective antigen were emerge from the recent sequencing of the
Microbiology and Molecular Genetics at mixed with normal forms, the heptamers code letters composing the B. anthracis ge-
Harvard Medical School. formed on cells as usual but were unable to nome. By finding genes that resemble those

Detecting Anthrax
Rapid sensing would save lives
By Rocco Casagrande
IF A TERRORIST GROUP spread anthrax spores into the open air, the onto the ends of DNA fragments unique to the pathogen. Next,
release could affect large numbers of people but would probably go through a procedure called the polymerase chain reaction (PCR),
unnoticed until victims showed up at hospitals. Many would the system makes many copies of the bound DNA, adding
undoubtedly seek help too late to be saved by current therapies. fluorescent labels to the new copies along the way. Within about
Much illness could be prevented, however, if future defenses 30 minutes GeneXpert can make enough DNA to reveal whether
against anthrax attacks included sensors that raised an alarm even a small amount of the worrisome organism inhabited the
soon after spores appeared in the environment. The needed original sample.
instruments are not yet ready for deployment, but various designs This system contains multiple PCR reaction chambers with
that incorporate cutting-edge technology are being developed. distinct primer sets to allow the detection of different pathogens
Environmental sensors must discriminate between disease- simultaneously. Furthermore, the GeneXpert system could be used
causing agents (pathogens) and the thousands of similar but to determine whether the anthrax bacterium is present in a nasal
harmless microorganisms that colonize air, water and soil. Most of swab taken from a patient in as little as half an hour, significantly
the tools being investigated work by detecting unique molecules on faster than the time it takes for conventional microbiological
the surface of the pathogens of interest or by picking out stretches techniques to yield results.
of DNA found only in those organisms. Instruments designed specifically to detect spores of the
The Canary, which is being developed at the Massachusetts anthrax bacterium or of closely related microbes (such as the one
Institute of Technology Lincoln Laboratory, is an innovative that causes botulism) can exploit the fact that such spores are
example of the devices that detect pathogens based on unique packed full of dipicolinic acid (DPA) a compound, rarely found
surface molecules. The sensors of the Canary consist of living elsewhere in nature, that helps them to survive harsh environ-
cells B cells of the immune system that have been genetically mental conditions. Molecules that fluoresce when bound to DPA
altered to emit light when their calcium levels change. Protruding have shown promise in chemically based anthrax detectors.
from these cells are receptors that will bind only to a unique part of Electronic noses, such as the Cyranose detection system made
a surface molecule on a particular pathogen. When the cells in the by Cyrano Sciences in Pasadena, Calif., could possibly smell the
sensor bind to their target, that binding triggers the release of presence of DPA in an air sample laced with anthrax spores.
calcium ions from stores within the cells, which in turn causes the The true danger of an anthrax release lies in its secrecy. If an
cells to give off light. The Canary can discern more than one type of attack is discovered soon after it occurs and if exposed individuals
pathogen by running a sample through several cell-filled modules, receive treatment promptly, victims have an excellent chance of
each of which reacts to a selected microorganism. surviving. By enhancing early detection, sensors based on the
The GeneXpert system, developed by Cepheid, in Sunnyvale, systems discussed above or on entirely different technologies could
Calif., is an example of a gene-centered approach. It begins its effectively remove a horrible weapon from a terrorists arsenal.
work by extracting DNA from microorganisms in a sample. Then, if
a pathogen of concern is present, small primers (strips of genetic ROCCO CASAGRANDE is a scientist at Surface Logix in Brighton, Mass., where he
material able to recognize specific short sequences of DNA) latch is developing methods and devices for detecting biological weapons.
of known functions in other organisms, tralize the toxin of concern as soon as it ed to prevent them from making people ill.
biologists are likely to discover addition- appears in the body, thus preventing dis- It is produced by growing the weakened
al information about how the anthrax bac- ease. Livestock in parts of the U.S. receive strain of B. anthracis in culture, filtering
terium causes disease and how to stop it. preparations consisting of B. anthracis the bacterial cells from the culture medi-
The continuing research should yield cells that lack the protective capsule and um, adsorbing the toxin proteins in the
several antitoxins. To be most effective, thus replicate poorly. A similar vaccine for remaining filtrate onto an adjuvant (a
such drugs will probably be used with an- humans has been used in the former Sovi- substance that enhances immune re-
tibiotics, much as cocktails of antiviral et Union. But preparations that contain sponses) and treating the mixture with
drugs are recommended for treating HIV whole microbes often cause side effects, formaldehyde to inactivate the proteins.
infection. and they raise the specter that renegade Injection of this preparation, known as
cells might at times give rise to the very dis- AVA (for anthrax vaccine adsorbed),
Promising Preventives eases they were meant to prevent. stimulates the immune system to produce
AS PLANS TO IMPROVE therapies pro- The only anthrax vaccine approved antibodies that specifically bind to and in-
ceed, so does work on better vaccines. for human use in the U.S. takes a differ- activate the toxins components. Most of
Vaccines against toxin-producing bacteria ent form. It consists primarily of toxin the antibodies act on protective antigen,
often prime the immune system to neu- molecules that have been chemically treat- however, which explains the proteins

ANTHRAX IN ACTION
Physicians classify anthrax according to the tissues that are
initially infected. The disease turns deadly when the causative
bacterium, Bacillus anthracis, reaches the bloodstream and
proliferates there, producing large amounts of a dangerous toxin.
Much research is now focused on neutralizing the toxin.
THREE TYPES
INHALATION ANTHRAX
Spores are breathed in
GASTROINTESTINAL ANTHRAX
Spores are ingested by eating contaminated meat
CUTANEOUS ANTHRAX
Spores penetrate the skin through a break
HOW INHALATION ANTHRAX ARISES

Inhalation anthrax is the most dangerous form,
probably because bacteria that land in the lungs are
more likely to reach the bloodstream and thus
disseminate their toxin through the body.
MACROPHAGE
1 Immune system cells called macrophages
ingest B. anthracis spores and carry them
SPORE to lymph nodes in the chest. En route, or in the
macrophages, the spores transform into
actively dividing cells
REPLICATING
BACTERIAL CELLS 2 Proliferating B. anthracis cells erupt from
macrophages and infiltrate the blood readily CELL
3 In the blood, the active bacteria evade

destruction by macrophages and other cells
of the immune system by producing a capsule
BACTERIUM (detail) that blocks the immune cells from
ingesting them and by producing a toxin that
CAPSULE enters immune cells and impairs their functioning
BACTERIA
IN BLOOD
4 Protected from immune destruction, the bacteria
multiply freely and spread through the body
MACROPHAGE
FILLED
WITH TOXIN
TOXIN
MOLECULES

HOW THE TOXIN INVADES CELLS ... AND HOW TO STOP IT LF
THE ANTHRAX TOXIN must enter cells to hurt the body. It consists of three collaborating proteins:
protective antigen (PA), edema factor (EF) and lethal factor (LF). The last two disrupt cellular
activities, but only after protective antigen delivers them to the cytosol the matrix surrounding
the cells intracellular compartments. Molecular understanding of how the factors reach the
EF
cytosol has led to ideas for blocking that journey and thus for neutralizing the toxin and saving
lives. The antitoxins depicted in the boxes have shown promise in laboratory studies.
TREATMENT IDEA
HEPTAMER
EF LF
4 Up to three copies of
EF or LF or a combination
INHIBITOR
of the two bind to the

heptamer Keep EF and LF from attaching
PA to their binding sites on PA
3 Seven copies combine,
forming a heptamer
heptamers. Plug those sites with
linked copies of a molecule that
also has affinity for the sites.
2 PA gets cleaved
HEPTAMER
5 The heptamer complexed
with EF and LF is delivered to a
COMPLEX
membrane-bound compartment
1 PA binds to its
receptor on a cell
ANTHRAX TOXIN
RECEPTOR (ATR) called an endosome
TREATMENT IDEA
ENDOSOME 6 Mild acidity in the endosome
causes the heptamer to inject EF
SOLUBLE RECEPTOR and LF into the cytosol
(sATR)
PA TREATMENT IDEA
ENDOSOME
HEPTAMER
DNI
Prevent PA from linking to its
receptor on cells. Induce it to CYTOSOL
bind instead to decoys, such as
soluble copies of the toxin Block transport of EF and LF from
receptors PA binding site. the endosome into the cytosol
by causing newly forming
8 LF is believed to be
important in causing
7 EF causes tissues
to swell and prevents
heptamers to incorporate a
version of PA known as a
CYTOSOL disease and death, but immune system cells dominant negative inhibitor
exactly how it does so from ingesting and (DNI). DNI-containing heptamers
is in question degrading bacteria cannot move EF and LF across
the endosomes membrane.

Medical Lessons report. Inhalation anthrax has two symptomatic phases an early
period marked by maladies common to a variety of ailments (such
Doctors now have a changed view of inhalation anthrax as fatigue, fever, aches and cough) and a later phase in which
By Ricki L. Rusting patients become critically ill with high fever, labored breathing and
shock. Six of the 10 patients received antibiotics active against the
THE RECENT CASES of inhalation anthrax in the U.S. have upended anthrax bacterium, Bacillus anthracis, while they were still
some old assumptions about that disease. When contaminated showing early symptoms of infection, and only they survived.
letters started appearing in September 2001, public health The types of antibiotics prescribed and the use of combinations
authorities initially believed that only those who received the of drugs might also have had a hand in the unexpectedly high
letters, and perhaps individuals nearby, were in danger. But spores survival rate. Nine of the people discussed in the review sought
clearly seeped out through the weave of the envelopes, care before the CDC published what it called interim guidelines for
contaminating postal facilities and jumping to other mail. Such treating inhalation anthrax on October 26, but most patients
cross contamination is a leading explanation for the deaths of received therapy consistent with those guidelines: ciprofloxacin
two of the 11 people confirmed to have contracted inhalation (the now famous Cipro) or doxycycline plus one or two other agents
anthrax last year. Also contrary to expectations, spores do not known to inhibit replication of B. anthracis (such as rifampin,
remain sedentary once they land. They can become airborne again vancomycin, penicillin, ampicillin, chloramphenicol, imipenem,
as people walk around in a tainted room. clindamycin and clarithromycin). Aggressive supportive care
One surprise was positive. Before October 2001 common including draining dangerous fluid from around the lungs probably
wisdom held that inhalation anthrax was almost always incurable helped as well, scientists say.
after symptoms appeared. But doctors beat those odds last fall, Even the survivors were very sick, however. Jernigan says they
saving six of the victims. What made the difference? Researchers are still being observed to see whether long-term complications will
cannot draw firm conclusions from so few cases. But some intriguing develop, although as of mid-January no obvious signs of such
patterns emerged when John A. Jernigan of the Centers for Disease problems had emerged. Researchers suspect that anthrax
Control and Prevention (CDC) and a team of others reviewed the antitoxins would ease the course of many people afflicted with
medical records of the first 10 patients. Their findings appear in the anthrax and might also rescue patients who could not be saved
November/December 2001 Emerging Infectious Diseases and with current therapies.
online at www.cdc.gov/ncidod/eid/vol7no6/jernigan.htm
Relatively prompt diagnosis may have helped, the researchers Ricki L. Rusting is a staff editor and writer.
name: it is the component that best elic- ful, less cumbersome and faster-acting ity to elicit immune responses. Hence,
its protective immunity. vaccine, many investigators are focusing they could do double duty: disarming the
AVA is given to soldiers and certain on developing inoculants composed anthrax toxin in the short run while
civilians but is problematic as a tool for primarily of protective antigen produced building up immunity that will persist lat-
shielding the general public against bio- by recombinant DNA technology. By er on.
logical warfare. Supplies are limited. And coupling the recombinant protein with a We have no doubt that the expanding
even if AVA were available in abundance, potent new-generation adjuvant, research on the biology of B. anthracis
it would be cumbersome to deliver on a scientists may be able to evoke good and on possible therapies and vaccines
large scale; the standard protocol calls for protective immunity relatively quickly will one day provide a range of effective
six shots delivered over 18 months fol- with only one or two injections. The anthrax treatments. We fervently hope
lowed by annual boosters. The vaccine dominant negative inhibitors discussed that these efforts will mean that nobody
has not been licensed for use in people al- earlier as possible treatments could be will have to die from anthrax acquired ei-
ready exposed to anthrax spores. But late useful forms of protective antigen to ther naturally or as a result of biological
last year officials, worried that spores choose. Those molecules retain their abil- terrorism.
might sometimes survive in the lungs for
MORE TO E XPLORE
BRYAN CHRISTIE DESIGN (preceding two pages)
a long time, began offering an abbreviat-

ed, three-course dose on an experimental Anthrax as a Biological Weapon: Medical and Public Health Management. Thomas V. Inglesby et al.
basis to postal workers and others who in Journal of the American Medical Association, Vol. 281, No. 18, pages 17351745; May 12, 1999.
had already taken 60 days of precaution- Dominant-Negative Mutants of a Toxin Subunit: An Approach to Therapy of Anthrax. Bret R.
Sellman, Michael Mourez and R. John Collier in Science, Vol. 292, pages 695697; April 27, 2001.
ary antibiotics. People who accepted the
offer were obliged to take antibiotics for Designing a Polyvalent Inhibitor of Anthrax Toxin. Michael Mourez et al. in Nature Biotechnology,
Vol. 19, pages 958961; October 2001.
an additional 40 days, after which the im-
Identification of the Cellular Receptor for Anthrax Toxin. Kenneth A. Bradley, Jeremy Mogridge,
munity stimulated by the vaccine would Michael Mourez, R. John Collier and John A. T. Young in Nature, Vol. 414, pages 225229;
presumably be strong enough to provide November 8, 2001.
adequate protection on its own. The U.S. Centers for Disease Control and Prevention maintain a Web site devoted to anthrax at
In hopes of producing a more power- www.cdc.gov/ncidod/dbmd/diseaseinfo/anthrax_g.htm

SHOOT
June 2003
THIS DEER
Chronic wasting disease, a cousin of mad cow disease, is
spreading among wild deer in parts of the U.S. Left unchecked,
the fatal sickness could threaten North American deer
populationsand maybe livestock and humans
By Philip Yam
A place called the eradication zone, lying about 40 miles west

of Madison, Wis., covers some 411 square miles. There thou-
sands of white-tailed deer liveor rather, used to live.
Last year the Wisconsin Department of culling can slow the spread. Currently no
Natural Resources instituted special hunt- practical live test exists to check whether an
ing periods to try to wipe out upward of apparently healthy, wild animal is actually
18,000 deer. During the fall, dead deer incubating the sickness; only a brain sam-
were taken to registration areas, where ple will do.
state employees in protective suits and The disease occurs because a pathogen
gloves dragged carcasses from pickup peppers neural tissue full of microscopic
trucks and lifted them onto plastic-covered holes and gums up the brain with toxic
picnic tables. With hacksaws, they severed clumps of protein called amyloid plaques.
the heads, double-bagged them and sent Long confined to a patch of land near the
them for testing; the bodies themselves Rocky Mountains, the disease has shown
were incinerated. up in 12 states and two Canadian prov-
The Dairy States massacre is an at- inces. The sickness passes readily from one
tempt to keep a fatal ailment known as deer to another no deer seem to have a
chronic wasting disease (CWD) from in- natural resistance. From everything weve
fecting its other 1.6 million deer. The test- seen, comments Michael W. Miller, a
ing enables wildlife officials to ascertain the CWD expert with the Colorado Division of
scope of the epidemic running at nearly Wildlife, it would persist. It would not go
1.6 percent and determine whether the away on its own.

The urgency also reflects concern evidence has accumulated to prove that expire within days and others in about a
about the nature of CWD, which belongs some proteins can in fact copy themselves year. The incubation period, during
to the same family as a better-known and that variants of PrP are essential play- which the animals show no symptoms,
scourge: bovine spongiform encephalo- ers in spongiform encephalopathies. ranges from about 20 to 30 months.
pathy (BSE), or mad cow disease. Spread That prions lack any DNA or RNA is The Fort Collins facility became a
by animal-based feed inadvertently con- also the prime reason why they are so CWD death trap. Between 1970 and
taining tissue from sick cows and sheep, tough. Germicidal light, formaldehyde 1981, 90 percent of the deer that stayed
BSE emerged in the U.K. in the 1980s and baths and boiling water all promptly dis- more than two years died from the disease
continues to plague that country at a low rupt bacterial and viral nucleic acids, yet or had to be euthanized. In 1980 the
level. (Nearly two dozen other countries such treatments have little effect on mal- scourge emerged outside Colorado, at the
have now also reported cases.) In 1996 formed prions. Researchers have exposed Sybille Research Unit in southeastern
scientists realized that BSE can pass to hu- prion-contaminated tissue to a dry heat of Wyoming, 120 miles northwest of Fort
mans who eat infected meat, leading to a 600 degrees Celsius and left it buried for Collins. The two facilities had exchanged
fatal condition: variant Creutzfeldt-Jakob three years, only to find that the materi- deer for breeding purposes, thus indicat-
disease, or vCJD (distinct from the more al, though greatly weakened, was still in- ing that the disease was infectious even
common sporadic CJD, which arises fectious. Indeed, physicians have unwit- to a different species: soon the elk at the
spontaneously in one in a million people). tingly passed prion diseases on to patients facilities contracted the disease. (Deer and
Researchers are now trying to figure out via surgical instruments and transplanted elk both belong to the cervid family.)
whether CWD could infect humans and organs that had undergone standard ster- For years, researchers thought CWD
livestock and thereby create an American ilization procedures. (Prion disinfection resulted from nutritional deficiencies, poi-
version of the U.K.s mad cow disaster. requires extended heating or corrosive soning, or stress from confinement. But in
chemicals such as sodium hydroxide.) 1977 Elizabeth S. Williams, studying for
Pathological Protein her doctorate at Colorado State Universi-
T H E D I S E A S E A G E N T C O M M O N to Foothold in the Foothills ty, discovered that this view was mistak-
all these maladies is the prion (PREE- THE RESILIENCE OF misfolded prions en. When Williams looked at brain slices
on), a term coined in 1982 by Stanley B. appears to be a key reason why chronic from infected animals, she saw that the
Prusiner of the University of California at wasting disease has persisted and spread tissue was full of microscopic holes. I
San Francisco. The prion is a protein that from its presumed starting point near Fort happened to be taking a course in neu-
exists in all animals, although the exact Collins, Colo. There, in 1967, at the ropathology and had studied a lot of brain
amino acid sequence depends on the spe- states Foothills Wildlife Research Facili- lesions, she recalls. The holes were un-
cies. It takes one of two shapes. Folded ty, CWD made its first recorded appear- mistakably like scrapie, the sheep sickness
correctly, it is the normal prion protein ance, in captive mule deer that were being that was the first documented spongiform
(PrP), which is especially abundant in maintained for nutritional studies (mule encephalopathy.
brain cells and may help process copper. deer are the most common type in the In fact, CWD appears to have origi-
Folded incorrectly, the prion protein be- West). As the name of the disease sug- nated from scrapie. Richard E. Race of
comes a pathogenic entity that kills. The gests, affected deer lose weight over the the National Institutes of Health Rocky
malformed protein has the ability to re- course of weeks or months. They often Mountain Laboratories in Hamilton,
fold copies of normal PrP in its own im- become thirsty, which drives them to Mont., conducted test tube studies that
age, thereby making more of itself. drink large amounts of water and, conse- revealed no distinction between the mal-
Prusiners conception of prions ini- quently, to urinate a great deal; they also formed PrP of scrapie sheep and CWD
tially met with great skepticism. That a start slobbering and drooling. They may cervids. Consistent with this discovery,
pathogen could replicate and pass on its stop socializing with fellow deer, become Amir Hamir of the U.S. Department of
traits without assistance from nucleic listless or hang their heads. Death typi- Agricultures National Animal Disease
acids (DNA or RNA) violated the ortho- cally ensues three to four months after Center in Ames, Iowa, found no differ-
doxy of molecular biology. But enough symptoms start, although some victims ence in the appearance of brain samples
from elk with CWD and elk experimen-
Overview/Chronic Wasting Disease tally infected with scrapie. (BSE also
probably arose from scrapie, after cows
Chronic wasting disease (CWD) is a fatal condition spreading among wild deer ate feed derived from infected sheep.)
in some parts of North America. It kills in part by making holes in the brain. But unlike BSE in cows (or vCJD in
Malformed proteins called prions trigger the disease. The extreme durability humans), the cervids were not getting ill
of prions and CWDs long incubation times make controlling the spread from their food. CWD behaves more like
of the sickness difficult. scrapie, in that the sickness spreads
Studies are under way to see whether CWD can infect humans and livestock. among individuals, although no one real-
ly knows how it does. The prions could

lurk in the urine. During rutting season, the University of Wyoming. More impor- idea is to find a fairly small focus and get
deer bucks lap up the urine of perhaps tant were the mountains and other natur- rid of all the animals in the area, in the
dozens of does to find out which are in al barriers, which scientists expected hopes of preventing CWD from attaining
heat. Elk females lick males that have would keep CWD from spreading rapid- a permanent hold in the region, Williams
sprayed themselves with urine. Saliva ly out of the endemic area. There was, says. A rapid spread is possible in Wis-
could be a vector, too; in both deer and however, an easy way past those natural consin because the deer population in the
elk, individuals meet and greet by licking barriers: along the roads, in a truck. states southwestern corner is dense:
each others mouths and noses, thus ex- Thomas Givnish, an expert on the ecolo-
changing drool. Ranched elk may swap Out and About gy of diseases at the University of Wis-
saliva when they feed in close quarters. It SOME 1 1 , 0 0 0 G A M E F A R M S and consinMadison, notes that it runs about
is also possible that animals take in the ranches holding hundreds of thousands of 50 to 100 deer per square mile, or 10
pathogen while grazing in areas where deer and elk dot the U.S. and Canada. Be- times that of the endemic area around
sick animals have shed prions on the sides harvesting the meat, ranchers can Fort Collins. The alternative is to do noth-
ground in their feces, urine and saliva. sell the antlers those from elk are mar- ing, Williams observes, and then you
By 1985 veterinarians discovered keted as a supplement in vitamin stores know its going to be established. By the
CWD in free-ranging deer and elk, gener- (velvet antler) and as an aphrodisiac in end of March, Wisconsin hunters had
ally within about 30 miles of the two Asia (velvet Viagra). To start such bagged 9,287 deerwhich will cut the fall
wildlife facilities. Whether the disease farms, ranchers must buy breeding cervids. population by 25 percent but will not
originated in the wild and spread to the Somewhere along the line, businesses must eliminate CWD, notes state wildlife biol-
captives, or vice versa, is not known. The have picked up incubating animals from ogist Tom Howard. A few more seasons
two populations had plenty of time to the endemic area. And the interstate trade of liberal hunting may be needed.
mingle. Especially during mating season, of cervids continued the spread, west Considering the persistence of prions,
wild cervids nosed up to captives through across the Continental Divide and east Wisconsin may have to live with CWD, as
the chain-link fences. Incubating deer across the Mississippi River. (These days Colorado does. The disease has been here
could also have escaped or been released. most states regulate such trade.) a long time, Miller comments of CWD
Both facilities tried hard to eradicate The first farmed cervid to display signs around northeastern Colorado. We cant
CWD. The Sybille center killed all the of CWD was an elk that fell ill in 1996 on get rid of it here. We try to get infection
deer and elk in the affected area and wait- a ranch in Saskatchewan. By 2001 some rates down so that it cant spread. Miller
ed a year to introduce new animals; four 20 ranches reported cases across six states says that Colorado had hoped to purge
years later deer and elk started coming (Colorado, Kansas, Montana, Nebraska, CWD through culling. But we discov-
down with CWD. The Fort Collins facil- Oklahoma and South Dakota) and one ered we were 10 to 20 years too late. It
ity acted more aggressively. Officials first other Canadian province (Alberta). Quick, was already out there; we didnt realize
killed off all the resident deer and elk; aggressive measures namely, killing off it. That statement may apply to other
then they turned several inches of soil and the herds appear to have eliminated the states that have found CWD among wild
repeatedly sprayed structures and pas- problems on the ranches. deer, including Illinois and New Mexico.
tures with swimming-pool chlorine, which Nevertheless, the transport of incu-
readily wipes out bacteria and viruses. Af- bating cervids may have carried CWD to Venison and Beyond
ter waiting a year, they brought in 12 elk wild populations in those states and be- NO ONE KNOWS whether CWD can
calves, but a few years afterward two of yondsuch as to white-tailed deer in Wis- pass to humans. A test tube study mixed
those elk contracted CWD. consins eradication zone. But precisely CWD prions with normal prion proteins
The diseases persistence has perma- when and how mule deer gave it to white- from cervids, humans, sheep and cows.
nently contaminated an area of about tailed deer, the most common type in the The CWD prions had a hard time con-
15,000 square miles in northeastern Col- eastern U.S., is unknown and may never verting normal human PrP less than
orado, southeastern Wyoming and (be- be clear. By the time these problems are 7 percent of the protein was changed.
ginning in 2001) southwestern Nebraska. discovered, Miller says, they have prob- The downside is that CWD prions con-
The incidence of CWD among the cervids ably been sitting there for decades, which verted human PrP about as efficiently as
in this so-called endemic area averages makes it difficult to go back and retrace BSE prions do. And because BSE has
about 4 to 5 percent but has reached 18 how things came about. Based on epi- infected humans, CWD might pose a
percent in some places. To help keep the demiological models and on Wisconsins similar risk. But because beef is far more
disease confined here, the research facili- roughly 1.6 percent incidence in the erad- popular than venison, CWD doesnt pre-
ties stopped trading captive animals with ication zone, Miller thinks CWD had sent quite the same public health threat.
each other. In fact, no captive cervids now probably been lurking there since the ear- To see if CWD has already infected
leave the endemic area alive: Theyre ly 1990s. people, the Centers for Disease Control
only allowed out to come to my necropsy Wisconsins approach makes sense to and Prevention investigated the deaths of
room, wryly remarks Williams, now at scientists studying prion diseases. The the three young venison eaters who suc-

cumbed to sporadic Creutzfeldt-Jakob eating food derived from any animal with The canine family is evidently immune to
disease. All were younger than 30 years, evidence of a spongiform encephalopathy. prion diseases, but felines can contract
which is exceedingly rare in CJD. In fact, Scientists are still trying to determine them. Transmission studies with moun-
through May 31, 2000, just one other U.S. if CWD poses a threat to livestock. In an tain lions have begun, and local lions that
case of sporadic CJD occurred in this age ongoing experiment begun in 1997, die for unknown reasons end up on the
group since surveillance began in 1979. Hamir and his colleagues injected brain pathology table, Williams says.
The first was a 28-year-old cashier, suspensions from CWD mule deer into Although many states have uncovered
who died in 1997; she had eaten deer and the brains of 13 Angus beef calves. Two CWD, other states are looking darn
elk as a child, from her fathers hunts in became ill about two years after inocula- hard, Miller says, but have not found it
Maine. The second was a 30-year-old tion, three others nearly five years after. among them, Arizona, Kansas, Michigan,
salesman from Utah who had been hunt- Hamir began repeating the experiment in Montana, Nevada and New Jersey. Ap-
ing regularly since 1985 and who died in November 2002, this time with the brains parently, only pockets of outbreaks exist.
1999. The third was a 27-year-old truck of CWD white-tailed deer. Wildlife managers therefore have a fight-
It is too early to conclude that chronic wasting disease

does not pose a HUMAN HEALTH HAZARD.
driver from Oklahoma who died in 2000; Under more natural conditions, bo- ing chance to keep CWD from gaining a
he had harvested deer at least once a year. vines have not contracted CWD. Williams permanent grip throughout the country,
Tests of the 1,037 deer and elk taken dur- has kept cows with infected cervids, and so long as control efforts begin promptly.
ing the 1999 hunting season from the re- more than five years on, the cows are still Unfortunately, not all states with CWD
gions where the victims meat originated healthy. Bovines kept with decomposing are as aggressive as Wisconsin when it
all turned up CWD negative (none of the CWD carcasses or isolated in pens that comes to surveillance and eradication.
meat came from the endemic area). The once housed CWD animals have also re- To stop or at least slow the spread of
victims brains showed no unique damage mained free of prion disease. (These re- the fatal sickness, extensive culling ap-
or distinct biochemical signs, as is the case ports are good news for pasture-grazing pears to be the best strategy. One could
with other prion diseases in humans. cows, which might find themselves in the hope that CWD occurs naturally in deer
Six other patients (all at least middle- company of wild deer.) To see whether and that the epidemics will run their
aged) raised suspicions about the CWD CWD might pose a danger when eaten, course and leave behind CWD-resistant
risk to humans. Three were outdoorsmen Williams has begun feeding CWD brain cervids. Some lines of sheep, for instance,
from the Midwest who had participated matter to calves. The long incubation of are immune to scrapie. But so far all
in wild deer and elk feasts and died in the these illnesses, however BSE incubates white-tailed and mule deer appear to be
1990s. The other cases were reported in for up to eight years means these exper- uniformly susceptible. I dont think ge-
April and include two from Washington iments must continue for several years. netics is going to save us on this, remarks
State who hunted together. Researchers, If U.S. livestock so far seem to be safe the NIHs Race. Sadly, the only way to save
however, could not find any connection from CWD, the same cannot be said of the deer, it seems, is to shoot them.
with CWD. And states with CWD have other animals. If an infected deer dies in
not discovered a higher incidence of the forest and nobody is there to see it, Philip Yam is Scientific Americans
Creutzfeldt-Jakob disease. plenty of coyotes, bobcats and other car- news editor. This article is adapted
These observations may seem reas- nivores will, and they will gladly scavenge from his book, The Pathological
suring, but it is too early to conclude that what remains of the wasted carcass. Protein: Mad Cow, Chronic Wasting,
CWD does not pose a human health haz- Moreover, during the clinical phase, CWD and Other Deadly Prion Diseases,
ard. The incubation period of prion dis- animals undoubtedly make easier prey. published in June.
eases may span upward of 40 years, and
CWD has been spreading noticeably in MORE TO E XPLORE
only the past 10. The rarity of prion dis-
Risk Analysis of Prion Diseases in Animals. Edited by Corinne I. Lasmzas and David B. Adams.
eases and the low national consumption
Scientific and Technical Review, Vol. 22, No. 1; April 2003.
of deer and elk (compared with beef) make
The Pathological Protein: Mad Cow, Chronic Wasting, and Other Deadly Prion Diseases.
it hard to draw any firm conclusions. Be- Philip Yam. Copernicus Books, 2003. (www.thepathologicalprotein.com)
cause of the uncertainties with CWD and
Chronic Wasting Disease Alliance Web site is at www.cwd-info.org/
the fact that animal prion diseases have
U.S. Department of Agriculture Web site on CWD is at www.aphis.usda.gov/lpa/issues/cwd/cwd.html
jumped to humans, the CDC warns against

June 2002
HOPE IN A
VIAL Will there be an AIDS

vaccine anytime soon?
By Carol Ezzell
It wasnt supposed to be this hard.
When HIV, the virus responsible for AIDS, was first
identified in 1984, Margaret M. Heckler, then secretary
of the U.S. Department of Health and Human Services,
predicted that a vaccine to protect against the scourge
would be available within two years. Would that it had
been so straightforward.
NOVEMBER 2003

Roughly 20 years into the pandemic, system or antiretroviral drugs the slip. would, such vaccines, they argue, might
40 million people on the planet are infect- Act One also spotlighted HIVs op- at least help prolong the lives of HIV-
ed with HIV, and three million died from position the bodys immune response infected people and delay the onset of the
it last year (20,000 in North America). Al- which consists of antibodies (Y-shaped symptomatic, AIDS phase of the disease.
though several potential AIDS vaccines molecules that stick to and tag invaders In the early 1990s scientists thought
are in clinical tests, so far none has lived such as viruses for destruction) and cyto- they could figure out the best vaccine
up to its early promise. Time and again re- toxic, or killer, T cells (white blood cells strategy for preventing AIDS by studying
searchers have obtained tantalizing pre- charged with destroying virus-infected long-term nonprogressors, people who
liminary results only to run up against a cells). For years after infection, the im- appeared to have harbored HIV for a
brick wall later. As recently as two years mune system battles mightily against decade or more but who hadnt yet fall-
ago, AIDS researchers were saying pri- HIV, pitting millions of new cytotoxic T en ill with AIDS. Sadly, many of the non-
vately that they doubted whether even a cells against the billions of virus particles progressors have become ill after all. The
partially protective vaccine would be hatched from infected cells every day. In key to their relative longevity seems to
available in their lifetime. addition, the immune system deploys have been a weakened virus and/or a
No stunning breakthroughs have oc- armies of antibodies targeted at HIV, at strengthened immune system, says John
curred since that time, but a trickle of en- least early in the course of HIV infection, P. Moore of Weill Medical College of
couraging data is prompting hope to although the antibodies prove relatively Cornell University. In other words, they
spring anew in the breasts of even jaded ineffectual against this particular foe. were lucky enough to have encountered a
AIDS vaccine hunters. After traveling As the curtain rises for Act Two, HIV slow-growing form of HIV at a time
down blind alleys for more than a decade, still has the stage. Results from the first when their bodies had the ammunition to
they are emerging battered but not beat- large-scale trial of an AIDS vaccine should keep it at bay.
en, ready to strike out in new directions. become available at the end of this year,
Its an interesting time for AIDS vaccine but few scientists are optimistic about it: Not Found in Nature?
research, observes Gregg Gonsalves, di- a preliminary analysis suggests that it A I D S V A C C I N E developers have strug-
rector of treatment and prevention advo- works poorly. Meanwhile controversy gled for decades to find the correlates of
cacy for Gay Mens Health Crisis in New surrounds a giant, U.S.-government-spon- immunity for HIV the magic combi-
York City. I feel like its Act Two now. sored trial of another potential vaccine nation of immune responses that, once in-
In the theater, Act One serves to in- slated to begin this September in Thailand. duced by a vaccine, would protect some-
troduce the characters and set the scene; But waiting in the wings are several ap- one against infection. But they keep com-
in Act Two, conflict deepens and the real proaches that are causing the AIDS re- ing up empty-handed, which leaves them
action begins. Act One of AIDS vaccine search community to sit up and take no- with no road map to guide them in the
research debuted HIV, one of the first so- tice. The strategies are reviving the debate search for an AIDS vaccine. Were try-
called retroviruses to cause a serious hu- about whether, to be useful, a vaccine ing to elicit an immune response not
man disease. Unlike most other viruses, must elicit immune responses that totally found in nature, admits Max Essex of
retroviruses insinuate their genetic mate- prevent HIV from colonizing a persons the Harvard School of Public Health. As
rial into that of the body cells they invade, cells or whether a vaccine that falls some- a result, the quest for an AIDS vaccine has
causing the viral genes to become a per- what short of that mark could be accept- been a bit scattershot.
manent fixture in the infected cells and in able. Some scientists see potential value in To be proved useful, a candidate
the offspring of those cells. Retroviruses vaccines that would elicit the kinds of im- AIDS vaccine must successfully pass
also reproduce rapidly and sloppily, pro- mune responses that kick in soon after a through three stages of human testing. In
viding ample opportunity for the emer- virus establishes a foothold in cells. By phase I, researchers administer the vac-
gence of mutations that allow HIV to shift constraining viral replication more effec- cine to dozens of people to assess its safe-
its identity and thereby give the immune tively than the bodys natural responses ty and to establish an appropriate dose.
Phase II involves hundreds of people and
Overview/AIDS Vaccines looks more closely at the vaccines im-
munogenicity, its ability to prompt an
Final results from the first large-scale test of a possible AIDS vaccine will be immune response. In phase III, the po-
available at the end of this year, but few researchers are optimistic it will work. tential vaccine is given to thousands of
Scientists are now aiming to generate potential AIDS vaccines that stimulate volunteers who are followed for a long
both arms of the immune system: killer cells and antibodies. time to see whether it protects them from
There are five main subtypes, or clades, of HIV. Researchers are debating infection. Phase III trials for any drug
whether it will be important to devise vaccines for a given area based on the tend to be costly and difficult to admin-
predominant clade infecting that area. ister. And the AIDS trials are especially
challenging because of an ironic require-

WORLD AIDS SNAPSHOT
MOST OF THE GLOBES 40 million people infected with HIV live in of HIV, which are also called clades. Although more than one clade
sub-Saharan Africa and South and Southeast Asia, as reflected in can usually be found in any given area, the map highlights the
the ranking below, which is based on 2001 data from the Joint predominant clade affecting each region. The boundaries
United Nations Program on HIV/AIDS. There are five major strains between prevailing clades are not exact; they change frequently.
PREDOMINANT
HIV CLADES
CLADE A
CLADE B
CLADE C
CLADE D
CLADE E
OTHER WORLD
NO INFORMATION Total Infected: 40,000,000
Newly Infected (in 2001): 5,000,000
Deaths (in 2001): 3,000,000
LAURIE GRACE; SOURCES: UNAIDS (statistics) AND VADIM ZALUNIN Los Alamos National Laboratory (clade boundaries)
1 SUB-SAHARAN AFRICA 3 LATIN AMERICA 5 E. EUROPE/C. ASIA 7 WESTERN EUROPE 9 CARIBBEAN

Total Infected: 28,100,000 Total Infected: 1,400,000 Total Infected: 1,000,000 Total Infected: 560,000 Total Infected: 420,000
Newly Infected: 3,400,000 Newly Infected: 130,000 Newly Infected: 250,000 Newly Infected: 30,000 Newly Infected: 60,000
Deaths: 2,300,000 Deaths: 80,000 Deaths: 23,000 Deaths: 6,800 Deaths: 30,000
2 SOUTH/SOUTHEAST ASIA 4 EAST ASIA/PACIFIC IS. 6 NORTH AMERICA 8 N. AFRICA/MIDDLE EAST 10 AUSTRALIA/NEW ZEALAND
Total Infected: 6,100,000 Total Infected: 1,000,000 Total Infected: 940,000 Total Infected: 440,000 Total Infected: 15,000
Newly Infected: 800,000 Newly Infected: 270,000 Newly Infected: 45,000 Newly Infected: 80,000 Newly Infected: 500
Deaths: 400,000 Deaths: 35,000 Deaths: 20,000 Deaths: 30,000 Deaths: 120
ment: subjects who receive the vaccine ing it to quickly mount an attack target- surface of the virus, and the companys
must be counseled extensively on how to ed to gp120 if HIV later finds its way into vaccine employs the molecule in its
reduce their chances of infection. They the body. monomeric, or single-molecule, form.
are told, for instance, to use condoms or, This vaccine, which is produced by Moreover, vaccines made of just protein
in the case of intravenous drug users, VaxGen in Brisbane, Calif. a spin-off of generally elicit only an antibody, or hu-
clean needles because HIV is spread biotech juggernaut Genentech in South moral, response, without greatly stimu-
through sex or blood-to-blood contact. San Francisco is being tested in more lating the cellular arm of the immune sys-
Yet the study will yield results only if than 5,400 people (mostly homosexual tem, the part that includes activity by cy-
some people dont heed the counseling men) in North America and Europe and totoxic T cells. A growing contingent of
and become exposed anyway. in roughly 2,500 intravenous drug users investigators suspect that an antibody re-
The first potential vaccine to have in Southeast Asia. The results from the sponse alone is not sufficient; a strong cel-
reached phase III consists of gp120, a pro- North American/European trial, which lular response must also be elicited to pre-
tein that studs the outer envelope of HIV began in 1998, are expected to be an- vent AIDS.
and that the virus uses to latch onto and nounced near the end of this year. Indeed, the early findings do not seem
infect cells. In theory, at least, the presence Many AIDS researchers are skeptical encouraging. Last October an indepen-
of gp120 in the bloodstream should acti- of VaxGens approach because gp120 dent data-monitoring panel did a prelim-
vate the recipients immune system, caus- normally occurs in clumps of three on the inary analysis of the results of the North

One AIDS Vaccine Strategy
A VACCINE APPROACH being pioneered by Merck involves an initial injec- to primarily arouse the cellular arm of the immune systemthe one
tion of a naked DNA vaccine followed months later by a booster shot of that uses cytotoxic T cells to destroy virus-infected cells. The naked
crippled, genetically altered adenovirus particles. Both are designed DNA vaccine also results in the production of antibody molecules
to elicit an immune response targeted to the HIV core protein, Gag, and against Gag, but such antibodies are not very useful in fighting HIV.
INITIAL INJECTION Naked DNA Muscle
Nucleus
Viral
core 1 Naked DNA vaccine
is injected
Gag gene
(encodes
viral core)
Cytoplasm
Human Immunodeficiency
Virus (HIV)
Gag gene
2 Naked DNA is taken up by
muscle tissue and by so-called
antigen-presenting cells (APCs)
BOOSTER SHOT,
MONTHS LATER Adenovirus
APC
Gag
protein
APC 3 APCs produce the Gag protein,

chop it and present bits of it to
immune cells, which communicate
using chemicals called cytokines
HUMORAL
CELLULAR IMMUNE IMMUNE
RESPONSE Gag protein
RESPONSE
Gag protein fragments
Inactive fragment
cytotoxic T cell Inactive Helper
cytotoxic T cell T cell (CD4)
Activated
cytotoxic T cell
Cytokines
5 An adenovirus booster reactivates

the cellular immune response Activated
B cell
4 The cytokines and the
Gag protein activate
immune cells that kill infected
cells or make antibodies
Antibodies
TERESE WINSLOW
Dying HIV-
infected cell

American/European data. Although the Allergy and Infectious Diseases (NIAID) colleagues reported that 42 percent of
panel conducted the analysis primarily to and the U.S. Department of Defense were volunteers who received the highest dose
ascertain that the vaccine was causing no scheduled to conduct essentially duplicate of the naked DNA vaccine raised cyto-
dangerous side effects in the volunteers, trials of the vaccine. But NIAID pulled the toxic T cells capable of attacking HIV-in-
the reviewers were empowered to recom- plug on its trial after an examination of fected cells.
mend halting the trial early if the vaccine the data from a phase II study showed The second trial employs the HIV gag
appeared to be working. They did not. that fewer than 30 percent of the volun- gene spliced into a crippled adenovirus,
For its part, VaxGen asserts that it teers generated cytotoxic T cells against the class responsible for many common
will seek U.S. Food and Drug Adminis- HIV. And in a bureaucratic twist, this colds. This altered adenovirus ferries the
tration approval to sell the vaccine even if past January the White House transferred gag gene into cells, which then make the
the phase III trials show that it reduces a the budget for the Defense Department HIV core protein and elicit an immune re-
persons likelihood of infection by as lit- trial over to NIAID as part of an effort to sponse targeted to that protein. Emini
tle as 30 percent. Company president and streamline AIDS research. told the conference that between 44 and
co-founder Donald P. Francis points out Peggy Johnston, assistant director of 67 percent of people who received injec-
that the first polio vaccine, developed by AIDS vaccines for NIAID, says she expects tions of the adenovirus-based vaccine
Jonas Salk in 1954, was only 60 percent there will be a trial of the vaccine but em- generated a cellular immune response
effective, yet it slashed the incidence of po- phasizes that it will be a Thai trial; we that varied in intensity according to the
lio in the U.S. quickly and dramatically. wont have any [NIAID] people there on size of the dose the subjects received and
This approach could backfire, though, the ground running things. how long ago they got their shots.
if people who receive a partially effective Critics cite these machinations as a Merck is now beginning to test a com-
AIDS vaccine believe they are then pro- case study of politics getting in the way of bination of the DNA and adenovirus ap-
tected from infection and can engage in progress against AIDS. Theres little sci- proaches because Emini predicts that the
risky behaviors. Karen M. Kuntz and ence involved in the trial, claims one vaccines will work best when adminis-
Elizabeth Bogard of the Harvard School skeptic, who wonders why the Thais tered as part of the same regimen. The
of Public Health have constructed a com- arent asking, If its not good enough concept, he says, is not that the DNA
puter model simulating the effects of such for America, how come its good enough vaccine will be a good vaccine on its own,
a vaccine in a group of injection drug for us? Others point out that the trial, but that it may work as a primer of the
users in Thailand. According to their mod- which was conceived by the Defense De- immune system, to be followed months
el, a 30 percent effective vaccine would partment, will answer only the question later by a booster shot of the adenovirus
not slow the spread of AIDS in a commu- of whether the vaccine works; it wont vaccine. A possible stumbling block is
nity if 90 percent of the people who recollect any data that scientists could use that most people have had colds caused
ceived it went back to sharing needles or to explain its potential failure. by adenoviruses. Accordingly, the im-
using dirty needles. They found that such mune systems of such individuals would
reversion to risky behavior would not Partial Protection already have an arsenal in place that
wash out the public health benefit if a vac- INTO THIS SCENE comes Merck, which could wipe out the adenovirus vaccine be-
cine were at least 75 percent effective. is completing separate phase I trials of fore it had a chance to deliver its payload
The controversial study set to begin two different vaccine candidates that it of HIV genes and stimulate AIDS immu-
in Thailand is also a large-scale phase III has begun to test together. In February, nity. Increasing the dose of the adenovirus
trial, involving nearly 16,000 people. It Emilio A. Emini, Mercks senior vice pres- vaccine could get around this obstacle.
combines the VaxGen vaccine with a ca- ident for vaccine research, wowed scien- Emini says he and his co-workers are
narypox virus into which scientists have tists attending the Ninth Conference on emphasizing cellular immunity in part be-
stitched genes that encode gp120 as well Retroviruses and Opportunistic Infec- cause of the disappointing results so far
as two other proteins one that makes tions in Seattle with the companys initial with vaccines designed to engender hu-
up the HIV core and one that allows it to data from the two trials. moral responses. Antibodies continue to
reproduce. Because this genetically engi- The first trial is investigating a poten- be a problem, he admits. There are a
neered canarypox virus (made by Aventis tial vaccine composed of only the HIV handful of reasonably potent antibodies
Pasteur, headquartered in Lyons, France) gag gene, which encodes the viruss core isolated from HIV-infected people, but
enters cells and causes them to display protein. It is administered as a so-called we havent figured out how to raise those
fragments of HIV on their surface, it naked DNA vaccine, consisting solely of antibodies using a vaccine.
stimulates the cellular arm of the immune DNA. Cells take up the gene and use it as Lawrence Corey of the Fred Hutchin-
system. a blueprint for making the viral protein, son Cancer Research Center in Seattle
Political wrangling and questions over which in turn stimulates a mild (and agrees: Youd like to have both [a cellu-
its scientific value have slowed wide- probably unhelpful) humoral response lar and an antibody response], but the
spread testing of the gp120/canarypox and a more robust cellular response [see greatest progress has been in eliciting a
vaccine. Initially the National Institute of illustration on page 40]. Emini and his cellular response, says Corey, who is

also principal investigator of the federal- types, or clades, that affect different areas Africa, together with Timothy Johan Paul
ly funded HIV Vaccine Trials Network. of the world. There are five major clades, Tucker of the South African AIDS Vac-
Antibodies are important, too, be- designated A through E [see illustration cine Initiative, wrote in the British Med-
cause they are the immune systems first on page 39]. Although clade B is the pre- ical Journal that the relevance of HIV
line of defense and are thought to be the dominant strain in North America and subtypes remains unresolved. They as-
key to preventing viruses from ever con- Europe, most of sub-Saharan Africa the sert that clades have assumed a political
tacting the cells they infect. Corey says that hardest-hit region of the globehas clade and national importance, which could in-
vaccines that are designed primarily to C. The ones primarily responsible for terfere with important international tri-
evoke cellular immunity (as are Mercks) AIDS in South and Southeast Asia the als of efficacy.
are not likely to prevent infection but second biggest AIDS hot spot are clades Early data from the Merck vaccine tri-
should give someone a head start in com- B, C and E. als suggest that clade differences blur
bating the virus if he or she does become Several studies indicate that anti- when it comes to cellular immunity. At
infected. Instead of progressing to AIDS bodies that recognize AIDS viruses from the retrovirus conference in February,
in eight years, you progress in 25 years, one clade might not bind to viruses from Emini reported that killer cells from 10 of
he predicts. But, Corey adds, it is unclear other clades, suggesting that a vaccine 13 people who received a vaccine based
whether a vaccine that only slowed disease made from the strain found in the U.S. on clade B also reacted in laboratory tests
progression would stem the AIDS pan- might not protect people in South Africa, to viral proteins from clade A or C virus-
demic, because people would still be able for example. But scientists disagree about es. There is a potential for a substantial
to spread the infection to others despite the significance of clade differences and cross-clade response in cellular immuni-
having less virus in their bloodstream. whether only strains that match the most ty, he says, but thats not going to hold
Finding a way to induce the produc- prevalent clade in a given area can be true for antibodies. Corey concurs that
tion of antibodies able to neutralize HIV tested in countries there. Essex, who is clade variation is likely to play much,
has been hard slogging for several reasons. gearing up to lead phase I tests of a clade much less of a role for killer cells than for
For one, the viruss shape-shifting ways al- Cbased vaccine in Botswana later this antibodies because most cytotoxic T cells
low it to stay one step ahead of the im- year, argues that unless researchers are recognize parts of HIV that are the same
mune response. The thing that distin- sure that a vaccine designed against one from clade to clade.
guishes HIV from all other human viruses clade can cross-react with viruses from Johnston of NIAID theorizes that one
is its ability to mutate so fast, Essex says. another, they must stick to testing vac- answer would be to use all five major
By the time you make a neutralizing an- cines that use the clade prevalent in the clades in every vaccine. Chiron in Emery-
tibody [against HIV], it is only against the populations being studied. Cross-reac- ville, Calif., is developing a multiclade
virus that was in you a month ago. tivity could occur under ideal circum- vaccine, which is in early clinical trials.
According to many scientists, vac- stances, but, he says, unless we know Such an approach could be overkill, how-
cines using a logical molecule, gp120 that, its important for us to use subtype- ever, Johnston says. It could be that pro-
the protein the virus uses to invade im- specific vaccines. teins from only one clade would be rec-
mune cells, as discussed above havent Using the corresponding clade also ognized and the other proteins would be
worked, probably because the antibodies avoids the appearance that people in de- wasted, she warns.
that such vaccines elicit bind to the wrong veloping countries are being used as Whatever the outcome on the clade
part of the molecule. Gp120 shields the guinea pigs for testing a vaccine that is de- question, Moore of Weill Medical Col-
precise binding site it uses to latch onto signed to work only in the U.S. or Europe. lege says he and fellow researchers are
CD4, its docking site on immune cells, VaxGens tests in Thailand are based on a more hopeful than they were a few years
until the last nanosecond, when it snaps combination of clades B and E, and in ago about their eventual ability to devise
open like a jackknife. One way to get April the International AIDS Vaccine Ini- an AIDS vaccine that would elicit both
around this problem, suggested in a paper tiative expanded tests of a clade Aderived killer cells and antibodies. The problem
published in Science three years ago by vaccine in Kenya, where clade A is found. is not impossible, he says, just ex-
Jack H. Nunberg of the University of But in January, Malegapuru William tremely difficult. SA
Montana and his colleagues, would be to Makgoba and Nandipha Solomon of the
make vaccines of gp120 molecules that Medical Research Council of South Carol Ezzell is a staff editor and writer.
have previously been exposed to CD4 and
therefore have already sprung open. But
MORE TO E XPLORE
HIV Vaccine Efforts Inch Forward. Brian Vastag in Journal of the American Medical Association,
those results have been difficult to repli- Vol. 286, No. 15, pages 18261828; October 17, 2001.
cate, according to Corey, making re-
For an overview of AIDS vaccine research, including the status of U.S.-funded AIDS clinical trials,
searchers pessimistic about the approach. visit www.niaid.nih.gov/daids/vaccine/default.htm
Another possible hurdle to getting an A global perspective on the AIDS pandemic and the need for a vaccine can be found at the
AIDS vaccine that elicits effective anti- International AIDS Vaccine Initiative Web site: www.iavi.org
HIV antibodies is the variety of HIV sub- Joint United Nations Program on HIV/AIDS: www.unaids.org


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GERM WARS ScientificAmerican.com

9 Behind Enemy Lines

20 The Unmet Challenges of Hepatitis C

33 Shoot This Deer

1 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

BEYOND By William A. Haseltine

2 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

Overview/Antiviral Drugs available or not effective. Antiviral treatments effect cures

4 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

CCR5 receptor CD4 receptor

Cell membrane Capsid 9 VIRUS ASSEMBLY

viral DNA into cellular DNA Cellular protein

5 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

WILLIAM A. HASELTINE, who has a doctorate in biophysics from Harvard

6 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

cidofovir Nucleotide analogue inhibitor of the Retinal inflammation caused by

delavardine, efavirenz Nonnucleoside, nonnucleotide inhibitors of HIV infection

lamivudine Nucleoside analogue inhibitor of reverse HIV, hepatitis B infections

ribavirin Synthetic nucleoside that induces mutations Hepatitis C infection

fomivirsen Antisense molecule that blocks translation Retinal inflammation caused by

interferon alpha Activator of intracellular immune defenses Hepatitis B and C infections

palivizumab Humanized monoclonal antibody that Respiratory syncytial infection

7 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

to other drugs. Recently, for instance, DuPont Pharmaceuticals

8 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

9 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

strains are not resistant to

10 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

11 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

12 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

AT WORK ria and can focus on those. Similarly, by comparing a targets

RIBOSOME The Chemistry, Biology, and Medicine of the Glycopeptide Antibiotics. K. C.

13 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

JOHNSON & FANCHER

FOODS UNDER STUDY as alternatives to injectable vac-

14 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

15 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

BACTERIAL CELL PLANT CELL

4 Allow callus to sprout

JARED SCHNEIDMAN DESIGN

16 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

I n short, the studies completed so far

ANTIGEN FROM ARRIVING

the antigen distant sites prod cytotoxic T CELL

INITIAL RESPONSE WHEN A DISEASE AGENT APPEARS

17 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

Moving against Malnutrition whether an orally delivered antigen will

18 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

JARED SCHNEIDMAN DESIGN

The Author Further Information

19 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

The Unmet Challenges of

by Adrian M. Di Bisceglie and Bruce R. Bacon

A s recently as the late 1980s few

20 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

21 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

R esearchers identified the hepati-

I f hepatitis persists for long enough

22 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

SERUM FROM CHIMPANZEE

23 SCIENTIFIC AMERICAN EXCLUSIVE ONLINE ISSUE NOVEMBER 2003

H epatitis C infection starts when vi-