Escolar Documentos
Profissional Documentos
Cultura Documentos
July 1, 2010
1
Abstract
HIV incidence has increase steadily over the time since it was first discovered in 1981.
Progress in treatment techniques is making what was once a death sentence into more of a
chronic disease. Studying the mechanisms of infection can give key insight into new targets for
treatment. Also looking at the mechanisms that allow both long term non-progression, and
immunity can illuminate new avenues in the development of new treatments and preventative
measures. Some key molecules involved in the process include antimicrobial polypeptides, and
chemokine receptors.
As more information is revealed about the structure of the virus and it replicative
process it allows for the development of new drugs to treat the disease. One of these new
targets is the chemokine receptor CCR5. The development of antagonists to this receptor may
block HIV entry. Prevalence data has also yielded some geographical areas of anomalously low
levels of the disease. This has lead to the study of what differs among these populations and
one popular theory is that it has to do with the amount of algae consumed in these areas. Algal
extracts can become a popular arena of study especially as they appear to block viral replication
in the gut where a large amount of lymphoid tissue is located. These have the potential not
only to be inexpensive and effective drugs but also to target an area of infection previously left
untouched.
2
It is commonly known that the human immunodeficiency virus (HIV) attacks the immune
system. It is this systematic destruction of the immune system that causes a person to become
immune deficient and vulnerable to diseases not seen in the immune competent (those with
functioning immune systems). In order to understand how HIV affects the immune system it is
h
The purpose of the immune system is to provide both immediate and long-term
immunity to foreign agents such as pathogens (v9). It does this through a variety of methods,
the first of which is physical barriers to infection, which include the intact skin and mucous
membranes (7, 17, 1v9). If the pathogen manages to breach these barriers it is then the innate
immune system that attacks the invaders. The innate immune system mounts a nonspecific
response to the foreign agents. It recognizes pathogen invasion by a set of conserved pattern
recognition receptors (PRRs). PRRs work by recognizing repetitive motifs called pathogen
associated molecular patterns (PAMPS) (142) that are common to potential pathogens, as well
as markers of cellular damage called danger associated molecular patterns (DAMPs) (v9). An
example of a PAMP would be lipopolysacccharide (LPS) which is found in the outer membrane
of gram negative bacteria (v4). DAMPS are host molecules released during times of stress or
damage, such as extracellular ATP, they are distinguished from PAMPS as they are derived from
The innate immune system includes cellular components such as natural killer cells and
neutrophils, and molecular components such as the complement cascade and interferons (v9).
v
Antimicrobial polypeptides (AMPs) may also be considered as being a part of innate immunity
as they are also mounted as part of the nonspecific responses (121). AMPs are produced by
macrophages, neutrophils, epithelial cells, haemocytes, fat body, reproductive tract and others.
Natural killer (NK) cells mediate the lysis of infected cells through the release of the cytotoxic
granules perforin and granzyme (106). Perforin, is a 70 kDa protein which is dependent on the
presence of Ca2+, the monomers bind and insert into the target membrane where they
polymerize to form aggregates, which form a pore of between 5-20nm (88, 116). Granzyme or
granule enzyme, a serine protease, is taken up into endosomes to enter the cell. Interaction
with perforin is necessary for its release into the cytosol from the endosomes (55, 148).
Interaction of perforin with granzyme triggers apoptosis of the affected cell (116). Neutrophils
The complement cascade is generated by the complement system; its two primary
functions are lysis and opsonization. The complement system relies on a series of protein
interactions in the blood to mediate its function (61). There are three pathways for the
activation of complement system, classical, mannose binding lectin (MBL), and alternative, all
converging in the activation of the membrane attack complex (MAC) that triggers cell lysis
(1v2). The MAC is a group of complement proteins that can lyse target cells, bacteria, virus
infected cells, or whole viruses, by penetrating their outer membranes (1v9). Opsonization
increases the effectiveness of the immune response by targeting cells for destruction (1v2). It
does this through the attachment of opsonins, surface bound proteins that identify foreign
material (1v9).
4
Interferons (IFNs), antiviral proteins formed by virus infected cells and in response to
immune interactions with microorganisms and chemicals, can be divided into two types, Type I
and Type II (15, 45). Type I are viral IFNs and include ɲ, ɴ, ʘ, and ʏ subtypes, whereas type II
includes only IFN-ɶ (15). These proteins modulate the immune response by interfering with the
The innate immune system focuses on immediate (hours to days) immunity, however, it
is insufficient for long term human survival so one of its key functions is to activate the acquired
immune system (v9). The innate immune response is also called the inflammatory response
and some cells that promote or enhance this process are known as inflammatory mediators
(v5). For long-term control of a pathogen the body mounts an adaptive (acquired) immune
to disease. Other AMPs include cathelicidins and histatins and they act against bacteria, fungi
and viruses. Many of these come into action when studying the mechanisms of immunity to
HIV. Most AMPs are < 10 kDa, have a net positive charge, and are membrane active and
hydrophobic (121). They have been identified in organisms ranging from insects and plants to
humans. Based on their structure they have been divided into five groups; V-helical, cysteine
rich, -sheet, rich in regular amino acids (AA), and those with rare AAs. While defensins are
5
classified as cysteine-rich, both cathelicidins and histatins fall under the category of rich in
Cathelicidin LLv7, for example, comes from neutrophils and epithelial cells and can
inhibit lentiviral replication as well as being chemotactic (attracting) for neutrophils, monocytes
?
Defensins are a class of antimicrobial peptides (AMP), mediators of innate host defense
that are primarily involved in membrane depolarization (12). Defensins, v-6 kDa non-
glycosylated peptides, are a potential innate HIV-1 inhibitor that have been found in mucosal
secretions and blood and are effectors that link innate and adaptive immune responses (12, 62,
oligomerization domain) signaling (v4). There are three categories of defensins in vertebrates ɲ,
ɴ, and ɽ defensins (146). The first two of these are found in humans and are differentiated
based on the structural pairing of their cysteine residues and disulfide bridges (12, 12v). The
third category ɽ-defensins were initially isolated in rhesus macaques (14v, 146).
ɲ-Defensins are further divided and named human neutrophil peptides 1-4 (HNP-1 to
HNP-4) because they are produced by neutrophils; though immature monocyte derived
dendritic cells have also been found to produce them (12). They are found circulating in the
plasma, within certain epithelial cells and are contained in neutrophilic granules (90). Some
6
studies only list the first three categories as divisions of ɲ-Defensins as HNP ʹ 4 was not isolated
until later (v0, 12v). HNPs1-v have been shown to be effective against Staphylococcus aureus,
Pseudomonas aeruginosa, Escherichia coli, and also against the fungus Cryptoccocus
ɴ- defensins are produced by epithelial tissues and there are four identified in humans
phospholipids (12). ɽ-defensins are 18 residue cyclic peptides that act as entry inhibitors; they
are homologues of the ɲ-defensins expressed by humans and some other mammals (146).
ɽ-defensin have been found encoded near the other defensin genes in humans. Their signal
sequences all contain a premature stop codon that prevents them from being transcribed, this
has also been found in bone marrow mRNA (109, 146). ɽ-defensins are also called retrocyclins
and are categorized as retrocyclins 1, 2, and v. Retrocyclin 1 has been shown to bind gp120 and
CD4 protecting cells from T- (T cell specific) and M- (macrophage specific) tropic strains of HIV-1
(146).
A study done on exposed but uninfected Kenyan sex workers found that HIV
neutralization was associated with HNPs1-v in vaginal secretions, though higher levels of these
were also associated with co-infections and higher HIV acquisition (81). This may seem a little
counter-intuitive but infection with other STIs (sexually transmitted infection) increases the
amount of these HNPs while at the same time making people more vulnerable to HIV infection.
7
Other proteins that have shown anti-HIV activity are the APOBEC family of proteins.
h
polypeptide) proteins are important as some members of this family provide protection against
viruses, especially HIV. The name is derived from the fact the first member of the family to be
discovered, APOBEC1, works in the intestine, and edits apoB mRNA producing apoB48 which is
the main structural component of chylomicrons (64, 111). APOBECs are the catalytic
changes a single cytosine to uracil to generate a premature translational stop codon. The
family contains APOBECs 1-4, cellular cytidine deaminases, and activation induced cytosine
deaminase (AID) (100, 111). The role of APOBEC2 is uncertain but its expression is specific to
The APOBECv family is unique to mammals and includes APOBECvA (hAvA) through
APOBECvH (hAvH), it also works specifically on single stranded DNA (ssDNA) rather than RNA
(58, 111). Of the seven APOBECv genes in humans hAvG has the most potent anti HIV activity
(100). Other members of the family have also been shown to have activity against other viruses
and retroelements (transposable DNA sequences) (100). The hAvG are incorporated into the
HIV virion during assembly and catalyze cytosine deamination during (-) strand synthesis. This
ultimately results in the degradation of the viral DNA and/or lethal hypermutagenesis (100).
Unfortunately, these effects are counteracted by the viral protein vif (68).
8
consider the types of cells involved in the immune process. Clusters of differentiation (CD) are
used to delineate between different types of immune cells. These are cell surface phenotype
antigen differences, of which there are over v50 (http://www.hcdm.org/) (2v). Within a
cluster, antibodies give identical cellular reaction patterns and identify the same molecular
species (74). Key in the discussion of HIV are the CD4+ T cells as the CD4 molecule is used as a
receptor for viral entry into cells. Further detail on immune cell function can be seen in Table 1.
Macrophage/Monocyte Phagocytosis, antigen presentation, secrete
interleukin-1 and interferons
Neutrophil Phagocytosis
Basophils Inflammatory mediator, release histamine and
heparin
Mast cells Inflammatory mediator
Eosinophils destroy antigen antibody immune complexes
Dendritic Follicular Sense changes in Bind opsonized virus
cell homeostasis and and trap it
Plasmocystoid target naïve T-cells Produce interferon ɲ
Natural Killer cell Induce apoptosis
T cells CD 4 helper secretion of interleukin-2, ɶ-IFN, and tumor
necrosis factor, proliferation of T and B cells
CD 8 cytotoxic Include suppressor T cells that reduce immune
function, and cytotoxic cells that release
cytokines to attract macrophages
Memory Found in lymphoid tissue, recognize invading
antigen from prior exposure
B cells Mature into plasma cells which produce
antibodies
(1v, 17, v9)
9
The acquired immune response is activated as follows. First an immunogen (a
antigen presenting cell (APC), generally a dendritic cell. APCs are differentiated into
͚professional͛ and ͚amateur͛ based on their ability to generate ͞signal 2͟ which involves the
expression of surface protein co-stimulatory molecules such as CD80 and CD86 that generate
this response (67, 99). Dendritic cells are considered professional due to their ability to elicit
this second signal and thus are the preferred APC (99). Dendritic cells respond to changes in
homeostasis by maturing and limiting surrounding cell activation and targeting naïve T-cells.
Activation of dendritic cells regulates antigen phagocytosis and presentation, also called ͞signal
1͟ in T cell activation (v9). The antigen, for example a viral protein, is cleaved into small
peptide fragments (representing specific viral epitopes, fragments of the antigen that will
generate an immune response) inside the APC. The APC then migrates to the lymphoid tissue
for antigen presentation (v5, 99). This epitope is then presented to B and T cells in association
with a cell surface protein called the major histocompatibility complex (MHC) (2v). MHC
molecules can be subdivided into classes I and II. B and T cells possess surface receptors that
respond to a specific epitope. In the case of T helper cells the binding of the MHC and epitope
along with the release of the cytokine interleukin 1 (IL-1) from the macrophage triggers the
activation of the cell. This activated T helper cell can then, with the aid of IL-4 and IL-5 which
are secreted by the activated T cell trigger the activation of B cells (v5). The T cell can then
mature and become a memory T helper cell (Table 1) or with the aid of interleukin 2 (IL-2) it can
trigger the activation of cytotoxic T cells. These cytotoxic T cells release cytotoxins, perforin
and granzyme that result in the death of virus-infected cells. They can also mature to become
10
memory cytotoxic cells. The activated B cells react with virus immunogen on the APC and
differentiate to become plasma cells that secrete antibodies. Viruses have many methods of
evading the immune response some of which will be discussed later in regards to HIV.
Function
!" activate T lymphocytes by enhancing the production of IL-2 and the expression of IL-2 receptors
!" generation and maintenance of T regulatory cells, activate NK cells and promote
the proliferation of B and T lymphocytes
!"# contribute to the activity of eosinophils in allergic inflammation through their capacities to
prolong eosinophil survival and to generate activated eosinophils, supports the growth of
precursors for a variety of hematopoietic cells
!"$ Induce antiparasitic and allergic immune responses, negative regulator of TH17, IL-19, IL-25, IL-
vv
!"% stimulating eosinophil production and release from the bone marrow, is chemotactic for
eosinophils and activates mature eosinophils, inducing eosinophil secretion and enhanced
cytotoxicity
!"& B lymphocytes differentiation into mature plasma cells and secrete immunoglobulins mediates
T-cell activation, growth, and differentiation, production of acute phase proteins
(28)
B cells are differentiated from hematopoietic stem cells in the bone marrow and are
defined by the production of immunoglobulin (Ig) antibodies. There are five classes of
immunoglobulin, IgA, IgC, IgD, IgE, and IgG. Naïve B cells express IgM and IgD on their cell
surfaces (2v). Stimulation by T cells triggers class switching which is characterized by the
production of IgG, IgA, or IgE antibodies (2v). Memory cells are formed in the late stages of
immune activation and require a smaller threshold for activation than non-memory cells. They
11
are key in providing a swift response upon re-exposure to a pathogen and are important in the
Antibody Function
IgA Provides local protection to mucous membranes;
present in tears, saliva, mucus, breast milk and other
mucosal secretions
lgD Activates B-cells; assists in differentiation of B-cells
IgE Involved in allergic reactions; defense against parasitic
infections
IgG Protects against bacteria and viruses; enhances
phagocytosis and triggers complement system; can cross
placenta from mother to fetus
IgM First antibodies secreted; cause lysis of pathogens
#
'
Viruses are biological entities that infect other cells and replicate. There are an
estimated 10v1 viruses on earth (16). When outside of cells they exist as free particles and their
general structure typically includes a RNA or DNA genome surrounded by a protein shell (16).
The majority of viruses are phages that attack bacteria (16). When a virus infects a eukaryotic
cell it generally triggers apoptosis of the infected cell by either directly or indirectly activating
cellular sensors that initiate cell death (42). Host mediated lysis is one of the mechanisms by
which viruses subvert the immune system, after replicating within the cell they use this lysis as
Viruses are classified into two major groups based on the type of nucleic acid they
12
further be divided by their genome type; double-stranded (ds) DNA, single-stranded (ss) DNA,
dsRNA, negative sense ssRNA, positive sense ssRNA and reverse transcribing (96, 119). The four
principle taxa (related clusters) of viruses as recognized by the international committee on the
Order (96). Family and order are not always used as there is not always the needed relatedness
between groups. These taxa can all be classified underneath their genome type. Of particular
(
Retroviruses are a family of encapsulated (within a viral envelope) RNA viruses (75).
They are about 90 -120 nm in size and consist of a two copies of the
ssRNA genome and number of enzyme molecules required for stable infection (89). One of
these is the reverse transcriptase, encoded by the pol gene, which is an RNA-directed DNA
polymerase that is essential for viral replication. All retroviruses contain three major coding
domains which are the gag, pol, and env genes. Gag, named for group specific antigen, directs
the synthesis of proteins that form the matrix, capsid, and nucleoprotein structures, pol
contains the information for the reverse transcriptase and integrase enzymes, and env codes
for the viral envelope proteins (26, 15v). There is also a smaller coding domain pro which
1v
encodes for the viral protease. The long terminal repeat (LTR) is also a part of the retroviral
genome and it contains the promoter and enhancer regions (75). Retroviruses can be classified
as simple or complex depending on whether they carry only this information or whether they
code for additional regulatory proteins as is the case with HIV (89).
The Retroviridae family is further divided into three subfamilies containing seven
glycoprotein which controls viral tropism and promotes the membrane fusion process (89, 107).
These genera are differentiated based on the shape and location of the inner protein core (26).
and epsilonretrovirus, the spumavirinae (foamy) spumavirus, and lentivirinae (slow) lentivirus
(120). The first five of these are retroviruses with oncogenic (cancer causing) potential, hence
the subfamily name oncovirinae, and are all simple retroviruses containing only the gag, pol,
and env genes (26). Oncogenic viruses are considered to be directly linked to cancer (155).
These seem to be associated with the incorporation of viral oncogenes into host DNA,
modification of viral oncogenes after integration, and the modification of host genes such as
human endogenous retroviruses (HERVs) integrated into viral genomes that then act as
14
oncogenes (155). All of these are pathogenic either in their natural host or in incidental hosts
(86). The lentivirus, meaning slowly developing, family is especially relevant as the virus HIV
!
Lentiviruses are found in felines, ungulates (sheep, cattle, etc.), avians, rodents, humans
and primates (79) (75). They cause disease primarily by killing or inducing the loss of function
of specific cells and tissues (26). Diseases included in this family encompass HIV, simian
immunodeficiency virus (SIV), feline immunodeficiency virus (FIV) and others (79). HIV is the
only one known to infect humans (72). These viruses are characterized as being nononcogenic,
immune cell infecting, remaining permanently associated with cells, causing slowly progressive,
chronic diseases, and the replication is generally species-specific. Common features in host
interaction with these viruses are long periods of clinical latency, only a small neutralizing (virus
specific) antibody response, extensive genetic mutation and drift, neuropathology and viral
15
0
0
immunodeficiency syndrome, AIDS (4v, 124). The symptoms of HIV infection may include;
swollen lymph nodes, fever, chills and night sweats, diarrhea, weight loss, coughing and
shortness of breath, persistent tiredness, skin sores, blurred vision and headaches, as wells as
the development of other infections (92, 1v1). It is spread through the transfer of bodily fluids
such as; blood (92), semen (57), vaginal secretions (1v1), and breast milk (10). Only in about 1-
5È of patients does saliva contain infectious HIV and tears contain only negligible amounts of
the virus (9, 108). The virus can survive outside the body for several days to weeks depending
on the environment, initial viral titer, and growth medium. Factors such as temperature,
humidity and sunlight affect the life of the virus outside the body (1v7) influencing survival
include. A person is generally considered to have progressed to AIDS if they have specific
clinical conditions indicative of severely impaired immune function, or a CD4 cell count of less
than 200/mmv (84). In the absence of treatment the onset of AIDS generally develops 8-10
HIV was first drawn to the attention of physicians in 1981 when the Centers for Disease
young homosexual men in Los Angeles (57, 102). Within a year the term AIDS was coined by
the CDC to describe the combination of opportunistic infections and tumors occurring in people
with markedly reduced helper (CD4+)T-cell count (table 1) (124). Initially the disease seemed to
16
be confined to a few high risk groups of people, (see table 4), however, in 198v a retrovirus
later named HIV was isolated from a patient with AIDS thus confirming suspicions that AIDS had
an infectious cause (102, 124). Finding this retrovirus led to the development of a test for HIV
(ELISA) to search for IgG antibodies specific to HIV in a patient͛s serum, which also allows for
the screening of donated blood for large scale epidemiological studies and for prevention of
infection from receipt of donated blood (115). Viral load is measured by assays such as the
Amplicor HIV-1 Monitor test (Roche Molecular Systems) which measures viral RNA by nucleic
acid amplification (69). There has been dissent over the naming of HIV as to the etiological
cause of AIDS, with some proposing that AIDS is caused not by HIV but rather by drugs and
other non-contagious risk factors, however, sufficient evidence to support alternate theories
The earliest documented case of HIV infection was identified from a stored plasma
sample from 1959 in the city now known as Kinshasa in the Democratic Republic of Congo
(154). The origin of the virus in humans is postulated to be the inadvertent introduction of
simian immunodeficiency virus (SIV) from a closely related primate species. In 1986 a second
strain of retrovirus called HIV-2 was isolated from patients with AIDS in West Africa (25, 124).
This strain shows a 40-60È homology at the RNA level with HIV-1 but differs significantly at the
protein level (25). Studies of HIV-2 show that it has a very close relationship with the SIV strain
that infects the primate known as the sooty mangabey (Cercocebus atys) in the same parts of
West Africa (77, 124). It is now thought that there have been at least three separate entries of
SIV into the human population. HIV-1 seems to have a greater degree of similarity with
17
chimpanzee (Pan troglodytes) strains of SIV in species whose habitat overlaps that of the
location where the HIV-1 is believed to have originated (124). Exactly how humans came to be
There are at least four different African primate species with their own
immunodeficiency virus variants. These include the Sooty mangabeys (Cercocebus torquatus
atys) SIVsm, African green monkeys (Cercopithecus aethiops) SIVagm, Sykes͛ monkeys
(Cercopithecus mitis) SIVsyk, and Mandrills (Mandrillus sphinx) SIVmnd (114). Also there have
been reported cases in chimpanzees (Pan troglodytes) SIVcpz and red-capped mangabeys
(Cercocebus torquatus torquatus) SIVrcm though it is unclear whether they are natural hosts
(114). In no case do these viruses appear to be pathogenic in these other primates and studies
of the mechanism behind this could give insight into dealing with HIV infection in humans (114).
It is important to note that this is not a function of viral load as there can be high viral loads in
these animals without progression of the disease to AIDS (9v). One study indicated that the
disease will eventually progress to classic AIDS if followed over a long enough period of time
(85). Despite this it is still apparent that non-human primates have some defense mechanism
The main immunologic feature of advanced stages of HIV is a decrease in the number of
circulating CD4+ T-cells, which are helper T cells. This feature quickly became the main
surrogate marker for HIV-related immunodeficiency. The normal CD4+ T-cell count is >500 T
cells per mmv, v50-500 T cells per mmv is slightly depressed, 200-v50 T cells per mmv is
moderately depressed and <200 T cells per mmv demonstrates severely compromised immune
18
function (97). Once absolute CD4+ T-cell count drops below 200 T cells per mmv in the
infections. The tendency of the virus to infect CD4+ T-cells as well as other CD4 expressing cells
was explained in 1984 when the CD4 molecule was shown to be a high affinity receptor for the
virus (v1).
The time course of HIV infection can be tracked by a combination of CD4+ T cell count
and viral load. In the first weeks after initial infection there is a drop in cell count and a rise in
viral load (amount of viral RNA detected) (128). This returns back to normal or near normal over
the course of the next weeks or months. The virus then enters a period of what is generally
considered to be latency, when viral load and cell count reach a stable set point (17). This set
point differs from person to person but looking at this can give an indication of how the disease
will progress. The set point for plasma viral load can range from as few as 100 copies/mL to as
many as 10,000 copies/mL. Patients on the higher end are at higher risk of disease progression
(17). During this time viral load and cell count remain relatively steady, and this can last for
years (17). Viral shedding can occur at any time during the course of the infection but other
19
The HIV virus is composed of a central core of cylindrical RNA which is surrounded by a
spherical bilayer membrane envelope with glycoprotein (gp) surface markers (84). Cell surface
markers enable the identification of specific cell types. The viral genome contains ~10,000
nucleotides and encodes nine genes. These include the three common to all retroviruses gag,
pol, and env, as well as tat, rev, vpu, nef, vpr, vif whose functions will be explained later. In HIV-
2 the vpr gene is replaced with the gene vpx (10v). HIV-1 entry into the cell requires interaction
with two separate membrane protein sites on target cells. These sites are the CD4+ receptor
and a 7 transmembrane (7-TM) chemokine receptor (84). This 7-TM chemokine receptor is
generally the CC-chemokine receptor 5 CCR5 or the CXC chemokine receptor 4 CXCR4 for
primary HIV-1 in vivo (127). These are named for the structure of the chemokines which they
have two adjacent cysteines (CC), while CXC have a variable amino acid between the two
cysteines (v2). HIV is classified as M-tropic, macrophage specific or T-tropic, T cell specific
dependent upon the type of cells it attacks. Viral tropism can change over the course of the
disease, initially infecting as M-tropic but changing to T-tropic as the virus progresses (84, 117).
CCR5 is primarily used by the M-tropic, viral strain whereas CXCR4 is more prevalent with the T-
tropic viral strain. This preferential targeting of the viral strains also gives the tropisms another
name: M-tropic is also designated R5 for use of the chemokine receptor CCR5 and T-tropic is
also designated X4 for use of the chemokine receptor CXCR4. The genes for chemokine
emergencies as signaled by on-site chemokine production (110). Specifically CCR5 aids memory
20
T cells by stimulating the up-regulation of adhesion receptors and allowing migration to
inflammatory sites. This function is also carried out by CCR2 and CCRv (40). CXCR4 is
chemotactic for T cells, and is involved in B cell development in the bone marrow (v2). It also
plays a role in vascularization of the gastrointestinal tract and formation of cerebellar tissue, as
defects have been seen in these areas when it is knocked out (v2).
There are two primary strains of HIV that are the focus of most studies, HIV-1, and HIV-
2. As mentioned earlier each of these is closely related to a specific SIV strain from primates.
The worldwide pandemic of HIV is primarily due to HIV-1 so this is the primary focus (129). HIV-
1 is further divided into three groups main (M) (not to be confused with the M-tropic (R5)
designation above), outlier (O), and non-M, non-O (N). The M group causes the majority of
infections and is divided into subtypes or clades given the letters A ʹ F (52, 129). These are
differentiated by their phylogenetic ancestry or the point at which they diverged. Variance in
subtype also denotes a variance in antigenic structure, transmissibility and infectivity (129).
21
)$'
(80)
As is shown in figure v there are several steps in viral replication: binding, fusion and
budding and maturation. Each of these steps involves particular proteins encoded by the viral
genome in order to proceed. The chemokine and CD4 receptors mentioned earlier and other
host proteins are also involved in this process. The following is a brief overview of the
replication process. Greater detail into the mechanics of this will be given as the proteins
In general the process of viral replication is as follows: HIV binds to the host cell
through the interaction of gp120 and CD4+ receptor site (17). Following this, interactions
between the virus and chemokine receptors trigger irreversible conformational changes (128).
After binding, gp41 facilitates the fusion of the viral envelope with the host cell membrane
22
through pore formation. Once fused the HIV viral core then uncoats releasing two single
strands of viral RNA into the cytoplasm (17). Reverse transcriptase then utilizes this ssRNA as a
template to make dsDNA. This dsDNA moves into the host nucleus where integrase promotes
its insertion into the transcriptionally active domains of the host cell DNA making proviral DNA
(17, 128). When the host cell is activated transcription of this proviral DNA into mRNA occurs.
Then mRNA exits the nucleus and aids in the production of viral polyproteins that will become
the HIV core and envelope. The enzyme protease modifies some of these polyproteins into
active proteins such as the envelope proteins gp120 and gp 41 (17). These are then assembled
into an HIV virion and then budding occurs to release HIV from the cell. Cleavage of the
budding virion is accomplished by the viral protease. It is this cleavage that marks the
Ë 34$
Entry of HIV into the cells is mediated by the envelope (env) gene coding for
glycoprotein gp120 (outer envelope) and gp41 (transmembrane) molecules, which are derived
from cleavage of gp 160 and are responsible for viral attachment to the target cell (92, 150). Gp
120 surface subunits are responsible for binding to cellular receptors and gp41 acts as an
anchor (v7). The first step in this entry is the binding of gp120 to CD4 followed by binding to a
receptor on the T-cell surface that triggers conformational changes in Env; this creates or
exposes a binding site for the 7TM chemokine receptor (v8). The identity of the receptor and
the specific mechanisms behind this viral entry are not clearly understood (29).
2v
polypeptides. Their general function involved the chemical attraction of various cells to specific
tissues (v2). They are traditionally classified according to the amino acid motif that occurs in
the first two N- terminal cysteines (v2). For the largest of these groups the amino acid motif
CXC. The CC or beta chemokines act on lymphocytes, monocytes, basophils, and eosinophils
and the CXC or alpha chemokines act mainly on lymphocytes and neutrophils (v2). The
receptors for these are in the family of seven transmembrane G ʹ protein coupled receptors
GCPRs which follow after the nomenclature of the chemokines that they respond to (v2).
These receptors range from vv9 to v7v amino acids in length (110). Their general functions
include cell migration, transcriptional activation, cell growth and differentiation (149).
As mentioned earlier viral tropism determines the type of cells that it infects and CCR5
and CXCR7 are the most common co-receptors for HIV. Studies done in vitro have
demonstrated that other chemokine receptors such as CCR2, CCRv, and CCR8 may also be used
as co-receptors for HIV (4). The M ʹtropic strain of HIV is thought to be critical in infection
establishment and maintenance and is the primary transmitting form of the virus (v2, 95).
CCR5, the primary co-receptor for the M-tropic (R5) strain, was first identified as the receptor
for CCLv, CCL4, and CCL5 (v2). It is expressed on memory T cells and IL-2 cultured T cells as well
as immature dendritic cells. The expression of CCR5 on dendritic cells is dramatically reduced
24
after lipopolysacccharide induced maturation of these cells (v2). Its normal function is to
CXCR4 is also known as Fusin, LESTR, and HUMSTR (v2). Expression of this receptor has
been demonstrated on T cells, B cells, and monocytes and mature dendritic cells, bone marrow
B-cell progenitors and in the embryonic brain (v2). The natural ligand for this receptor is
CXCL12 (v2). Exposure of resting T cells to IL-4 has been reported to increase expression of this
receptor. In mice, knockouts of the gene for this receptor or its ligand result in lethal mutations
and in abnormalities in gastrointestinal tract vascularization (v2). HIV strains using CXCR4 are
rarely transmitted but evolve as the disease progressed and are associated with poor outcomes
(105).
-
A v2 base pair deletion in the gene that codes for the CCR5 receptor results in a loss of
expression of functional receptors (v2). This defect is present in about 1È of the Caucasian
population (v2). People homozygous for the CCR5 ɷ v2 mutation have a high degree of
immune protection from HIV due to the lack of expression of any functional CCR5 on their cell
surfaces whereas those who are heterozygous have partial protection (4, 50). This partial
protection is conveyed in an apparent delayed disease progression (22). CCR5 also seems to
play a role in the lack of pathogenicity of the SIV infection; sooty mangabeys down-regulate the
expression of CCR5 on their CD4+ T cells (50). In red caped mangabeys there is a similar
deletion in the gene coding for CCR5 and this 24 bp deletion is present in 98È of all red-caped
mangabeys (50).
25
Other mutations in the chemokine receptors that have been studied are the CCR2-64I
point mutation and the SDF1-v͛A mutation, a single nucleotide polymorphism, in the SDF1 gene
coding for the chemokine ligand for CXCR4. These mutations confer favorable prognosis to
HAART treatment and protection against the development of AIDS respectively (4). An
additional mutation in chemokine receptors is the CCR5-P1 mutation that plays a role in slowing
-.
Matrix protein (MA) is one of the karyophillic (attracted to the nucleus) elements
present in the viral proteome, and it is important in the nuclear import of HIV. MA, one of the
first viral proteins of HIV-1 to be related to nuclear import, utilizes nuclear localization signals
(NLS) to mediate its action (122). NLS target molecules to the nucleus of the cell, commonly
using a short stretch of basic amino acids that give an overall net positive charge (20). The NLS
connects the preintegration complex (PIC) with karyopherin ʹ ɲ (importin ɲ), and cellular
import machinery and this drives import into nucleus (56). Karyopherin ʹ ɲ is a host protein
that is part of the nuclear import machinery (122). The PIC is a nucleoprotein complex that
contains viral proteins (reverse transcriptase, MA, NC, and Vpr) and cellular factors that are
26
(
Reverse transcriptase (RT) is one of three proteins derived from the pol protein from
the pol gene in retroviruses (92). It is an RNA- and DNA-dependent DNA polymerase and also
has ribonuclease H (RNase H) activity (54). Its function is to make a dsDNA copy of the viral
RNA (75). RNAse H activity triggers the degradation of the RNA genome (126). This
transcription takes place in the cytoplasm of the host cell (Figure v). The RT enzyme lacks a
proof-reading mechanism which, along with its rapid replication rate, makes the virus easily
mutated (124). This is one of the key targets for antiretroviral therapy.
)
Integrase (IN) is a v2 kDa enzyme necessary for the integration of viral DNA into the host
cell chromosome (11). It is part of a large nucleoprotein complex, the PIC, which includes viral
DNA, Vpr, the integrase interactor protein, and several cellular proteins, such as cellular lens
epithelium derived growth factor (11, 140). IN binds to a recognition sequence found at the
ends of the viral LTRs. It catalyzes the integration of viral DNA through a v͛end processing and
strand transfer (11, 140). The third step in integration, gap repair, is mediated by cellular
proteins (11). Inhibitors have been developed that interfere with the strand transfer process
which could be beneficial in treatment of HIV (11). The lack of a functional, naturally occurring
equivalent of the enzyme against integrase makes it less likely for toxicity to develop (11).
Transactivator of transcription (Tat) and Rev are additional viral cofactors that are
essential for replication. Tat promotes elongation of the viral genome by binding to HIV͛s
transactivation response element (TAR) (6). TAR recruits positive transcription elongation
27
factor b (P-TEFb) which is necessary for HIV transcription as well as the transcription of other
cellular genes (6). Rev is involved with the nuclear export of unspliced viral RNA. Absence of
tat and cellular activation signals in resting cells is responsible for viral latency (5v). There is
currently no way to detect the difference between uninfected cells and latently infected cells.
'
Viral protein R (vpr) encoded by the vpr gene is a small (96 amino acid) nuclear protein
that is expressed late in the virus replication cycle. It is responsible for the nuclear import of
the preintegration complex (PIC), apoptosis of cells, and aids in the reverse transcription
process, G2 cell cycle arrest and transactivation of the LTR and/or target genes. Its actions in
the transactivation process control the gene expression of HIV (140). When vpr is deleted in
vitro the virus replicates effectively in cell lines and activated CD4+ T cells but not as well in
macrophages and monocytoid (resembling monocytes) dendritic cells (58). Mutations in the
gene have been associated with long-term non-progression resulting from a decrease in
proapoptotic activity. The protein also has neurotoxic effects and defects in the gene have
'
The gene vpu codes for viral protein U (vpu). It has two primary functions, degradation
of the CD4 molecule in the endoplasmic reticulum and is involved with virion release from cells
28
(Figure v) (11v). This role in virion release is cell type specific. It works by countering an
interferon-induced restriction factor, called B cell stromal factor 2 (BST-2) or tetherin, that
would normally prevent retrovirus release. Theories as to how it does this include:
proteosomal degradation of tetherin, and acting in a manner similar to nef and down regulating
the expression of tetherin (91). As tetherin works to inhibit virion release it is being looked at
A few of the proteins encoded by the viral genome are involved specifically in immune
evasion. Immune evasion is the process by which viruses ͚trick͛ the immune system into
helping them spread or prevent host mechanisms from destroying the virus.
'
Viral infectivity factor (vif) is a basic, 2v kDa protein that works to counteract the effect
of APOBEC by decreasing the intracellular concentration of hAvG and hAvF, preventing their
incorporation into HIV virion (100). Vif is essential for HIV propagation in vivo. It is only in the
absence of vif that the APOBEC proteins are allowed to function. Since vif plays a critical role in
eliminating an intrinsic defense mechanism it could be an attractive target for future therapy.
Vif functions by utilizing the ubiquitin-proteasome pathway to degrade APOBEC (68, 91).
Ubiquitination is a general cellular mechanism for marking damaged proteins for destruction by
the proteasome by covalently attaching a small protein called ubiquitin. This ligation is
catalyzed by a series of complex reactions utilizing ubiquitin activating (E1), conjugating (E2),
29
and ligating (Ev) enzymes (68). Ubiquitin is activated forming an E1 thiol-ester ubiquitin
intermediate. E2 catalyze the attachment to target proteins and then Ev binds and ligates that
target protein. The HIV Vif protein hijacks an Ev enzyme, specifically the Cul5-Ev inducing
(91).
The nef gene, one of the six small genes specific to HIV encodes the protein nef. Nef
(negative factor) has many varied functions which makes it somewhat of a misnomer for it to
be named negative factor. In vivo it is required for efficient viral replication and pathogenicity
(1vv). In vitro it affects cellular signal transduction pathways and down regulates expression of
both surface CD4 (the HIV receptor), and MHC I and II molecules (24, 1vv). Defects in the nef
and/or long terminal repeat (LTR) region (figure2) have been associated with the absence of
disease progression (140). Nef also binds and mediates the redistribution, and removal from
the cell surface, of the immune co-stimulatory molecules CD80 and CD86. These molecules in
the B7 family act as the second signal in T cell activation, triggering CD28, an immune co-
stimulatory molecule, which is expressed on naïve T cells (67). The B7 family is a family of
immunoglobulins that includes B7-1 (CD-80), B7-2 (CD-86), B7-Hv, PD-L1, PD-L2, and GL50.
They are primarily expressed on activated B cells, and macrophages (1v4), and their functions
include T cell priming, IL-2, IL-4, IFNɶ production, and inhibition of T cell proliferation and
response (8v).
v0
The use of antiretroviral drugs for the treatment of HIV was introduced in the mid
1980͛s (14). Treatment typically consisted of a nucleoside reverse transcriptase inhibitor, NRTI,
monotherapy (single drug). Monotherapy did not adequately suppress the virus which led to
the emergence of drug resistant HIV variants (49). The goal of antiretroviral treatment is to
reduce mortality and morbidity rates (128). Soon trials were done that experimented with
multiple drug therapy. These studies combined two NRTIs and showed a reduction in mortality
rates and progression to AIDS (14). With the advent of new classes of antiretroviral drugs such
as protease inhibitors and non-nucleoside reverse transcriptase inhibitors new multiple drug
treatments could be used. Combination therapies reduce the incidence of drug resistance and
toxicity. These multiple drug therapies are known as ͚highly active antiretroviral therapy͛, or
HAART (14).
inhibitors (NRTIs and NNRTIs), CCR5 receptor antagonists, protease inhibitors, fusion inhibitors,
and integrase inhibitors (14, v7). HAART generally includes a combination of two different
reverse transcriptase inhibitors and a protease inhibitor (1v5). Research into new
short/long term side effects are problems with the current drugs. Fusion inhibitors are
generally reserved for patients who have been treated for long periods of time (11). They have
been approved along with other drugs to be used in patients who despite treatment still have
residual viremia (48). Figure three shows where these drugs act in disrupting the infectivity of
v1
HIV. Though NRTIs and NNRTIs both act on reverse transcriptase they differ in their binding
sites, NNRTIs interact with the non-substrate binding site and NRTIs interact with the substrate
reverse transcriptase binding site (vv). Binding in this non-substrate site seems to lock the
reverse transcriptase into an inactive conformation. These two sites are functionally and
spatially associated and thus using a combination therapy of the two RTIs is more effective (vv).
While these therapies are good at decreasing the viral load, they have little effect on
renewing CD4+ T cell levels or immune reconstitution. This is why there is also the need for
adjuvant therapies. Adjuvant therapies work to complement the work that the antiretrovirals
do, by directly targeting the immune system (94). The most promising of these is interleukin-2
(IL-2) immunotherapy. Studies show that in conjunction with HAART there is considerable
v2
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times the patient has to switch treatment regimens. In 2002 the average drug cost for one to
six switches was ~$5,978/yr or ~$498/mth and for seven to thirteen switches it was ~$7,412/yr
or ~$618/mth (101). Decisions on changing treatment are based not only on T cell counts and
viral load, but also on whether the patient is experiencing side effects from the therapy (44).
These costs have risen as new drugs are developed and treatment regimens expand beyond the
As more information is learned about the structures involved in viral entry and
replication new drugs can be made to target them. Some of the new avenues for treatment
include CCR5 receptor antagonists, stem cell transplant from a person with the CCR5 ɷv2
mutation, fusion inhibitors, and algal extracts. It has been speculated, based on prevalence
data and in vitro and limited in vivo evidence of the anti-HIV activity of algae, that algae
consumption is related to lower rates of HIV (1v6). HIV/AIDS prevalence in eastern Asia is
about 0.1È in adults, (Figure 4) whereas in Africa rates may be 40È or higher (1, 1v0). Some
other explanations for the low prevalence rates include; recent introduction of the virus to the
population, recorded infections confined to high risk groups and early aggressive response to
the disease (1). An exception to the high rates in Africa is Chad where there is a higher
consumption of algae and prevalence rates of about 2.vÈ-v.6È have been reported. Other
locations in Africa showing lower prevalence rates include Niger and Senegal though there is no
data for whether there is a higher consumption of algae in these areas (141). Extracts from
seaweeds and Spirulina a cyanobacteria have been shown in vitro to inhibit a variety of
v4
enveloped viruses including HIV (1v6, 151). Its action seems to be mediated by direct
interaction with the HIV binding site of the CD4 receptor (v).
Algae are one of the first sources of natural compounds with in vitro anti-HIV activity.
They are grouped into six main classes based mainly on color. These classes are Chlorophyceae
Antibacterial activity of these plants has been reported as early as 1940 (125). Compounds
isolated from algae include steroids and sulfoglycolipids, but most research has centered on
natural and synthetic sulfated polysaccharides. These polysaccharides are able to block HIV
replication in cell culture in a manner that is dependent upon molecular weight and degree of
sulfation (125). They are found on the external surface of cell membranes, in pericellular
locations, in the extracellular matrix and at the mucous membrane. Their primary functions
seem to be cell to cell communication, to act as a barrier between tissues, cell adhesion, as a
reservoir for growth factors and also to protect against pathogens (1v8). Though they have
been studied primarily in blue green algae, extracts from over 60 species including brown and
red algae have been tested (145). Fucoidans, another group of sulfated polysaccharides, found
in brown seaweed stimulate immunoreactions, both humoral and cellular, and enhance
v5
Polysaccharide ID
CC50 (ʅ ) C50 (ʅ )
C 0 (ʅ ) SI
GLP 10
0.002 -.0001
0.v6 -.025 5000
G P 10
0.006 -.0006 0.vv-.01
166
GLP A
.6±0.1 0.01-.0004
0.8 -.045 60
G PA 10 0.006-.0005 0.54-.0v
166
DS
. ±0.18 0.001-.0006
0.1 -.00 00
%"0'
%31.%32) %35
,%31 %32) %350'
$10'
2 ,631 632 635
$
.%34%3 )
0'
(145)
Carbohydrate-binding proteins (CBP), ~ 8-50 kDa proteins, can also be isolated from
algae, specifically from blue green algae, as well as sea corals, plants, invertebrates and
vertebrates (8). These show anti-HIV activity in cell culture perhaps due in some way to their
interacting with glycoproteins found on the viral envelop (8). Sulfated polysaccharides function
sulfated polysaccharides exert their influence by providing direct inhibition of viral uptake in
the gut, and also by immune enhancement such as increased interleukin-12 and interferon-1a
production, and stimulation of NK and B cells (1v6). Gut associated lymphoid tissue (GALT)
takes up large molecules which are presented to local immune cells and then may be
v6
transported to the lymph (1v6). The gut contains the majority of the body͛s lymphocytes and
as it an important route for HIV entry and potentially dominant site of HIV replication it is
most transmission of HIV occurs at the mucosal surfaces of the cervicovaginal and
gastrointestinal tracts, topical antimicrobicides can be applied to prevent entry (144). One
molecule that has shown promising results in vitro at preventing HIV entry is
cyclotriazadisulfonamide (CADA). It functions by decreasing the amount of the cell surface CD4
receptor which is necessary for viral entry (144). While this is not necessarily a treatment it
could be useful to examine the mechanisms behind this in order to provide more reliable
therapies.
Some people are homozygous for a v2 nucleotide deletion in the gene coding for the co-
receptor CCR5. This is naturally occurring in about 1È of the Caucasian population (6). The
CCR5 ɷv2 mutation confers some degree of immunity to HIV without significant immunological
disruption. A stem cell transplant from a person with this mutation has been shown to return
CD4+ T cell counts to normal in a patient with acute myeloid leukemia (65). These transplants
carry significant risks as they require the elimination of the patients͛ immune system (82).
Methods that could confer this mutation without having to rebuild the immune system would
carry much less risk. Research is being done into a CCR5 receptor antagonist. One such therapy
is to utilize modified forms of CCL5/RANTES (see below) that competitively bind gp120,
v7
reducing binding to CCR5 (6). It is important to note that the T-tropic (X4) strain of the virus, as
Despite treatment, progression of the disease eventually leads to AIDS. This is defined
by demonstrating a CD4+ T cell count of less than 200 cells per mmv or being diagnosed with
certain characteristic infections. AIDS defining infections include; AIDS dementia complex,
salmonella, sepsis and wasting due to infection (92). Decay of the immune system is
-
Multiple studies have been done on people who appear to have some resistance to HIV
infection. These people typically fall into one of two groups; serologically discordant couples
and sex workers. Serologically discordant couple is a term that means that one partner is
positive for infection and the other is not (76). There are many terms used to described these
individual some of these include exposed seronegative (ESN), highly exposed persistently
seronegative (HEPS), and exposed uninfected EU (76). By studying these individuals insight into
not only the infectivity of the virus but also clues as to how to combat it can be gained. Some
of the factors that have been shown to be involved in this immunity include chemokine
v8
It was initially believed that the CD4 receptor was all that was required for virus entry
into the cell (92). After observing the slow progression of the disease in a certain population of
patients, it was discovered that a deletion in the gene responsible for chemokine receptors
CCR5 (ɷv2 mutation) rendered them less susceptible to the disease (92). This mutation results
in a nonfunctional chemokine receptor. As binding to both the CD4 receptor and a chemokine
receptor is required for viral entry this blocks the entry of the M-tropic (R5) strain of the virus.
As mentioned earlier this mutation is present in ~1È of the Caucasian population (6).
It has also been shown that in some groups despite repeated exposure to the virus there
was no infection (21, 59, 62, 6v, 66, 70). As sexual transmission of the virus is inefficient,
(99.5È of exposures do not result in infection), any method that decreases this could be highly
beneficial (66). This is important in that studying how these groups resist infection could lead
to a vaccination and/or antimicrobicides that will work to prevent infection. After ruling out
the CCR5 ɷv2 mutation as a cause of this resistance, it was observed that the mucosal layer
played a role in this resistance (21). It appears to have some protective factors such as HIV
specific IgA, cellular immune responses, and inhibitory factors like the protein RANTES
alpha/beta-defensins, lysozyme, lactoferrin, calprotectin, cystatin, and histone H2A (21, 81).
Higher levels of IFN-ɲ secretion which interferes with viral infection of cells was also noted in
While these mucosal factors have some inhibitory effects on HIV, that is not their
primary function. RANTES is a 68 amino acid chemokine that recruits leukocytes to the site of
v9
inflammation, activates lymphocytes and neutrophils. In HIV it can suppress viral replication in
vitro (5). SLP1 is in the family of carboxy terminal (C) -type tandem C2 proteins; they are
thought to be involved with vesicle transport and regulated endocytosis (47). Defensins were
and attacks the cell walls of bacteria (1v9). Lactoferrin interacts with reactive oxygen
intermediates (ROI) to facilitate the generation of hydroxyl radicals (1v9). It is a member of the
immune response (147). Cystatins are known to inhibit cysteine proteinases and may be
Another study found that a small molecular weight protein elafin/trappin-2 was
elevated in secretions from resistant women. This protein is also called skin-derived
40
antileukoproteinase (SKALP) (66). This protein is also related to the SLP1 found in the previous
)
In 2005 there were 4.5 million new infections and 2.8 million deaths worldwide (128).
Infection rates have skewed to include more women and increasingly, new infections are
diagnosed in those under 25 (between 25-40È) (1). Sub-Saharan Africa continues to have a
disproportionately large number of infected (table 6) and 70È of those are women. This has to
do with not only sexual mores but also the fact that male to female transmission rates are
higher than female to male rates. Transmission risk varies with viral load, infection stage,
treatment status, circumcision status, presence of other sexually transmitted infections, and
41
4
There are many more mechanism of immunity that are potentially interesting to explore
further. Mechanisms to inhibit, deactivate, or block the CCR5 receptors would make for an
mechanism that would allow the preferential blocking of the CXCR4 co-receptor only where it
interacts with HIV would make for a viable combined therapy. As the structure of these cell
surface glycoproteins gp120 and gp41 is studied perhaps drugs can be developed to interfere
with the binding of these structures thus blocking viral entry. More study should be given to
the role of ɽ-defensins in the long term control of SIV. SIV rarely if ever progresses into full
blown AIDS and this may be a function of defective CCR5 receptors but it could also be related
also be a good avenue of study. In places where access to care is unavailable or limited, being
polysaccharides are also important as they are the only mechanism that has been shown to
prevent HIV entry into cells of the gut. As this is an important area of viral replication these
42
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