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Insilico Prediction of Vaccine Candidates against Botulism caused by Clostridium

botulinium by Reverse Vaccinology
Selvaraj C1, Shobana K2, Thiagarajan B3,
1
selnikraj@gmail.com, 2shobana_bioinf@gmail.com, 3thiagarajan.b@grd.edu.in
1 2 3
+91-9894579639, +91-9944936040, +91-9944944356,
Department of Bioinformatics, Dr G R Damodaran College of Science, Coimbatore – 641 014.

Abstract
Botulism is a neuroparalytic disease caused by neurotoxins produced by the bacteria Clostridium
botulinium. Botulinium neurotoxins (BoNTs) are amongst the most potent naturally occurring toxins and are
biological threat agents. The seven toxin serotypes of BoNTs (serotypes A–G) have different toxicities, act
through three different intracellular protein targets and exhibit different durations of effect. Botulism may
follow ingestion of food contaminated with BoNT, from toxin production of Clostridium botulinium present
in the intestines or wounds or from the inhalation of aerosolized toxin. Intoxication classically presents as an
acute, symmetrical, descending flaccid paralysis.
The objective of the work is to predict the vaccine candidates against the disease Botulism caused by
Clostridium botulinium that could be used as a biological weapon against the civilian population. Predicting
vaccine candidates against Clostridium botulinium is possible with various reverse vaccinology methods,
such as genome analysis, localization prediction, predicting surface associated proteins and comparing with
human genome. The usage of various servers in various platforms could predict the description of the
proteome by the screening techniques. The epitope mapping could define the binding predictions and

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homology mapping. The results would encourage the researchers in selecting the antigens from the
predicted proteome of pathogens for which cell culture is difficult or mere impossibility.

1.1 Clostridium Botulism

Clostridium botulinium will only grow at low Eh values which are normally correlated with the absence of
oxygen. There are a number of neurotoxin types which differ markedly in their characteristics and ability to
cause disease in humans. Two groups are important in food:
• Group I-Types A, B and F (proteolytic strains)
• Group II-Types B, E and F (nonproteolytic strains)
Because group I organisms are proteolytic their growth generally causes spoilage of the
Contaminated food. There are two manifestations of disease relevant to food; classic food borne botulism
and infant botulism
There are five clinical categories of botulism: 1) foodborne botulism; 2) wound botulism; 3) infant botulism;
4) adult infectious botulism; 5) inadvertent, following botulinium toxin injection, Botulism is caused by a
group of anaerobic spore-forming organisms called Clostridium botulinum. This is classified as a single
species but consists of at least three genetically distinguishable groups of organisms that have been
recognized as toxic for humans. They share the ability to produce neurotoxins with similar pharmacological
activities but diverse serologic properties. The toxin types are classified as A, B, C, D, E, F and G. Human
botulism has been described with the strains of Clostridium botulinum that produce toxin types A, B and E.
Less frequently, cases involving type F toxin produced by C. baratii and type E toxin produced by C.
butyricum have been published

1.2 Clostridium as Bioweapon

Clostridium botulinum is a spore forming , obligate anaerobe whose natural habitat is soil, from which it can
be isolated without undue difficulty. The species C botulinum consists of 4 genetically diverse groups that
would not otherwise be designated as a single species except for their common characteristic of producing
botulinum toxin.33,34 Botulinum toxin exists in 7 distinct antigenic types that have been assigned the letters
A through G. The toxin types are defined by their absence of cross neutralization (eg, anti-A antitoxin does
not neutralize toxin types B-G). The toxin types also serve as convenient epidemiological markers. In
addition to C botulinum, unique strains of Clostridium baratii and Clostridium butyricum have the capacity
to produce botulinum toxin.35-37 Botulinum toxin is a simple di chain polypeptide that consists of a 100-kd
“heavy” chain joined by a single disulfide bond to a 50-kd “light” chain; its 3-dimensional structure was
recently resolved to 3.3 A.38 The toxin’s light chain is a Zn++- containing endopeptidase that blocks
acetylcholine-containing vesicles from fusing with the terminal membrane of the motor neuron, resulting in

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flaccid muscle paralysis.8 Thelethal dose of botulinum toxin for humans is not known but can be estimated
from primate studies. By extrapolation, the lethal amounts of crystalline type A toxin for a 70-kg human
would be approximately 0.09-0.15 μg intravenously or intramuscularly, 0.70-0.90 μg inhalationally, and 70
μg orally.10,39-41 Therapeutic botulinum toxin represents an impractical bioterrorist weapon because a vial
of the type A preparation currently licensed in the United States containsonly about 0.3% of the estimated

Fig 1.2.1 Mechanism of Action of Botulinum Toxin

1.3 Reverse vaccinology

Reverse vaccinology is one of the best examples of how bioinformatics can boost molecular immunology,
the conventional approach to design vaccines requires pathogen’s cultivation and dissection of its main
components before testing their ability to elicit protective immunity, reverse vaccinology’s novelty consists

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in starting the search for immunogenic antigens from Insilico analyses of the pathogens genome instead of
culturing the microorganism. This allow scientist to save time and money while facing pathogens for which
cell culture is difficult and impossible. Reverse vaccinology potentially permits researchers to select ,in
addition to most in vivo expressed antigens, any proteins encoded by the genome of the pathogen. Reverse
vaccinology is less helpful with eukaryotes due to complexities of cellular and tissue organization, and it is
more effective with prokaryotes, extra and intracellular, indeed, the production of specific antibodies can
boost immunity not only against extra cellular pathogens , usually controlled by Th2-polorized responses,
but also against the either obligate or facultative intracellular ones, usually controlled by Th1-polorised
responses, even these later pathogens are susceptible to humoral immunity during the extra cellular phases
of their infectious cycle and are made vulnerable by antibody cross-linking that modifies the intracellular
millleu through signalling.
The focus of the reverse vaccinology is one of the in silico vaccine candidates from the list of predicted
proteins to address two issues such as the possiblities (i) of auto matizing the process with new criteria of
analyses and (ii) of reducing the percentage of vaccine candidates to less than 20 – 30 % reported until now,
indeed, the real goal of Insilico selection is to choose the minimal number of vaccine candidate sufficient to
find protective antigens during during experimental phases, thus designing a vaccine that conserves time
and money, therefore, it has to be stressed that obtaining a high recall of protective antigens in a very small
number of selected vaccine candidates is more convenient than trying to find all possible protective
antigens, according to this approach it is more productive to select the protective antigens that will most
likely be easily expressed, this reduces the risk of experimental troubles, the extra cellular loops of
multispanning membrane proteins can actually be significant targets especially if reasonably large, hence
one may focus on fragments likely to be exposed and accessible to antibodies rather than an entire protein in
order to overcome eventual cloning and expression problems, yet discarding non surface exposed predicting
antigens, thereby forwarding further bioinformatic analysis on selected ones, proved successful, however to
minimise the risk of loosing good vaccine candidates, the information concerning all other proteins is
retained, vaccine candidate are presented with corresponding integrated analyses. In order to rationalize
selection step, it is been taken into account the important of avoiding antigens that can potentially cause
autoimmunity in man. Since major histocompatiblity complex(MHC) ligands can be really short and as few
as eight residues. This problem may rise also from antigens sharing weak global similarity with host
proteins.

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Fig 1.3.1 Reverse Vaccinology

The first proteome scanning assign each sequence seven predictions. They are (i) subcellular localization of
proteins using PSORTb (ii) transmembrane topology prediction using using HMMMTOP (iii)signal peptide
prediction using signal P (iv) prediction of lip protein signal peptides using LipoP (v) prediction of non
classical protein secretion using secretomeP (vi) Tatsignal peptide prediction using TatP (vii) predicting
topology of beta barrel outer membrane proteins such as outer membrane using Pred TMBB, indeed,
surface- exposed proteins and especially adhesins are ideal targets for vaccine development. The second
step was determining the virulence of short listed candidates, this was done using virulentPred server.
The step addresses the problem of sharing similarity regions between pathogen and host proteins, it is well
known that in vaccine development this can either cause low immune response, tolerance or autoimmunity.

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The algorithm BLAST was chosen to compare each pathogen’s sequence as a query against the human
proteome, local alignment is suitable for this task because potential MHC ligands that help in predicting
potential interenference such as tolerance or autoimmunity with the immune system can be very short, in
this way it is not only taken in to account MHC II, commonly involed in presentation of exogenes antigens,
but also MHC I ,involved in cross prediction

2, Tools used for screening techniques

2. 1. PSORTb 2.0
Computational prediction of the subcellular localization of proteins is a valuble tool for genome analysis
and annotation, since proteins subcellular localization can provide clues regarding its function in an
organism, for bacterial pathogens, the prediction of proteins on the cell surface is of particular intrest due to
potential of such proteins to be primary drug or vaccine targets, the protein’s subcellular localization is
influenced by several feautures present within the proteins primary structure, such as a presence of a signal
peptide or trans membrane spanning alpha helices,PSORT family of programs analyzes several feautures at
once, using information obtained from each analysis to generate an overall prediction of localization site,
developed by kenta nakai in 1991, PSORT is an algorithm which assigns a probable localization site to a
protein given an aminoacid sequence alone, originally developed for prediction for protein localization in
gram negative bacteria ,PSORT was expanded into a suite of programs i.e. PSORT, PSORT II ipSORT
capable of handling proteins from all classes of organisms.
2. 2. HMMTOP 2.0
Hidden markov’s model for topology prediction(HMMTOP) method is based on the principle that the
topology of the transmembrane proteins are determined by the maximum divergence of aminoacid
composition of sequence segments in various structural parts of these proteins rather than by specific amino
acid composition of sequence segments in various in various structural parts of these proteins rather than by
specific aminoacid composition of these parts, it predicts both the localization of helical trans membrane
proteins, this hidden markovs model with special architecture was developed to search transmembrane
topology corresponding to the maximum likelihood among all the possible topologies of the given protein

2. 3. LipoP 1.0
The LipoP 1.0 server produces predictions of lipoproteins and discriminates between lipoprotein signal
peptides, other signal peptides and n-terminal membrane helices in Gram negative bacteria.
2. 4. SecretomeP 2.0
The SecretomeP 2.0 server produces ab initio predictions of non-classical i.e. not signal peptide triggered
protein secretion. The method queries a large number of other feature prediction servers to obtain

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information on various post-translational and localizational aspects of the protein, which are integrated into
the final secretion prediction. It represents the overview of bacterial non classical secretion and a prediction
method for identification of proteins following signal peptide independent secretion pathways, they have
compiled a list of proteins found extracellularly despite the absence of signal peptide
2. 5. VirulentPred
VirulentPred is a bacterial virulent protein prediction method based on bi-layer cascade Support Vector
Machine (SVM). The first layer SVM classifiers were trained and optimized with different individual
protein sequence features like amino acid composition, dipeptide composition (occurrences of the possible
pairs of i and i+1 amino acid residues), higher order dipeptide composition (pairs of i and i+2 residues) and
remote evolutionary relationships by use of Position-Specific Iterated BLAST (PSI-BLAST) generated
Position Specific Scoring Matrix (PSSM). A five-fold cross-validation technique was used for the
evaluation of various prediction strategies in the current work. The results from the first layer (SVM scores
and PSI-BLAST output) were cascaded to the second layer SVM classifier to train and generate the final
classifier. The cascade SVM classifier was able to accomplish a significantly higher accuracy of 81.8%,
covering 86% area in the Receiver Operator Characteristic (ROC) plot, better than that of either of the layer
one SVM classifiers based either on single or multiple sequence features.
VirulentPred can be used to serene virulent proteins in proteomes, together with experimentally verified
virulent proteins, several putative, non annotated and hypothetical protein sequences have been predicted to
be high scoring virulent proteins by the prediction method.
The prediction of virulent proteins will aid studies aimed at knowing more about bacterial virulence and
annotation of virulent genes for the identification of novel antimicrobial targets, hence in the present study
an attempt has been made to develop reliable SVM based on feautures such as compositions of amino acids,
dipeptides and higher order dipeptides , evolutionary information in the form of multiple sequence
alignment and similarity search , finally, the performance of cascade SVM module, developed using
individual feature modules were found to be much more efficient, hence used as default option for the
VirulentPred Server.
2. 6. BlastP
After finishing the first proteome screenings the fourth step address the problem of the sharing similarity
regions between pathogen and host proteins, it is well known that in vaccine development this can either
cause low immune response or autoimmunity. The algorithm BlastP is used

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3. Sequence Information
3.1. Source from Pedant database
The pedant genome database provides exhaustive automatic analysis of genomic sequences by a large
variety of bioinformatics tools. For example the following pre-computed analyses are available to analyse
protein function: Blast similarity searches against the non-redundant protein sequence database, motif
searches against the Pfam, BLOCKS and PROSITE databases. Prediction of cellular roles and functions are
made based on high stringency BLAST searches against protein sequences. Out of 1715 entries the 24
proteins are selected for the outlet as selected proteins and this protein is based on the membrane protein,
the membrane protein is selected because the membrane protein will efficiently bind to the host protein, thus
the membrane protein molecule is selected.
3.2. Clostridium botulinum A str. ATCC 3502

Table 3.1 selected protein information based on membrane protein

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4. Results and Discussion

4.1 Predicted vaccine candidates using reverse vaccinology

Using the reverse vaccinology methods the genome of Clostridium botulinum A strain was analysed, the
screening was done with the help of different servers such as VirulentPred, LipoP, SecretomeP, SignalP,
PredTMBB etc, after the screening the proteins with significant scores are selected for comparing with
human genome with BLASTP, the proteins which shows more than 80% similarity were omitted
The criteria for selection are
• Should be virulent
• Should have minimum secretomeP of 0.5
• Should contain signal peptide cleavage sites
• Should be a membrane protein
• Transmembrane helices must be minimum
• Presence of Beta barrels are significant
• Non secretory proteins are omitted

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4.2 Epitope mapping results

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Three proteins were selected for the mapping experiments using IEDB tools, the selected proteins are given
below

Table 5.2 Selected Proteins List

4.3 Peptide binding to MHC Class II Molecules

This tool will take in an amino acid sequence, or set of sequences and determine each subsequence's ability
to bind to a specific MHC class I molecule

Table 4.3 Peptide binding to MHC Class II molecules

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Fig 4.1Epitope Prediction by Chow & Fasman Beta Turn Prediction

GI: 148378204 NC_009495 238008238652

Fig 4.2 Epitope Prediction by Bepipred Linear Epitope Prediction

GI: 148378204 NC_009495 238008238652

Fig 4.3 through < 50 filter in Bepipred linear epitope Prediction

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5. Conclusion
The clostridium botulism is taken as the source organism and the sequence information is collected from the
pedant databases, and from that 24 protein sequence are collected on the basis of the membrane protein,
from that the tools are utilised and the virulent vaccine candidate has predicted, from the best three vaccine
candidates and examined for the experimental determinations, The predicted vaccine candidates alone are
cloned and expressed using PCR, the resulting protein are purified and immunization experiments are done
with mice, this is followed by ELISA and Western blot test, after testing them with in vitro and in vivo assay
identifications are done with many experiments ,and the out of final vaccine development, out of these only
few are predicted as efficient vaccine candidates

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6. Bibliography

1. G Franciosa, J L Ferreira and C L Hatheway et al, Detection of type A, B, and E botulism neurotoxin
genes in Clostridium botulinum and other Clostridium species by PCR. 1994 August; 32(8): 1911-1917

2. Cherington M, Department of Neurology, University of Colorado School of Medicine, Denver, USA.

Clinical spectrum of botulism.1998 Jun;21(6):701-10

3. V. R. Dowell Jr, L. M. McCroskey, C. L. Hatheway, G. L. Lombard, J. M. Hughes and M. H. Merson,

Coproexamination for botulinal toxin and clostridium botulinum. A new procedure for laboratory diagnosis
of botulism, 2004; 70:2919-2927

4. Dodds, KL, Diseases Caused by Bacteria; Clostridium botulinum, 1994

5. Takeshi K, Fujinaga y, Inoue K, Kanakjima H. Oguma K, Ueno T, Sunagawa H,Ohyama T, Simple

method for detection of Clostridium botulinum type A to F neurotoxin genes by polymerase chain reaction,
1996, vol. 40, no1, pp. 5-12

6. Heike Schoepe, Christian Pache, Axel Neubauer, Heidrun Potschka, Tobias Schlapp, Lothar H. Wieler,
and Georg Baljer, Naturally Occurring Clostridium perfringens Nontoxic Alpha-Toxin Variant as a
Potential Vaccine Candidate against Alpha-Toxin-Associated Diseases, November 2001, p. 7194-7196, Vol.
69, No. 11

7. Woodruff BA, Griffin PM, McCroskey LM, Smart JF, Wainwright RB, Bryant RG, Hutwagner LC,

Hatheway CL. Clinical and laboratory comparison of botulism from toxin types A, B, and E in the United
States, 1992 Dec;166(6):1281-6.

8. Ernest C. Dickson M.D.1 and Richard Shevky Ph.D, Botulism. studies on the manner in which the Toxin

of Clostridium botulinum acts upon the Body, Vol 37, 711-731

9. Russell Kutz and Ogi Okwumabua, Differentiation of Highly Virulent Strains of Streptococcus suis
Serotype 2 by the GDH Electrophoretic and Sequence Types, 10.1128/JCM.02309-07,2008

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10. R . Villar , S . Elliott , K . Davenport, Botulism: The Many Faces of Botulinum Toxin and its Potential
for Bioterrorism, Infectious Disease Clinics of North America, Volume 20 , Issue 2 , Pages 313 - 327

7. Websites

1, http://bioinfo.icgeb.res.in/virulent/
2, http://www.cbs.dtu.dk/services/SecretomeP/
3, http://www.cbs.dtu.dk/services/LipoP/
4, http://www.enzim.hu/hmmtop/html/submit.html
5, http://blast.ncbi.nlm.nih.gov/Blast.cgi
6, http://www.immuneepitope.org
7, http://www.ncbi.nlm.nih.gov/pubmed/
8, http://pedant.gsf.de/index.jsp

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