Case Study

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Case Study 2 How Bioinformatics Aids in Vaccine
Development / Peptide Vaccine Development Using
Bionformatics Approaches

• Ashok Kolaskar
• University of Pune, Pune 411 007, India.
• puvc_at_unipune.ernet.in

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Emerging and re-emerging infectious diseases
threats, 1980-2001

• Viral
• Bolivian hemorrhagic fever-1994,Latin America
• Bovine spongiform encephalopathy-1986,United
  Kingdom
• Creulzfeldt-Jackob disease(a new variant
  V-CID)/mad cow disease-1995-96, UK/France
• Dengue fever-1994-97,Africa/Asia/Latin
  America/USA
• Ebola virus-1994,Gabon1995,Zaire1996,United
  States(monkey)
• Hantavirus-1993,United States 1997, Argentina
• HIV subtype O-1994,Africa
• Influenza A/Beijing/32/92, A/Wuhan/359/95,
  HSN1-1993,United States 1995,China 1997,
  Hongkong
• Japanese Encephalitis-1995, Australia
• Lassa fever-1992,Nigeria
• Measles-1997, Brazil
• Monkey pox-1997,Congo
• Morbillivirus 1994, Australia
• Onyong-nyong fever-1996,Uganda
• Polio-1996,Albania
• Rift Valley fever-1993,Sudan
• Venezuelan equine encephalitis-1995-96,Venezuela/C
  olombia
• West Nile Virus-1996,Romania

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Emerging and re-emerging infectious diseases
threats contd.,

• Parasitic
• African trypanosomiasis-1997,Sudan
• Ancylcostoma caninum(eosinophilic
  enteritis)-1990s,Australia
• Cryptosporiadiasis-1993,United States
• Malaria-1995-97,Africa/Asia/Latin America/United
  states
• Metorchis-1996,Canada
• Microsporidiosis-Worldwide
• Fungal
• Coccidiodomycosis-1993,United States
• Penicillium marneffi

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Emerging and re-emerging infectious diseases
threats contd.

• Bacterial
• Anthrax-1993,Caribbean
• Cat scratch disease/Bacillary angiomatosis(Bartone
  lla henseiae)-1900s, USA
• Chlamydia pneumoniae(Pneumonia/Coronary artery
  disease?)-1990s, USA(discovered 1983)
• Cholera-1991,Latin America
• Diphtheria-1993,Former Soviet Union
• Ehrlichia chaffeensis,Human monocytic
  ahrlichiosis(HME)-United States
• Ehrlichia phagocytophilia,Human Granulocytic
  ehrlichis(HGE)-United States
• Escherichia coli O157-1982-1997,United
  States1996,Japan
• Gonorrhea(drug resistant)-1995,United States
• Helicobacter pylori(ulcers/cancer_-worldwide(disco
  vered 1983)
• Leptospirosis-195,Nicaragun
• Lyme disease(Borrelia burgdorferi)-1990s,United
  states
• Meningococcal meningitis(serogroup
  A)-1995-1997,West Africa
• Pertussis-1994,UK/Netherlands1996,USA
• Plague-1994,India
• Salmonella typhimurium DT104(drug
  resistant)-1995,USA
• Staphylococcus aureus(drug resistant)-1997,United
  States/Japan
• Toxic strep-United States

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Types of Vaccines

• Killed virus vaccines
• Live-attenuated vaccines
• Recombinant DNA vaccines
• Genetic vaccines
• Subunit vaccines
• Polytope/multi-epitope vaccines
• Synthetic peptide vaccines

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Molecular vaccines in phase III clinical trials
and beyond
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Contd../
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Contd../
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Systems with potential use as T-cell vaccines
CD4 T-cell vaccines CD8 T-cell
vaccines Killed microbe Live attenuated
microbe Live attenuated microbe - Synthetic
peptide coupled Synthetic peptide to
protein delivered in liposomes or
ISCOMsRecombinant microbial protein -bearing
CD4 T-cell epitope Chimeric virus
expressing Chimeric virus expressing CD4
T-cell epitope CD8 T-cell epitope Chimeric
Ig Self-molecule expressing CD8 T-cell
epitope Chimeric-peptide-MHC Chimeric
peptide-MHCclass II complex Class I
complex Receptor-linked peptide - Naked DNA
expressing Naked DNA expressing CD4 T-cell
epitope CD8 T-cell epitope Abbreviations Ig,
Immunoglobulin, ISCOM, immune-stimulating
complex MHC,Major histocompability complex.
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Why Synthetic Peptide Vaccines?

• Chemically well defined, selective and safe.
• Stable at ambient temperature.
• No cold chain requirement hence cost effective in
  tropical countries.
• Simple and standardised production facility.

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Epitopes
B-cell epitopes
Th-cell epitopes
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Antigenic determinants of Egp of JEV Kolaskar
Tongaonkar approach
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Peptide vaccines to be launched in near future

• Foot Mouth Disease Virus (FMDV)
• Human Immuno Deficiency Virus (HIV)
• Metastatic Breast Cancer
• Pancreatic Cancer
• Melanoma
• Malaria

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Retro-Inverso peptides -Potential Synthetic
peptide Vaccines

• A retro structure is obtained by synthesizing
  peptides in reverse order, in which the direction
  of peptide bond is reversed and side-chains are
  oriented in the manner similar to that in
  D-enantiomer, which explains why these two
  analogs cross-react immunochemically.
• When both of these transformations are combined
  in the form of an all-D-retro or Retro-Inverso
  (RI) peptide, the side-chains are oriented as in
  original L-peptide.
• As a result, antibodies raised against the L- or
  the all-D-retro form cross-react strongly with
  both structures.
• Ex RI peptide as vaccine candidate for FMDV
  (Muller, S., Brown, F, MHV Van Regenmortel)

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Various transformations on side-chain orientation
in a model tetrapeptide
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Mirror symmetry between L- and D-forms of
peptidesNote that the retro modification does
not take the peptide through an axis of symmetry.
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Schematic representation of the natural(L) IRGERA
peptide of histone H3 1 Cys and 2Gly were added
at Nterm for conjugation with carrier.
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• Vaccine development
• In Post-genomic era
• Reverse Vaccinology
• Approach.

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Reverse Vaccinology

• Advantages
• Fast access to virtually every antigen
• Non-cultivable can be approached
• Non abundant antigens can be identified
• Antigens not expressed in vitro can be
  identified.
• Non-structural proteins can be used
• Disadvantages
• Non proteinous antigens like polysaccharides,
  glycolipids cannot be used.

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Genome Sequence
Proteomics Technologies
In silico analysis
IVET, STM, DNA microarrays
High throughput Cloning and expression
In vitro and in vivo assays for Vaccine candidate
identification
Global genomic approach to identify new vaccine
candidates
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In Silico Analysis
Peptide Multitope vaccines
VACCINOME
Candidate Epitope DB
Epitope prediction
Disease related protein DB
Gene/Protein Sequence Database
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Genome sequencing Identification of vaccine
Candidates of Neisseria meningitidis

• Complete Genome Sequence of Neisseria
  meningitidis Serogroup B Strain MC58.
• Tettelin, et al., (2000). Science. 2871089-1815.
• Identification of Vaccine Candidates Against
  Serogroup B Meningococcus by Whole-Genome
  Sequencing.
• Pizza, et al., (2000). Science. 2871816-1820.

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Genome analysis of Neisseria meningitidis
serogroup B strain MC58

• Genome size 2,272,351 bp
• Predicted coding regions 2158
• Function annotations 1158 (53.7)
• 3 major islands of horizontal transfer have been
  observed.
• 2 contains genes encoding proteins involved in
  pathogenicity
• 1 contains a hypothetical protein
• It contains more genes that undergo phase
  variation than any pathogen studied to date, a
  mechanism that controls their expression and
  contributes to the evasion of the host immune
  system.

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Neisseria meningitidis (Meningococcus)

• Is a gram negative ? Proteobacterium.
• Is a cause of life threatening invasive bacterial
  infections especially in infants.
• Causes Meningitis Septicemia, which are
  significant public health problem.
• The fatality rate ranges from 5-15 and up to 25
  of survivors are left with neurological sequelae.
  
• There are 5 pathogenic serogroups (A,B,C,Y and
  W135) based on chemical composition of
  distinctive capsular polysaccharide typing.
• Although the vaccine exists, its impact has been
  limited.

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Comparative Genomics key to identify the genes
responsible forpathogenesis of meningitis

• Complete genomes of 2 strains of Neisseria
  meningitidis are available.
• Serogroup B strain MC58
• Serogroup A strain Z2491
• Complete genome of Haemophilus influenzae,
  another pathogen responsible for meningitis and
  the first organism to be sequenced completely is
  also available.
• Comparative genomics of these three genomes
  provides an opportunity to define a common subset
  of genes that are responsible for pathogenesis of
  this disease.

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Comparison of ORFs of two strains of
N.meningitidis
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Dendogram showing genetic relation ship among 107
N.meningitidis stains
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Synthetic Peptide Vaccine Case Study Design
and Development of Synthetic Peptide vaccine
against Japanese encephalitis virus
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Egp of JEV as an Antigen

• Is a major structural antigen.
• Responsible for viral haemagglutination.
• Elicits neutralising antibodies.
• 500 amino acids long.
• Structure of extra-cellular domain (399) was
  predicted using knowledge-based homology modeling
  approach.

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Multiple alignment of Predicted TH-cell epitope
in the JE_Egp with corresponding epitopes in
Egps of other Flaviviruses 426 457 JE
DFGSIGGVFNSIGKAVHQVFGGAFRTLFGGMS MVE
DFGSVGGVFNSIGKAVHQVFGGAFRTLFGGMS WNE
DFGSVGGVFTSVGKAIHQVFGGAFRSLFGGMS KUN
DFGSVGGVFTSVGKAVHQVFGGAFRSLFGGMS SLE
DFGSIGGVFNSIGKAVHQVFGGAFRTLFGGMS DEN2
DFGSLGGVFTSIGKALHQVFGAIYGAAFSGVS YF
DFSSAGGFFTSVGKGIHTVFGSAFQGLFGGLN TBE
DFGSAGGFLSSIGKAVHTVLGGAFNSIFGGVG COMM DF S GG S
GK H V G F G Multiple alignment of
JE_Egp with Egps of other Flaviviruses in the
YSAQVGASQ region. 151
183 JE SENHGNYSAQVGASQAAKFTITPNAPSITLKLG MVE
STSHGNYSTQIGANQAVRFTISPNAPAITAKMG WNE
VESHG----KIGATQAGRFSITPSAPSYTLKLG KUN
VESHGNYFTQTGAAQAGRFSITPAAPSYTLKLG SLE
STSHGNYSEQIGKNQAARFTISPQAPSFTANMG DEN2
HAVGNDTG-----KHGKEIKITPQSSTTEAELT YF
QENWN--------TDIKTLKFDALSGSQEVEFI TBE
VAANETHS----GRKTASFTIS--SEKTILTMG

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STEPS in Homology Modeling

• Template structure (PDB entry 1SVB). (Rey et
  al., 1995).
• Alignment of Egp of JEV and Egp of TBEV.
• Definition of SCRs and Loops.
• Assignment of Initial co-ordinates to Backbone
  Side-chains.
• Rotamer search for the favored side-chain
  conformations.

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Model RefinementPARAMETERS USED

• force field AMBER all atom
• Dielectric const Distance dependent
• Optimisation Steepest Descents
• Conjugate Gradients.
• rms derivative 0.1 kcal/mol/A for SD
• rms derivative 0.001 kcal/mol/A for CG
• Biosym from InsightII, MSI and modules therein

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Model For Solvated Protein

• Egp of JEV molecule was soaked in the water layer
  of 10A?.
• 4867 water molecules were added.
• The system size was increased to 20,648 atoms
  from 6047.

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Model Evaluation IEnergy Profile
TBE JEV
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Model Evaluation II Ramachandran Plot
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Peptide Modeling
Initial random conformation Force field
Amber Distance dependent dielectric constant
4rij Geometry optimization Steepest descents
Conjugate gradients Molecular dynamics at 400 K
for 1ns Peptides are SENHGNYSAQVGASQ
NHGNYSAQVGASQ YSAQVGASQ YSAQVGASQAAKFT
NHGNYSAQVGASQAAKFT SENHGNYSAQVGASQAAKFT 149
168
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Prediction of conformations of the antigenic
peptides

• Lowest energy Allowed conformations were
  obtained using multiple MD simulations
• Initial conformation random, allowed
• Amber force field with distance dependent
  dielectric constant of 4rij
• Geometry optimization using Steepest descents
  Conjugate gradient
• 10 cycles of molecular dynamics at 400 K each of
  1ns duration, with an equilibration for 500 ps
• Conformations captured at 10ps intervals,
  followed by energy minimization of each
• Analysis of resulting conformations to identify
  the lowest energy, geometrically and
  stereochemically allowed conformations

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MD simulations of following peptides were carried
out

• B Cell Epitopes
• SENHGNYSAQVGASQ
• NHGNYSAQVGASQ
• YSAQVGASQ
• YSAQVGASQAAKFT
• NHGNYSAQVGASQAAKFT
• 149 168
• SENHGNYSAQVGASQAAKFT

T-helper Cell Epitope 436 445 SIGKAVHQVF

• Chimeric BTh Cell Epitope With Spacer
• SENHGNYSAQVGASQAAKFTSIGKAVHQVF

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Structural comparison of Egps of Nakayama and Sri
Lanka strains of JEV. Single amino acid
differences are highlighted.