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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.
[email protected]
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,Gabon;1995,Zaire;1996,United States(monkey)
Hantavirus-1993,United States; 1997, Argentina
HIV subtype O-1994,Africa
Influenza A/Beijing/32/92, A/Wuhan/359/95, HS:N1-1993,United States; 1995,China;
1997, Hongkong
Japanese Encephalitis-1995, Australia
Lassa fever-1992,Nigeria
Measles-1997, Brazil
Monkey pox-1997,Congo
Morbillivirus – 1994, Australia
O’nyong-nyong fever-1996,Uganda
Polio-1996,Albania
Rift Valley fever-1993,Sudan
Venezuelan equine encephalitis-1995-96,Venezuela/Colombia
West Nile Virus-1996,Romania
Yellow fever-1993,Kenya;1995,Peru
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
Emerging and re-emerging infectious diseases threats contd.
• Bacterial
– Anthrax-1993,Caribbean
– Cat scratch disease/Bacillary angiomatosis(Bartonella 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 States;1996,Japan
– Gonorrhea(drug resistant)-1995,United States
– Helicobacter pylori(ulcers/cancer_-worldwide(discovered 1983)
– Leptospirosis-195,Nicaragun
– Lyme disease(Borrelia burgdorferi)-1990s,United states
– Meningococcal meningitis(serogroup A)-1995-1997,West Africa
– Pertussis-1994,UK/Netherlands;1996,USA
– Plague-1994,India
– Salmonella typhimurium DT104(drug resistant)-1995,USA
– Staphylococcus aureus(drug resistant)-1997,United States/Japan
– Toxic strep-United States
– Trench fever(Barnionella quintana)-1990s,United States
– Tuberculosis(highly transmissible)-1995,United states
– Vibrio cholerae 0139-1992,Southern Asia
Types of Vaccines
•
•
•
•
•
•
•
Killed virus vaccines
Live-attenuated vaccines
Recombinant DNA vaccines
Genetic vaccines
Subunit vaccines
Polytope/multi-epitope vaccines
Synthetic peptide vaccines
Molecular vaccines in phase III
clinical trials and beyond
Company
Product/Type
Status
Aphton
Indication
Gastrimmune
Phase III
Blocks
hormone
Gastrin 17
Avax Technologies M-Vax Vaccine
Phase III
Inc.
Autologous cellular
vaccine
AVI
Biopharma Avicin (CTP-7)
Phase II/III
Inc/Immunotherapy Peptide conjugate
Corp
caccine
GI Cancers
Biomira Inc/Chiron
Corp
Phase III
Metastatic
breast cancer
Phase III
e1999
Phase III
e2000
Prostate cancer
Theratope vaccine
Synthetic cancer
vaccine
Jenner Biotherapies OncoVax-P Vaccine
Inc.
Jenner Biotherapies OncoVax-CL
Inc.
Vaccine
Melanoma
Pancreatic
Colorectal and
lung cancer
Contd../
Company
Progenics
Pharmaceuticals/B
MS
Ribi Immunochem
Product/Type
Status
GMK
Phase III
Ganglioside
conjugate vaccine
Melacine Allogeneic BLA
tumour
specific filing e1999
vaccine
Agouron
Remune Inactivated Phase III
Pharmaceuticals Inc HIV
therapeutic
vaccine
Aviron
FluMist Intranasal flu PLA
submitted
(but refused)
mid-98
Bio-Technology
Bio-Hep-B
Phase III trials
General Corp
Generally engineered Completed
vacine
Chiron Corp
Pertugen (acellular MAA filed
pertussis)
Subunit
vaccine
Indication
Melanoma
Melanoma
HIV/AIDS
Influenza
in
children/adults
Hepatitis B
Pertussis
Contd../
Company
Product/Type
Status
Indication
Chiron Corp
Influenza + MF-59 Phase III
Subunit vaccine
Hepatitis A
Chiron Copr
Hepatitis A Vaccine
Phase III
(Europe)
Hepatitis A
NABI
StaphVAX
Polysaccharide
conjugate vaccine
Phase III
Nosocomial
Staphylococc
us
aureus
bacteremia
OraVAX
Inc Arilvax
Live Phase III 1999 Yellow fever
(Peptide
attenuated vaccine
Therapeutics Ltd)
SB
LYMErix
FDA approval Lyme disease
Recombinanat OspA 5/98
vaccine
SB
Recombinant subunit Phase III
Genital
vaccine
herpes
prophylaxis
VaxGen Inc
AIDSVax
Phase III
HIV/AIDS
Recombinant gp 120
SB
Recombinant subunit Phase III end Chronic
hepatitis B
Therapeutic vaccine 1998
Systems with potential use as T-cell vaccines
CD4 + T-cell vaccines
Killed microbe
Live attenuated microbe
Synthetic peptide coupled
to protein
Recombinant microbial protein
bearing CD4+ T-cell epitope
CD8+ T-cell vaccines
Live attenuated microbe
Synthetic peptide
delivered in liposomes
or ISCOMs
-
Chimeric virus expressing
CD4+ T-cell epitope
Chimeric virus expressing
CD8+ T-cell epitope
Chimeric Ig
Self-molecule expressing
CD8+ T-cell epitope
Chimeric-peptide-MHC
class II complex
Chimeric peptide-MHC
Class I complex
Receptor-linked peptide
Naked DNA expressing
CD4+ T-cell epitope
Naked DNA expressing
CD8+ T-cell epitope
Abbreviations: Ig, Immunoglobulin, ISCOM, immune-stimulating complex;
MHC,Major histocompability complex.
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.
Epitopes …
B-cell epitopes
Th-cell epitopes
What Are Epitopes?
Antigenic determinants or Epitopes are the
portions of the antigen molecules which are
responsible for specificity of the antigens in
antigen-antibody (Ag-Ab) reactions and
that combine with the antigen binding site
of Ab, to which they are complementary.
Epitopes could be contiguous (when Ab binds to a
contiguous sequence of amino acids)
non-contiguous (when Ab binds to
non-contiguous residues, brought
together by folding).
Sequential epitopes are contiguous
epitopes.
Conformational epitopes are noncontiguous antigenic determinants.
Properties of Amino Acids: predictors for
Epitopes
Sequential epitope prediction methods
Theoretical methods are based on properties of amino
acids and their propensity scales.
Hopp & Woods, 1981.
Parker et al., 1986
Kolaskar & Tongaonkar, 1990.
The accuracy of prediction: 50-75%.
Conformational epitope prediction method
Kolaskar & Kulkarni-Kale, 1999.
Antigenic determinants of Egp of JEV
Kolaskar & Tongaonkar approach
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
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 Lpeptide.
• As a result, antibodies raised against the L- or the allD-retro form cross-react strongly with both structures.
• Ex: RI peptide as vaccine candidate for FMDV
(Muller, S., Brown, F, MHV Van Regenmortel)
Various transformations on side-chain
orientation in a model tetrapeptide
Mirror
symmetry
between L- and Dforms of peptides
Note that the retro
modification
does
not take the peptide
through an axis of
symmetry.
Schematic
representation
of the
natural(L)
IRGERA
peptide of
histone H3
1 Cys and 2Gly
were added at
N’term
for
conjugation with
carrier.
Vaccine development
In Post-genomic era:
Reverse Vaccinology
Approach.
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.
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
In Silico Analysis
Peptide
Multitope
vaccines
VACCINOME
Candidate Epitope DB
Epitope prediction
Disease related protein DB
Gene/Protein Sequence Database
Genome sequencing & Identification
of vaccine Candidates of
Neisseria meningitidis
• Complete Genome Sequence of Neisseria
meningitidis Serogroup B Strain MC58.
– Tettelin, et al., (2000). Science. 287:1089-1815.
• Identification of Vaccine Candidates
Against Serogroup B Meningococcus by
Whole-Genome Sequencing.
– Pizza, et al., (2000). Science. 287:1816-1820.
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.
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.
Comparative Genomics: key to
identify the genes responsible for
pathogenesis 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.
Comparison of ORFs of two
strains of N.meningitidis
Dendogram showing genetic relation ship
among 107 N.meningitidis stains
Synthetic Peptide Vaccine
Case Study:
Design and Development of
Synthetic Peptide vaccine against
Japanese encephalitis virus
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.
MULTIPLE SEQUENCE ALIGNMENT OF FLAVIVIRUSES.
PREDICTED ANTIGENIC DETERMINANTS
USING KOLASKAR & TONGAONKAR, 1990 ARE MARKED.
PART 1/5
JE
FNCLGMGNRDFIEGASGATWVDLVLEGDSCLTIMANDKPTLDVRMINIEASQLAEVRSYC
MVE FNCLGMSSRDFIEGASGATWVDLVLEGDSCITIMAADKPTLDIRMMNIEATNLALVRNYC
WNE FNCLGMSNRDFLEGVSGATWVDLVLEGDSCVTIMSKDKPTIDVKMMNMEAANLADVRSYC
KUN FNCLGMSNRDFLEGVSGATWVDLVLEGDSCVTIMSKDKPTIDVKMMNMEAANLAEVRSYC
SLE FNCLGTSNRDFVEGASGATWIDLVLEGGSCVTVMAPEKPTLDFKVMKMEATELATVRKYC
DEN2 MRCIGMSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYC
YF
AHCIGITDRDFIEGVHGGTWVSATLEQDKCVTVMAPDKPSLDISLETVAIDRPAEVRKVC
TBE SRCTHLENRDFVTGTQGTTRVTLVLELGGCVTITAEGKPSMDVWLDAIYQENPAKTREYC
*
***. * * . .
**
*.* . **..* .
* * *
JE
MVE
WNE
KUN
SLE
DEN2
YF
TBE
YHASVTDISTVARCPTTGEAHNEKRADSSYVCKQGFTDRGWGNGCGLFGKGSIDTCAKFS
YAATVSDVSTVSNCPTTGESHNTKRADHNYLCKRGVTDRGWGNGCGLFGKGSIDTCAKFT
YLASVSDLSTRAACPTMGEAHNEKRADPAFVCKQGVVDRGWGNGCGLFGKGSIDTCAKFA
YLATVSELSTKAACPTMGEAHNDKRADPSFVCKQGVVDRGWGNGCGLFGKGSIDTCAKFA
YEATLDTLSTVARCPTTGEAHNTKRSDPTFVCKRDVVDRGWGNGCGLFGKGSIDTCAKFT
IEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFR
YNAVLTHVKINDKCPSTGEAHLAEENEGDNACKRTYSDRGWGNGCGLFGKGSIVACAKFT
LHAKLSDTKVAARCPTMGPATLAEEHQGGTVCKRDQSDRGWGNHCGLFGKGSIVACVKAA
* .
**. *
**.
****** ******* * .*
PART 2/5
JE
CTS--KAIGRTIQPENIKYEVGIFVHGTTTSENHGNYSAQVGASQAAKFTITPNAPSITL
MVE CSN--SAAGRLILPEDIKYEVGVFVHGSTDSTSHGNYSTQIGANQAVRFTISPNAPAITA
WNE CTT--KATGWIIQKENIKYEVAIFVHGPTTVESHG----KIGATQAGRFSITPSAPSYTL
KUN CST--KATGRTILKENIKYEVAIFVHGPTTVESHGNYFTQTGAAQAGRFSITPAAPSYTL
SLE CKN--KATGKTILRENIKYEVAIFVHGSTDSTSHGNYSEQIGKNQAARFTISPQAPSFTA
DEN2 CKK--NMEGKVVQPENLEYTIVITPHSGEEHAVGNDTG-----KHGKEIKITPQSSTTEA
YF
CAK--SMSLFEVDQTKIQYVIRAQLHVGAKQENWN--------TDIKTLKFDALSGSQEV
TBE CEAKKKATGHVYDANKIVYTVKVEPHTGDYVAANETHS----GRKTASFTIS--SEKTIL
. * .
.
*
*
JE
MVE
WNE
KUN
SLE
DEN2
YF
TBE
KLGDYGEVTLDCEPRSGLNTEAFYVMTVGS------KSFLVHREWFHDLALPWTSPSSTKMGDYGEVTVECEPRSGLNTEAYYVMTIGT------KHFLVHREWFNDLLLPWTSPASTKLGEYGEVTVDCEPRSGIDTSAYYVMSVGE------KSFLVHREWFMDLNLPWSSAGSTKLGEYGEVTVDCEPRSGIDTSAYYVMTVGT------KTFLVHREWFMDLNLPWSSAESNNMGEYGTVTIDCEARSGINTEDYYVFTVKE------KSWLVNRDWFHDLNLPWTSPATTELTGYGTVTMECSPRTGLDFNEMVLLQMEN------KAWLVHRQWFLDLPLPWLPGADTQ
EFIGYGKATLECQVQTAVDFGNSYIAEMET------ESWIVDRQWAQDLTLPWQSGSGGTMGEYGDVSLLCRVASGVDLAQTVILELDKTVEHLPTAWQVHRDWFNDLALPWKHEGAQ** .. *
. .
. .
* * * ** ***
PART 3/5
JE
--AWRNRELLMEFEEAHATKQSVVALGSQEGGLHQALAGAIVVEYSSS----VKLTSGHL
MVE --EWRNREILVEFEEPHATKQSVVALGSQEGALHQALAGAIPVEFSSST---LKLTSGHL
WNE --TWRNRETLMEFEEPHATKQSVVALGSQEGALHQALAGAIPVEFSSNT---VKLTSGHL
KUN --VWRNRETLMEFEEPHATKQSVIALGSQEGALHQALAGAIPVEFSSNT---VKLTSGHL
SLE --DWRNRETLVEFEEPHATKQTVVALGSQEGRPATALAGAIPATVSSST---LTLQSGHL
DEN2 GSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSG----NLLFTGHL
YF
--VWREMHHLVEFEPPHAATIRVLALGNQEGSLKTALTGAMRVTKDTNDNNLYKLHGGHV
TBE --NWNNAERLVEFGAPHAVKMDVYNLGDQTGVLLKALAGVPVAHIEGTK---YHLKSGHV
*
*. *
**
* ** * *
**.*
* **.
JE
MVE
WNE
KUN
SLE
DEN2
YF
TBE
KCRLKMDKLALKGTTYGMCTE-KFSFAKNPADTGHGTVVIELSYSGSDGPCKIPIVSVAS
KCRVKMEKLKLKGTTYGMCTE-KFTFSKNPADTGHGTVVLELQYTGSDGPCKIPISSVAS
KCRVKMEKLQLKGTTYGVCSK-AFKFARTPADTGHGTVVLELQYTGTDGPCKVPISSVAS
KCRVKMEKLQLKGTTYGVCSK-AFRFLGTPADTGHGTVVLELQYTGTDGPCKIPISSVAS
KCRAKLDKVKIKGTTYGMCDS-AFTFSKNPTDTGHGTVIVELQYTGSNGPCRVPISVTAN
KCRLRMDKLQLKGMSYSMCTG-KFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDSCRVKLSALTLKGTSYKICTD-KMFFVKNPTDTGHGTVVMQVKVS-KGAPCRIPVIVADD
TCEVGLEKLKMKGLTYTMCDKTKFTWKRAPTDSGHDTVVMEVTFS-GTKPCRIPVRAVAH
*
. . .** .* .*
... * *... .
**..*
PART 4/5
JE
LNDMTPVGRLVTVNPFVATSSANSKVLVEMEPPFGDSYIVVGRGDKQINHHWHKAGSTLG
MVE LNDMTPVGRMVTANPYVASSTANAKVLVEIEPPFGDSYIVVGRGDKQINHHWHKEGSSIG
WNE LNDLTPVGRLVTVNPFVSVATANSKVLIELEPPFGDSYIVVGRGEQQINHHWHKSGSSIG
KUN LNDLTPVGRLVTVNPFVSVSTANAKVLIELEPPFGDSYIVVGRGEQQINHHWHKSGSSIG
SLE LMDLTPVGRLVTVNPFISTGGANNKVMIEVEPPFGDSYIVVGRGTTQINYHWHKEGSSIG
DEN2 LEKRHVLGRLITVNPIVTE--KDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKGSSIG
YF
LTAAINKGILVTVNPIASTN--DDEVLIEVNPPFGDSYIIVGRGDSRLTYQWHKEGSSIG
TBE GSPDVNVAMLITPNPTIEN---NGGGFIEMQLPPGDNIIYVG----ELSHQWFQKGSSIG
..* **
.*
* ** * .*
.
* . **..*
JE
MVE
WNE
KUN
SLE
DEN2
YF
TBE
KAFSTTLKGAQRLAALGDTAWDFGSIGGVFNSIGKAVHQVFGGAFRTLFGGMSWITQGLM
KAFSTTLKGAQRLAALGDTAWDFGSVGGVFNSIGKAVHQVFGGAFRTLFGGMSWISQGLL
KAFTTTLRGAQRLAALGDTAWDFGSVGGVFTSVGKAIHQVFGGAFRSLFGGMSWITQGLL
KAFTATLKGAQRLAALGDTAWDFGSVGGVFTSVGKAVHQVFGGAFRSLFGGMSWITQGLL
KALATTWKGAQRLAVLGDTAWDFGSIGGVFNSIGKAVHQVFGGAFRTLFGGMSWITQGLL
QMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILI
KLFTQTMKGVERLAVMGDTAWDFSSAGGFFTSVGKGIHTVFGSAFQGLFGGLNWITKVIM
RVFQKTKKGIERLTVIGEHAWDFGSAGGFLSSIGKAVHTVLGGAFNSIFGGVGFLPKLLL
.
* .* *.. .*. **** * **
*.** .* * * .
* *.
. ..
PART 5/5
JE
GALLLWMGVNARDRSIALAFLATGGVLVFLATNVHA
MVE GALLLWMGVNARDKSIALAFLATGGVLLFLATNVHA
WNE GALLLWMGINARDRSIAMTFLAVGGVLLFLSVNVHA
KUN GALLLWMGINARDRSIALTFLAVGGVLLFLSVNVHA
SLE GALLLWMGLQARDRSISLTLLAVGGILIFLATSVQA
DEN2 GVIITWIGMNSRSTSLSVTLVLVGIVTLYLGVMVQA
YF
GAVLIWVGINTRNMTMSMSMILVGVIMMFLSLGVGA
TBE GVALAWLGLNMRNPTMSMSFLLAGGLVLAMTLGVGA
* . *.*.. * ..... . * . . .
* *
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
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.
Model Refinement
PARAMETERS USED
• force field:
• Dielectric const:
• Optimisation:
AMBER all atom
Distance dependent
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
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.
Model Evaluation I:Energy Profile
TBE
JEV
Model Evaluation II:
Ramachandran Plot
An Algorithm to Identify
Conformational Epitopes
Calculate the percent accessible surface
area (ASA) of the amino acid residues.
If ASA  30%, then residue was termed
as accessible residues.
A contiguous stretch of more than three
accessible residues was termed as the
antigenic determinant.
…Cont.
A determinant is extended to N- and Cterminals, only if, accessible amino
acid(s) are present after an
inaccessible amino acid residue.
A list of sequential antigenic
determinants was prepared.
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
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 4*rij
– 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
MD simulations of following peptides were carried out
B Cell Epitopes:
SENHGNYSAQVGASQ
NHGNYSAQVGASQ
YSAQVGASQ
YSAQVGASQAAKFT
NHGNYSAQVGASQAAKFT
149
T-helper Cell Epitope:
436
445
SIGKAVHQVF
168
SENHGNYSAQVGASQAAKFT
Chimeric B+Th Cell Epitope With Spacer:
SENHGNYSAQVGASQAAKFTSIGKAVHQVF
Structural comparison of Egps of Nakayama and Sri Lanka
strains of JEV.
Single amino acid differences are highlighted.