1. DEPT.OF BIOINFORMATICS SUMAN MISHRA & PRITAM KUMAR PANDA
Centre for Biological Research Hyderabad
Dept. of Biotechnology
DISSERTATION OF IMSc BIOINFORMATICS PROTEOME SCREENING, IDENTIFICATION, DESIGNING OF POTENT ANTIGENIC PEPTIDES AND CHEMOINFORMATICS APPROACH OF NEISSERIA MENINGITIDES: A CAUSE OF MENINGITIS
BUXI JAGABANDHU BIDYADHAR (AUTONOMOUS) COLLEGE
DEPT.OF BIOINFORMATICS
SUMAN MISHRA & PRITAM KUMAR PANDA

2. ABSTRACT
Meningitis is an acute inflammation of the protective membranes covering the brain and spinal cord, known collectively as the meninges.
Meningitis can be life-threatening because of the inflammation's proximity to the brain and spinal cord; therefore, the condition is classified as a medical emergency.
The scope of the current work is to predict and design a potent antigenic peptide of the bacteria Neisseria Meningitides so as to develop a potential vaccine.
The bacterial proteome is collected from NCBI database followed by the screening for the foreign proteins. The proteins were further screened for the identification of most antigenic sites within them.
The selected antigenic peptides were designed using Argus Lab software followed by the geometry optimization and energy evaluation. Based on the energy values the best antigenic peptides were selected and can be further processed for the production of potential vaccine to eradicate the disease.
Further the Chemo-informatics approach may leads to the medication of this epidemic disease and to identify the Binding affinity of the peptides for the purpose of eradication of Meningitis using HEX and in addition to that several parametric calculations and simulation can be done for designing of attenuated vaccines.

3. Meningitis is an acute inflammation of the protective membranes covering the brain and spinal cord, known collectively as the meninges. The inflammation may be caused by infection with viruses bacteria or other microorganisms and less commonly by certain drugs Meningitis can be life- threatening because of the inflammation's proximity to the brain and spinal cord; therefore, the condition is classified as a medical emergency.
The most common symptoms of meningitis are headache and neck stiffness associated with fever, confusion or altered consciousness, vomiting, and an inability to tolerate light (photophobia) or loud noises (phonophobia). Children often exhibit only nonspecific symptoms, such as irritability and drowsiness. If a rash is present, it may indicate a particular cause of meningitis; for instance, meningitis caused b meningococcal bacteria may be accompanied by a characteristic rash.

4. Causes:
Meningitis is typically caused by an infection with microorganisms. Most infections are due to viruses, with bacteria, fungi, and protozoa being the next most common causes.It may also result from various non-infectious causes.
The term aseptic meningitis refers to cases of meningitis in which no bacterial infection can be demonstrated. This type of meningitis is usually caused by viruses but it may be due to bacterial infection that has already been partially treated, when bacteria disappear from the meninges, or pathogens infect a space adjacent to the meninges (e.g. sinusitis).
Endocarditis (an infection of the heart valves which spreads small clusters of bacteria through the bloodstream) may cause aseptic meningitis. Aseptic meningitis may also result from infection with spirochetes, a type of bacteria that includes Treponemapallidum (the cause of syphilis) and Borreliaburgdorferi (known for causing Lyme disease).
Meningitis may be encountered in cerebral malaria (malaria infecting the brain) or amoebic meningitis, meningitis due to infection with amoebae such as Naegleriafowleri, contracted from freshwater sources.

5. Listeria monocytogenes
TYPES OF MENINGITIS
Bacterial:-
Organisms
Children
Adults
Elderly (>65)
GroupB Streptococcus
Escherichia coli
Listeria monocytogenes
Streptococcus pneumoniae (pneumococcus)
Neisseria meningitis (meningococcus)
Haemophilusinfluenzaetyp e B (less common now with the advent of the HiB vaccination)
Streptococcus pneumoniae
Neisseria meningitis
(these two organisms cause 80% of cases)
Streptococcus pneumoniae
Neisseria meningitis

6. TYPES
Viral-
Viruses that cause meningitis include enteroviruses, herpes simplex virus type 2 (and less commonly type 1), varicella zoster virus (known for causing chickenpox and shingles), mumps virus, HIV, and LCMV.
Fungal:
immunosuppressants (such as after organ transplantation), HIV/AIDS,and the loss of immunity associated with aging
Parasitic:
A parasitic cause is often assumed when there is a predominance of eosinophils (a type of white blood cell) in the CSF. The most common parasites implicated are Angiostrongyluscantonensis, Gnathostomaspinigerum, Schistosoma, as well as the conditions cysticercosis, toxocariasis, baylisascariasis, paragonimiasis, and a number of rarer infections and no infective conditions-
Non-infectious-
Meningitis may occur as the result of several non-infectious causes: spread of cancer to the meninges (malignant or neoplastic meningitis)and certain drugs (mainly non-steroidal anti-inflammatory drugs, antibiotics and intravenous immunoglobulins)

7. GLIMPSES

8. OVERVIEW OF PLAN OF ACTION:

9. WORK FLOW

10. WORKFLOW
VISUALISATION
HEX DOCKING

11. MATERIALS AND METHODS:
Protein FASTA Sequences of Neisseria Meningitides:-

12. Identification of Antigenic Sites using EMBOSS ANTIGENIC
SAA= Total no of Leucine+Valine+Cystine/ length of the peptide
 Sl.No.
   Protein name
Antigenic sites/ epitopes
PEPTIDE
SAA (SURFACE ACCECIBILITY AREA)
START POSITION
END POSITION 1.
Structure Of A N. Meningitides Protein 
(7)
GGSYALRVQGE 
2/11= 0.18
54-G
64-E 2.
catabolite repression control protein
(12)
EYIYCGSLYVAHQKM 
2/15= 0.13
142-E
156-M 3.
exonuclease III
ARLHQLQAVIDK
3/12= 0.25
13-A
24-K 4.
Exodeoxyribonuclease III
DHAPVVVD 
3/8= 0.38
262-D
269-D

13. Antigenic sites obtained from Protein Variability Server

14. Peptide with Surface accessibility area (SAA)
Method
used to
Prot
predict
Sequence
SAA
ein
antigenic
site
Antigenic
GGSYALRVQGE 
2/11= 0.18 1.
Emboss
Protein
2/11=0.18
Variability
PGGSYALRVQG   
Server 2.
2/15= 0.13
EYIYCGSLYVAHQKM  
3/15=0.2
REYIYCGSLYVAHQK 3.
3/12= 0.25
ARLHQLQAVIDK
3/12=0.25
RARLHQLQAVID 4.
DHAPVVVD
3/8= 0.38
SDHAPVVV
3/8=0.37

15. Designing and Energy Optimization:

16. ENERGY CALCULATION USING ARGUS LAB

17. ENEGY MINIMISATION USING SWISS PDB VIEWER

18. ENERGY MINIMISATION PEPTIDE NO. PEPTIDE SEQUENCE ORGANISM LINE SEARCH
GEOMETRY OPTIMIZATION
ENERGY MINIMISATION 4
GGSYALRVQGE
Structure of A N. Meningitides Protein
BFGS
78.78kcal/mol.... 8
EYIYCGSLYVAHQKM
Catabolite repression control protein:
STEEPEST
161.26kcal/mol 10
DHAPVVVD
Exodeoxyribonuclease III:
96.72kcal/mol 13
ARLHQLQAVIDK
Exonuclease III:
129.83kcal/mol

19. Peptide Properties Calculation Using Innovagen:
Number of residues: 11
Molecular weight:
g/mol
Extinction coefficient:
1280 M-1cm-1
Iso-electric point:
pH 6.89
Net charge at pH 7:
Estimated solubility:
Good water solubility.

20. Visualization Of Peptide And Result Analysis

21. ANALYSIS OF ANTIGENIC SITE OF HUMAN MENINGITIS
HLA class II histocompatibility antigen, DRB1-15 beta chain.
Binds peptides derived from antigens that access the endocytic route of antigen presenting cells (APC) and presents them on the cell surface for recognition by the CD4 T-cells. The peptide binding cleft accommodates peptides of residues.
The peptides presented by MHC class II molecules are generated mostly by degradation of proteins that access the endocytic route, where they are processed by lysosomal proteases and other hydrolases. Exogenous antigens that have been endocytosed by the APC are thus readily available for presentation via MHC II molecules, and for this reason this antigen presentation pathway is usually referred to as exogenous.
As membrane proteins on their way to degradation in lysosomes as part of their normal turn-over are also contained in the endosomal/lysosomal compartments, exogenous antigens must compete with those derived from endogenous components. Autophagy is also a source of endogenous peptides, autophagosomes constitutively fuse with MHC class II loading compartments. In addition to APCs, other cells of the gastrointestinal tract, such as epithelial cells, express MHC class II molecules and CD74 and act as APCs, which is an unusual trait of the GI tract. To produce a MHC class II molecule that presents an antigen, three MHC class II molecules (heterodimers of an alpha and a beta chain) associate with a CD74 trimer in the ER to form a heterononamer. Soon after the entry of this complex into the endosomal/lysosomal system where antigen processing occurs, CD74 undergoes a sequential degradation by various proteases, including CTSS and CTSL, leaving a small fragment termed CLIP (class-II-associated invariant chain peptide). The removal of CLIP is facilitated by HLA-DM via direct binding to the alpha- beta-CLIP complex so that CLIP is released. HLA-DM stabilizes MHC class II molecules until primary high affinity antigenic peptides are bound. The MHC II molecule bound to a peptide is then transported to the cell membrane surface. In B-cells, the interaction between HLA-DM and MHC class II molecules is regulated by HLA-DO. Primary dendritic cells (DCs) also to express HLA-DO. Lysosomal microenvironment has been implicated in the regulation of antigen loading into MHC II molecules, increased acidification produces increased proteolysis and efficient peptide loading.

22. FASTA Sequence:

23. GENOMIC LOCATION

24. SMART:

25. STRING:

26. BLAST ANALYSIS:

27. DOCKING:
Energy e+002, RMS

28. DOCKING ENERGY Sl.no. PEPTIDE NO. ENERGY 1 PEPTIDE 4
e+002, RMS 2
PEPTIDE 8
e+002, RMS 3
PEPTIDE 10
e+002, RMS 4
PEPTIDE 13
e+002, RMS

29. CONCLUSION
In the above work the complete proteins of the Neisseria Meningitides were identified and the sequences were collected from NCBI Database.
The sequences were further screened to identify the foreign proteins to the human proteome.
These foreign proteins were further tested for the presence of antigenic sites using EMBOSS Antigenic. From among the selected epitopes some of them having maximum antigenic propensity were screened based on PVS server. The final selected peptides were designed in Argus Lab.
The geometry of the peptides was optimized and the energy evaluation was performed. Based on the energy values the best peptide selected for the Vaccine development was Structure of A.N Meningitides. The peptide can be synthesized and attenuated so as to use it for vaccination against the Meningitis.

30. CONCLUSION
The designed peptides were docked with specific antigen of human which causes meningitis using HEX and their energy has been calculated.
Out of the four peptides peptide 8 has been shown the minimum energy and more stability when binds to the antigenic protein of human.
Thus it is concluded that the peptides thus designed can be imparted for the development of vaccines and thus further parametric calculations and simulations can be made to develop vaccines for the treatment of meningitis.