Pratip Shil

Molecular basis of antigenic drift in Influenza A/H3N2 strains (1968-2007) in the light of antigenantibody interactions.

The emergence of new strains of Influenza virus have caused several pandemics over the last hundred years with the latest being the H1N1 Swine flu pandemic of 2009. The Hemagglutinin (HA) protein of the Influenza virus is the primary target of human immune system and is responsible for generation of protective antibodies in humans. Mutations in this protein results in change in antigenic regions (antigenic drift) which consequently leads to loss of immunity in hosts even in vaccinated population (herd immunity). This necessitates periodic changes in the Influenza vaccine composition. In this paper, we investigate the molecular basis of the reported loss of herd immunity in vaccinated population (vaccine component: Influenza A/X-31/1968 (H3N2)) which resulted in the outbreak due to strain Influenza A/Port Chalmers/1/1973 (H3N2). Also, the effects of antigenic drift in HA protein (H3N2 vaccine strains 1968-2007) on the 3D structures as well as interactions with BH151, a 1968 antibody, has been studied. Rigid body molecular docking protocol has been used to study the antigen-antibody interactions. We believe that the present study will help in better understanding of host-pathogen interactions at the molecular level.

Induction of virus-specific neutralizing immune response against West Nile and Japanese encephalitis viruses by chimeric peptides representing T-helper and B-cell epitopes.

West Nile virus (WNV) and Japanese encephalitis virus (JEV), the members of JEV serocomplex group are pathogens of global health concern. The co-circulation of these viruses poses challenges in effective diagnostics due to antigenic similarity between the E-protein of these viruses. The present study aimed to design chimeric peptides and study the immune response against the same. B-cell epitopes were predicted on structural proteins of WNV and JEV based on bioinformatics tools. The peptides representing to these B-cell epitopes were synthesized and subjected to ELISA. Two peptides, one each from WNV (named WE147) and JEV (named JE40) E-protein, showed virus-specific and strong reactivity to the immune mice sera and human clinical samples. The chimeric peptides for WNV and JEV were constructed by synthesizing the B-cell epitope of WNV (WE147) or JEV (JE40) with T-helper epitope (JM17) separated by diglycine spacer in between. The immune response generated against these chimeric peptides was found to be specific to the respective B-cell epitopes. The anti-peptide sera showed virus-specific reactivity in ELISA and in immunofluorescence assay with no cross-reactivity. Also, the anti-peptide sera could neutralize JE and WN viruses in an in vitro virus neutralization assay. The B-cell epitopes identified in the present study may be used as diagnostic markers for differentiating between WN and JE virus infections. The present study can form a basis for future design of vaccines.

Antigenic variability in Neuraminidase protein of Influenza A/H3N2 vaccine strains (1968 - 2009).

Antigenic drift and shift involving the surface proteins of Influenza virus gave rise to new strains that caused epidemics affecting millions of people worldwide over the last hundred years. Variations in the membrane proteins like Hemagglutinin (HA) and Neuraminidase (NA) necessitates new vaccine strains to be updated frequently and poses challenge to effective vaccine design. Though the HA protein, the primary target of the human immune system, has been well studied, reports on the antigenic variability in the other membrane protein NA are sparse. In this paper we investigate the molecular basis of antigenic drift in the NA protein of the Influenza A/H3N2 vaccine strains between 1968 and 2009 and proceed to establish correlation between antigenic drift and antigen-antibody interactions. Sequence alignments and phylogenetic analyses were carried out and the antigenic variability was evaluated in terms of antigenic distance. To study the effects of antigenic drift on the protein structures, 3D structure of NA from various strains were predicted. Also, rigid body docking protocol has been used to study the interactions between these NA proteins and antibody Mem5, a 1998 antibody.

Zika virus: Indian perspectives.

The emergence of Zika virus (ZiV), a mosquito borne Flavivirus like dengue (DEN) and chikungunya (CHIK), in Brazil in 2014 and its spread to various countries have led to a global health emergency. Aedes aegypti is the major vector for ZiV. Fast dissemination of this virus in different geographical areas posses a major threat especially to regions where the population lacks herd immunity against the ZiV and there is abundance of Aedes mosquitoes. In this review, we focus on current global scenario, epidemiology, biology, diagnostic challenges and remedial measures for ZiVconsidering the Indian perspective.

Design and characterization of polytope construct with multiple B and TH epitopes of Japanese encephalitis virus.

Japanese encephalitis (JE) remains a major public health threat with vaccination as the only measure for its prevention. Epitope-based vaccination is a promising approach for achieving protective immunity and avoid immunopathology in Japanese encephalitis virus (JEV) infection due to flavivirus cross-reactivity. We have mapped B-cell epitopes from JEV envelope protein, responsible for elicitation of neutralizing antibodies. Incorporation of T helper (T(H)) epitopes, along with these, imparted protective immunity to the host. In the present study, based on in silico epitope selection we optimized and proposed a polytope DNA construct (P-JEV) consisting B-cell and T(H) epitopes from the JEV envelope (E) protein as well as non-structural protein-1 (NS1). The immunogenicity and protective efficacy of P-JEV was assessed by in vitro and in vivo experiments. The expressed P-JEV showed reactivity in in vitro assays with JEV monoclonal antibodies. Protective efficacy of P-JEV was assessed in BALB/c mice. Our findings indicate that P-JEV may be a candidate vaccine for the prevention of JEV infection.

Antigen-Antibody docking reveals the molecular basis for cross-reactivity of the 1918 and 2009 Influenza A/H1N1 pandemic viruses.

To understand the reported cross-reactivity of the 2009 H1N1 and the 1918 H1N1 pandemic viruses we docked the crystal structure of 2D1, an antibody derived from a survivor of the 1918 pandemic, to the structures of hemaglutinin (HA) of the 2009 strain and seasonal H1 vaccine strains. Our studies revealed that 2D1 binds to the 2009 HA at antigenic site ‘Sa’, with stabilizing contacts, similar to that in an available co-crystal structure of 2D1-1918 HA. However, 2D1 failed to bind to the known antigenic sites in the HAs of seasonal strains. Our study thus reveals the molecular basis for pre-existing immunity in elderly people to the 2009 pandemic virus.

Differences in Type I interferon response in human lung epithelial cells infected by highly pathogenic H5N1 and low pathogenic H11N1 avian influenza viruses

Influenza A virus infection induces type I interferons (IFNs α/β) which activate host antiviral responses through a cascade of IFN signaling events. Herein, we compared highly pathogenic H5N1 and low pathogenic H11N1 avian influenza viruses isolated from India, for their replication kinetics and ability to induce IFN-β and interferon-stimulating genes (ISGs). The H5N1 virus showed a higher replication rate and induced less IFN-β and ISGs compared to the H11N1 virus when grown in the human lung epithelial A549 cells, reflecting the generation of differential innate immune responses during infection by these viruses. The non-structural 1 (NS1) protein, a major IFN-antagonist, known to help the virus in evading host innate immune response was compared from both the strains using bioinformatics tools. Analyses revealed differences in the composition of the NS1 proteins from the two strains that may have an impact on the modulation of the innate immune response. Intriguingly, H5N1 virus attenuated IFN-β response in a non-NS1 manner, suggesting the possible involvement of other viral proteins (PB2, PA, PB1/PB1-F2) of H5N1 in synergy with NS1. Preliminary analyses of the above proteins of the two strains by sequence comparison show differences in charged residues. The insight gained will be useful in designing experimental studies to elucidate a probable role of the polymerase protein(s) in association with NS1 in inhibiting the IFN signaling and understanding the molecular mechanism governing the difference.

Bioinformatics characterization of envelope glycoprotein from Kyasanur Forest disease virus

Background & objectives: Kyasanur Forest disease (KFD) is a febrile illness characterized by haemorrhages and caused by KFD virus (KFDV), which belongs to the Flaviviridae family. It is reported to be an endemic disease in Shimoga district of Karnataka State, India, especially in forested and adjoining areas. Several outbreaks have been reported in newer areas, which raised queries regarding the changing nature of structural proteins if any. The objective of the study was to investigate amino acid composition and antigenic variability if any, among the envelope glycoprotein (E-proteins) from old and new strains of KFDV. Methods: Bioinformatic tools and techniques were used to predict B-cell epitopes and three-dimensional structures and to compare envelope glycoprotein (E-proteins) between the old strains of KFDV and those from emerging outbreaks till 2015. Results: The strain from recent outbreak in Thirthahalli, Karnataka State (2014), was similar to the older strain of KFDV (99.2%). Although mutations existed in strains from 2015 in Kerala KFD sequences, these did not alter the epitopes. Interpretation & conclusions: The study revealed that though mutations existed, there were no drastic changes in the structure or antigenicity of the E-proteins from recent outbreaks. Hence, no correlation could be established between the mutations and detection in new geographical areas. It seems that KFDV must be present earlier also in many States and due to availability of testing system and alertness coming into notice now.

Identification of a conserved neutralizing epitope in the G-protein of Chandipura virus

Chandipura virus (CHPV), associated with an encephalitic illness in humans, has caused multiple outbreaks with high mortality in central and western India in recent years. The present study compares surface glycoprotein (G-protein) from prototype and recent outbreak strains using in silico tools and in vitro experiments. In silico epitope predictions (B-cell and T-helper cell) for the sequences, 3D structure prediction and comparison of the G-proteins of the strains: I653514 (Year 1965), CIN0327 (Year 2003) and 148974 (Year 2014) revealed that the CHPV G-protein is stable and antigenic determinants are conserved. A monoclonal antibody developed against strain CIN0327 (named NAbC) was found to neutralize prototype I653514 as well as the currently circulating strain 148974. In silico antigen-antibody interaction studies using molecular docking of predicted structures of NAbC and G-proteins of various CHPV strains led to the identification of a conserved neutralizing epitope in the fusion domain of G-protein, which also contained a putative T-helper peptide. The identification of a conserved neutralizing epitope in domain IV (fusion domain amino acids 53 to 172) of CHPV G-protein is an important finding that may have the scope towards the development of protective targets against CHPV infection.