PhD Graduate: Janice OH Hsueh Ling (Collaborative Anti-Viral Research Laboratory)
Thesis Title: Understanding the adaptive immune responses against newly emerged viruses, SARS coronavirus and avian H5N1 influenza.
With increasing opportunities for animal-to-human and human-to-human transmission of infectious pathogens, many new strains of viruses have emerged. Two recent newly-emerged viruses are the severe acute respiratory syndrome coronavirus (SARS-CoV) and the H5N1 influenza A virus. Together with innate immunity, the host adaptive immune responses are crucial for the clearance of the virus during an infection.
In the first part of this study, the longevity and functionality of SARS-specific memory T cells were examined. The SARS accessory 3a protein was first demonstrated to elicit humoral and T cell response in a mouse model. The study was extended to using peripheral blood mononuclear cells (PBMCs) from 16 SARS-recovered individuals, 4 years post-infection. An unbiased approach, which is independent of HLA types, was utilized to identify putative T cell epitopes in the accessory 3a and the nucleocapsid (NP) protein. The IFNγ
ELISPOT and intracellular cytokine staining assays showed that approximately 50% of them had positive memory T cell responses against the two proteins tested. CD4+ T and CD8+ T cell responses were observed following stimulation with a pool of overlapping peptides spanning the entire NP and 3a proteins. Five potential CD8+ T cell epitopes were identified. Among them, peptide NP44 was the most frequently recognized peptide and 3a2, which displayed both CD4+ and CD8+ T cell response, was the only peptide identified in the 3a protein. Cytokine analysis of these T cells revealed polyfunctional activity. Interestingly, all the CD8+ T cell epitopes were restricted by HLA-B subtype. These data can be useful in designing vaccines against SARS as well as understanding memory T cell responses against novel acute infections.
In the second part of the study, a neutralizing monoclonal antibody (mAb) 9F4 against the hemagglutinin (HA) protein of the influenza A/chicken/hatay/2004 H5N1 virus (clade 1) was generated and characterized. Firstly, the baculovirus-expressed and purified recombinant HA (rHA) was shown to elicit neutralizing humoral and T cell responses in the mouse model. This was followed by the production of mAb 9F4 using the rHA. MAb 9F4 binds both the denatured and native forms of HA and recognizes the HA proteins of three other heterologous strains of H5N1 viruses. By using lentiviral pseudotyped particles carrying HA on the surface, mAb 9F4 was shown to effectively neutralize the homologous and other H5N1 strains belonging to clade 1, 2.1 and 2.2 but not other subtypes of the influenza A virus. Epitope mapping analysis revealed that mAb 9F4 binds a previously uncharacterized and well-conserved epitope below the globular head of the HA1 subunit. MAb 9F4 does not block the interaction between HA and its receptor but prevents pH-mediated conformation change of HA which is necessary for the fusion step during viral entry. It was also found to be protective, both prophylactically and therapeutically, against lethal viral challenge of mice. Our data suggest that mAb 9F4 could be a potential candidate for immunotherapy. It also provided new information on a novel neutralizing epitope, yielding a new avenue for the design and development of a universal vaccine against H5N1.
Figure Legend: MAb 9F4 prevents the entry of HApp of other clades of H5N1 virus, but not that of HApp from other subtypes (H1N1 and H3N2) of influenza virus. (A) Pseudotyped lentiviral particles harboring the HA proteins from the respective H5N1 influenza viruses, namely clade 1 (VN04-H5pp), clade 2.1 (Indo5-H5pp) and clade 2.2 (IND06-H5pp) were incubated with different concentrations of mAb 9F4 at 4 oC for 2 h before inoculation onto MDCK cells. (B) Pseudotyped lentiviral particles harboring the HA proteins from 2 other subtypes of the influenza virus, namely H1N1 (H1-HApp) and H3N2 (H3-HApp) were incubated with different concentrations of mAb 9F4 at 4 oC for 2 h before inoculation onto MDCK cells. Luciferase activity in the cell lysates was determined 72 h post-infection. Percentage of viral entry, as indicated by the luciferase activity, was expressed as a relative percentage of the reading obtained in the absence of antibody (No Ab), which was set as 100%. A control mAb of the same isotype (IgG control) was used at the highest concentration tested. The experiments were repeated three times and data shown is a representative. Each histogram is the mean value of duplicate wells with standard deviation bars shown.