Evaluation of influenza-specific antibody-dependent cellular cytotoxicity (ADCC) in human and non-human primates

© 2013 Dr. Sinthujan JegaskandaPublications included in thesis:Jegaskanda, S., Job, E. R., Kramski, M., Laurie, K., Isitman, G., De Rose, R., et al. (2012). Cross-reactive influenza-specific antibody-dependent cellular cytotoxicity antibodies in the absence of neutralizing antibodies. Journal of Immunology, 190(3), 1837-1848. DOI: 10.4049/jimmunol.1201574Jegaskanda, S., Weinfurter, J. T., Friedrich, T. C., & Kent, S. J. (2013). Antibody-dependent cellular cytotoxicity is associated with control of pandemic H1N1 influenza virus infection of macaques. Journal of Virology, 87(10), 5512-5522. DOI: 10.1128/JVI.03030-12Jegaskanda, S., Laurie, K., Amarasena, T. H., Winnall, W., Kramski, M., De Rose, R., et al. (in press 2013). Age-associated cross-reactive antibody-dependent cellular cytotoxicity toward 2009 pandemic influenza A virus subtype H1N1. Journal of Infectious Diseases. DOI: 10.1093/infdis/jit294Jegaskanda, S., Vandenberg, K., Rockman, S., & Kent, S. J. Cross-reactive influenza-specific antibody-dependent cellular cytotoxicity present in intravenous immunoglobulin. (Submitted to Journal of Immunology, August 2013)Jegaskanda, S., Amarasena, T. H., Laurie, K. L., Tan, H-X., Butler, J., Parsons, M. S., et al. (2013). Standard trivalent influenza protein vaccination does not prime antibody-dependent cellular cytotoxicity (ADCC) in macaques. Journal of Virology, 87(24), 13706-13718. DOI: 10.1128/JVI.01666-13Influenza virus is a major cause of morbidity and mortality worldwide. Yearly trivalent influenza vaccination partially reduces the severity of influenza infection. Seasonal influenza vaccines mediate their protective activity through the induction of neutralising antibodies towards the influenza virus surface hemagglutinin (HA) and neuraminidase (NA) glycoproteins. HA- and NA-specific antibodies act by either inhibiting virus entry or preventing the release of new virions from host cells, respectively. However, influenza-specific neutralising antibodies are susceptible to loss of recognition by accumulation of point mutations in the HA and NA over time (antigenic drift), or by the introduction of new viral HA and NA glycoproteins (antigenic shift). Fortunately, antibodies have a number of non-neutralising effector functions including antibody-dependent cellular cytotoxicity (ADCC). Non-neutralising antibody effector functions rely on binding of the antibody fragment antigen-binding (Fab) region to surface antigens, but mediate their effector activity through the fragment crystallisable (Fc) region of the antibody. ADCC in particular may play an important role in protection from influenza infection, but few studies have been undertaken in this area for over 3 decades. Previous studies on influenza-specific ADCC were performed using either chromium-release assays or in in vivo using FcR-knockout mice. Until recently no flow-cytometry based assay was available to measure and characterise influenza-specific ADCC-mediating antibodies in human and non-human primate sera samples. Using newly developed assays we showed that healthy individuals aged between 18-43 years of age did not have neutralising antibodies to a 1968 H3N2 influenza virus, but they did possess binding antibodies. Furthermore, a portion of these non-neutralising antibodies mediated ADCC effector functions including activating NK cells and eliminating virus-infected respiratory epithelial cells (Chapter 2). This highlighted that ADCC-mediating antibodies have broad cross-reactive capacity that may play an important role in protection against influenza virus infections. In April 2009, a swine-origin H1N1 influenza (A(H1N1)pdm09) virus caused a pandemic in the human population. The pandemic was estimated to have resulted in 4 million cases worldwide, but only lead to approximately 106,700-395,699 deaths. This was a comparatively milder influenza pandemic, as seasonal influenza epidemics generally lead to approximately 350,000-500,000 deaths worldwide. Moreover, yearly influenza epidemics generally cause deaths in younger (65 years old), particularly targeting individuals with major risk factors such as cardiovascular disease. In contrast, the 2009 H1N1 pandemic had a lower mortality in older individuals (>65 years old), with younger healthy individuals experiencing greater morbidity. These two separate observations suggested that there were two levels of protection detected in the population prior to the 2009 H1N1 pandemic: (1) the reduced severity of the 2009 H1N1 pandemic on the population and (2) the lower incidence of mortality in older individuals in the population. The potential role of cross-reactive ADCC-mediating antibodies in providing protection during the 2009 H1N1 pandemic was investigated. To determine if there was ADCC-mediated protection against A(H1N1)pdm09 virus in older individuals, we obtained human sera samples collected from separate individuals (aged between 1-72 years) prior to or following the 2009 H1N1 pandemic. We showed that individuals greater than 45 years of age had a moderate level of cross-reactive A(H1N1)pdm09-specific ADCC-mediating antibodies prior to the 2009 H1N1 pandemic (Chapter 3). To understand the role that ADCC-mediating antibodies may have played in reducing the severity of A(H1N1)pdm09 virus on the whole population, we obtained intravenous immunoglobulin (IVIG) manufactured either prior to the 2009 or post 2009 pandemic. We showed that there was a level of cross-reactive A(H1N1)pdm09-specific ADCC-mediating antibodies in the whole population prior to the 2009 H1N1 pandemic (Chapter 4). Lastly, to examine the mechanism by which cross-reactive A(H1N1)pdm09-specific ADCC-mediating antibodies are induced, we measured ADCC-mediating antibodies in naïve rhesus macaques (Macaca mulatta) infected with seasonal H1N1 influenza virus and subsequently challenged with the A(H1N1)pdm09 virus (Chapter 5). We showed that seasonal H1N1 infection of macaques induced cross-reactive ADCC-mediating antibodies towards the A(H1N1)pdm09 virus. Collectively, of these studies implicate ADCC in the protection observed during the 2009 H1N1 pandemic. The induction of influenza-specific ADCC-mediating antibodies by trivalent vaccines remains unknown. To investigate whether trivalent influenza vaccines can prime or induce ADCC-mediating antibodies we vaccinated influenza naïve pigtail macaques (Macaca nemestrina) with two doses of trivalent influenza vaccine (2012, A/California/07/2009 (H1N1), A/Perth/16/2009 (H3N2) and B/Brisbane/60/2008 (Type B), Sanofi Pasteur) and subsequently serially infected them with either H1N1 or H3N2 influenza strains. Our results showed that ADCC-mediating antibodies and cytotoxic T lymphocytes (CTL) responses were not primed or induced by vaccinations with seasonal trivalent influenza vaccine (Chapter 6). These studies suggested that the induction of ADCC-mediating antibodies by trivalent vaccination most likely relied on a background of previous influenza exposures. The design of new vaccines that induce broad ADCC-mediating antibodies may provide greater protection from divergent influenza strains and should be an important direction for future studies. Together, these studies serve as a basis for future research into influenza-specific ADCC immunity. The association of cross-reactive ADCC mediating antibodies with the protection observed during the 2009 H1N1 pandemic has important implications for the control of potential future influenza pandemics. The induction of broadly cross-reactivity ADCC mediating antibodies in nearly all individuals could be utilised for universal influenza vaccine design