Clinical Studies of Vaccines for Pandemic Influenza

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

Seasonal epidemics and sporadic pandemics of influenza A viruses (IAV) pose a global public health burden. Influenza A viruses are divided into subtypes on the basis of the antigenicity and nucleotide sequence relatedness of their surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Hemagglutinin-specific antibodies directly bind the virus and prevent its entry into host cells, providing narrow immunity from reinfection by closely related strains. CD8 T cell responses to IAV generated against highly conserved viral proteins/epitopes contribute to clearance of virus during primary IAV infection and also confer broad heterosubtypic protection in animal models. Recent evidence links the cross-reactive CD8 T cell response in man to reduced viral replication and protection from severe illness in pandemic H1N1 infections in European populations, and H7N9 infections in China. Because pre-existing T-cell immunity, independent of baseline antibodies, protects against symptoms and viral shedding associated with influenza, influenza vaccines that stimulate broadly reactive CD8 T cell responses may have the capacity to protect against any pandemic influenza A virus. Human infections with H5N1 and H7N9 avian IAV and the 2009 H1N1 pandemic have spurred an interest in the development of vaccines against IAV with pandemic potential. Major challenges to this effort include our inability to predict which virus will emerge and rapid production and deployment of vaccine if the virus spreads rapidly and vaccine yield is not optimal. In addition, the number of doses of vaccine required depends on whether the population is immunologically naive. We have previously shown that pandemic live attenuated influenza viruses (pLAIV), prime for a rapid and robust antibody response on subsequent administration of pandemic inactivated subunit vaccine (pISV). This is observed even in individuals who had undetectable antibody responses following the initial vaccination. To define the mechanistic basis of pLAIV priming, we turned to a non-human primate model and performed a detailed analysis of B cell responses in systemic and local lymphoid tissues following prime-boost vaccination with pLAIV and pISV. We vaccinated groups of 12 African green monkeys (AGMs) with H5N1 pISV or pLAIV alone or H5N1 pLAIV followed by pISV and examined immunity systemically and in local draining lymph nodes (LN). The AGM model recapitulated the serologic observations from clinical studies. Interestingly, H5N1 pLAIV induced robust germinal center B cell responses in the mediastinal LN (MLN). Subsequent boosting with H5N1 pISV drove increases in H5-specific B cells in the axillary LNs, spleen and circulation in H5N1 pLAIV-primed animals. Thus, H5N1 pLAIV primes localized B cell responses in the MLN that are recalled systemically following pISV boost. These data provide mechanistic insights for the generation of robust humoral responses via prime-boost vaccination. Two master donor viruses (MDVs) are licensed for the generation of seasonal LAIVs and both have been used as backbones to generate candidate vaccines for avian influenza viruses belonging to the H5 subtype. They have been evaluated in humans, but have differed in their infectivity and immunogenicity. To understand these differences, we generated four H5N2 candidate pLAIVs derived from either MDV and compared their biological characteristics in vitro and in vivo. We demonstrated that all candidate pLAIVs, regardless of gene constellation and derivation, were comparable with respect to infectivity, immunogenicity, and protection from challenge in the ferret model of influenza. These observations suggest that differences in clinical performance of H5 pLAIVs may be due to factors other than inherent biological properties of the two MDVs. Influenza viruses of the H1N1, H2N2, and H3N2 subtypes have caused previous pandemics. H2 influenza viruses represent a pandemic threat due to continued circulation in wild birds and limited immunity in the human population. In the event of a pandemic, anti-viral agents are the mainstay for treatment, but broadly neutralizing antibodies (bNAbs) may be a viable alternative for short-term prophylaxis or treatment. The HA stem binding bNAbs CR6261 and CR9114 have been shown to protect mice from severe disease following challenge with H1N1 and H5N1, and H1N1, H3N2, and influenza B viruses, respectively. Early studies with CR6261 and CR9114 showed weak in vitro activity against human H2 influenza viruses, but the in vivo efficacy against H2 viruses was unknown. Therefore, in collaboration with scientists from Crucell, we evaluated these antibodies against human and animal origin H2 viruses: A/Ann Arbor/6/1960 (H2N2) AA60 and A/swine/MO/4296424/06 (H2N3) Sw06. In vitro, CR6261 neutralized both H2 viruses, while CR9114 only neutralized Sw06. To evaluate prophylactic efficacy, mice were given CR6261 or CR9114 and intranasally challenged 24 hours later with lethal doses of AA60 or Sw06. Both antibodies reduced mortality, weight loss, airway inflammation, and pulmonary viral load. Using engineered bNAb variants, antibody dependent cell cytotoxicity reporter assays, and Fc receptor deficient (Fcer1g-/-) mice, we show that the in vivo efficacy of CR9114 against AA60 is mediated by Fc receptor-dependent mechanisms. Collectively, these findings demonstrate the in vivo efficacy of CR6261 and CR9114 against H2 viruses, and emphasize the need for in vivo evaluation of bNAbs. The stem of the influenza A virus hemagglutinin (HA) is highly conserved and represents an attractive target for a universal influenza vaccine. The 18 HA subtypes of influenza A are phylogenetically divided into 2 groups, and while protection with group 1 HA stem vaccines has been demonstrated in animal models, studies on group 2 stem vaccines are limited. Thus, we engineered group 2 HA stem-immunogen (SI) vaccines targeting the epitope for the broadly neutralizing monoclonal antibody CR9114 and evaluated vaccine efficacy in mice and ferrets. Immunization induced antibodies that bound to recombinant HA protein and viral particles, and competed with CR9114 for binding to the HA stem. Mice vaccinated with H3 and H7-SI were protected from lethal homologous challenge with X-79 (H3N2) or A/Anhui/1/2013 (H7N9), and displayed moderate heterologous protection. In ferrets, H7-SI vaccination did not significantly reduce weight loss or nasal wash titers after robust H7N9 virus challenge. Epitope mapping revealed ferrets developed lower titers of antibodies that bound a narrow range of HA stem epitopes compared to mice, and this likely explains the lower efficacy in ferrets. Collectively, these findings indicate that while group 2 SI vaccines show promise, their immunogenicity and efficacy are reduced in larger outbred species, and will have to be enhanced for successful translation to a universal vaccine. We have previously evaluated a broadly protective influenza vaccine (BPIV) candidate called S-FLU developed by Professor Alain Townsend at the University of Oxford that is limited to a single cycle of replication. This vaccine induces a strong cross-reactive T cell response, but a minimal antibody response to HA. We tested whether an H3N2 S-FLU can protect pigs and ferrets from heterosubtypic H1N1 influenza challenge. Aerosol administration of S-FLU to pigs induced lung tissue resident memory T cells and reduced lung pathology, but not the viral load. In contrast, in ferrets S-FLU reduced viral replication and aerosol transmission. Our data show that S-FLU has different protective efficacy in pigs and ferrets, and that in the absence of antibody, lung T cell immunity can reduce disease severity without reducing challenge viral replication.