Development of Bordetella pertussis as a live vehicles for heterologous antigens delivery, and its application as a universal influenza A vaccine

Abstract

In this study, H5N1 specific antigen candidate NA, and several other antigens that are highly conserved among influenza A viruses, namely the ectodomain of M2 protein (M2e), 3 conserved neutralizing epitopes in H5 and the nucleocapsid protein (NP) were selected and expressed in attenuated B. pertussis BPZE1. These antigens were either expressed in the cytoplasm of B. pertussis or secreted into the external milieu using the filamentous hemagglutinin (FHA) as carrier. Unexpectedly, we observed in this study that prior nasal administration of an attenuated strain of BPZE1 provided effective and sustained protection against lethal challenge with mouse-adapted H3N2 influenza A virus. Surprisingly, no significant difference in the viral load was observed between BPZE1-immunized and non-immunized mice, indicating that the processes involved in the cross-protection do not directly target the viral particles and/or infected cells. This hypothesis is further supported by the observation that no cross-reactive antibodies and T-cells were found in mice pre-treated with BPZE1 and then stimulated with heat-killed H3N2 influenza virus.Instead, mice pre-treated with BPZE1 were protected from influenza-induced-lymphocyte depletion in the lungs and displayed markedly reduced lung inflammation and tissue damage as well as decreased neutrophilic infiltration. and a significantly lower production of the major pro-inflammatory cytokines and chemokines in their broncho-alveolar lavage fluid samples. In addition, we showed that nasal pre-treatment with BPZE1 also protected against H1N1 virus challenge, although with a lesser efficacy. These observations thus pointed to nonspecific anti-inflammatory properties of BPZE1 and suggested a potential prophylactic application to protect against highly pathogenic influenza A viruses.

We also investigated the potential of recombinant B. pertussis strains expressing antigen candidates from influenza virus as live recombinant vaccines against influenza virus, thus combining the nonspecific anti-inflammatory properties of BPZE1 and the specific anti-influenza immune responses. In particular, the universal influenza vaccine candidate M2e was expressed in BPZE1 as a FHA-(M2e)3 chimera and the nasal administration of the recombinant BPLR3 bacteria triggered significant production of anti-M2e IgA antibodies in the respiratory tract. Although BPLR3 immunization did not provide further protection compared with BPZE1 in the challenge experiments, BPLR3 still represents a promising universal vaccine against influenza A viruses and deserves future improvement. In addition, we showed for the first time that the cytoplasmic expression of heterologous antigens such as NP and truncated NA in BPZE1 may not be an effective approach to prime the host immune system. In conclusion, our data indicates that BPZE1 represents a promising vehicle for the nasal delivery of subunit vaccine candidates against influenza virus by combining the non-specific anti-inflammatory properties of the vehicle BPZE1 with the specific adaptive immunity induced by the production of the foreign antigens.