Single-Dose Mucosal Immunization with a Candidate Universal Influenza Vaccine Provides Rapid Protection from Virulent H5N1, H3N2 and H1N1 Viruses

The sudden emergence of novel influenza viruses is a global public health concern. Conventional influenza vaccines targeting the highly variable surface glycoproteins hemagglutinin and neuraminidase must antigenically match the emerging strain to be effective. In contrast, "universal" vaccines targeting conserved viral components could be used regardless of viral strain or subtype. Previous approaches to universal vaccination have required protracted multi-dose immunizations. Here we evaluate a single dose universal vaccine strategy using recombinant adenoviruses (rAd) expressing the conserved influenza virus antigens matrix 2 and nucleoprotein.In BALB/c mice, administration of rAd via the intranasal route was superior to intramuscular immunization for induction of mucosal responses and for protection against highly virulent H1N1, H3N2, or H5N1 influenza virus challenge. Mucosally vaccinated mice not only survived, but had little morbidity and reduced lung virus titers. Protection was observed as early as 2 weeks post-immunization, and lasted at least 10 months, as did antibodies and lung T cells with activated phenotypes. Virus-specific IgA correlated with but was not essential for protection, as demonstrated in studies with IgA-deficient animals.Mucosal administration of NP and M2-expressing rAd vectors provided rapid and lasting protection from influenza viruses in a subtype-independent manner. Such vaccines could be used in the interval between emergence of a new virus strain and availability of strain-matched vaccines against it. This strikingly effective single-dose vaccination thus represents a candidate off-the-shelf vaccine for emergency use during an influenza pandemic

Cold-Adapted Influenza and Recombinant Adenovirus Vaccines Induce Cross-Protective Immunity against pH1N1 Challenge in Mice

The rapid spread of the 2009 H1N1 pandemic influenza virus (pH1N1) highlighted problems associated with relying on strain-matched vaccines. A lengthy process of strain identification, manufacture, and testing is required for current strain-matched vaccines and delays vaccine availability. Vaccines inducing immunity to conserved viral proteins could be manufactured and tested in advance and provide cross-protection against novel influenza viruses until strain-matched vaccines became available. Here we test two prototype vaccines for cross-protection against the recent pandemic virus.BALB/c and C57BL/6 mice were intranasally immunized with a single dose of cold-adapted (ca) influenza viruses from 1977 or recombinant adenoviruses (rAd) expressing 1934 nucleoprotein (NP) and consensus matrix 2 (M2) (NP+M2-rAd). Antibodies against the M2 ectodomain (M2e) were seen in NP+M2-rAd immunized BALB/c but not C57BL/6 mice, and cross-reacted with pH1N1 M2e. The ca-immunized mice did not develop antibodies against M2e. Despite sequence differences between vaccine and challenge virus NP and M2e epitopes, extensive cross-reactivity of lung T cells with pH1N1 peptides was detected following immunization. Both ca and NP+M2-rAd immunization protected BALB/c and C57BL/6 mice against challenge with a mouse-adapted pH1N1 virus.Cross-protective vaccines such as NP+M2-rAd and ca virus are effective against pH1N1 challenge within 3 weeks of immunization. Protection was not dependent on recognition of the highly variable external viral proteins and could be achieved with a single vaccine dose. The rAd vaccine was superior to the ca vaccine by certain measures, justifying continued investigation of this experimental vaccine even though ca vaccine is already available. This study highlights the potential for cross-protective vaccines as a public health option early in an influenza pandemic

Pre-Clinical Evaluation of a Replication-Competent Recombinant Adenovirus Serotype 4 Vaccine Expressing Influenza H5 Hemagglutinin

Influenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4) vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA) gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn). Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis.The Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus.Several non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine

Vaccination to Conserved Influenza Antigens in Mice Using a Novel Simian Adenovirus Vector, PanAd3, Derived from the Bonobo <em>Pan paniscus</em>

<div><p>Among approximately 1000 adenoviruses from chimpanzees and bonobos studied recently, the Pan Adenovirus type 3 (PanAd3, isolated from a bonobo, <i>Pan paniscus</i>) has one of the best profiles for a vaccine vector, combining potent transgene immunogenicity with minimal pre-existing immunity in the human population. In this study, we inserted into a replication defective PanAd3 a transgene expressing a fusion protein of conserved influenza antigens nucleoprotein (NP) and matrix 1 (M1). We then studied antibody and T cell responses as well as protection from challenge infection in a mouse model. A single intranasal administration of PanAd3-NPM1 vaccine induced strong antibody and T cell responses, and protected against high dose lethal influenza virus challenge. Thus PanAd3 is a promising candidate vector for vaccines, including universal influenza vaccines.</p> </div

NPM1 fusion protein insert.

<p>a) Design of the insert showing CMV promoter, NPM1 transgene, and BGH-polyadenylation cassettes. b) Complete amino acid sequence of the consensus NPM1 fusion protein. NP is indicated in red, linker sequence is shown in black, and M1 is green. The deletion of the nuclear localization signal by mutation of TKR to AAA in NP is indicated in bold text.</p

Antibody responses to different doses of PanAd3NPM1.

<p>Mice were vaccinated with indicated doses. Four weeks later mice were sacrificed, and serum and BAL were collected for antibody analysis. Error bars indicate mean ± SEM. a) ELISA for IgG antibodies to rNP in the serum and BAL. b) ELISA for IgA antibodies to rNP in the serum and BAL. c) ELISA for IgG antibodies to rM1 in the serum and BAL.</p

T cell responses to different doses of PanAd3-NPM1.

<p>Mice were immunized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055435#pone-0055435-g003" target="_blank"><b>Figure 3</b></a>. Four weeks post-immunization, T -cells of three mice per group were assayed by IFN-γ ELISPOT. Results shown are for lung T cells, and are reported as number of IFN-γ secreting cells per 10⁶ cells. Bars show mean ± SEM of results for lungs of individual mice.</p

Protection against lethal influenza challenge by PanAd3-NPM1.

<p>Mice were immunized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055435#pone-0055435-g003" target="_blank"><b>Figure 3</b></a>. Approximately 9 weeks post-immunization, mice were challenged with influenza virus A/FM at a dose of 10⁴ TCID₅₀ (100 LD₅₀) per mouse, and monitored for body weight and mortality. Survival of the PanAd3-NPM1 group at the dose of 10⁹ vp differs significantly (p<0.05) from the PanAd3-RSV control group. Error bars indicate mean ± SEM.</p

Antibody and T cell responses to PanAd3NPM1.

<p>Mice were vaccinated with 10⁹ or 10⁷ total vp/mouse. Immunizations were by either the i.n. or i.m. route, as indicated. Four weeks later mice were sacrificed, and serum and BAL were collected for antibody analysis. Lung T cells were also collected for IFN-γ ELISPOT analysis. Error bars indicate mean ± SEM. a) ELISA for IgG antibodies to rNP in the serum. b) ELISA for IgG antibodies to rNP in the BAL. c) ELISA for IgA antibodies to rNP in the BAL. d) ELISPOT for IFN-γ T cell responses. Results for three mice per group are reported as number of IFN-γ secreting cells per 10⁶ cells. Black bars, stimulation with NP_147–155 peptide. Gray bars, stimulation with SARS_209–221 peptide. Top: T cells in the lungs. Bottom: T cells in the spleen. Bars show mean ± SEM of pooled group triplicates, not individual animals.</p