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Neutralizing antibody is associated with the prevention and clearance of influenza virus infection. Microneutralization (MN) and hemagglutination inhibition (HI) assays are currently used to evaluate neutralizing antibody responses against human and avian influenza viruses, including H5N1. The MN assay is somewhat labor intensive, while HI is a surrogate for neutralization. Moreover, use of replication competent viruses in these assays requires biosafety level 3 (BSL-3) containment. Therefore, a neutralization assay that does not require BSL-3 facilities would be advantageous. Toward this goal, we generated a panel of pseudotypes expressing influenza hemagglutinin (HA) and neuraminidase (NA) and developed a pseudotype-based neutralization (PN) assay. Here we demonstrate that HA/NA pseudotypes mimic release and entry of influenza virus and that the PN assay exhibits good specificity and reveals quantitative difference in neutralizing antibody titers against different H5N1 clades and subclades. Using immune ferret sera, we demonstrated excellent correlation between the PN, MN, and HI assays. Thus, we conclude that the PN assay is a sensitive and quantifiable method to measure neutralizing antibodies against diverse clades and subclades of H5N1 influenza virus.

The development of a successful vaccine against human immunodeficiency virus type 1 (HIV-1) likely requires immunogens that elicit both broadly neutralizing antibodies against envelope spikes and T cell responses that recognize multiple viral proteins. HIV-1 virus-like particles (VLP), because they display authentic envelope spikes on the particle surface, may be developed into such immunogens. However, in one way or the other current systems for HIV-1 VLP production have many limitations. To overcome these, in the present study we developed a novel strategy to produce HIV-1 VLP using stably transfected Drosophila S2 cells. We cotransfected S2 cells with plasmids encoding HIV-1 envelope, Gag, and Rev proteins and a selection marker. After stably transfected S2 clones were established, HIV-1 VLP and their immunogenicity in mice were carefully evaluated. Here, we report that HIV-1 envelope proteins are properly cleaved, glycosylated, and incorporated into VLP with Gag. The amount of VLP released into culture supernatants is comparable to those produced by insect cells infected with recombinant baculoviruses. Moreover, cryo-electron microscopy tomography revealed average 17 spikes per purified VLP, and antigenic epitopes on the spikes were recognized by the broadly neutralizing antibodies 2G12, b12, VRC01, and 4E10 but not by PG16. Finally, mice primed with DNA and boosted with VLP in the presence of CpG exhibited anti-envelope antibody responses, including ELISAbinding, neutralizing, antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated viral inhibition, as well as envelope and Gag-specific CD8 T cell responses. Thus, we conclude that HIV-1 VLP produced by the S2 expression system has many desirable features to be developed into a vaccine component against HIV-1.

BackgroundAlthough DNA plasmid and virus-like particle (VLP) vaccines have been individually tested against highly pathogenic avian influenza (HPAI) H5N1 viruses, the combination of both vaccines into a heterologous prime-boost strategy against HPAI H5N1 viruses has not been reported before.Methodology/Principal FindingsWe constructed DNA plasmid encoding H5HA (A/Shenzhen/406H/06, subclade 2.3.4) and generated VLP expressing the same H5HA and N1NA. We then compared neutralizing antibody responses and immune protection elicited with heterologous DNA-VLP, homologous DNA-DNA and VLP-VLP prime-boost strategies against HPAI H5N1 viruses in mice. We demonstrate that DNA-VLP elicits the highest neutralizing antibody titers among the three prime-boost strategies, whereas DNA-DNA elicits higher neutralizing antibody titers than VLP-VLP. We show that although all three prime-boost strategies protect mice from death caused by 10 MLD50 of homologous and heterologous H5N1 challenge, only DNA-VLP and DNA-DNA protect mice from infection as manifested by no weight loss and no lung pathology. In addition, we show that although DNA-VLP and DNA-DNA protect mice from death caused by 1,000 MLD50 of homologous H5N1 challenge, only DNA-VLP protects mice from infection. Moreover, we show that after 1,000 MLD50 of heterologous H5N1 challenge, while all mice in PBS, VLP-VLP and DNA-DNA died, 3 of 6 mice in DNA-VLP actually survived. Finally, we show that DNA-VLP completely protects mice from infection after 1,000 MLD50 of homologous H5N1 challenge even when the challenge was administrated at 60 days post the boost.Conclusions/SignificanceThese results provide strong support for clinical evaluation of heterologous DNA-VLP prime-boost strategy as a public health intervention against a possible H5N1 pandemic.