Influenza virus infection predisposes to secondary bacterial pneumonia. Currently licensed influenza vaccines aim at the induction of neutralizing antibodies and are less effective if the induction of neutralizing antibodies is low and/or the influenza virus changes its antigenic surface. We investigated the effect of suboptimal vaccination on the outcome of post-influenza bacterial superinfection.

Fc-dependent effector functions are an important determinant of the in vivo potency of therapeutic antibodies. Effector function is determined by the combination of FcRs bound by the antibody and the cell expressing the relevant FcRs, leading to antibody-dependent cellular cytotoxicity (ADCC). A number of ADCC assays have been developed; however, they suffer from limitations in terms of throughput, reproducibility, and in vivo relevance. Existing assays measure NK cell-mediated ADCC activity; however, studies suggest that macrophages mediate the effector function of many antibodies in vivo. Here, we report the development of a macrophage-based ADCC assay that relies on luciferase expression in target cells as a measure of live cell number. In the presence of primary mouse macrophages and specific antibodies, loss of luciferase signal serves as a surrogate for ADCC-dependent killing. We show that the assay functions for a variety of mouse and human isotypes with a model antigen/antibody complex in agreement with the known effector function of the isotypes. We also use this assay to measure the activity of a number of influenza-specific antibodies and show that the assay correlates well with the known in vivo effector functions of these antibodies.

Human epidermal growth factor receptor-2 (HER2) is upregulated in 20% to 30% of breast cancers and is a marker of a poor outcome. Due to the development of resistance to passive immunotherapy with Trastuzumab, active anti-HER2 vaccination strategies that could potentially trigger durable tumor-specific immune responses have become an attractive research area. Recently, we have shown that budded virus-like particles (VLPs) produced in Sf9 insect cells are an ideal platform for the expression of complex membrane proteins. To assess the efficacy of antigen-displaying VLPs as active cancer vaccines, BALB/c mice were immunized with insect cell glycosylated and mammalian-like glycosylated HER2-displaying VLPs in combination with two different adjuvants and were challenged with HER2-positive tumors. Higher HER2-specific antibody titers and effector functions were induced in mice vaccinated with insect cell glycosylated HER2 VLPs compared to mammalian-like glycosylated counterparts. Moreover, insect cell glycosylated HER2 VLPs elicited a protective effect in mice grafted with HER2-positive mammary carcinoma cells. Interestingly, no protection was observed in mice that were adjuvanted with Poly (I:C). Here, we show that antigen-displaying VLPs produced in Sf9 insect cells were able to induce robust and durable immune responses in vivo and have the potential to be utilized as active cancer vaccines.

Epidemic or pandemic influenza can annually cause significant morbidity and mortality in humans. We developed novel chimeric hemagglutinin (cHA)-based universal influenza virus vaccines, which contain a conserved HA stalk domain from a 2009 pandemic H1N1 (pH1N1) strain combined with globular head domains from avian influenza A viruses. Our previous reports demonstrated that prime-boost sequential immunizations induced robust antibody responses directed toward the conserved HA stalk domain in ferrets. Herein, we further followed vaccinated animals for one year to compare the efficacy and durability of these vaccines in the preclinical ferret model of influenza. Although all cHA-based immunization regimens induced durable HA stalk-specific and heterosubtypic antibody responses in ferrets, sequential immunization with live-attenuated influenza virus vaccines (LAIV-LAIV) conferred the best protection against upper respiratory tract infection by a pH1N1 influenza A virus. The findings from this study suggest that our sequential immunization strategy for a cHA-based universal influenza virus vaccine provides durable protective humoral and cellular immunity against influenza virus infection.

The stalk domain of the hemagglutinin has been identified as a target for induction of protective antibody responses due to its high degree of conservation among numerous influenza subtypes and strains. However, current assays to measure stalk-based immunity are not standardized. Hence, harmonization of assay readouts would help to compare experiments conducted in different laboratories and increase confidence in results. Here, serum samples from healthy individuals (n = 110) were screened using a chimeric cH6/1 hemagglutinin enzyme-linked immunosorbent assay (ELISA) that measures stalk-reactive antibodies. We identified samples with moderate to high IgG anti-stalk antibody levels. Likewise, screening of the samples using the mini-hemagglutinin (HA) headless construct #4900 and analysis of the correlation between the two assays confirmed the presence and specificity of anti-stalk antibodies. Additionally, samples were characterized by a cH6/1N5 virus-based neutralization assay, an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and competition ELISAs, using the stalk-reactive monoclonal antibodies KB2 (mouse) and CR9114 (human). A "pooled serum" (PS) consisting of a mixture of selected serum samples was generated. The PS exhibited high levels of stalk-reactive antibodies, had a cH6/1N5-based neutralization titer of 320, and contained high levels of stalk-specific antibodies with ADCC activity. The PS, along with blinded samples of varying anti-stalk antibody titers, was distributed to multiple collaborators worldwide in a pilot collaborative study. The samples were subjected to different assays available in the different laboratories, to measure either binding or functional properties of the stalk-reactive antibodies contained in the serum. Results from binding and neutralization assays were analyzed to determine whether use of the PS as a standard could lead to better agreement between laboratories. The work presented here points the way towards the development of a serum standard for antibodies to the HA stalk domain of phylogenetic group 1.

The antigenically conserved hemagglutinin stalk region is a target for universal influenza virus vaccines since antibodies against it can provide broad protection against influenza viruses of different subtypes. We tested a universal influenza virus vaccination regimen based on sequential immunization with chimeric hemagglutinin (HA) containing viruses in a swine influenza virus pig model with maternal antibodies against pandemic H1N1. Vaccines were administered as live attenuated virus or inactivated influenza virus split vaccine (+/- Emulsigen adjuvant). As controls, we included groups that received trivalent inactivated influenza vaccine that contained pandemic H1N1 antigens, inactivated adjuvanted H1N2 vaccine (control group for vaccine associated enhanced respiratory disease in the pig model) or mock-vaccination. No induction of H1 head or stalk-specific antibody responses was observed upon vaccination, while responses against H3 and influenza B HA were elicited in the group vaccinated with the trivalent vaccine. Four weeks post vaccination, pigs were intratracheally challenged with pandemic H1N1 virus and euthanized 5 days after challenge. Despite the lack of detectable anti-stalk immunity, the chimeric hemagglutinin vaccine resulted in better clinical outcomes compared to control groups.

A successful severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine must not only be safe and protective, but must also meet the demand on a global scale at a low cost. Using the current influenza virus vaccine production capacity to manufacture an egg-based inactivated Newcastle disease virus (NDV)/SARS-CoV-2 vaccine would meet that challenge. Here, we report pre-clinical evaluations of an inactivated NDV chimera stably expressing the membrane-anchored form of the spike (NDV-S) as a potent coronavirus disease 2019 (COVID-19) vaccine in mice and hamsters. The inactivated NDV-S vaccine was immunogenic, inducing strong binding and/or neutralizing antibodies in both animal models. More importantly, the inactivated NDV-S vaccine protected animals from SARS-CoV-2 infections. In the presence of an adjuvant, antigen-sparing could be achieved, which would further reduce the cost while maintaining the protective efficacy of the vaccine.