The hemagglutinin (HA) surface protein is the primary immune target for most influenza vaccines. The neuraminidase (NA) surface protein is often a secondary target for vaccine designs. In this study, computationally optimized broadly reactive antigen methodology was used to generate the N1-I NA vaccine antigen that was designed to cross-react with avian, swine, and human influenza viruses of N1 NA subtype. The elicited antibodies bound to NA proteins derived from A/California/07/2009 (H1N1)pdm09, A/Brisbane/59/2007 (H1N1), A/Swine/North Carolina/154074/2015 (H1N1) and A/Viet Nam/1203/2004 (H5N1) influenza viruses, with NA-neutralizing activity against a broad panel of HXN1 influenza strains. Mice vaccinated with the N1-I COBRA NA vaccine were protected from mortality and viral lung titers were lower when challenged with four different viral challenges: A/California/07/2009, A/Brisbane/59/2007, A/Swine/North Carolina/154074/2015 and A/Viet Nam/1203/2004. Vaccinated mice had little to no weight loss against both homologous, but also cross-NA genetic clade challenges. Lung viral titers were lower compared to the mock vaccinated mice, and at times, equivalent to the homologous control. Thus, the N1-I COBRA NA antigen has the potential to be a complimentary component in a multi-antigen universal influenza virus vaccine formulation that also contains HA antigens. Importance The development and distribution of a universal influenza vaccines would alleviate global economic and public health stress from annual influenza virus outbreaks. The influenza virus NA vaccine antigen allows for protection from multiple HA subtypes and virus host origins, but it has not been the focus of vaccine development. The N1-I NA antigen described here protected mice from direct challenge of four distinct influenza viruses and inhibited the enzymatic activity of a N1 influenza virus panel. The use of the NA antigen in combination with the HA widens the breadth of protection against various virus strains. Therefore, this research opens the door to the development of a longer lasting vaccine with increased protective breadth.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and seasonal influenza viruses are co-circulating in the human population. However, only a few cases of viral co-infection with these two viruses have been documented in humans with some people having severe disease and others mild disease. In order to examine this phenomenon, ferrets were co-infected with SARS-CoV-2 and human seasonal influenza A viruses (IAVs) (H1N1 or H3N2) and were compared to animals that received each virus alone. Ferrets were either immunologically naïve to both viruses or vaccinated with the 2019-2020 split-inactivated influenza virus vaccine. Co-infected naive ferrets lost significantly more body weight than ferrets infected with each virus alone and induced more severe inflammation in both the nose and lungs than ferrets single-infected with each virus. Co-infected naïve animals had predominantly higher IAV titers than SARS-CoV-2 titers, and IAVs efficiently transmitted to the co-housed ferrets by direct contact. Comparatively, SARS-CoV-2 failed to transmit to the ferrets that co-housed with co-infected ferrets by direct contact. Moreover, vaccination significantly reduced IAVs virus titers and shortened the viral shedding, but did not completely block influenza virus direct contact transmission. Notably, vaccination significantly ameliorated the influenza associated disease by protecting vaccinated animals from severe morbidity after IAV single infection or IAV and SARS-CoV-2 co-infection, suggesting that seasonal influenza virus vaccination is pivotal to prevent severe disease induced by IAVs and SARS-CoV-2 co-infection during the COVID-19 pandemic. Importance Influenza A viruses cause severe morbidity and mortality during each influenza virus season. The emergence of SARS-CoV-2 infection in the human population offers the opportunity to potential co-infections of both viruses. The development of useful animal models to asses pathogenesis, transmission, and viral evolution of these viruses as the co-infect a host is of critical importance for the development of vaccines and therapeutics. The ability to prevent the most severe effects of viral co-infections can be studied using effect co-infection ferret models described in this report.
Influenza viruses infect millions of people each year, resulting in significant morbidity and mortality in the human population. Therefore, generation of a universal influenza virus vaccine is an urgent need and would greatly benefit public health. Recombinant protein technology is an established vaccine platform and has resulted in several commercially available vaccines. Herein, we describe the approach for developing stable transfected human cell lines for the expression of recombinant influenza virus hemagglutinin (HA) and recombinant influenza virus neuraminidase (NA) proteins for the purpose of in vitro and in vivo vaccine development. HA and NA are the main surface glycoproteins on influenza virions and the major antibody targets. The benefits for using recombinant proteins for in vitro and in vivo assays include the ease of use, high level of purity and the ability to scale-up production. This work provides guidelines on how to produce and purify recombinant proteins produced in mammalian cell lines through either transient transfection or generation of stable cell lines from plasmid creation through the isolation step via Immobilized Metal Affinity Chromatography (IMAC). Collectively, the establishment of this pipeline has facilitated large-scale production of recombinant HA and NA proteins to high purity and with consistent yields, including glycosylation patterns that are very similar to proteins produced in a human host.
Influenza viruses have caused numerous pandemics throughout human history. The 1957 influenza pandemic was initiated by an H2N2 influenza virus. This H2N2 influenza virus was the result of a reassortment event between a circulating H2N2 avian virus and the seasonal H1N1 viruses in humans. Previously, our group has demonstrated the effectiveness of HA antigens derived using computationally optimized broadly reactive antigen (COBRA) methodology against H1N1, H3N2, and H5N1 viruses. Using the COBRA methodology, H2 HA COBRA antigens were designed using sequences from H2N2 viruses isolated from humans in the 1950s and 1960s, as well as, H2Nx viruses isolated from avian and mammalian species between the 1950s and 2016. In this manuscript, the effectiveness of H2 COBRA HA antigens (Z1, Z3, Z5, and Z7) were evaluated in DBA/2J mice and compared to wild-type H2 HA antigens. The COBRA HA vaccines elicited neutralizing antibodies to the majority of viruses in our H2 HA panel and across all three clades as measured by HAI and neutralization assays. Comparatively, several wild-type HA vaccines elicited antibodies against a majority of the viruses in the H2 HA panel. DBA/2J mice vaccinated with COBRA vaccines showed increase survival for all three viral challenges as compared to the wild-type H2 vaccines. In particular, the Z1 COBRA is a promising candidate for future work towards a pandemic H2 influenza vaccine. IMPORTANCE H2N2 influenza has caused at least one pandemic in the past. Given that individuals born after 1968 have not been exposed to H2N2 influenza viruses, a future pandemic caused by H2 influenza is likely. An effective H2 influenza vaccine would need to elicit broadly cross-reactive antibodies to multiple H2 influenza viruses. Choosing a wild-type virus to create a vaccine may elicit a narrow immune response and not protect against multiple H2 influenza viruses. COBRA H2 HA vaccines were developed and evaluated in mice along with wild-type H2 HA vaccines. Multiple COBRA H2 HA vaccines protected mice from all three viral challenges and produced broadly cross-reactive neutralizing antibodies to H2 influenza viruses.
Infection and inflammation of the middle ears that characterizes acute and chronic o titis m edia (OM), is a major reason for doctor visits and antibiotic prescription, particularly among children. Nasopharyngeal pathogens that are commonly associated with OM in humans do not naturally colonize the middle ears of rodents, and experimental models in most cases involve directly injecting large numbers of human pathogens into the middle ear bullae of rodents, where they induce a short-lived acute inflammation but fail to persist. Here we report that Bordetella pseudohinzii , a respiratory pathogen of mice, naturally, efficiently and rapidly ascends the eustachian tubes to colonize the middle ears, causing acute and chronic histopathological changes with progressive decrease in hearing acuity that closely mimics otitis media in humans. Laboratory mice experimentally inoculated intranasally with very low numbers of bacteria consistently have their middle ears colonized and subsequently transmit the bacterium to cage mates. Taking advantage of the specifically engineered and well characterized immune deficiencies available in mice we conducted experiments to uncover different roles of T and B cells in controlling bacterial numbers in the middle ear during chronic OM. The iconic mouse model provides significant advantages for elucidating aspects of host-pathogen interactions in otitis media that are currently not possible using other animal models. This natural model of otitis media permits the study of transmission between hosts, efficient early colonization of the respiratory tract, ascension of the eustachian tube, as well as colonization, pathogenesis and persistence in the middle ear. It also allows the combination of the powerful tools of mouse molecular immunology and bacterial genetics to determine the mechanistic basis for these important processes.
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