BackgroundIn the face of impending influenza pandemic, a rapid vaccine production and mass vaccination is the most effective approach to prevent the large scale mortality and morbidity that was associated with the 1918 “Spanish Flu”. The traditional process of influenza vaccine production in eggs is time consuming and may not meet the demands of rapid global vaccination required to curtail influenza pandemic.Methodology/Principal FindingsRecombinant technology can be used to express the hemagglutinin (HA) of the emerging new influenza strain in a variety of systems including mammalian, insect, and bacterial cells. In this study, two forms of HA proteins derived from the currently circulating novel H1N1 A/California/07/2009 virus, HA1 (1–330) and HA (1–480), were expressed and purified from E. coli under controlled redox refolding conditions that favoured proper protein folding. However, only the recombinant HA1 (1–330) protein formed oligomers, including functional trimers that bound receptor and caused agglutination of human red blood cells. These proteins were used to vaccinate ferrets prior to challenge with the A/California/07/2009 virus. Both proteins induced neutralizing antibodies, and reduced viral loads in nasal washes. However, the HA1 (1–330) protein that had higher content of multimeric forms provided better protection from fever and weight loss at a lower vaccine dose compared with HA (1–480). Protein yield for the HA1 (1–330) ranged around 40 mg/Liter, while the HA (1–480) yield was 0.4–0.8 mg/Liter.Conclusions/SignificanceThis is the first study that describes production in bacterial system of properly folded functional globular HA1 domain trimers, lacking the HA2 transmembrane protein, that elicit potent neutralizing antibody responses following vaccination and protect ferrets from in vivo challenge. The combination of bacterial expression system with established quality control methods could provide a mechanism for rapid large scale production of influenza vaccines in the face of influenza pandemic threat.

The impending influenza virus pandemic requires global vaccination to prevent large-scale mortality and morbidity, but traditional influenza virus vaccine production is too slow for rapid responses.

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Acute viral infections can have durable functional impacts on the immune system long after recovery, but how they affect homeostatic immune states and responses to future perturbations remain poorly understood [1][2][3][4] . Here we use systems immunology approaches, including longitudinal multimodal single cell analysis (surface proteins, transcriptome, and V(D)J sequences), to comparatively assess baseline immune statuses and responses to influenza vaccination in 33 healthy individuals after recovery from mild, non-hospitalized COVID-19 (mean: 151 days after diagnosis) and 40 age-and sex-matched controls who never had COVID-19. At baseline and independent of time since COVID-19, recoverees had elevated T-cell activation signatures and lower expression of innate immune genes in monocytes.COVID-19-recovered males had coordinately higher innate, influenza-specific plasmablast, and antibody responses after vaccination compared to healthy male and COVID-19-recovered females, partly because male recoverees had monocytes with higher IL-15 responses early after vaccination coupled with elevated pre-vaccination frequencies of "virtual memory" like CD8+ T-cells poised to produce more IFNγ upon IL-15 stimulation. In addition, the expression of the repressed innate immune genes in monocytes increased by day 1 through day 28 post-vaccination in recoverees, thus moving towards the pre-vaccination baseline of healthy controls. In contrast, these genes decreased on day 1 and returned to the baseline by day 28 in controls. Our study reveals sex-dimorphic impacts of prior mild COVID-19 and suggests that viral infections in humans can establish new set-points impacting future immune responses in an antigen-agnostic manner.

The large, uniformly performed clinical investigations with influenza A/New Jersey vaccines provided an opportunity to correlate results of laboratory tests of vaccine with human reactivity and antibody responses. These vaccines were given to large numbers of subjects under code, and significant differences in immunogenicity and reactivity were observed in unprimed individuals. A single, relatively large dose of intact virus was more immunogenic and reactive than split-virus vaccines in unprimed subjects. Differences in immunogenicity and reactivity in unprimed subjects correlated with the amount of intact virus in the vaccines (measured by column chromatography or electron microscopy) and with the amount of viral hemagglutinin in the vaccine (measured by immunodiffusion), but not with the number of chick cell-agglutinating units.