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Here, we describe a serological enzyme-linked immunosorbent assay for the screening and identification of human SARS-CoV-2 seroconverters. This assay does not require the handling of infectious virus, can be adjusted to detect different antibody types in serum and plasma and is amenable to scaling. Serological assays are of critical importance to help define previous exposure to SARS-CoV-2 in populations, identify highly reactive human donors for convalescent plasma therapy and investigate correlates of protection. Development of an enzyme-linked immunosorbent assay to detect antibodies to the SARS-CoV-2 spike protein in human sera and plasma.

Seasonal influenza viruses constantly change through antigenic drift and the emergence of pandemic influenza viruses through antigenic shift is unpredictable. Conventional influenza virus vaccines induce strain-specific neutralizing antibodies against the variable immunodominant globular head domain of the viral hemagglutinin protein. This necessitates frequent re-formulation of vaccines and handicaps pandemic preparedness. In this completed, observer-blind, randomized, placebo-controlled phase I trial (NCT03300050), safety and immunogenicity of chimeric hemagglutinin-based vaccines were tested in healthy, 18–39-year-old US adults. The study aimed to test the safety and ability of the vaccines to elicit broadly cross-reactive antibodies against the hemagglutinin stalk domain. Participants were enrolled into five groups to receive vaccinations with live-attenuated followed by AS03-adjuvanted inactivated vaccine ( n  = 20), live-attenuated followed by inactivated vaccine ( n  = 15), twice AS03-adjuvanted inactivated vaccine ( n  = 16) or placebo ( n  = 5, intranasal followed by intramuscular; n  = 10, twice intramuscular) 3 months apart. Vaccination was found to be safe and induced a broad, strong, durable and functional immune response targeting the conserved, immunosubdominant stalk of the hemagglutinin. The results suggest that chimeric hemagglutinins have the potential to be developed as universal vaccines that protect broadly against influenza viruses. New influenza virus vaccines tested in humans elicit broadly cross-reactive antibodies that bind the stalk of the viral hemagglutinin protein and may serve as templates to design a universal influenza vaccine.

There is a need for improved influenza vaccines. In this study we compared the antibody responses in humans after vaccination with an AS03-adjuvanted versus nonadjuvanted H5N1 avian influenza virus inactivated vaccine. Healthy young adults received two doses of either formulation 3 wk apart. We found that AS03 significantly enhanced H5 hemagglutinin (HA)-specific plasmablast and antibody responses compared to the nonadjuvanted vaccine. Plasmablast response after the first immunization was exclusively directed to the conserved HA stem region and came from memory B cells. Monoclonal antibodies (mAbs) derived from these plasmablasts had high levels of somatic hypermutation (SHM) and recognized the HA stem region of multiple influenza virus subtypes. Second immunization induced a plasmablast response to the highly variable HA head region. mAbs derived from these plasmablasts exhibited minimal SHM (naive B cell origin) and largely recognized the HA head region of the immunizing H5N1 strain. Interestingly, the antibody response to H5 HA stem region was much lower after the second immunization, and this suppression was most likely due to blocking of these epitopes by stem-specific antibodies induced by the first immunization. Taken together, these findings show that an adjuvanted influenza vaccine can substantially increase antibody responses in humans by effectively recruiting preexisting memory B cells as well as naive B cells into the response. In addition, we show that high levels of preexisting antibody can have a negative effect on boosting. These findings have implications toward the development of a universal influenza vaccine.

The aim of candidate universal influenza vaccines is to provide broad protection against influenza A and B viruses. Studies have demonstrated that broadly reactive antibodies require Fc–Fc gamma receptor interactions for optimal protection; however, the innate effector cells responsible for mediating this protection remain largely unknown. Here, we examine the roles of alveolar macrophages, natural killer cells, and neutrophils in antibody-mediated protection. We demonstrate that alveolar macrophages play a dominant role in conferring protection provided by both broadly neutralizing and non-neutralizing antibodies in mice. Our data also reveal the potential mechanisms by which alveolar macrophages mediate protection in vivo, namely antibody-induced inflammation and antibody-dependent cellular phagocytosis. This study highlights the importance of innate effector cells in establishing a broad-spectrum antiviral state, as well as providing a better understanding of how multiple arms of the immune system cooperate to achieve an optimal antiviral response following influenza virus infection or immunization. Broadly reactive antibodies that recognize influenza A virus HA can be protective, but the mechanism is not completely understood. Here, He et al. show that the inflammatory response and phagocytosis mediated by the interaction between protective antibodies and macrophages are essential for protection.

Antibodies that bind to both H1 and H3 influenza strains exist in the pre-vaccination serum repertoire of healthy adults; most vaccine-elicited clonotypes bind either H1 or H3 strains. Molecular understanding of serological immunity to influenza has been confounded by the complexity of the polyclonal antibody response in humans. Here we used high-resolution proteomics analysis of immunoglobulin (referred to as Ig-seq) coupled with high-throughput sequencing of transcripts encoding B cell receptors (BCR-seq) to quantitatively determine the antibody repertoire at the individual clonotype level in the sera of young adults before and after vaccination with trivalent seasonal influenza vaccine. The serum repertoire comprised between 40 and 147 clonotypes that were specific to each of the three monovalent components of the trivalent influenza vaccine, with boosted pre-existing clonotypes accounting for ∼60% of the response. An unexpectedly high fraction of serum antibodies recognized both the H1 and H3 monovalent vaccines. Recombinant versions of these H1 + H3 cross-reactive antibodies showed broad binding to hemagglutinins (HAs) from previously circulating virus strains; several of these antibodies, which were prevalent in the serum of multiple donors, recognized the same conserved epitope in the HA head domain. Although the HA-head-specific H1 + H3 antibodies did not show neutralization activity in vitro , they protected mice against infection with the H1N1 and H3N2 virus strains when administered before or after challenge. Collectively, our data reveal unanticipated insights regarding the serological response to influenza vaccination and raise questions about the added benefits of using a quadrivalent vaccine instead of a trivalent vaccine.

A panel of influenza virus-like sequences were recently documented in fish and amphibians. Of these, the Wuhan spiny eel influenza virus (WSEIV) was found to phylogenetically cluster with influenza B viruses as a sister clade. Influenza B viruses have been documented to circulate only in humans, with certain virus isolates found in harbor seals. It is therefore interesting that a similar virus was potentially found in fish. Here we characterize the putative hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins of the WSEIV. Functionally, we show that the WSEIV NA-like protein has sialidase activity comparable to B/Malaysia/2506/2004 influenza B virus NA, making it a bona fide neuraminidase that is sensitive to NA inhibitors. We tested the functionality of the HA by addressing the receptor specificity, stability, preferential airway protease cleavage, and fusogenicity. We show highly specific binding to monosialic ganglioside 2 (GM2) and fusogenicity at a range of different pH conditions. In addition, we found limited antigenic conservation of the WSEIV HA and NA relative to the B/Malaysia/2506/2004 virus HA and NA. In summary, we perform a functional and antigenic characterization of the glycoproteins of WSEIV to assess if it is indeed a bona fide influenza virus potentially circulating in ray-finned fish. The recently identified Wuhan spiny eel influenza virus (WSEIV) sequence is more closely related to influenza B than A viruses. Here, the authors functionally characterize the putative surface glycoproteins of WSEIV and show that its NA-like protein has sialidase activity and its HA-like protein binds monosialic ganglioside 2.

Because of the broad neutralization and in vivo protection across influenza A and influenza B virus strains, monoclonal antibody CR9114 is widely used in influenza virus research as a positive control in many experiments. To produce amounts sufficient for the demand requires regular transient transfections, resulting in varying yield as well as differing batch to batch quality. Here, we report the development of a serum-free CHO DG44 cell line, stably producing a CR9114-like antibody with a potential to become a useful influenza virus research tool.

A substantial proportion of influenza-related childhood deaths are due to infection with influenza B viruses, which co-circulate in the human population as two antigenically distinct lineages defined by the immunodominant receptor binding protein, haemagglutinin. While broadly cross-reactive, protective monoclonal antibodies against the haemagglutinin of influenza B viruses have been described, none targeting the neuraminidase, the second most abundant viral glycoprotein, have been reported. Here, we analyse a panel of five murine anti-neuraminidase monoclonal antibodies that demonstrate broad binding, neuraminidase inhibition, in vitro antibody-dependent cell-mediated cytotoxicity and in vivo protection against influenza B viruses belonging to both haemagglutinin lineages and spanning over 70 years of antigenic drift. Electron microscopic analysis of two neuraminidase–antibody complexes shows that the conserved neuraminidase epitopes are located on the head of the molecule and that they are distinct from the enzymatic active site. In the mouse model, one therapeutic dose of antibody 1F2 was more protective than the current standard of treatment, oseltamivir, given twice daily for six days. This study reports the identification of broadly protective antibodies targeting the influenza B neuraminidase away from its active site. One dose of antibody therapy was more protective in mice than multiple doses of the current standard of care.

Fc gamma receptor (FcyR)-mediated antibody functions play a crucial role in preventing HIV infection. One such function, antibody-dependent phagocytosis (ADP), is thought to be involved in controlling other viral infections, but its role in HIV infection is unknown. We measured the ability of HIV-specific polyclonal and monoclonal antibodies (mAbs) to mediate the internalization of HIV-1 virions and HIV-1-decorated cells by phagocytes. To measure ADP of virions, we primarily used a green-fluorescent protein-expressing molecular clone of HIV-1JRFL, an R5, clinical isolate, in combination with polyclonal HIVIG or mAbs known to capture and/or neutralize HIV-1. THP-1 and U937 cells, as well as freshly isolated primary monocytes from healthy individuals, were used as phagocytic effector cells, and uptake of virions was measured by cytometry. We surprisingly found minimal or no ADP of virions with any of the antibodies. However, after coating virions with gp41 or with gp41-derived peptides, gp41- (but not gp120-) specific mAbs efficiently mediated phagocytosis. We estimated that a minimum of a few hundred gp41 molecules were needed for successful phagocytosis, which is similar to the number of envelope spikes on viruses that are readily phagocytosed (e.g. influenza virus). Furthermore, by employing fluorescence correlation spectroscopy, a well-established technique to measure particle sizes and aggregation phenomena, we found a clear association between virus aggregation and ADP. In contrast to virions themselves, virion-decorated cells were targets for ADP or trogocytosis in the presence of HIV-specific antibodies. Our findings indicate that ADP of virions may not play a role in preventing HIV infection, likely due to the paucity of trimers and the consequent inability of virion-bound antibody to cross-link FcyRs on phagocytes. However, ADP or trogocytosis could play a role in clearing HIV-infected cells and cells on the verge of infection.

The global health burden of influenza B viruses, especially in children, has long been underappreciated. Although two antigenically distinct influenza B virus lineages cocirculated before the coronavirus disease 2019 (COVID-19) pandemic, the commonly used trivalent seasonal vaccines contain antigens from only one influenza B virus, providing limited cross-protection against viruses of the other lineage. Current influenza virus vaccines and antivirals have limitations, some of which disproportionately affect their utilization against influenza B viruses. To inform ongoing efforts to address the considerable global burden of influenza B viruses, we previously described five murine monoclonal antibodies that broadly bind conserved epitopes on the neuraminidase of influenza B viruses and protect against lethal challenge in a mouse model when delivered via intraperitoneal injection. Here, we validate the continued relevance of these antibodies by demonstrating that their protective effects extend to lethal challenge with mouse-adapted influenza B viruses recently isolated from humans. We also found that humanization of murine antibodies 1F2 and 4F11 resulted in molecules that retain the ability to protect mice from lethal challenge when administered prophylactically. Intranasal administration as an alternative route of 1F2 delivery revealed no differences in the mouse challenge model compared to intraperitoneal injection, supporting further assessment of this more targeted and convenient administration method. Lastly, we evaluated the potential for intranasal 1F2 administration initiated 1 day after infection to prevent transmission of an influenza B virus between cocaged guinea pigs. Here, we observed a 40% rate of transmission with the 1F2 antibody administered to the infected donor compared to 100% transmission with administration of an irrelevant control antibody. These data suggest that intranasal administration could be a viable route of administration for antibody therapeutics. Collectively, these findings demonstrate the potential of broad antineuraminidase antibodies as therapeutics to prevent and treat infections caused by influenza B viruses. IMPORTANCE The global health burden of influenza B viruses, especially in children, has long been underappreciated. Although two antigenically distinct influenza B virus lineages cocirculated before the coronavirus disease 2019 (COVID-19) pandemic, the commonly used trivalent seasonal vaccines contain antigens from only one influenza B virus, providing limited cross-protection against viruses of the other lineage. Additionally, studies have called into question the clinical effectiveness of the neuraminidase inhibitors that comprise the majority of available antivirals in treating influenza B virus infections. We previously described antibodies that bind broadly to neuraminidases of influenza B viruses across decades of antigenic evolution and potently protect mice against lethal challenge. Here we appraise additional factors to develop these antineuraminidase antibodies as antivirals to prevent and treat infections caused by an extensive range of influenza B viruses. In addition this work assesses recent clinical isolates belonging to the two influenza B virus lineages, finding evidence supporting the development of these antibodies for prophylactic and therapeutic use.