Protective human monoclonal antibodies target conserved sites of vulnerability on the underside of influenza virus neuraminidase.

Continuously evolving influenza viruses cause seasonal epidemics and pose global pandemic threats. Although viral neuraminidase (NA) is an effective drug and vaccine target, our understanding of the NA antigenic landscape still remains incomplete. Here, we describe NA-specific human antibodies that target the underside of the NA globular head domain, inhibit viral propagation of a wide range of human H3N2, swine-origin variant H3N2, and H2N2 viruses, and confer both pre- and post-exposure protection against lethal H3N2 infection in mice. Cryo-EM structures of two such antibodies in complex with NA reveal non-overlapping epitopes covering the underside of the NA head. These sites are highly conserved among N2 NAs yet inaccessible unless the NA head tilts or dissociates. Our findings help guide the development of effective countermeasures against ever-changing influenza viruses by identifying hidden conserved sites of vulnerability on the NA underside.

Progress in vaccine development for infectious diseases-a Keystone Symposia report.

The COVID-19 pandemic has taught us many things, among the most important of which is that vaccines are one of the cornerstones of public health that help make modern longevity possible. While several different vaccines have been successful at stemming the morbidity and mortality associated with various infectious diseases, many pathogens/diseases remain recalcitrant to the development of effective vaccination. Recent advances in vaccine technology, immunology, structural biology, and other fields may yet yield insight that will address these diseases; they may also help improve societies' preparedness for future pandemics. On June 1-4, 2022, experts in vaccinology from academia, industry, and government convened for the Keystone symposium "Progress in Vaccine Development for Infectious Diseases" to discuss state-of-the-art technologies, recent advancements in understanding vaccine-mediated immunity, and new aspects of antigen design to aid vaccine effectiveness.

Breathing and tilting: mesoscale simulations illuminate influenza glycoprotein vulnerabilities.

Influenza virus has resurfaced recently from inactivity during the early stages of the COVID-19 pandemic, raising serious concerns about the nature and magnitude of future epidemics. The main antigenic targets of influenza virus are two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Whereas the structural and dynamical properties of both glycoproteins have been studied previously, the understanding of their plasticity in the whole-virion context is fragmented. Here, we investigate the dynamics of influenza glycoproteins in a crowded protein environment through mesoscale all-atom molecular dynamics simulations of two evolutionary-linked glycosylated influenza A whole-virion models. Our simulations reveal and kinetically characterize three main molecular motions of influenza glycoproteins: NA head tilting, HA ectodomain tilting, and HA head breathing. The flexibility of HA and NA highlights antigenically relevant conformational states, as well as facilitates the characterization of a novel monoclonal antibody, derived from human convalescent plasma, that binds to the underside of the NA head. Our work provides previously unappreciated views on the dynamics of HA and NA, advancing the understanding of their interplay and suggesting possible strategies for the design of future vaccines and antivirals against influenza.

Breathing and Tilting: Mesoscale Simulations Illuminate Influenza Glycoprotein Vulnerabilities.

Influenza virus has resurfaced recently from inactivity during the early stages of the COVID-19 pandemic, raising serious concerns about the nature and magnitude of future epidemics. The main antigenic targets of influenza virus are two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Whereas the structural and dynamical properties of both glycoproteins have been studied previously, the understanding of their plasticity in the whole-virion context is fragmented. Here, we investigate the dynamics of influenza glycoproteins in a crowded protein environment through mesoscale all-atom molecular dynamics simulations of two evolutionary-linked glycosylated influenza A whole-virion models. Our simulations reveal and kinetically characterize three main molecular motions of influenza glycoproteins: NA head tilting, HA ectodomain tilting, and HA head breathing. The flexibility of HA and NA highlights antigenically relevant conformational states, as well as facilitates the characterization of a novel monoclonal antibody, derived from convalescent human donor, that binds to the underside of the NA head. Our work provides previously unappreciated views on the dynamics of HA and NA, advancing the understanding of their interplay and suggesting possible strategies for the design of future vaccines and antivirals against influenza.

Structure-based design of stabilized recombinant influenza neuraminidase tetramers.

Influenza virus neuraminidase (NA) is a major antiviral drug target and has recently reemerged as a key target of antibody-mediated protective immunity. Here we show that recombinant NAs across non-bat subtypes adopt various tetrameric conformations, including an "open" state that may help explain poorly understood variations in NA stability across viral strains and subtypes. We use homology-directed protein design to uncover the structural principles underlying these distinct tetrameric conformations and stabilize multiple recombinant NAs in the "closed" state, yielding two near-atomic resolution structures of NA by cryo-EM. In addition to enhancing thermal stability, conformational stabilization improves affinity to protective antibodies elicited by viral infection, including antibodies targeting a quaternary epitope and the broadly conserved catalytic site. Stabilized NAs can also be integrated into viruses without affecting fitness. Our findings provide a deeper understanding of NA structure, stability, and antigenicity, and establish design strategies for reinforcing the conformational integrity of recombinant NA proteins.

A comprehensive influenza reporter virus panel for high-throughput deep profiling of neutralizing antibodies.

Broadly neutralizing antibodies (bnAbs) have been developed as potential countermeasures for seasonal and pandemic influenza. Deep characterization of these bnAbs and polyclonal sera provides pivotal understanding for influenza immunity and informs effective vaccine design. However, conventional virus neutralization assays require high-containment laboratories and are difficult to standardize and roboticize. Here, we build a panel of engineered influenza viruses carrying a reporter gene to replace an essential viral gene, and develop an assay using the panel for in-depth profiling of neutralizing antibodies. Replication of these viruses is restricted to cells expressing the missing viral gene, allowing it to be manipulated in a biosafety level 2 environment. We generate the neutralization profile of 24 bnAbs using a 55-virus panel encompassing the near-complete diversity of human H1N1 and H3N2, as well as pandemic subtype viruses. Our system offers in-depth profiling of influenza immunity, including the antibodies against the hemagglutinin stem, a major target of universal influenza vaccines.

Glycan repositioning of influenza hemagglutinin stem facilitates the elicitation of protective cross-group antibody responses.

The conserved hemagglutinin (HA) stem has been a focus of universal influenza vaccine efforts. Influenza A group 1 HA stem-nanoparticles have been demonstrated to confer heterosubtypic protection in animals; however, the protection does not extend to group 2 viruses, due in part to differences in glycosylation between group 1 and 2 stems. Here, we show that introducing the group 2 glycan at Asn38HA1 to a group 1 stem-nanoparticle (gN38 variant) based on A/New Caledonia/20/99 (H1N1) broadens antibody responses to cross-react with group 2 HAs. Immunoglobulins elicited by the gN38 variant provide complete protection against group 2 H7N9 virus infection, while the variant loses protection against a group 1 H5N1 virus. The N38HA1 glycan thus is pivotal in directing antibody responses by controlling access to group-determining stem epitopes. Precise targeting of stem-directed antibody responses to the site of vulnerability by glycan repositioning may be a step towards achieving cross-group influenza protection.

Immunogenicity and protective capacity of a virosomal respiratory syncytial virus vaccine adjuvanted with monophosphoryl lipid A in mice.

Respiratory Syncytial Virus (RSV) is a major cause of viral brochiolitis in infants and young children and is also a significant problem in elderly and immuno-compromised adults. To date there is no efficacious and safe RSV vaccine, partially because of the outcome of a clinical trial in the 1960s with a formalin-inactivated RSV vaccine (FI-RSV). This vaccine caused enhanced respiratory disease upon exposure to the live virus, leading to increased morbidity and the death of two children. Subsequent analyses of this incident showed that FI-RSV induces a Th2-skewed immune response together with poorly neutralizing antibodies. As a new approach, we used reconstituted RSV viral envelopes, i.e. virosomes, with incorporated monophosphoryl lipid A (MPLA) adjuvant to enhance immunogenicity and to skew the immune response towards a Th1 phenotype. Incorporation of MPLA stimulated the overall immunogenicity of the virosomes compared to non-adjuvanted virosomes in mice. Intramuscular administration of the vaccine led to the induction of RSV-specific IgG2a levels similar to those induced by inoculation of the animals with live RSV. These antibodies were able to neutralize RSV in vitro. Furthermore, MPLA-adjuvanted RSV virosomes induced high amounts of IFNγ and low amounts of IL5 in both spleens and lungs of immunized and subsequently challenged animals, compared to levels of these cytokines in animals vaccinated with FI-RSV, indicating a Th1-skewed response. Mice vaccinated with RSV-MPLA virosomes were protected from live RSV challenge, clearing the inoculated virus without showing signs of lung pathology. Taken together, these data demonstrate that RSV-MPLA virosomes represent a safe and efficacious vaccine candidate which warrants further evaluation.