Epstein-Barr virus (EBV) represents a major global health problem. Though it is associated with infectious mononucleosis and ∼200,000 cancers annually worldwide, a vaccine is not available. The major ...target of immunity is EBV glycoprotein 350/220 (gp350) that mediates attachment to B cells through complement receptor 2 (CR2/CD21). Here, we created self-assembling nanoparticles that displayed different domains of gp350 in a symmetric array. By focusing presentation of the CR2-binding domain on nanoparticles, potent neutralizing antibodies were elicited in mice and non-human primates. The structurally designed nanoparticle vaccine increased neutralization 10- to 100-fold compared to soluble gp350 by targeting a functionally conserved site of vulnerability, improving vaccine-induced protection in a mouse model. This rational approach to EBV vaccine design elicited potent neutralizing antibody responses by arrayed presentation of a conserved viral entry domain, a strategy that can be applied to other viruses.
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•Self-assembling nanoparticles present the conserved gp350 receptor-binding domain•The nanoparticles elicit more potent neutralizing antibodies than soluble gp350•These neutralizing antibodies predominantly target the CR2-binding site on gp350•The nanoparticles elicit potent neutralizing antibodies in mice and non-human primates
Structurally designed EBV vaccine candidates based on self-assembling nanoparticles elicit potent and durable virus-neutralizing antibodies that target the receptor-binding site on the viral envelope protein gp350, a site of vulnerability, serving as a template to develop an EBV vaccine and providing a basis for immunofocusing through rational vaccine design.
The present vaccine against influenza virus has the inevitable risk of antigenic discordance between the vaccine and the circulating strains, which diminishes vaccine efficacy. This necessitates new ...approaches that provide broader protection against influenza. Here we designed a vaccine using the hypervariable receptor-binding domain (RBD) of viral hemagglutinin displayed on a nanoparticle (np) able to elicit antibody responses that neutralize H1N1 influenza viruses spanning over 90 years. Co-display of RBDs from multiple strains across time, so that the adjacent RBDs are heterotypic, provides an avidity advantage to cross-reactive B cells. Immunization with the mosaic RBD-np elicited broader antibody responses than those induced by an admixture of nanoparticles encompassing the same set of RBDs as separate homotypic arrays. Furthermore, we identified a broadly neutralizing monoclonal antibody in a mouse immunized with mosaic RBD-np. The mosaic antigen array signifies a unique approach that subverts monotypic immunodominance and allows otherwise subdominant cross-reactive B cell responses to emerge.
Antibodies capable of neutralizing divergent influenza A viruses could form the basis of a universal vaccine. Here, from subjects enrolled in an H5N1 DNA/MIV-prime-boost influenza vaccine trial, we ...sorted hemagglutinin cross-reactive memory B cells and identified three antibody classes, each capable of neutralizing diverse subtypes of group 1 and group 2 influenza A viruses. Co-crystal structures with hemagglutinin revealed that each class utilized characteristic germline genes and convergent sequence motifs to recognize overlapping epitopes in the hemagglutinin stem. All six analyzed subjects had sequences from at least one multidonor class, and—in half the subjects—multidonor-class sequences were recovered from >40% of cross-reactive B cells. By contrast, these multidonor-class sequences were rare in published antibody datasets. Vaccination with a divergent hemagglutinin can thus increase the frequency of B cells encoding broad influenza A-neutralizing antibodies. We propose the sequence signature-quantified prevalence of these B cells as a metric to guide universal influenza A immunization strategies.
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•Isolation of group 1 and group 2 influenza A-neutralizing antibodies from H5N1 vaccinees•Discovery of three classes of broadly neutralizing antibodies directed to the HA stem•Delineation of sequence signatures specific for broadly neutralizing antibodies•Antibody quantification by NGS to guide the development of a universal vaccine
Quantifying B cells capable of producing broadly neutralizing antibodies against influenza serves as a metric to guide the development of a universal influenza vaccine.
Influenza A virus (IAV) employs diverse strategies to circumvent type I interferon (IFN) responses, particularly by inhibiting the synthesis of type I IFNs. However, it is poorly understood if and ...how IAV regulates the type I IFN receptor (IFNAR)-mediated signaling mode. In this study, we demonstrate that IAV induces the degradation of IFNAR subunit 1 (IFNAR1) to attenuate the type I IFN-induced antiviral signaling pathway. Following infection, the level of IFNAR1 protein, but not mRNA, decreased. Indeed, IFNAR1 was phosphorylated and ubiquitinated by IAV infection, which resulted in IFNAR1 elimination. The transiently overexpressed IFNAR1 displayed antiviral activity by inhibiting virus replication. Importantly, the hemagglutinin (HA) protein of IAV was proved to trigger the ubiquitination of IFNAR1, diminishing the levels of IFNAR1. Further, influenza A viral HA1 subunit, but not HA2 subunit, downregulated IFNAR1. However, viral HA-mediated degradation of IFNAR1 was not caused by the endoplasmic reticulum (ER) stress response. IAV HA robustly reduced cellular sensitivity to type I IFNs, suppressing the activation of STAT1/STAT2 and induction of IFN-stimulated antiviral proteins. Taken together, our findings suggest that IAV HA causes IFNAR1 degradation, which in turn helps the virus escape the powerful innate immune system. Thus, the research elucidated an influenza viral mechanism for eluding the IFNAR signaling pathway, which could provide new insights into the interplay between influenza virus and host innate immunity.
Influenza A virus (IAV) infection causes significant morbidity and mortality worldwide and remains a major health concern. When triggered by influenza viral infection, host cells produce type I interferon (IFN) to block viral replication. Although IAV was shown to have diverse strategies to evade this powerful, IFN-mediated antiviral response, it is not well-defined if IAV manipulates the IFN receptor-mediated signaling pathway. Here, we uncovered that influenza viral hemagglutinin (HA) protein causes the degradation of type I IFN receptor subunit 1 (IFNAR1). HA promoted phosphorylation and polyubiquitination of IFNAR1, which facilitated the degradation of this receptor. The HA-mediated elimination of IFNAR1 notably decreased the cells' sensitivities to type I IFNs, as demonstrated by the diminished expression of IFN-induced antiviral genes. This discovery could help us understand how IAV regulates the host innate immune response to create an environment optimized for viral survival in host cells.
Gene based delivery of immunoglobulins promises to safely and durably provide protective immunity to individuals at risk of acquiring infectious diseases such as HIV. We used a rhesus macaque animal ...model to optimize delivery of naturally-arising, autologous anti-SIV neutralizing antibodies expressed by Adeno-Associated Virus 8 (AAV8) vectors. Vectored transgene expression was confirmed by quantitation of target antibody abundance in serum and mucosal surfaces. We tested the expression achieved at varying doses and numbers of injections. Expression of the transgene reached a saturation at about 2 x 10(12) AAV8 genome copies (gc) per needle-injection, a physical limitation that may not scale clinically into human trials. In contrast, expression increased proportionately with the number of injections. In terms of anti-drug immunity, anti-vector antibody responses were universally strong, while those directed against the natural transgene mAb were detected in only 20% of animals. An anti-transgene antibody response was invariably associated with loss of detectable plasma expression of the antibody. Despite having atypical glycosylation profiles, transgenes derived from AAV-directed muscle cell expression retained full functional activity, including mucosal accumulation, in vitro neutralization, and protection against repeated limiting dose SIVsmE660 swarm challenge. Our findings demonstrate feasibility of a gene therapy-based passive immunization strategy against infectious disease, and illustrate the potential for the nonhuman primate model to inform clinical AAV-based approaches to passive immunization.
Vaccine-induced memory B cell responses to evolving viruses like influenza A involve activation of pre-existing immunity and generation of new responses. To define the contribution of these two types ...of responses, we analyzed the response to H7N9 vaccination in H7N9-naive adults. We performed comprehensive comparisons at the single-cell level of the kinetics, Ig repertoire, and activation phenotype of established pre-existing memory B cells recognizing conserved epitopes and the newly generated memory B cells directed toward H7 strain-specific epitopes. The recall response to conserved epitopes on H7 HA involved a transient expansion of memory B cells with little observed adaptation. However, the B cell response to newly encountered epitopes was phenotypically distinct and generated a sustained memory population that evolved and affinity matured months after vaccination. These findings establish clear differences between newly generated and pre-existing memory B cells, highlighting the challenges in achieving long-lasting, broad protection against an ever-evolving virus.
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•Newly generated memory B cells evolve and affinity mature over several months•Long-term pre-existing memory B cells evolve little upon re-vaccination•Newly generated memory B cells transiently become atypical, T-bethi CD21lo CD27−•T-betlo CD21hi CD27−, but not CD27+, resting memory B cells are maintained long-term
Influenza vaccination occurs in the context of pre-existing immunity. Andrews et al. compare the pre-existing memory IgG B cell response recognizing conserved epitopes on influenza hemagglutinin with the newly generated response to strain-specific epitopes upon H7N9 vaccination. The differences in magnitude, phenotype, and affinity maturation between the two responses highlight the challenges in achieving long-lasting, broad protection against an ever-evolving virus.
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.
Immunodominance (ID) defines the hierarchical immune response to competing antigens in complex immunogens. Little is known regarding B cell and antibody ID despite its importance in immunity to ...viruses and other pathogens. We show that B cells and serum antibodies from inbred mice demonstrate a reproducible ID hierarchy to the five major antigenic sites in the influenza A virus hemagglutinin globular domain. The hierarchy changed as the immune response progressed, and it was dependent on antigen formulation and delivery. Passive antibody transfer and sequential infection experiments demonstrated 'original antigenic suppression', a phenomenon in which antibodies suppress memory responses to the priming antigenic site. Our study provides a template for attaining deeper understanding of antibody ID to viruses and other complex immunogens.
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