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The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coupled with a lack of therapeutics, has paralyzed the globe. Although significant effort has been invested in identifying antibodies that block infection, the ability of antibodies to target infected cells through Fc interactions may be vital to eliminate the virus. To explore the role of Fc activity in SARS-CoV-2 immunity, the functional potential of a cross-SARS-reactive antibody, CR3022, was assessed. CR3022 was able to broadly drive antibody effector functions, providing critical immune clearance at entry and upon egress. Using selectively engineered Fc variants, no protection was observed after administration of WT IgG1 in mice or hamsters. Conversely, the functionally enhanced Fc variant resulted in increased pathology in both the mouse and hamster models, causing weight loss in mice and enhanced viral replication and weight loss in the more susceptible hamster model, highlighting the pathological functions of Fc-enhancing mutations. These data point to the critical need for strategic Fc engineering for the treatment of SARS-CoV-2 infection.

► We tested a MHC I/peptide single chain DNA vaccine against Listeria infection in mice. ► This approach was designed to optimize antigen presentation to CD8+ T cells. ► Kd/LLO single chain DNA vaccine elicited CD8+ T cells and reduced bacterial loads. ► Helper T cell responses improved memory but not primary anti-bacterial responses. ► Single chain approaches should be considered as part of a composite vaccine. To circumvent limitations of poor antigen presentation and immunogenicity of DNA vaccines that target induction of CD8+ T cell immunity, we have generated single chain MHC I trimers (MHC I SCTs) composed of a single polypeptide chain with a linear composition of antigenic peptide, β2-microglobulin, and heavy chain of a MHC class I molecule connected by flexible linkers. Because of its pre-assembled nature, the SCT presents enhanced expression and presentation of the antigenic peptide/MHC complexes at the cell surface. Furthermore, DNA vaccination with a plasmid DNA encoding an SCT incorporating an immunodominant viral epitope elicited protective CD8+ T cell responses against lethal virus infection. To extend these findings, here we tested the efficacy of SCT DNA vaccines against bacterial infections. In a mouse infection model of Listeria monocytogenes, the SCT DNA vaccine encoding H-2Kd and the immunodominant peptide LLO 91–99 generated functional primary and memory peptide-specific CD8+ T cells that confer partial protection against L. monocytogenes infection. DNA immunization of Kd/LLO91–99 SCTs generated functional memory CD8+ T cells independently of CD4+ T cells, although the expression of cognate or non-cognate CD4+ helper T cell epitopes further enhanced the protective efficacy of SCTs. Our study further demonstrates that the SCT serves as a potent platform for DNA vaccines against various infectious diseases.

Components of the endoplasmic-reticulum-associated signal peptidase complex is required for infection by numerous flaviviruses, including West Nile, dengue and Zika viruses, but is not required for infection by other types of virus or for host protein synthesis. Flaviviruses infect hundreds of millions of people annually, and no antiviral therapy is available 1 , 2 . We performed a genome-wide CRISPR/Cas9-based screen to identify host genes that, when edited, resulted in reduced flavivirus infection. Here, we validated nine human genes required for flavivirus infectivity, and these were associated with endoplasmic reticulum functions including translocation, protein degradation, and N -linked glycosylation. In particular, a subset of endoplasmic reticulum-associated signal peptidase complex (SPCS) proteins was necessary for proper cleavage of the flavivirus structural proteins (prM and E) and secretion of viral particles. Loss of SPCS1 expression resulted in markedly reduced yield of all Flaviviridae family members tested (West Nile, dengue, Zika, yellow fever, Japanese encephalitis, and hepatitis C viruses), but had little impact on alphavirus, bunyavirus, or rhabdovirus infection or the surface expression or secretion of diverse host proteins. We found that SPCS1 dependence could be bypassed by replacing the native prM protein leader sequences with a class I major histocompatibility complex (MHC) antigen leader sequence. Thus, SPCS1, either directly or indirectly via its interactions with host proteins, preferentially promotes the processing of specific protein cargo, and Flaviviridae have a unique dependence on this signal peptide processing pathway. SPCS1 and other signal processing pathway members could represent pharmacological targets for inhibiting infection by the expanding number of flaviviruses of medical concern.

Ig heavy chain (IgH) isotypes (e.g., IgM, IgG, and IgE) are generated as secreted/soluble antibodies (sIg) or as membrane-bound (mIg) B cell receptors (BCRs) through alternative RNA splicing. IgH isotype dictates soluble antibody function, but how mIg isotype influences B cell behavior is not well defined. We examined IgH isotype-specific BCR function by analyzing naturally switched B cells from wild-type mice, as well as by engineering polyclonal Ighγ1/γ1 and Ighε/ε mice, which initially produce IgG1 or IgE from their respective native genomic configurations. We found that B cells from wild-type mice, as well as Ighγ1/γ1 and Ighε/ε mice, produce transcripts that generate IgM, IgG1, and IgE in an alternative splice form bias hierarchy, regardless of cell stage. In this regard, we found that mIgμ > mIgγ1 > mIgε, and that these BCR expression differences influence respective developmental fitness. Restrained B cell development from Ighγ1/γ1 and Ighε/ε mice was proportional to sIg/mIg ratios and was rescued by enforced expression of the respective mIgs. In addition, artificially enhancing BCR signal strength permitted IgE⁺ memory B cells—which essentially do not exist under normal conditions—to provide long-lived memory function, suggesting that quantitative BCR signal weakness contributes to restraint of IgE B cell responses. Our results indicate that IgH isotype-specific mIg/BCR dosage may play a larger role in B cell fate than previously anticipated.

Eastern equine encephalitis virus (EEEV) is a mosquito-transmitted alphavirus with a high case mortality rate in humans. EEEV is a biodefence concern because of its potential for aerosol spread and the lack of existing countermeasures. Here, we identify a panel of 18 neutralizing murine monoclonal antibodies (mAbs) against the EEEV E2 glycoprotein, several of which have ‘elite’ activity with 50 and 99% effective inhibitory concentrations (EC 50 and EC 99 ) of less than 10 and 100 ng ml −1 , respectively. Alanine-scanning mutagenesis and neutralization escape mapping analysis revealed epitopes for these mAbs in domains A or B of the E2 glycoprotein. A majority of the neutralizing mAbs blocked infection at a post-attachment stage, with several inhibiting viral membrane fusion. Administration of one dose of anti-EEEV mAb protected mice from lethal subcutaneous or aerosol challenge. These experiments define the mechanistic basis for neutralization by protective anti-EEEV mAbs and suggest a path forward for treatment and vaccine design. Neutralizing murine monoclonal antibodies against the Eeastern equine encephalitis virus target the E2 glycoprotein, block infection at a post-attachment stage by inhibiting viral membrane fusion and protect mice from lethal challenge.

Highlights * We tested a MHC I/peptide single chain DNA vaccine againstListeriainfection in mice. * This approach was designed to optimize antigen presentation to CD8+T cells. * Kd/LLO single chain DNA vaccine elicited CD8+T cells and reduced bacterial loads. * Helper T cell responses improved memory but not primary anti-bacterial responses. * Single chain approaches should be considered as part of a composite vaccine.

Hepatitis C virus (HCV) represents a major global health burden. Treatment with direct-acting antiviral agents results in viral clearance from a large majority of HCV infected individuals, but no preventative vaccine is available. While natural HCV infection results in highly diverse quasispecies over time, mutations accumulate more slowly in tissue culture, in part because of the inefficiency of replication in cell culture. To create a highly diverse population of HCV particles in vitro we engineered a library of infectious HCV with all codons represented at most positions in the ectodomain of the E2 gene. Using this E2 mutant library, we aimed to identify and characterize novel growth enhancing mutations and to select for neutralizing monoclonal antibody (NMAb) escape mutations. Via limited serial passage we identified numerous putative growth adaptive mutations and selected nine highly represented E2 mutants for further study: Q412R, T416R, S449P, T563V, A579R, L619T, V626S, K632T, and L644I. We evaluated these mutants for changes in particle to infectious unit ratio, sensitivity to NMAb inhibition, CD81 binding site exposure, entry factor usage, and buoyant density profiles. Remarkably, all nine variants showed reduced dependence on scavenger receptor class B type I (SR-BI) for infection. To identify antibody escape mutations, we performed antibody selection on the E2 gene library with 4 NMAbs targeting different epitopes in E2. If outgrowth of virus was observed following selection we used amplicon sequencing to identify putative escape mutations. Four substitutions, H405L, N417S, N417S, and G418E, were confirmed to alter sensitivity of HCV to one or more antibodies, but a large majority of the other mutations did not alleviate NMAb inhibition. Interestingly, two hypervariable region 1 (HVR1) mutants, T387L and H405L, appeared to be somewhat resistant to multiple NMAbs supporting a model in which single amino acid changes in HVR1 can modulate access to multiple conserved epitopes within E2. Despite encountering challenges in selecting escape mutations for HCV, we suggest the methods described herein could be better applied to rapidly growing viruses in the future. In summary, our results demonstrate that residues influencing SR-BI usage are distributed across E2 and support the development of large scale mutagenesis studies to identify viral variants with unique functional properties.

Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with an unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded monoclonal antibodies (mAbs) from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found 7 major mAb competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of mAb-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. mAbs that competed for binding the original S isolate bound differentially to S variants, suggesting the protective importance of otherwise-redundant recognition. The results furnish a global atlas of the S-specific memory B cell repertoire and illustrate properties conferring robustness against emerging SARS-CoV-2 variants.

Optimal immune responses furnish long-lasting (durable) antibodies protective across dynamically mutating viral variants (broad). To assess robustness of immunity induced by mRNA vaccination, we compared durability and breadth after SARS-CoV-2 infection and vaccination. While vaccination delivered robust initial virus-specific antibodies with some cross-variant coverage, pre-variant SARS-CoV-2 infection-induced antibodies, while modest in magnitude, showed highly stable long-term antibody dynamics. Vaccination after infection induced maximal antibody magnitudes with enhanced longitudinal stability while infection-naïve vaccinee antibodies fell with time to post-infection-alone levels. The composition of antibody neutralizing activity to variant relative to original virus also differed between groups, with infection-induced antibodies demonstrating greater relative breadth. Differential antibody durability trajectories favored COVID-19-recovered subjects with dual memory B cell features of greater early antibody somatic mutation and cross-coronavirus reactivity. By illuminating an infection-mediated antibody breadth advantage and an anti-SARS-CoV-2 antibody durability-enhancing function conferred by recalled immunity, these findings may serve as guides for ongoing vaccine strategy enhancement. Competing Interest Statement The authors have declared no competing interest.