Influenza chimeric hemagglutinin structures in complex with broadly protective antibodies to the stem and trimer interface.

Influenza virus hemagglutinin (HA) has been the primary target for influenza vaccine development. Broadly protective antibodies targeting conserved regions of the HA unlock the possibility of generating universal influenza immunity. Two group 2 influenza A chimeric HAs, cH4/3 and cH15/3, were previously designed to elicit antibodies to the conserved HA stem. Here, we show by X-ray crystallography and negative-stain electron microscopy that a broadly protective antistem antibody can stably bind to cH4/3 and cH15/3 HAs, thereby validating their potential as universal vaccine immunogens. Furthermore, flexibility was observed in the head domain of the chimeric HA structures, suggesting that antibodies could also potentially interact with the head interface epitope. Our structural and binding studies demonstrated that a broadly protective antihead trimeric interface antibody could indeed target the more open head domain of the cH15/3 HA trimer. Thus, in addition to inducing broadly protective antibodies against the conserved HA stem, chimeric HAs may also be able to elicit antibodies against the conserved trimer interface in the HA head domain, thereby increasing the vaccine efficacy.

Immunity to Seasonal Coronavirus Spike Proteins Does Not Protect from SARS-CoV-2 Challenge in a Mouse Model but Has No Detrimental Effect on Protection Mediated by COVID-19 mRNA Vaccination.

Seasonal coronaviruses have been circulating widely in the human population for many years. With increasing age, humans are more likely to have been exposed to these viruses and to have developed immunity against them. It has been hypothesized that this immunity to seasonal coronaviruses may provide partial protection against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and it has also been shown that coronavirus disease 2019 (COVID-19) vaccination induces a back-boosting effects against the spike proteins of seasonal betacoronaviruses. In this study, we tested if immunity to the seasonal coronavirus spikes from OC43, HKU1, 229E, or NL63 would confer protection against SARS-CoV-2 challenge in a mouse model, and whether pre-existing immunity against these spikes would weaken the protection afforded by mRNA COVID-19 vaccination. We found that mice vaccinated with the seasonal coronavirus spike proteins had no increased protection compared to the negative controls. While a negligible back-boosting effect against betacoronavirus spike proteins was observed after SARS-CoV-2 infection, there was no negative original antigenic sin-like effect on the immune response and protection induced by SARS-CoV-2 mRNA vaccination in animals with pre-existing immunity to seasonal coronavirus spike proteins. IMPORTANCE The impact that immunity against seasonal coronaviruses has on both susceptibility to SARS-CoV-2 infection as well as on COVID-19 vaccination is unclear. This study provides insights into both questions in a mouse model of SARS-CoV-2.

A single-shot adenoviral vaccine provides hemagglutinin stalk-mediated protection against heterosubtypic influenza challenge in mice.

Conventional influenza vaccines fail to confer broad protection against diverse influenza A viruses with pandemic potential. Efforts to develop a universal influenza virus vaccine include refocusing immunity towards the highly conserved stalk domain of the influenza virus surface glycoprotein, hemagglutinin (HA). We constructed a non-replicating adenoviral (Ad) vector, encoding a secreted form of H1 HA, to evaluate HA stalk-focused immunity. The Ad5_H1 vaccine was tested in mice for its ability to elicit broad, cross-reactive protection against homologous, heterologous, and heterosubtypic lethal challenge in a single-shot immunization regimen. Ad5_H1 elicited hemagglutination inhibition (HI+) active antibodies (Abs), which conferred 100% sterilizing protection from homologous H1N1 challenge. Furthermore, Ad5_H1 rapidly induced H1-stalk-specific Abs with Fc-mediated effector function activity, in addition to stimulating both CD4+ and CD8+ stalk-specific T cell responses. This phenotype of immunity provided 100% protection from lethal challenge with a head-mismatched, reassortant influenza virus bearing a chimeric HA, cH6/1, in a stalk-mediated manner. Most importantly, 100% protection from mortality following lethal challenge with a heterosubtypic avian influenza virus, H5N1, was observed following a single immunization with Ad5_H1. In conclusion, Ad-based influenza vaccines can elicit significant breadth of protection in naive animals and could be considered for pandemic preparedness and stockpiling.

A natriuretic peptide molecule from Vigna angularis, VaEG45, confers rust resistance by inhibiting fungal development.

KEY MESSAGE: Novel function and mechanism of a PNP molecule VaEG45 from adzuki bean involved in plant immunity. Plant natriuretic peptides (PNPs) can affect a broad spectrum of physiological responses in plants acting as peptidic signaling molecules. However, PNPs may play additional roles in plant immunity. Our previous transcriptome data of adzuki bean (Vigna angularis) in response to Uromyces vignae infection revealed association of PNP-encoding gene VaEG45 with U. vignae resistance. To determine the function of VaEG45 in disease resistance, we cloned the 589 bp nucleotide sequence of VaEG45 containing 2 introns, encoding a putative 13.68 kDa protein that is 131 amino acids in length. We analyzed expression in different resistant cultivars of V. angularis and found significant induction of VaEG45 expression after U. vignae infection. Transient expression of VaEG45 improved tobacco resistance against Botrytis cinerea. We next analyzed the mechanism by which VaEG45 protects plants from fungal infection by determination of the biological activity of the prokaryotic expressed VaEG45. The results showed that the fusion protein VaEG45 can significantly inhibit urediospores germination of U. vignae, mycelial growth, and the infection of tobacco by B. cinerea. Further analysis revealed that VaEG45 exhibits ß-1, 3-glucanase activity. These findings uncover the function of a novel PNP molecule VaEG45 and provide new evidence about the mechanism of PNPs in plant immunity.

Enhancing Neuraminidase Immunogenicity of Influenza A Viruses by Rewiring RNA Packaging Signals.

Humoral immune protection against influenza virus infection is mediated largely by antibodies against hemagglutinin (HA) and neuraminidase (NA), the two major glycoproteins on the virus surface. While influenza virus vaccination efforts have focused mainly on HA, NA-based immunity has been shown to reduce disease severity and provide heterologous protection. Current seasonal vaccines do not elicit strong anti-NA responses-in part due to the immunodominance of the HA protein. Here, we demonstrate that by swapping the 5' and 3' terminal packaging signals of the HA and NA genomic segments, which contain the RNA promoters, we are able to rescue influenza viruses that express more NA and less HA. Vaccination with formalin-inactivated "rewired" viruses significantly enhances the anti-NA antibody response compared to vaccination with unmodified viruses. Passive transfer of sera from mice immunized with rewired virus vaccines shows better protection against influenza virus challenge. Our results provide evidence that the immunodominance of HA stems in part from its abundance on the viral surface, and that rewiring viral packaging signals-thereby increasing the NA content on viral particles-is a viable strategy for improving the immunogenicity of NA in an influenza virus vaccine.IMPORTANCE Influenza virus infections are a major source of morbidity and mortality worldwide. Increasing evidence highlights neuraminidase as a potential vaccination target. This report demonstrates the efficacy of rewiring influenza virus packaging signals for creating vaccines with more neuraminidase content which provide better neuraminidase (NA)-based protection.

Combined Intranasal Nanoemulsion and RIG-I Activating RNA Adjuvants Enhance Mucosal, Humoral, and Cellular Immunity to Influenza Virus.

Current influenza virus vaccines are focused on humoral immunity and are limited by the short duration of protection, narrow cross-strain efficacy, and suboptimal immunogenicity. Here, we combined two chemically and biologically distinct adjuvants, an oil-in-water nanoemulsion (NE) and RNA-based agonists of RIG-I, to determine whether the diverse mechanisms of these adjuvants could lead to improved immunogenicity and breadth of protection against the influenza virus. NE activates TLRs, stimulates immunogenic apoptosis, and enhances cellular antigen uptake, leading to a balanced TH1/TH2/TH17 response when administered intranasally. RIG-I agonists included RNAs derived from Sendai and influenza viral defective interfering RNAs (IVT DI, 3php, respectively) and RIG-I/TLR3 agonist, poly(I:C) (pIC), which induce IFN-Is and TH1-polarized responses. NE/RNA combined adjuvants potentially allow for costimulation of multiple innate immune receptor pathways, more closely mimicking patterns of activation occurring during natural viral infection. Mice intranasally immunized with inactivated A/Puerto Rico/8/1934 (H1N1) (PR/8) adjuvanted with NE/IVT DI or NE/3php (but not NE/pIC) showed synergistic enhancement of systemic PR/8-specific IgG with significantly greater avidity and virus neutralization activity than the individual adjuvants. Notably, NE/IVT DI induced protective neutralizing titers after a single immunization. Hemagglutinin stem-specific antibodies were also improved, allowing recognition of heterologous and heterosubtypic hemagglutinins. All NE/RNAs elicited substantial PR/8-specific sIgA. Finally, a unique cellular response with enhanced TH1/TH17 immunity was induced with the NE/RNAs. These results demonstrate that the enhanced immunogenicity of the adjuvant combinations was synergistic and not simply additive, highlighting the potential value of a combined adjuvant approach for improving the efficacy of vaccination against the influenza virus.

Development of Influenza B Universal Vaccine Candidates Using the "Mosaic" Hemagglutinin Approach.

Influenza B viruses cause seasonal epidemics and are a considerable burden to public health. However, protection by current seasonal vaccines is suboptimal due to the antigenic changes of the circulating strains. In this study, we report a novel universal influenza B virus vaccination strategy based on "mosaic" hemagglutinins. We generated mosaic B hemagglutinins by replacing the major antigenic sites of the type B hemagglutinin with corresponding sequences from exotic influenza A hemagglutinins and expressed them as soluble trimeric proteins. Sequential vaccination with recombinant mosaic B hemagglutinin proteins conferred cross-protection against both homologous and heterologous influenza B virus strains in the mouse model. Of note, we rescued recombinant influenza B viruses expressing mosaic B hemagglutinins, which could serve as the basis for a universal influenza B virus vaccine.IMPORTANCE This work reports a universal influenza B virus vaccination strategy based on focusing antibody responses to conserved head and stalk epitopes of the hemagglutinin. Recombinant mosaic influenza B hemagglutinin proteins and recombinant viruses have been generated as novel vaccine candidates. This vaccine strategy provided broad cross-protection in the mouse model. Our findings will inform and drive development toward a more effective influenza B virus vaccine.

Extending the Stalk Enhances Immunogenicity of the Influenza Virus Neuraminidase.

Influenza viruses express two surface glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA). Anti-NA antibodies protect from lethal influenza virus challenge in the mouse model and correlate inversely with virus shedding and symptoms in humans. Consequently, the NA is a promising target for influenza virus vaccine design. Current seasonal vaccines, however, poorly induce anti-NA antibodies, partly because of the immunodominance of the HA over the NA when the two glycoproteins are closely associated. To address this issue, here we investigated whether extending the stalk domain of the NA could render it more immunogenic on virus particles. Two recombinant influenza viruses based on the H1N1 strain A/Puerto Rico/8/1934 (PR8) were rescued with NA stalk domains extended by 15 or 30 amino acids. Formalin-inactivated viruses expressing wild-type NA or the stalk-extended NA variants were used to vaccinate mice. The virus with the 30-amino-acid stalk extension induced significantly higher anti-NA IgG responses (characterized by increased in vitro antibody-dependent cellular cytotoxicity [ADCC] activity) than the wild-type PR8 virus, while anti-HA IgG levels were unaffected. Similarly, extending the stalk domain of the NA of a recent H3N2 virus enhanced the induction of anti-NA IgGs in mice. On the basis of these results, we hypothesize that the subdominance of the NA can be modulated if the protein is modified such that its height surpasses that of the HA on the viral membrane. Extending the stalk domain of NA may help to enhance its immunogenicity in influenza virus vaccines without compromising antibody responses to HA.IMPORTANCE The efficacy of influenza virus vaccines could be improved by enhancing the immunogenicity of the NA protein. One of the reasons for its poor immunogenicity is the immunodominance of the HA over the NA in many seasonal influenza virus vaccines. Here we demonstrate that, in the mouse model, extending the stalk domain of the NA protein can enhance its immunogenicity on virus particles and overcome the immunodominance of the HA without affecting antibody responses to the HA. The antibody repertoire is broadened by the extended NA and includes additional ADCC-active antibodies. Our findings may assist in the efforts toward more effective influenza virus vaccines.

Antibody Responses toward the Major Antigenic Sites of Influenza B Virus Hemagglutinin in Mice, Ferrets, and Humans.

The influenza B virus hemagglutinin contains four major antigenic sites (the 120 loop, the 150 loop, the 160 loop, and the 190 helix) within the head domain. These immunodominant antigenic sites are the main targets of neutralizing antibodies and are subject to antigenic drift. Yet little is known about the specific antibody responses toward each site in terms of antibody prevalence and hemagglutination inhibition activity. In this study, we used modified hemagglutinins of influenza B virus which display only one or none of the major antigenic sites to measure antibody responses toward the classical as well as the noncanonical epitopes in mice, ferrets, and humans. With our novel reagents, we found that both hemagglutination inhibition antibodies and total IgGs were mostly induced by the major antigenic sites. However, in human adults, we observed high hemagglutination inhibition antibody responses toward the noncanonical epitopes. By stratifying the human samples into age groups, we found that the noncanonical antibody responses appeared to increase with age.IMPORTANCE This study dissected the specific antibody responses toward the major antigenic sites and the noncanonical epitopes of influenza B virus hemagglutinin in animals and humans using novel reagents. These findings will guide the design of the next generation of influenza virus vaccines.

The Influenza B Virus Hemagglutinin Head Domain Is Less Tolerant to Transposon Mutagenesis than That of the Influenza A Virus.

Influenza A and B viruses can continuously evade humoral immune responses by developing mutations in the globular head of the hemagglutinin (HA) that prevent antibody binding. However, the influenza B virus HA over time displays less antigenic variation despite being functionally and structurally similar to the influenza A virus HA. To determine if the influenza B virus HA is under constraints that limit its antigenic variation, we performed a transposon screen to compare the mutational tolerance of the currently circulating influenza A virus HAs (H1 and H3 subtypes) and influenza B virus HAs (B/Victoria87 and B/Yamagata88 antigenic lineages). A library of insertional mutants for each HA was generated and deep sequenced after passaging to determine where insertions were tolerated in replicating viruses. The head domains of both viruses tolerated transposon mutagenesis, but the influenza A virus head was more tolerant to insertions than the influenza B virus head domain. Furthermore, all five of the known antigenic sites of the influenza A virus HA were tolerant of 15 nucleotide insertions, while insertions were detected in only two of the four antigenic sites in the influenza B virus head domain. Our analysis demonstrated that the influenza B virus HA is inherently less tolerant of transposon-mediated insertions than the influenza A virus HA. The reduced insertional tolerance of the influenza B virus HA may reveal genetic restrictions resulting in a lower capacity for antigenic evolution.IMPORTANCE Influenza viruses cause seasonal epidemics and result in significant human morbidity and mortality. Influenza viruses persist in the human population through generating mutations in the hemagglutinin head domain that prevent antibody recognition. Despite the similar selective pressures on influenza A and B viruses, influenza A virus displays a higher rate and breadth of antigenic variability than influenza B virus. A transposon mutagenesis screen was used to examine if the reduced antigenic variability of influenza B virus was due to inherent differences in mutational tolerance. This study demonstrates that the influenza A virus head domain and the individual antigenic sites targeted by humoral responses are more tolerant to insertions than those of influenza B virus. This finding sheds light on the genetic factors controlling the antigenic evolution of influenza viruses.

Immunodominance of Antigenic Site B in the Hemagglutinin of the Current H3N2 Influenza Virus in Humans and Mice.

The hemagglutinin protein of H3N2 influenza viruses is the major target of neutralizing antibodies induced by infection and vaccination. However, the virus frequently escapes antibody-mediated neutralization due to mutations in the globular head domain. Five topologically distinct antigenic sites in the head domain of H3 hemagglutinin, A to E, have been previously described by mapping the binding sites of monoclonal antibodies, yet little is known about the contribution of each site to the immunogenicity of modern H3 hemagglutinins, as measured by hemagglutination inhibition activity, which is known to correlate with protection. To investigate the hierarchy of antibody immunodominance, five Δ1 recombinant influenza viruses expressing hemagglutinin of the A/Hong Kong/4801/2014 (H3N2) strain with mutations in single antigenic sites were generated. Next, the Δ1 viruses were used to determine the hierarchy of immunodominance by measuring the hemagglutination inhibition reactivity of mouse antisera and plasma from 18 human subjects before and after seasonal influenza vaccination in 2017-2018. In both mice and humans, mutations in antigenic site B caused the most significant decrease in hemagglutination inhibition titers compared to wild-type hemagglutinin. This study revealed that antigenic site B is immunodominant in the H3N2 influenza virus strain included in the current vaccine preparations.IMPORTANCE Influenza viruses rapidly evade humoral immunity through antigenic drift, making current vaccines poorly effective and antibody-mediated protection short-lived. The majority of neutralizing antibodies target five antigenic sites in the head domain of the hemagglutinin protein that are also the most sequence-variable regions. A better understanding of the contribution of each antigenic site to the overall antibody response to hemagglutinin may help in the design of improved influenza virus vaccines.

Mutations in the Transmembrane Domain and Cytoplasmic Tail of Hendra Virus Fusion Protein Disrupt Virus-Like-Particle Assembly.

Hendra virus (HeV) is a zoonotic paramyxovirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral fusion protein (F). Here we investigated how endosomal F trafficking affects HeV assembly. We found that the HeV matrix (M) and F proteins each induced particle release when they were expressed alone but that their coexpression led to coordinated assembly of virus-like particles (VLPs) that were morphologically and physically distinct from M-only or F-only VLPs. Mutations to the F protein transmembrane domain or cytoplasmic tail that disrupted endocytic trafficking led to failure of F to function with M for VLP assembly. Wild-type F functioned normally for VLP assembly even when its cleavage was prevented with a cathepsin inhibitor, indicating that it is endocytic F trafficking that is important for VLP assembly, not proteolytic F cleavage. Under specific conditions of reduced M expression, we found that M could no longer induce significant VLP release but retained the ability to be incorporated as a passenger into F-driven VLPs, provided that the F protein was competent for endocytic trafficking. The F and M proteins were both found to traffic through Rab11-positive recycling endosomes (REs), suggesting a model in which F and M trafficking pathways converge at REs, enabling these proteins to preassemble before arriving at plasma membrane budding sites.IMPORTANCE Hendra virus and Nipah virus are zoonotic paramyxoviruses that cause lethal infections in humans. Unlike that for most paramyxoviruses, activation of the henipavirus fusion protein occurs in recycling endosomal compartments. In this study, we demonstrate that the unique endocytic trafficking pathway of Hendra virus F protein is required for proper viral assembly and particle release. These results advance our basic understanding of the henipavirus assembly process and provide a novel model for the interplay between glycoprotein trafficking and paramyxovirus assembly.

Chimeric Hemagglutinin Constructs Induce Broad Protection against Influenza B Virus Challenge in the Mouse Model.

Seasonal influenza virus epidemics represent a significant public health burden. Approximately 25% of all influenza virus infections are caused by type B viruses, and these infections can be severe, especially in children. Current influenza virus vaccines are an effective prophylaxis against infection but are impacted by rapid antigenic drift, which can lead to mismatches between vaccine strains and circulating strains. Here, we describe a broadly protective vaccine candidate based on chimeric hemagglutinins, consisting of globular head domains from exotic influenza A viruses and stalk domains from influenza B viruses. Sequential vaccination with these constructs in mice leads to the induction of broadly reactive antibodies that bind to the conserved stalk domain of influenza B virus hemagglutinin. Vaccinated mice are protected from lethal challenge with diverse influenza B viruses. Results from serum transfer experiments and antibody-dependent cell-mediated cytotoxicity (ADCC) assays indicate that this protection is antibody mediated and based on Fc effector functions. The present data suggest that chimeric hemagglutinin-based vaccination is a viable strategy to broadly protect against influenza B virus infection.IMPORTANCE While current influenza virus vaccines are effective, they are affected by mismatches between vaccine strains and circulating strains. Furthermore, the antiviral drug oseltamivir is less effective for treating influenza B virus infections than for treating influenza A virus infections. A vaccine that induces broad and long-lasting protection against influenza B viruses is therefore urgently needed.

Matrix proteins of Nipah and Hendra viruses interact with beta subunits of AP-3 complexes.

UNLABELLED: Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. IMPORTANCE: Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people, and smaller outbreaks have since occurred in Bangladesh and India. In this study, we have defined, for the first time, host factors that interact with henipavirus M proteins and contribute to viral particle assembly. We have also defined a new host protein-viral protein binding interface that can potentially be targeted for the inhibition of paramyxovirus infections.

An in vitro experimental pipeline to characterize the epitope of a SARS-CoV-2 neutralizing antibody.

IMPORTANCE: The COVID-19 pandemic remains a significant public health concern for the global population; the development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread so long as they recognize and interact with circulating variants. The epitope and binding specificity of a neutralizing anti-SARS-CoV-2 Spike receptor-binding domain antibody clone against many SARS-CoV-2 variants of concern were characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis and VSV-spike neutralization studies. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

The immunodominance of antigenic site Sb on the H1 influenza virus hemagglutinin increases with high immunoglobulin titers of the cohorts and with young age, but not sex.

The head domain of the hemagglutinin of influenza viruses plays a dominant role in the antibody response due to the presence of immunodominant antigenic sites that are the main targets of host neutralizing antibodies. For the H1 hemagglutinin, five major antigenic sites defined as Sa, Sb, Ca1, Ca2, and Cb have been described. Although previous studies have focused on defining the hierarchy of the antigenic sites of the hemagglutinin in different human cohorts, it is still unclear if the immunodominance profile of the antigenic sites might change with the antibody levels of individuals or if other demographic factors (such as exposure history, sex, or age) could also influence the importance of the antigenic sites. The major antigenic sites of influenza viruses hemagglutinins are responsible for eliciting most of the hemagglutination inhibition antibodies in the host. To determine the antibody prevalence towards each major antigenic site, we evaluated the hemagglutination inhibition against a panel of mutant H1 viruses, each one lacking one of the "classic" antigenic sites. Our results showed that the individuals from the Stop Flu NYU cohort had an immunodominant response towards the sites Sb and Ca2 of H1 hemagglutinin. A simple logistic regression analysis of the immunodominance profiles and the hemagglutination inhibition titers displayed by each donor revealed that individuals with high hemagglutination inhibition titers against the wild-type influenza virus exhibited higher probabilities of displaying an immunodominance profile dominated by Sb, followed by Ca2 (Sb > Ca2 profile), while individuals with low hemagglutination inhibition titers presented a higher chance of displaying an immunodominance profile in which Sb and Ca2 presented the same level of immunodominance (Sb = Ca2 profile). Finally, while age exhibited an influence on the immunodominance of the antigenic sites, biological sex was not related to displaying a specific immunodominance profile.

Intranasal SARS-CoV-2 Omicron variant vaccines elicit humoral and cellular mucosal immunity in female mice.

BACKGROUND: In order to prevent the emergence and spread of future variants of concern of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), developing vaccines capable of stopping transmission is crucial. The SARS-CoV-2 vaccine NDV-HXP-S can be administered live intranasally (IN) and thus induce protective immunity in the upper respiratory tract. The vaccine is based on Newcastle disease virus (NDV) expressing a stabilised SARS-CoV-2 spike protein. NDV-HXP-S can be produced as influenza virus vaccine at low cost in embryonated chicken eggs. METHODS: The NDV-HXP-S vaccine was genetically engineered to match the Omicron variants of concern (VOC) BA.1 and BA.5 and tested as an IN two or three dose vaccination regimen in female mice. Furthermore, female mice intramuscularly (IM) vaccinated with mRNA-lipid nanoparticles (LNPs) were IN boosted with NDV-HXP-S. Systemic humoral immunity, memory T cell responses in the lungs and spleens as well as immunoglobulin A (IgA) responses in distinct mucosal tissues were characterised. FINDINGS: NDV-HXP-S Omicron variant vaccines elicited high mucosal IgA and serum IgG titers against respective SARS-CoV-2 VOC in female mice following IN administration and protected against challenge from matched variants. Additionally, antigen-specific memory B cells and local T cell responses in the lungs were induced. Host immunity against the NDV vector did not interfere with boosting. Intramuscular vaccination with mRNA-LNPs was enhanced by IN NDV-HXP-S boosting resulting in improvement of serum neutralization titers and induction of mucosal immunity. INTERPRETATION: We demonstrate that NDV-HXP-S Omicron variant vaccines utilised for primary immunizations or boosting efficiently elicit humoral and cellular immunity. The described induction of systemic and mucosal immunity has the potential to reduce infection and transmission. FUNDING: This work was partially funded by the NIAIDCenters of Excellence for Influenza Research and Response (CEIRR) and by the NIAID Collaborative Vaccine Innovation Centers and by institutional funding from the Icahn School of Medicine at Mount Sinai. See under Acknowledgements for details.

Protective effect and molecular mechanisms of human non-neutralizing cross-reactive spike antibodies elicited by SARS-CoV-2 mRNA vaccination.

Neutralizing antibodies correlate with protection against SARS-CoV-2. Recent studies, however, show that binding antibody titers, in the absence of robust neutralizing activity, also correlate with protection from disease progression. Non-neutralizing antibodies cannot directly protect from infection but may recruit effector cells thus contribute to the clearance of infected cells. Also, they often bind conserved epitopes across multiple variants. We characterized 42 human mAbs from COVID-19 vaccinated individuals. Most of these antibodies exhibited no neutralizing activity in vitro but several non-neutralizing antibodies protected against lethal challenge with SARS-CoV-2 in different animal models. A subset of those mAbs showed a clear dependence on Fc-mediated effector functions. We determined the structures of three non-neutralizing antibodies with two targeting the RBD, and one that targeting the SD1 region. Our data confirms the real-world observation in humans that non-neutralizing antibodies to SARS-CoV-2 can be protective.

Newcastle disease virus vector-based SARS-CoV-2 vaccine candidate AVX/COVID-12 activates T cells and is recognized by antibodies from COVID-19 patients and vaccinated individuals.

Introduction: Several effective vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed and implemented in the population. However, the current production capacity falls short of meeting global demand. Therefore, it is crucial to further develop novel vaccine platforms that can bridge the distribution gap. AVX/COVID-12 is a vector-based vaccine that utilizes the Newcastle Disease virus (NDV) to present the SARS-CoV-2 spike protein to the immune system. Methods: This study aims to analyze the antigenicity of the vaccine candidate by examining antibody binding and T-cell activation in individuals infected with SARS-CoV-2 or variants of concern (VOCs), as well as in healthy volunteers who received coronavirus disease 2019 (COVID-19) vaccinations. Results: Our findings indicate that the vaccine effectively binds antibodies and activates T-cells in individuals who received 2 or 3 doses of BNT162b2 or AZ/ChAdOx-1-S vaccines. Furthermore, the stimulation of T-cells from patients and vaccine recipients with AVX/COVID-12 resulted in their proliferation and secretion of interferon-gamma (IFN-γ) in both CD4+ and CD8+ T-cells. Discussion: The AVX/COVID-12 vectored vaccine candidate demonstrates the ability to stimulate robust cellular responses and is recognized by antibodies primed by the spike protein present in SARS-CoV-2 viruses that infected patients, as well as in the mRNA BNT162b2 and AZ/ChAdOx-1-S vaccines. These results support the inclusion of the AVX/COVID-12 vaccine as a booster in vaccination programs aimed at addressing COVID-19 caused by SARS-CoV-2 and its VOCs.

The impact of automatic speech recognition technology on second language pronunciation and speaking skills of EFL learners: a mixed methods investigation.

Introduction: This study employed an explanatory sequential design to examine the impact of utilizing automatic speech recognition technology (ASR) with peer correction on the improvement of second language (L2) pronunciation and speaking skills among English as a Foreign Language (EFL) learners. The aim was to assess whether this approach could be an effective tool for enhancing L2 pronunciation and speaking abilities in comparison to traditional teacher-led feedback and instruction. Methods: A total of 61 intermediate-level Chinese EFL learners were randomly assigned to either a control group (CG) or an experimental group (EG). The CG received conventional teacher-led feedback and instruction, while the EG used ASR technology with peer correction. Data collection involved read-aloud tasks, spontaneous conversations, and IELTS speaking tests to evaluate L2 pronunciation and speaking skills. Additionally, semi-structured interviews were conducted with a subset of the participants to explore their perceptions of the ASR technology and its impact on their language learning experience. Results: The quantitative analysis of the collected data demonstrated that the EG outperformed the CG in all measures of L2 pronunciation, including accentedness and comprehensibility. Furthermore, the EG exhibited significant improvements in global speaking skill compared to the CG. The qualitative analysis of the interviews revealed that the majority of the participants in the EG found the ASR technology to be beneficial in enhancing their L2 pronunciation and speaking abilities. Discussion: The results of this study suggest that the utilization of ASR technology with peer correction can be a potent approach in enhancing L2 pronunciation and speaking skills among EFL learners. The improved performance of the EG compared to the CG in pronunciation and speaking tasks demonstrates the potential of incorporating ASR technology into language learning environments. Additionally, the positive feedback from the participants in the EG underscores the value of using ASR technology as a supportive tool in language learning classrooms.