Influenza viruses expressing chimeric hemagglutinins (HAs) are important tools in the quest for a universal vaccine. Using cryo-electron tomography, we have determined the structures of a chimeric HA variant that comprises an H1 stalk and an H5 globular head domain (cH5/1 HA) in native and antibody-bound states. We show that cH5/1 HA is structurally different from native HA, displaying a 60° rotation between the stalk and head groups, leading to a novel and unexpected "open" arrangement of HA trimers. cH5/1N1 viruses also display higher glycoprotein density than pH1N1 or H5N1 viruses, but despite these differences, antibodies that target either the stalk or head domains of hemagglutinins still bind to cH5/1 HA with the same consequences as those observed with native H1 or H5 HA. Our results show that a large range of structural plasticity can be tolerated in the chimeric spike scaffold without disrupting structural and geometric aspects of antibody binding.

Hantaviruses are the etiological agent of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). The latter is associated with case fatality rates ranging from 30% to 50%. HCPS cases are rare, with approximately 300 recorded annually in the Americas. Recently, an HCPS outbreak of unprecedented size has been occurring in and around Epuyén, in the southwestern Argentinian state of Chubut. Since November of 2018, at least 29 cases have been laboratory confirmed, and human-to-human transmission is suspected. Despite posing a significant threat to public health, no treatment or vaccine is available for hantaviral disease. Here, we describe an effort to identify, characterize, and develop neutralizing and protective antibodies against the glycoprotein complex (Gn and Gc) of Andes virus (ANDV), the causative agent of the Epuyén outbreak. Using murine hybridoma technology, we generated 19 distinct monoclonal antibodies (MAbs) against ANDV GnGc. When tested for neutralization against a recombinant vesicular stomatitis virus expressing the Andes glycoprotein (GP) (VSV-ANDV), 12 MAbs showed potent neutralization and 8 showed activity in an antibody-dependent cellular cytotoxicity reporter assay. Escape mutant analysis revealed that neutralizing MAbs targeted both the Gn and the Gc. Four MAbs that bound different epitopes were selected for preclinical studies and were found to be 100% protective against lethality in a Syrian hamster model of ANDV infection. These data suggest the existence of a wide array of neutralizing antibody epitopes on hantavirus GnGc with unique properties and mechanisms of action.IMPORTANCE Infections with New World hantaviruses are associated with high case fatality rates, and no specific vaccine or treatment options exist. Furthermore, the biology of the hantaviral GnGc complex, its antigenicity, and its fusion machinery are poorly understood. Protective monoclonal antibodies against GnGc have the potential to be developed into therapeutics against hantaviral disease and are also great tools to elucidate the biology of the glycoprotein complex.

Current seasonal influenza virus vaccines do not induce robust immune responses to neuraminidase. Several factors, including immunodominance of hemagglutinin over neuraminidase, instability of neuraminidase in vaccine formulations, and variable, nonstandardized amounts of neuraminidase in the vaccines, may contribute to this effect. However, vaccines that induce strong antineuraminidase immune responses would be beneficial, as they are highly protective. Furthermore, antigenic drift is slower for neuraminidase than for hemagglutinin, potentially providing broader coverage. Here, we designed stabilized recombinant versions of neuraminidase by replacing the N-terminal cytoplasmic domain, transmembrane, and extracellular stalk with tetramerization domains from the measles or Sendai virus phosphoprotein or from an Arabidopsis thaliana transcription factor. The measles virus tetramerization domain-based construct, termed N1-MPP, was chosen for further evaluation, as it retained antigenicity, neuraminidase activity, and structural integrity and provided robust protection in vivo against lethal virus challenge in the mouse model. We tested N1-MPP as a standalone vaccine, admixed with seasonal influenza virus vaccines, or given with seasonal influenza virus vaccines but in the other leg of the mouse. Admixture with different formulations of seasonal vaccines led to a weak neuraminidase response, suggesting a dominant effect of hemagglutinin over neuraminidase when administered in the same formulation. However, administration of neuraminidase alone or with seasonal vaccine administered in the alternate leg of the mouse induced robust antibody responses. Thus, this recombinant neuraminidase construct is a promising vaccine antigen that may enhance and broaden protection against seasonal influenza viruses. IMPORTANCE Influenza virus infections remain a high risk to human health, causing up to 650,000 deaths worldwide every year, with an enormous burden on the health care system. Since currently available seasonal vaccines are only partially effective and often mismatched to the circulating strains, a broader protective influenza virus vaccine is needed. Here, we generated a recombinant influenza virus vaccine candidate based on the more conserved neuraminidase surface glycoprotein in order to induce a robust and broader protective immune response against a variety of circulating influenza virus strains.

Despite efforts to control influenza virus infection and transmission, influenza viruses still cause significant morbidity and mortality in the global human population each year. Most of the current vaccines target the immunodominant hemagglutinin surface glycoprotein of the virus. However, reduced severity of disease and viral shedding have also been linked to antibodies targeting the second viral surface glycoprotein, the neuraminidase. Importantly, antineuraminidase immunity was shown to be relatively broad, in contrast to vaccine-induced antibodies to the hemagglutinin head domain. In this study, we assessed recombinant neuraminidase protein vaccination for its ability to prevent or limit virus transmission. We vaccinated guinea pigs either intramuscularly or intranasally with a recombinant influenza B virus neuraminidase to assess whether neuraminidase vaccination via these routes could prevent transmission of the homologous virus to a naive recipient. Guinea pigs vaccinated with neuraminidase showed reduced virus titers; however, only vaccination via the intranasal route fully prevented virus transmission to naive animals. We found high levels of antineuraminidase antibodies capable of inhibiting neuraminidase enzymatic activity in the nasal washes of intranasally vaccinated animals, which may explain the observed differences in transmission. We also determined that mucosal immunity to neuraminidase impaired the transmission efficiency of a heterologous influenza B virus, although to a lesser extent. Finally, we found that neuraminidase-vaccinated animals were still susceptible to infection via the airborne and contact transmission routes. However, significantly lower virus titers were detected in these vaccinated recipients. In summary, our data suggest that supplementing vaccine formulations with neuraminidase and vaccinating via the intranasal route may broadly prevent transmission of influenza B viruses.IMPORTANCE Recently, the protective effect of anti-neuraminidase immunity has been highlighted by several studies in humans and animal models. However, so far the role that anti-neuraminidase immunity plays in inhibition of virus transmission has not been explored. In addition, neuraminidase has been ignored as an antigen for influenza virus vaccines. We show here that neuraminidase-based vaccines can inhibit the transmission of influenza virus. Therefore, neuraminidase should be considered as an antigen for improved influenza virus vaccines that not only protect individuals from disease but also inhibit further spread of the virus in the population.

Current seasonal influenza virus vaccines target regions of the hemagglutinin (HA) head domain that undergo constant antigenic change, forcing the painstaking annual reformulation of vaccines. The development of broadly protective or universal influenza virus vaccines that induce cross-reactive, protective immune responses could circumvent the need to reformulate current seasonal vaccines. Many of these vaccine candidates target the HA stalk domain, which displays epitopes conserved within and across influenza virus subtypes, including those with pandemic potential. While HA head-mediated antigenic drift is well understood, the potential for antigenic drift in the stalk domain is understudied. Using a panel of HA stalk-specific monoclonal antibodies (MAbs), we applied selection pressure to the stalk domain of A/Netherlands/602/2009 (pdmH1N1) to determine fitness and phenotypes of escape mutant viruses (EMVs). We found that HA stalk MAbs with lower cross-reactivity caused single HA stalk escape mutations, whereas MAbs with broader cross-reactivity forced multiple mutations in the HA. Each escape mutant virus greatly decreased mAb neutralizing activity, but escape mutations did not always ablate MAb binding or Fc-Fc receptor-based effector functions. Escape mutant viruses were not attenuated in vitro but showed attenuation in an in vivo mouse model. Importantly, mice vaccinated with a chimeric HA universal vaccine candidate were protected from lethal challenge with EMVs despite these challenge viruses containing escape mutations in the stalk domain. Our study indicates that while the HA stalk domain can mutate under strong MAb selection pressure, mutant viruses may have attenuated phenotypes and do not evade a polyclonal, stalk-based vaccine-induced response.IMPORTANCE Broadly protective or universal influenza virus vaccines target viral epitopes that appear to be conserved. However, it is unclear whether the virus will be able to escape once immunological pressure is applied to these epitopes through vaccination of large proportions of the population. Studies that investigate the fitness and antigenic characteristics of viruses that escape immunological pressure on these conserved epitopes are therefore urgently needed.

Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were developed in record time and show excellent efficacy and effectiveness against coronavirus disease 2019 (COVID-19). However, currently approved vaccines cannot meet the global demand. In addition, none of the currently used vaccines is administered intranasally to potentially induce mucosal immunity. Here, we tested the safety and immunogenicity of a second-generation SARS-CoV-2 vaccine that includes a stabilized spike antigen and can be administered intranasally. The vaccine is based on a live Newcastle disease virus vector expressing a SARS-CoV-2 spike protein stabilized in a prefusion conformation with six beneficial proline substitutions (AVX/COVID-12-HEXAPRO; Patria). Immunogenicity testing in the pig model showed that both intranasal and intramuscular application of the vaccine as well as a combination of the two induced strong serum neutralizing antibody responses. Furthermore, substantial reactivity to B.1.1.7, B.1.351, and P.1 spike variants was detected. Finally, no adverse reactions were found in the experimental animals at any dose level or delivery route. These results indicate that the experimental vaccine AVX/COVID-12-HEXAPRO (Patria) is safe and highly immunogenic in the pig model. IMPORTANCE Several highly efficacious vaccines for SARS-CoV-2 have been developed and are used in the population. However, the current production capacity cannot meet the global demand. Therefore, additional vaccines-especially ones that can be produced locally and at low cost-are urgently needed. This work describes preclinical testing of a SARS-CoV-2 vaccine candidate which meets these criteria.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the prime target for vaccine development. The spike protein mediates both binding to host cells and membrane fusion and is also so far the only known viral target of neutralizing antibodies. Coronavirus spike proteins are large trimers that are relatively unstable, a feature that might be enhanced by the presence of a polybasic cleavage site in SARS-CoV-2 spike. Exchange of K986 and V987 for prolines has been shown to stabilize the trimers of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus spike proteins. Here, we test multiple versions of a soluble spike protein for their immunogenicity and protective effect against SARS-CoV-2 challenge in a mouse model that transiently expresses human angiotensin-converting enzyme 2 via adenovirus transduction. Variants tested include spike proteins with a deleted polybasic cleavage site, proline mutations, or a combination thereof, besides the wild-type protein. While all versions of the protein were able to induce neutralizing antibodies, only the antigen with both a deleted cleavage site and the K986P and V987P (PP) mutations completely protected from challenge in this mouse model.IMPORTANCE A vaccine for SARS-CoV-2 is urgently needed. A better understanding of antigen design and attributes that vaccine candidates need to have to induce protective immunity is of high importance. The data presented here validate the choice of antigens that contain the PP mutations and suggest that deletion of the polybasic cleavage site may lead to a further-optimized design.

Influenza remains a major global health burden. Seasonal vaccines offer protection but can be rendered less effective when the virus undergoes extensive antigenic drift. Antibodies that target the highly conserved hemagglutinin stalk can protect against drifted viruses, and vaccine constructs designed to induce such antibodies form the basis for a universal influenza virus vaccine approach. In this study, we analyzed baseline and postvaccination serum samples of children (6 to 59 months), adults (18 to 49 years), and elderly individuals (≥65 years) who participated in clinical trials with a recombinant hemagglutinin-based vaccine. We found that baseline IgG and IgA antibodies against the H1 stalk domain correlated with the ages of patients. Children generally had very low baseline titers and did not respond well to the vaccine in terms of making stalk-specific antibodies. Adults showed the highest induction of stalk-specific antibodies, but the elderly had the highest absolute antibody titers against the stalk. Importantly, the stalk antibodies measured by enzyme-linked immunosorbent assay (ELISA) showed neutralizing activity in neutralization assays and protected mice in a passive-transfer model in a stalk titer-dependent manner. Finally, we found similar patterns of stalk-specific antibodies directed against the H3 and influenza B virus hemagglutinins, albeit at lower levels than those measured against the H1 stalk. The relatively high levels of stalk-specific antibodies in the elderly patients may explain the previously reported low influenza virus infection rates in this age group. (This study has been registered at ClinicalTrials.gov under registration no. NCT00336453, NCT00539981, and NCT00395174.)

The immunogenicity of current influenza virus vaccines is assessed by measuring an increase of influenza virus-specific antibodies in a hemagglutination inhibition assay. This method exclusively measures antibodies against the hemagglutinin head domain. While this domain is immunodominant, it has been shown that hemagglutination inhibition titers do not always accurately predict protection from disease. In addition, several novel influenza virus vaccines that are currently under development do not target the hemagglutinin head domain, but rather more conserved sites, including the hemagglutinin stalk. Importantly, antibodies against the hemagglutinin stalk do not show activity in hemagglutination inhibition assays and will require different methods for quantification. In this study, we tested human serum samples from a seasonal influenza virus vaccination trial and an avian H5N1 virus vaccination trial for antibody activities in multiple types of assays, including binding assays and also functional assays. We then performed serum transfer experiments in mice which then received an H1N1 virus challenge to assess the in vivo protective effects of the antibodies. We found that hemagglutinin-specific antibody levels measured in an enzyme-linked immunosorbent assay (ELISA) correlated well with protection from weight loss in mice. In addition, we found that weight loss was also inversely correlated with the level of serum antibody-dependent cellular cytotoxicity (ADCC) as measured in a reporter assay. These findings indicate that protection is in part conferred by Fc-dependent mechanisms. In conclusion, ELISAs can be used to measure hemagglutinin-specific antibody levels that could serve as a surrogate marker of protection for universal influenza virus vaccines.IMPORTANCE Influenza viruses are a serious concern for public health and cause a large number of deaths worldwide every year. Current influenza virus vaccines can confer protection from disease, but they often show low efficacy due to the ever-changing nature of the viruses. Novel vaccination approaches target conserved epitopes of the virus, including the hemagglutinin stalk domain, to elicit universally protective antibodies that also bind to mutated viruses or new subtypes of viruses. Importantly, the hemagglutination inhibition assay-the only assay that has been accepted as a correlate of protection by regulatory authorities-cannot measure antibodies against the hemagglutinin stalk domain. Therefore, novel correlates of protection and assays to measure vaccine immunogenicity need to be developed. In this study, we correlated the results from multiple assays with protection in mice after transfer of human serum and a lethal virus challenge to investigate potential novel serological surrogate markers for protection.

In contrast to influenza virus vaccination, natural infection induces long-lived and relatively broad immune responses. However, many aspects of the antibody response to natural infection are not well understood. Here, we assessed the immune response after H1N1 influenza virus infection in children and adults in a Nicaraguan household transmission study using an influenza virus protein microarray (IVPM). This technology allows us to simultaneously measure IgG and IgA antibody responses to hemagglutinins of many different virus strains and subtypes quantitatively with a high throughput. We found that children under 6 years of age responded to natural infection with a relatively narrow response that targeted mostly the hemagglutinin of the strain that caused the infection. Adults, however, have a much broader response, including a boost in antibodies to many group 1 subtype hemagglutinins. Also, a strong recall response against historic H1 hemagglutinins that share the K133 epitope with the pandemic H1N1 virus was observed. Of note, some children, while responding narrowly within H1 and group 1 hemagglutinins, induced a boost to H3 and other group 2 hemagglutinins when infected with H1N1 when they had experienced an H3N2 infection earlier in life. This is an interesting phenomenon providing evidence for immune imprinting and a significant new insight which might be leveraged in future universal influenza virus vaccine strategies. Finally, preexisting immunity to pandemic H1 hemagglutinins was significantly associated with protection from infection in both children and adults. In adults, preexisting immunity to non-H1 group 1 hemagglutinins was also significantly associated with protection from infection.IMPORTANCE It is known since Thomas Francis, Jr. published his first paper on original antigenic sin in 1960 that the first infection(s) with influenza virus leaves a special immunological imprint which shapes immune responses to future infections with antigenically related influenza virus strains. Imprinting has been implicated in both protective effects as well as blunting of the immune response to vaccines. Despite the fact that this phenomenon was already described almost 60 years ago, we have very little detailed knowledge of the characteristics and breadth of the immune response to the first exposure(s) to influenza virus in life and how this compares to later exposure as adults. Here, we investigate these immune responses in detail using an influenza virus protein microarray. While our findings are mostly descriptive in nature and based on a small sample size, they provide a strong basis for future large-scale studies to better understand imprinting effects.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing a global pandemic. The antigen specificity of the antibody response mounted against this novel virus is not understood in detail. Here, we report that subjects with a more severe SARS-CoV-2 infection exhibit a larger antibody response against the spike and nucleocapsid protein and epitope spreading to subdominant viral antigens, such as open reading frame 8 and nonstructural proteins. Subjects with a greater antibody response mounted a larger memory B cell response against the spike, but not the nucleocapsid protein. Additionally, we revealed that antibodies against the spike are still capable of binding the D614G spike mutant and cross-react with the SARS-CoV-1 receptor binding domain. Together, this study reveals that subjects with a more severe SARS-CoV-2 infection exhibit a greater overall antibody response to the spike and nucleocapsid protein and a larger memory B cell response against the spike.IMPORTANCE With the ongoing pandemic, it is critical to understand how natural immunity against SARS-CoV-2 and COVID-19 develops. We have identified that subjects with more severe COVID-19 disease mount a more robust and neutralizing antibody response against SARS-CoV-2 spike protein. Subjects who mounted a larger response against the spike also mounted antibody responses against other viral antigens, including the nucleocapsid protein and ORF8. Additionally, this study reveals that subjects with more severe disease mount a larger memory B cell response against the spike. These data suggest that subjects with more severe COVID-19 disease are likely better protected from reinfection with SARS-CoV-2.

After first emerging in late 2019 in China, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized, but the supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the full-length spike protein of SARS-CoV-2. In this study, we generated mouse monoclonal antibodies (MAbs) against different epitopes on the RBD and assessed binding and neutralization of authentic SARS-CoV-2. We demonstrate that antibodies with neutralizing activity, but not nonneutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the MAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variant in vitro. IMPORTANCE Cross-neutralization of SARS-CoV-2 variants by RBD-targeting antibodies is still not well understood, and very little is known about the potential protective effect of nonneutralizing antibodies in vivo. Using a panel of mouse monoclonal antibodies, we investigate both of these points.

Antibody responses to influenza virus hemagglutinin provide protection against infection and are well studied. Less is known about the human antibody responses to the second surface glycoprotein, neuraminidase. Here, we assessed human antibody reactivity to a panel of N1, N2, and influenza B virus neuraminidases in different age groups, including children, adults, and the elderly. Using enzyme-linked immunosorbent assays (ELISA), we determined the breadth, magnitude, and isotype distribution of neuraminidase antibody responses to historic, current, and avian strains, as well as to recent isolates to which these individuals have not been exposed. It appears that antibody levels against N1 neuraminidases were lower than those against N2 or B neuraminidases. The anti-neuraminidase antibody levels increased with age and were, in general, highest against strains that circulated during the childhood of the tested individuals, providing evidence for "original antigenic sin." Titers measured by ELISA correlated well with titers measured by the neuraminidase inhibition assays. However, in the case of the 2009 pandemic H1N1 virus, we found evidence of interference from antibodies binding to the conserved stalk domain of the hemagglutinin. In conclusion, we found that antibodies against the neuraminidase differ in magnitude and breadth between subtypes and age groups in the human population. (This study has been registered at ClinicalTrials.gov under registration no. NCT00336453, NCT00539981, and NCT00395174.)IMPORTANCE Anti-neuraminidase antibodies can afford broad protection from influenza virus infection in animal models and humans. However, little is known about the breadth and magnitude of the anti-neuraminidase response in the human population. Here we assessed antibody levels of children, adults, and the elderly against a panel of N1, N2, and type B influenza virus neuraminidases. We demonstrated that antibody levels measured by ELISA correlate well with functional neuraminidase inhibition titers. This is an important finding since ELISA is a simpler method than functional assays and can be implemented in high-throughput settings to analyze large numbers of samples. Furthermore, we showed that low titers of broadly cross-reactive antibodies against neuraminidase are prevalent in humans. By the use of an appropriate vaccination strategy, these titers could potentially be boosted to levels that might provide broad protection from influenza virus infection.

Zika virus is a mosquito-borne flavivirus which can cause severe disease in humans, including microcephaly and other congenital malformations in newborns and Guillain-Barré syndrome in adults. There are currently no approved prophylactics or therapeutics for Zika virus; the development of a safe and effective vaccine is an urgent priority. Preclinical studies suggest that the envelope glycoprotein can elicit potently neutralizing antibodies. However, such antibodies are implicated in the phenomenon of antibody-dependent enhancement of disease. We have previously shown that monoclonal antibodies targeting the Zika virus nonstructural NS1 protein are protective without inducing antibody-dependent enhancement of disease. Here, we investigated whether the NS1 protein itself is a viable vaccine target. Wild-type mice were vaccinated with an NS1-expressing DNA plasmid followed by two adjuvanted protein boosters, which elicited high antibody titers. Passive transfer of the immune sera was able to significantly protect STAT2 knockout mice against lethal challenge by Zika virus. In addition, long-lasting NS1-specific IgG responses were detected in serum samples from patients in either the acute or the convalescent phase of Zika virus infection. These NS1-specific antibodies were able to functionally engage Fcγ receptors. In contrast, envelope-specific antibodies did not activate Fc-mediated effector functions on infected cells. Our data suggest that the Zika virus NS1 protein, which is expressed on infected cells, is critical for Fc-dependent cell-mediated immunity. The present study demonstrates that the Zika virus NS1 protein is highly immunogenic and can elicit protective antibodies, underscoring its potential for an effective Zika virus vaccine.IMPORTANCE Zika virus is a global public health threat that causes microcephaly and congenital malformations in newborns and Guillain-Barré syndrome in adults. Currently, no vaccines or treatments are available. While antibodies targeting the envelope glycoprotein can neutralize virus, they carry the risk of antibody-dependent enhancement of disease (ADE). In contrast, antibodies generated against the NS1 protein can be protective without eliciting ADE. The present study demonstrates the effectiveness of an NS1-based vaccine in eliciting high titers of protective antibodies against Zika virus disease in a mouse model. Sera generated by this vaccine can elicit Fc-mediated effector functions against Zika virus-infected cells. Lastly, we provide human data suggesting that the antibody response against the Zika virus NS1 protein is long-lasting and functionally active. Overall, our work will inform the development of a safe and effective Zika virus vaccine.

Influenza virus infections leave a signature of immune memory that influences future responses to infections with antigenically related strains. It has been hypothesized that the first exposure in life to H1N1 influenza virus imprints the host immune system, potentially resulting in protection from severe infection with H5N1 later in life through hemagglutinin (HA) stalk-specific antibodies. To study the specific role of the HA on protection against infection without interference of cellular immunity or humoral antineuraminidase immunity, we primed mice with influenza B viruses that express an H1 HA (group 1; B-H1), H3 HA (group 2; B-H3), or wild-type influenza B virus and subsequently challenged them at different time points with an H5N1 virus. Weight loss and survival monitoring showed that the B-H1-primed mice exhibited better protection against H5N1 compared to the control mice. Analysis of H5-specific serum IgG, before and 21 days after H5N1 challenge, evidenced the presence of anti-stalk H5 cross-reactive antibodies in the BH-1 group that were boosted by H5N1 infection. The increased immune responses and protection induced by priming with the B-H1 viruses lasted at least up to 1 year. Hence, a single HA priming based on natural infection induces long-lasting protective immunity against heterosubtypic strains from the same phylogenetic HA group in mice. This study gives mechanistic support to the earlier finding in humans that imprinting by H1 HA protects against H5N1 infections and that highly conserved regions on the HA, like the stalk, are involved in this phenomenon.IMPORTANCE Current studies point out that an HA-mediated immunological imprint is established early in life during the first exposure to influenza viruses, which critically shapes and biases future immune responses. However, studies in animal models are limited and the precise mechanisms of this phenomenon are under investigation. Studies that explore the effect of HA-specific immunity induced during natural infection on future exposures to heterosubtypic influenza strains are needed.

The ongoing concurrent outbreaks of Zika, Chikungunya, and dengue viruses in Latin America and the Caribbean highlight the need for development of broad-spectrum antiviral treatments. The type I interferon (IFN) system has evolved in vertebrates to generate tissue responses that actively block replication of multiple known and potentially zoonotic viruses. As such, its control and activation through pharmacological agents may represent a novel therapeutic strategy for simultaneously impairing growth of multiple virus types and rendering host populations resistant to virus spread. In light of this strategy's potential, we undertook a screen to identify novel interferon-activating small molecules. Here, we describe 1-(2-fluorophenyl)-2-(5-isopropyl-1,3,4-thiadiazol-2-yl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione, which we termed AV-C. Treatment of human cells with AV-C activates innate and interferon-associated responses that strongly inhibit replication of Zika, Chikungunya, and dengue viruses. By utilizing genome editing, we investigated the host proteins essential to AV-C-induced cellular states. This showed that the compound requires a TRIF-dependent signaling cascade that culminates in IFN regulatory factor 3 (IRF3)-dependent expression and secretion of type I interferon to elicit antiviral responses. The other canonical IRF3-terminal adaptor proteins STING and IPS-1/MAVS were dispensable for AV-C-induced phenotypes. However, our work revealed an important inhibitory role for IPS-1/MAVS, but not TRIF, in flavivirus replication, implying that TRIF-directed viral evasion may not occur. Additionally, we show that in response to AV-C, primary human peripheral blood mononuclear cells secrete proinflammatory cytokines that are linked with establishment of adaptive immunity to viral pathogens. Ultimately, synthetic innate immune activators such as AV-C may serve multiple therapeutic purposes, including direct antimicrobial responses and facilitation of pathogen-directed adaptive immunity.IMPORTANCE The type I interferon system is part of the innate immune response that has evolved in vertebrates as a first line of broad-spectrum immunological defense against an unknowable diversity of microbial, especially viral, pathogens. Here, we characterize a novel small molecule that artificially activates this response and in so doing generates a cellular state antagonistic to growth of currently emerging viruses: Zika virus, Chikungunya virus, and dengue virus. We also show that this molecule is capable of eliciting cellular responses that are predictive of establishment of adaptive immunity. As such, this agent may represent a powerful and multipronged therapeutic tool to combat emerging and other viral diseases.

The global coronavirus disease 2019 (COVID-19) pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent-phase plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous biosafety level 3 (BSL3) conditions, which limits high-throughput screening of patient and vaccine sera. Myriad BSL2-compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making intergroup comparisons difficult. To address these limitations, we developed a standardized VNA using CoV2-S pseudotyped particles (CoV2pp) based on vesicular stomatitis virus bearing the Renilla luciferase gene in place of its G glycoprotein (VSVΔG); this assay can be robustly produced at scale and generate accurate neutralizing titers within 18 h postinfection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S enzyme-linked immunosorbent assay (ELISA) results and live-virus neutralizations in confirmed convalescent-patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n > 120). Our data (i) show that absolute 50% inhibitory concentration (absIC50), absIC80, and absIC90 values can be legitimately compared across diverse cohorts, (ii) highlight the substantial but consistent variability in neutralization potency across these cohorts, and (iii) support the use of the absIC80 as a more meaningful metric for assessing the neutralization potency of a vaccine or convalescent-phase sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2 plus TMPRSS2. When these are used in combination with our CoV2pp, we can produce CoV2pp sufficient for 150,000 standardized VNAs/week.IMPORTANCE Vaccines and antibody-based therapeutics like convalescent-phase plasma therapy are premised upon inducing or transferring neutralizing antibodies that inhibit SARS-CoV-2 entry into cells. Virus neutralization assays (VNAs) for measuring neutralizing antibody titers (NATs) are an essential part of determining vaccine or therapeutic efficacy. However, such efficacy testing is limited by the inherent dangers of working with the live virus, which requires specialized high-level biocontainment facilities. We therefore developed a standardized replication-defective pseudotyped particle system that mimics the entry of live SARS-CoV-2. This tool allows for the safe and efficient measurement of NATs, determination of other forms of entry inhibition, and thorough investigation of virus entry mechanisms. Four independent labs across the globe validated our standardized VNA using diverse cohorts. We argue that a standardized and scalable assay is necessary for meaningful comparisons of the myriad of vaccines and antibody-based therapeutics becoming available. Our data provide generalizable metrics for assessing their efficacy.

As demonstrated by severe acute respiratory syndrome coronavirus 2, coronaviruses pose a significant pandemic threat. Here, we show that coronavirus disease 2019 mRNA vaccination can induce significant levels of cross-reactive antibodies against diverse coronavirus spike proteins. While these antibodies are binding antibodies that likely have little neutralization capacity and while their contribution to cross-protection is unclear, it is possible that they may play a role in protection from progression to severe disease with novel coronaviruses.

The PARIS (Protection Associated with Rapid Immunity to SARS-CoV-2) cohort follows health care workers with and without documented coronavirus disease 2019 (COVID-19) since April 2020. We report our findings regarding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-binding antibody stability and protection from infection in the pre-variant era. We analyzed data from 400 health care workers (150 seropositive and 250 seronegative at enrollment) for a median of 84 days. The SARS-CoV-2 spike-binding antibody titers were highly variable with antibody levels decreasing over the first 3 months, followed by a relative stabilization. We found that both more advanced age (>40 years) and female sex were associated with higher antibody levels (1.6-fold and 1.4-fold increases, respectively). Only six percent of the initially seropositive participants "seroreverted." We documented a total of 11 new SARS-CoV-2 infections (10 naive participants and 1 previously infected participant without detectable antibodies; P < 0.01), indicating that spike antibodies limit the risk of reinfection. These observations, however, only apply to SARS-CoV-2 variants antigenically similar to the ancestral SARS-CoV-2 ones. In conclusion, SARS-CoV-2 antibody titers mounted upon infection are stable over several months and provide protection from infection with antigenically similar viruses. IMPORTANCE SARS-CoV-2 is the cause of one of the largest noninfluenza pandemics of this century. This exceptional public health crisis highlights the urgent need for better understanding of the correlates of protection from infection and severe COVID-19. We established the PARIS cohort to determine durability and effectiveness of SARS-CoV-2 immune responses. Here, we report on the kinetics of SARS-CoV-2 spike-binding antibody after SARS-CoV-2 infection as well as reinfection rates using data collected between April 2020 and August 2021. We found that antibody levels stabilized at individual steady state levels after an initial decrease with seroreversion being found in only 6% of the convalescent participants. SARS-CoV-2 infections only occurred in participants without detectable spike-binding antibodies, indicating significant protection from reinfection with antigenically similar viruses. Our data indicate the importance of spike-binding antibody titers in protection prior to vaccination and the wide circulation of antigenically diverse variants of concern.

In an attempt to assess the cross-protective potential of the influenza virus neuraminidase (NA) as a vaccine antigen, different subtypes of recombinant NA were expressed in a baculovirus system and used to vaccinate mice prior to lethal challenge with homologous, heterologous, or heterosubtypic viruses. Mice immunized with NA of subtype N2 were completely protected from morbidity and mortality in a homologous challenge and displayed significantly reduced viral lung titers. Heterologous challenge with a drifted strain resulted in morbidity but no mortality. Similar results were obtained for challenge experiments with N1 NA. Mice immunized with influenza B virus NA (from B/Yamagata/16/88) displayed no morbidity when sublethally infected with the homologous strain and, importantly, were completely protected from morbidity and mortality when lethally challenged with the prototype Victoria lineage strain or a more recent Victoria lineage isolate. Upon analyzing the NA content in 4 different inactivated-virus vaccine formulations from the 2013-2014 season via Western blot assay and enzyme-linked immunosorbent assay quantification, we found that the amount of NA does indeed vary across vaccine brands. We also measured hemagglutinin (HA) and NA endpoint titers in pre- and postvaccination human serum samples from individuals who received a trivalent inactivated seasonal influenza vaccine from the 2004-2005 season; the induction of NA titers was statistically less pronounced than the induction of HA titers. The demonstrated homologous and heterologous protective capacity of recombinant NA suggests that supplementing vaccine formulations with a standard amount of NA may offer increased protection against influenza virus infection.