Understanding "Hybrid Immunity": Comparison and Predictors of Humoral Immune Responses to Severe Acute Respiratory Syndrome Coronavirus 2 Infection (SARS-CoV-2) and Coronavirus Disease 2019 (COVID-19) Vaccines.

BACKGROUND: Comparison of humoral responses in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccinees, those with SARS-CoV-2 infection, or combinations of vaccine/ infection ("hybrid immunity") may clarify predictors of vaccine immunogenicity. METHODS: We studied 2660 US Military Health System beneficiaries with a history of SARS-CoV-2 infection-alone (n = 705), vaccination-alone (n = 932), vaccine-after-infection (n = 869), and vaccine-breakthrough-infection (n = 154). Peak anti-spike-immunoglobulin G (IgG) responses through 183 days were compared, with adjustment for vaccine product, demography, and comorbidities. We excluded those with evidence of clinical or subclinical SARS-CoV-2 reinfection from all groups. RESULTS: Multivariable regression results indicated that vaccine-after-infection anti-spike-IgG responses were higher than infection-alone (P < .01), regardless of prior infection severity. An increased time between infection and vaccination was associated with greater post-vaccination IgG response (P < .01). Vaccination-alone elicited a greater IgG response but more rapid waning of IgG (P < .01) compared with infection-alone (P < .01). BNT162b2 and mRNA-1273 vaccine-receipt was associated with greater IgG responses compared with JNJ-78436735 vaccine-receipt (P < .01), regardless of infection history. Those with vaccine-after-infection or vaccine-breakthrough-infection had a more durable anti-spike-IgG response compared to infection-alone (P < .01). CONCLUSIONS: Vaccine-receipt elicited higher anti-spike-IgG responses than infection-alone, although IgG levels waned faster in those vaccinated (compared to infection-alone). Vaccine-after-infection elicits a greater humoral response compared with vaccine or infection alone; and the timing, but not disease severity, of prior infection predicted these post-vaccination IgG responses. While differences between groups were small in magnitude, these results offer insights into vaccine immunogenicity variations that may help inform vaccination timing strategies.

Evaluating SARS-CoV-2 Saliva and Dried Blood Spot Surveillance Strategies in a Congregate Population.

The optimal approach to COVID-19 surveillance in congregate populations remains unclear. Our study at the US Naval Academy in Annapolis, Maryland, USA, assessed the concordance of antibody prevalence in longitudinally collected dried blood spots and saliva in a setting of frequent PCR-based testing. Our findings highlight the utility of salivary-based surveillance.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) mRNA Vaccine-Breakthrough Infection Phenotype Includes Significant Symptoms, Live Virus Shedding, and Viral Genetic Diversity.

Little is known about severe acute respiratory syndrome coronavirus 2 "vaccine-breakthrough" infections (VBIs). Here we characterize 24 VBIs in predominantly young healthy persons. While none required hospitalization, a proportion endorsed severe symptoms and shed live virus as high as 4.13 × 103 plaque-forming units/mL. Infecting genotypes included both variant-of-concern (VOC) and non-VOC strains.

Novel Hendra Virus Variant Detected by Sentinel Surveillance of Horses in Australia.

We identified and isolated a novel Hendra virus (HeV) variant not detected by routine testing from a horse in Queensland, Australia, that died from acute illness with signs consistent with HeV infection. Using whole-genome sequencing and phylogenetic analysis, we determined the variant had ≈83% nt identity with prototypic HeV. In silico and in vitro comparisons of the receptor-binding protein with prototypic HeV support that the human monoclonal antibody m102.4 used for postexposure prophylaxis and current equine vaccine will be effective against this variant. An updated quantitative PCR developed for routine surveillance resulted in subsequent case detection. Genetic sequence consistency with virus detected in grey-headed flying foxes suggests the variant circulates at least among this species. Studies are needed to determine infection kinetics, pathogenicity, reservoir-species associations, viral-host coevolution, and spillover dynamics for this virus. Surveillance and biosecurity practices should be updated to acknowledge HeV spillover risk across all regions frequented by flying foxes.

Durability of Antibody Response and Frequency of SARS-CoV-2 Infection 6 Months after COVID-19 Vaccination in Healthcare Workers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies decay but persist 6 months postvaccination; lower levels of neutralizing titers persist against Delta than wild-type virus. Of 227 vaccinated healthcare workers tested, only 2 experienced outpatient symptomatic breakthrough infections, despite 59/227 exhibiting serologic evidence of SARS-CoV-2 infection, defined as presence of nucleocapsid protein antibodies.

Structural and functional analyses reveal promiscuous and species specific use of ephrin receptors by Cedar virus.

Cedar virus (CedV) is a bat-borne henipavirus related to Nipah virus (NiV) and Hendra virus (HeV), zoonotic agents of fatal human disease. CedV receptor-binding protein (G) shares only ∼30% sequence identity with those of NiV and HeV, although they can all use ephrin-B2 as an entry receptor. We demonstrate that CedV also enters cells through additional B- and A-class ephrins (ephrin-B1, ephrin-A2, and ephrin-A5) and report the crystal structure of the CedV G ectodomain alone and in complex with ephrin-B1 or ephrin-B2. The CedV G receptor-binding site is structurally distinct from other henipaviruses, underlying its capability to accommodate additional ephrin receptors. We also show that CedV can enter cells through mouse ephrin-A1 but not human ephrin-A1, which differ by 1 residue in the key contact region. This is evidence of species specific ephrin receptor usage by a henipavirus, and implicates additional ephrin receptors in potential zoonotic transmission.

Clinical, Immunological, and Virological SARS-CoV-2 Phenotypes in Obese and Nonobese Military Health System Beneficiaries.

BACKGROUND: The mechanisms underlying the association between obesity and coronavirus disease 2019 (COVID-19) severity remain unclear. After verifying that obesity was a correlate of severe COVID-19 in US Military Health System (MHS) beneficiaries, we compared immunological and virological phenotypes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in both obese and nonobese participants. METHODS: COVID-19-infected MHS beneficiaries were enrolled, and anthropometric, clinical, and demographic data were collected. We compared the SARS-CoV-2 peak IgG humoral response and reverse-transcription polymerase chain reaction viral load in obese and nonobese patients, stratified by hospitalization, utilizing logistic regression models. RESULTS: Data from 511 COVID-19 patients were analyzed, among whom 24% were obese and 14% severely obese. Obesity was independently associated with hospitalization (adjusted odds ratio [aOR], 1.91; 95% confidence interval [CI], 1.15-3.18) and need for oxygen therapy (aOR, 3.39; 95% CI, 1.61-7.11). In outpatients, severely obese had a log10 (1.89) higher nucleocapsid (N1) genome equivalents (GE)/reaction and log10 (2.62) higher N2 GE/reaction than nonobese (P = 0.03 and P < .001, respectively). We noted a correlation between body mass index and peak anti-spike protein IgG in inpatients and outpatients (coefficient = 5.48, P < .001). CONCLUSIONS: Obesity is a strong correlate of COVID-19 severity in MHS beneficiaries. These findings offer new pathophysiological insights into the relationship between obesity and COVID-19 severity.

Durability of SARS-CoV-2-Specific T-Cell Responses at 12 Months Postinfection.

BACKGROUND: Characterizing the longevity and quality of cellular immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enhances understanding of coronavirus disease 2019 (COVID-19) immunity that influences clinical outcomes. Prior studies suggest SARS-CoV-2-specific T cells are present in peripheral blood 10 months after infection. Analysis of the function, durability, and diversity of cellular response long after natural infection, over a range of ages and disease phenotypes, is needed to identify preventative and therapeutic interventions. METHODS: We identified participants in our multisite longitudinal, prospective cohort study 12 months after SARS-CoV-2 infection representing a range of disease severity. We investigated function, phenotypes, and frequency of T cells specific for SARS-CoV-2 using intracellular cytokine staining and spectral flow cytometry, and compared magnitude of SARS-CoV-2-specific antibodies. RESULTS: SARS-CoV-2-specific antibodies and T cells were detected 12 months postinfection. Severe acute illness was associated with higher frequencies of SARS-CoV-2-specific CD4 T cells and antibodies at 12 months. In contrast, polyfunctional and cytotoxic T cells responsive to SARS-CoV-2 were identified in participants over a wide spectrum of disease severity. CONCLUSIONS: SARS-CoV-2 infection induces polyfunctional memory T cells detectable at 12 months postinfection, with higher frequency noted in those who experienced severe disease.

Prospective Assessment of SARS-CoV-2 Seroconversion (PASS) study: an observational cohort study of SARS-CoV-2 infection and vaccination in healthcare workers.

BACKGROUND: SARS-CoV-2 is a recently emerged pandemic coronavirus (CoV) capable of causing severe respiratory illness. However, a significant number of infected people present as asymptomatic or pauci-symptomatic. In this prospective assessment of at-risk healthcare workers (HCWs) we seek to determine whether pre-existing antibody or T cell responses to previous seasonal human coronavirus (HCoV) infections affect immunological or clinical responses to SARS-CoV-2 infection or vaccination. METHODS: A cohort of 300 healthcare workers, confirmed negative for SARS-CoV-2 exposure upon study entry, will be followed for up to 1 year with monthly serology analysis of IgM and IgG antibodies against the spike proteins of SARS-CoV-2 and the four major seasonal human coronavirus - HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63. Participants will complete monthly questionnaires that ask about Coronavirus Disease 2019 (COVID-19) exposure risks, and a standardized, validated symptom questionnaire (scoring viral respiratory disease symptoms, intensity and severity) at least twice monthly and any day when any symptoms manifest. SARS-CoV-2 PCR testing will be performed any time participants develop symptoms consistent with COVID-19. For those individuals that seroconvert and/or test positive by SARS-CoV-2 PCR, or receive the SARS-CoV-2 vaccine, additional studies of T cell activation and cytokine production in response to SARS-CoV-2 peptide pools and analysis of Natural Killer cell numbers and function will be conducted on that participant's cryopreserved baseline peripheral blood mononuclear cells (PBMCs). Following the first year of this study we will further analyze those participants having tested positive for COVID-19, and/or having received an authorized/licensed SARS-CoV-2 vaccine, quarterly (year 2) and semi-annually (years 3 and 4) to investigate immune response longevity. DISCUSSION: This study will determine the frequency of asymptomatic and pauci-symptomatic SARS-CoV-2 infection in a cohort of at-risk healthcare workers. Baseline and longitudinal assays will determine the frequency and magnitude of anti-spike glycoprotein antibodies to the seasonal HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63, and may inform whether pre-existing antibodies to these human coronaviruses are associated with altered COVID-19 disease course. Finally, this study will evaluate whether pre-existing immune responses to seasonal HCoVs affect the magnitude and duration of antibody and T cell responses to SARS-CoV-2 vaccination, adjusting for demographic covariates.

Rousettus aegyptiacus Bats Do Not Support Productive Nipah Virus Replication.

Nipah virus (NiV) is a bat-borne zoonotic pathogen that can cause severe respiratory distress and encephalitis upon spillover into humans. NiV is capable of infecting a broad range of hosts including humans, pigs, ferrets, dogs, cats, hamsters, and at least 2 genera of bats. Little is known about the biology of NiV in the bat reservoir. In this study, we evaluate the potential for the Egyptian fruit bat (EFB), Rousettus aegyptiacus, to serve as a model organism for studying NiV in bats. Our data suggest that NiV does not efficiently replicate in EFBs in vivo. Furthermore, we show no seroconversion against NiV glycoprotein and a lack of viral replication in primary and immortalized EFB-derived cell lines. Our data show that despite using a conserved target for viral entry, NiV replication is limited in some bat species. We conclude that EFBs are not an appropriate organism to model NiV infection or transmission in bats.

Serological Evidence for Henipa-like and Filo-like Viruses in Trinidad Bats.

Bat-borne zoonotic pathogens belonging to the family Paramxyoviridae, including Nipah and Hendra viruses, and the family Filoviridae, including Ebola and Marburg viruses, can cause severe disease and high mortality rates on spillover into human populations. Surveillance efforts for henipaviruses and filoviruses have been largely restricted to the Old World; however, recent studies suggest a potentially broader distribution for henipaviruses and filoviruses than previously recognized. In the current study, we screened for henipaviruses and filoviruses in New World bats collected across 4 locations in Trinidad near the coast of Venezuela. Bat tissue samples were screened using previously established reverse-transcription polymerase chain reaction assays. Serum were screened using a multiplex immunoassay to detect antibodies reactive with the envelope glycoprotein of viruses in the genus Henipavirus and the family Filoviridae. Serum samples were also screened by means of enzyme-linked immunosorbent assay for antibodies reactive with Nipah G and F glycoproteins. Of 84 serum samples, 28 were reactive with ≥1 henipavirus glycoprotein by ≥1 serological method, and 6 serum samples were reactive against ≥1 filovirus glycoproteins. These data provide evidence of potential circulation of viruses related to the henipaviruses and filoviruses in New World bats.

Serologic Evidence of Fruit Bat Exposure to Filoviruses, Singapore, 2011-2016.

To determine whether fruit bats in Singapore have been exposed to filoviruses, we screened 409 serum samples from bats of 3 species by using a multiplex assay that detects antibodies against filoviruses. Positive samples reacted with glycoproteins from Bundibugyo, Ebola, and Sudan viruses, indicating filovirus circulation among bats in Southeast Asia.

Rescue and characterization of recombinant cedar virus, a non-pathogenic Henipavirus species.

BACKGROUND: Hendra virus and Nipah virus are zoonotic viruses that have caused severe to fatal disease in livestock and human populations. The isolation of Cedar virus, a non-pathogenic virus species in the genus Henipavirus, closely-related to the highly pathogenic Hendra virus and Nipah virus offers an opportunity to investigate differences in pathogenesis and receptor tropism among these viruses. METHODS: We constructed full-length cDNA clones of Cedar virus from synthetic oligonucleotides and rescued two replication-competent, recombinant Cedar virus variants: a recombinant wild-type Cedar virus and a recombinant Cedar virus that expresses a green fluorescent protein from an open reading frame inserted between the phosphoprotein and matrix genes. Replication kinetics of both viruses and stimulation of the interferon pathway were characterized in vitro. Cellular tropism for ephrin-B type ligands was qualitatively investigated by microscopy and quantitatively by a split-luciferase fusion assay. RESULTS: Successful rescue of recombinant Cedar virus expressing a green fluorescent protein did not significantly affect virus replication compared to the recombinant wild-type Cedar virus. We demonstrated that recombinant Cedar virus stimulated the interferon pathway and utilized the established Hendra virus and Nipah virus receptor, ephrin-B2, but not ephrin-B3 to mediate virus entry. We further characterized virus-mediated membrane fusion kinetics of Cedar virus with the known henipavirus receptors ephrin-B2 and ephrin-B3. CONCLUSIONS: The recombinant Cedar virus platform may be utilized to characterize the determinants of pathogenesis across the henipaviruses, investigate their receptor tropisms, and identify novel pan-henipavirus antivirals. Moreover, these experiments can be conducted safely under BSL-2 conditions.

Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5.

BACKGROUND: Australian bat lyssavirus (ABLV), a rhabdovirus of the genus Lyssavirus which circulates in both pteropid fruit bats and insectivorous bats in mainland Australia, has caused three fatal human infections, the most recent in February 2013, manifested as acute neurological disease indistinguishable from clinical rabies. Rhabdoviruses infect host cells through receptor-mediated endocytosis and subsequent pH-dependent fusion mediated by their single envelope glycoprotein (G), but the specific host factors and pathways involved in ABLV entry have not been determined. METHODS: ABLV internalization into HEK293T cells was examined using maxGFP-encoding recombinant vesicular stomatitis viruses (rVSV) that express ABLV G glycoproteins. A combination of chemical and molecular approaches was used to investigate the contribution of different endocytic pathways to ABLV entry. Dominant negative Rab GTPases were used to identify the endosomal compartment utilized by ABLV to gain entry into the host cell cytosol. RESULTS: Here we show that ABLV G-mediated entry into HEK293T cells was significantly inhibited by the dynamin-specific inhibitor dynasore, chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and the actin depolymerizing drug latrunculin B. Over expression of dominant negative mutants of Eps15 and Rab5 also significantly reduced ABLV G-mediated entry into HEK293T cells. Chemical inhibitors of caveolae-dependent endocytosis and macropinocytosis and dominant negative mutants of Rab7 and Rab11 had no effect on ABLV entry. CONCLUSIONS: The predominant pathway utilized by ABLV for internalization into HEK293T cells is clathrin-and actin-dependent. The requirement of Rab5 for productive infection indicates that ABLV G-mediated fusion occurs within the early endosome compartment.

An in vivo BSL-2 model for henipavirus infection based on bioluminescence imaging of recombinant Cedar virus replication in mice.

Henipaviruses are enveloped single-stranded, negative-sense RNA viruses of the paramyxovirus family. Two henipaviruses, Nipah virus and Hendra virus, cause a systemic respiratory and/or neurological disease in humans and ten additional species of mammals, with a high fatality rate. Because of their highly pathogenic nature, Nipah virus and Hendra virus are categorized as BSL-4 pathogens, which limits the number and scope of translational research studies on these important human pathogens. To begin to address this limitation, we are developing a BSL-2 model of authentic henipavirus infection in mice, using the non-pathogenic henipavirus, Cedar virus. Notably, wild-type mice are highly resistant to Hendra virus and Nipah virus infection. However, previous work has shown that mice lacking expression of the type I interferon receptor (IFNAR-KO mice) are susceptible to both viruses. Here, we show that luciferase-expressing recombinant Cedar virus (rCedV-luc) is also able to replicate and establish a transient infection in IFNAR-KO mice, but not in wild-type mice. Using longitudinal bioluminescence imaging (BLI) of luciferase expression, we detected rCedV-luc replication as early as 10 h post-infection. Viral replication peaks between days 1 and 3 post-infection, and declines to levels undetectable by BLI by 7 days post-infection. Immunohistochemistry is consistent with viral infection and replication in endothelial cells and other non-immune cell types within tissue parenchyma. Serology analyses demonstrate significant IgG responses to the Cedar virus surface glycoprotein with potent neutralizing activity in IFNAR-KO mice, whereas antibody responses in wild-type animals were non-significant. Overall, these data suggest that rCedV-luc infection of IFNAR-KO mice represents a viable platform for the study of in vivo henipavirus replication, anti-henipavirus host responses and henipavirus-directed therapeutics.

Serological evidence of zoonotic filovirus exposure among bushmeat hunters in Guinea.

Human Ebola virus (EBOV) outbreaks caused by persistent EBOV infection raises questions on the role of zoonotic spillover in filovirus epidemiology. To characterise filovirus zoonotic exposure, we collected cross-sectional serum samples from bushmeat hunters (n = 498) in Macenta Prefecture Guinea, adjacent to the index site of the 2013 EBOV-Makona spillover event. We identified distinct immune signatures (20/498, 4.0%) to multiple EBOV antigens (GP, NP, VP40) using stepwise ELISA and Western blot analysis and, live EBOV neutralisation (5/20; 25%). Using comparative serological data from PCR-confirmed survivors of the 2013-2016 EBOV outbreak, we demonstrated that most signatures (15/20) were not plausibly explained by prior EBOV-Makona exposure. Subsequent data-driven modelling of EBOV immunological outcomes to remote-sensing environmental data also revealed consistent associations with intact closed canopy forest. Together our findings suggest exposure to other closely related filoviruses prior to the 2013-2016 West Africa epidemic and highlight future surveillance priorities.

Antibody targeting of conserved sites of vulnerability on the SARS-CoV-2 spike receptor-binding domain.

Given the continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoCs), immunotherapeutics that target conserved epitopes on the spike (S) glycoprotein have therapeutic advantages. Here, we report the crystal structure of the SARS-CoV-2 S receptor-binding domain (RBD) at 1.95 Å and describe flexibility and distinct conformations of the angiotensin-converting enzyme 2 (ACE2)-binding site. We identify a set of SARS-CoV-2-reactive monoclonal antibodies (mAbs) with broad RBD cross-reactivity including SARS-CoV-2 Omicron subvariants, SARS-CoV-1, and other sarbecoviruses and determine the crystal structures of mAb-RBD complexes with Ab246 and CR3022 mAbs targeting the class IV site, WRAIR-2134, which binds the recently designated class V epitope, and WRAIR-2123, the class I ACE2-binding site. The broad reactivity of class IV and V mAbs to conserved regions of SARS-CoV-2 VoCs and other sarbecovirus provides a framework for long-term immunotherapeutic development strategies.

Immune and behavioral correlates of protection against symptomatic post-vaccination SARS-CoV-2 infection.

Introduction: We sought to determine pre-infection correlates of protection against SARS-CoV-2 post-vaccine inzfections (PVI) acquired during the first Omicron wave in the United States. Methods: Serum and saliva samples from 176 vaccinated adults were collected from October to December of 2021, immediately before the Omicron wave, and assessed for SARS-CoV-2 Spike-specific IgG and IgA binding antibodies (bAb). Sera were also assessed for bAb using commercial assays, and for neutralization activity against several SARS-CoV-2 variants. PVI duration and severity, as well as risk and precautionary behaviors, were assessed by questionnaires. Results: Serum anti-Spike IgG levels assessed by research assay, neutralization titers against Omicron subvariants, and low home risk scores correlated with protection against PVIs after multivariable regression analysis. Commercial assays did not perform as well as research assay, likely due to their lower dynamic range. Discussion: In the 32 participants that developed PVI, anti-Spike IgG bAbs correlated with lower disease severity and shorter duration of illness.

Recombinant Cedar Virus: A Henipavirus Reverse Genetics Platform.

The isolation of Cedar virus, a nonpathogenic henipavirus that is closely related to the highly pathogenic Nipah virus and Hendra virus, provides a new platform for henipavirus experimentation and a tool to investigate biological differences among these viruses under less stringent biological containment. Here, we detail a reverse genetics system used to rescue two replication-competent, recombinant Cedar virus variants: a recombinant wild-type Cedar virus and a recombinant Cedar virus that express a green fluorescent protein from an open reading frame inserted between the phosphoprotein and matrix genes. This recombinant Cedar virus platform may be utilized to characterize the determinants of pathogenesis across the henipaviruses, investigate their receptor tropisms, and identify novel pan-henipavirus antivirals safely under biosafety level-2 conditions.