Comparative analysis of SARS-CoV-2 neutralization titers reveals consistency between human and animal model serum and across assays.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires ongoing monitoring to judge the ability of newly arising variants to escape the immune response. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal serum samples. We compared 18 datasets generated using human, hamster, and mouse serum and six different neutralization assays. Datasets using animal model serum samples showed higher titer magnitudes than datasets using human serum samples in this comparison. Fold change in neutralization of variants compared to ancestral SARS-CoV-2, immunodominance patterns, and antigenic maps were similar among serum samples and assays. Most assays yielded consistent results, except for differences in fold change in cytopathic effect assays. Hamster serum samples were a consistent surrogate for human first-infection serum samples. These results inform the transition of surveillance of SARS-CoV-2 antigenic variation from dependence on human first-infection serum samples to the utilization of serum samples from animal models.

Ipsilateral and contralateral coadministration of influenza and COVID-19 vaccines produce similar antibody responses.

BACKGROUND: World Health Organisation (WHO) and USA Centers for Disease Control and Prevention (U.S. CDC) recommendations now allow simultaneous administration of COVID-19 and other vaccines. We compared antibody responses after coadministration of influenza and bivalent COVID-19 vaccines in the same (ipsilateral) arm vs. different (contralateral) arms. METHODS: Pre- and post-vaccination serum samples from individuals in the Prospective Assessment of COVID-19 in a Community (PACC) cohort were used to conduct haemaglutination inhibition (HI) assays with the viruses in the 2022-2023 seasonal influenza vaccine and focus reduction neutralisation tests (FRNT) using a BA.5 SARS-CoV-2 virus. The effect of ipsilateral vs. contralateral vaccination on immune responses was inferred in a model that accounted for higher variance in vaccine responses at lower pre-vaccination titers. FINDINGS: Ipsilateral vaccination did not cause higher influenza vaccine responses compared to contralateral vaccination. The response to SARS-CoV-2 was slightly increased in the ipsilateral group, but equivalence was not excluded. INTERPRETATION: Coadministration of influenza and bivalent COVID-19 vaccines in the same arm or different arms did not strongly influence the antibody response to either vaccine. FUNDING: This work was supported by the U.S. CDC (grant number: 75D30120C09259).

A compendium of multi-omics data illuminating host responses to lethal human virus infections.

Human infections caused by viral pathogens trigger a complex gamut of host responses that limit disease, resolve infection, generate immunity, and contribute to severe disease or death. Here, we present experimental methods and multi-omics data capture approaches representing the global host response to infection generated from 45 individual experiments involving human viruses from the Orthomyxoviridae, Filoviridae, Flaviviridae, and Coronaviridae families. Analogous experimental designs were implemented across human or mouse host model systems, longitudinal samples were collected over defined time courses, and global multi-omics data (transcriptomics, proteomics, metabolomics, and lipidomics) were acquired by microarray, RNA sequencing, or mass spectrometry analyses. For comparison, we have included transcriptomics datasets from cells treated with type I and type II human interferon. Raw multi-omics data and metadata were deposited in public repositories, and we provide a central location linking the raw data with experimental metadata and ready-to-use, quality-controlled, statistically processed multi-omics datasets not previously available in any public repository. This compendium of infection-induced host response data for reuse will be useful for those endeavouring to understand viral disease pathophysiology and network biology.

Tracing the origin of SARS-CoV-2 omicron-like spike sequences detected in an urban sewershed: a targeted, longitudinal surveillance study of a cryptic wastewater lineage.

BACKGROUND: The origin of novel SARS-CoV-2 spike sequences found in wastewater, without corresponding detection in clinical specimens, remains unclear. We sought to determine the origin of one such cryptic wastewater lineage by tracking and characterising its persistence and genomic evolution over time. METHODS: We first detected a cryptic lineage, WI-CL-001, in municipal wastewater in Wisconsin, USA, in January, 2022. To determine the source of WI-CL-001, we systematically sampled wastewater from targeted sub-sewershed lines and maintenance holes using compositing autosamplers. Viral concentrations in wastewater samples over time were measured by RT digital PCR. In addition to using metagenomic 12s rRNA sequencing to determine the virus's host species, we also sequenced SARS-CoV-2 spike receptor binding domains, and, where possible, whole viral genomes to identify and characterise the evolution of this lineage. FINDINGS: We traced WI-CL-001 to its source at a single commercial building. There we detected the cryptic lineage at concentrations as high as 2·7 × 109 genome copies per L. The majority of 12s rRNA sequences detected in wastewater leaving the identified source building were human. Additionally, we generated over 100 viral receptor binding domain and whole-genome sequences from wastewater samples containing the cryptic lineage collected over the 13 consecutive months this virus was detectable (January, 2022, to January, 2023). These sequences contained a combination of fixed nucleotide substitutions characteristic of Pango lineage B.1.234, which circulated in humans in Wisconsin at low levels from October, 2020, to February, 2021. Despite this, mutations in the spike gene and elsewhere resembled those subsequently found in omicron variants. INTERPRETATION: We propose that prolonged detection of WI-CL-001 in wastewater indicates persistent shedding of SARS-CoV-2 from a single human initially infected by an ancestral B.1.234 virus. The accumulation of convergent omicron-like mutations in WI-CL-001's ancestral B.1.234 genome probably reflects persistent infection and extensive within-host evolution. People who shed cryptic lineages could be an important source of highly divergent viruses that sporadically emerge and spread. FUNDING: The Rockefeller Foundation, Wisconsin Department of Health Services, Centers for Disease Control and Prevention, National Institute on Drug Abuse, and the Center for Research on Influenza Pathogenesis and Transmission.

Characterization of Omicron BA.4.6, XBB, and BQ.1.1 subvariants in hamsters.

During the Omicron wave, previous variants such as BA.2, BA.4, and BA.5 were replaced by newer variants with additional mutations in the spike protein. These variants, BA.4.6, BQ.1.1, and XBB, have spread in different countries with different degrees of success. Here, we evaluated the replicative ability and pathogenicity of BA.4.6, BQ1.1, and XBB clinical isolates in male Syrian hamsters. Although we found no substantial differences in weight change among hamsters infected with these Omicron subvariants, the replicative ability of BQ.1.1 and XBB in lung tissue was higher than that of BA.4.6 and BA.5. Of note, BQ.1.1 was lethal in both male and female transgenic human ACE2 hamsters. In competition assays, XBB replicated better than BQ.1.1 in the nasal turbinate tissues of female hamsters previously infected with Omicron BA.2. These results suggest that newer Omicron subvariants in the XBB family are still evolving and should be closely monitored.

Persistent SARS-CoV-2 infection: significance and implications.

SARS-CoV-2 causes persistent infections in a subset of individuals, which is a major clinical and public health problem that should be prioritised for further investigation for several reasons. First, persistent SARS-CoV-2 infection often goes unrecognised, and therefore might affect a substantial number of people, particularly immunocompromised individuals. Second, the formation of tissue reservoirs (including in non-respiratory tissues) might underlie the pathophysiology of the persistent SARS-CoV-2 infection and require new strategies for diagnosis and treatment. Finally, persistent SARS-CoV-2 replication, particularly in the setting of suboptimal immune responses, is a possible source of new, divergent virus variants that escape pre-existing immunity on the individual and population levels. Defining optimal diagnostic and treatment strategies for patients with persistent virus replication and monitoring viral evolution are therefore urgent medical and public health priorities.

Broad protection against clade 1 sarbecoviruses after a single immunization with cocktail spike-protein-nanoparticle vaccine.

The 2002 SARS outbreak, the 2019 emergence of COVID-19, and the continuing evolution of immune-evading SARS-CoV-2 variants together highlight the need for a broadly protective vaccine against ACE2-utilizing sarbecoviruses. While updated variant-matched formulations are a step in the right direction, protection needs to extend beyond SARS-CoV-2 and its variants to include SARS-like viruses. Here, we introduce bivalent and trivalent vaccine formulations using our spike protein nanoparticle platform that completely protect female hamsters against BA.5 and XBB.1 challenges with no detectable virus in the lungs. The trivalent cocktails elicit highly neutralizing responses against all tested Omicron variants and the bat sarbecoviruses SHC014 and WIV1. Finally, our 614D/SHC014/XBB trivalent spike formulation completely protects human ACE2-transgenic female hamsters against challenges with WIV1 and SHC014 with no detectable virus in the lungs. Collectively, these results illustrate that our trivalent protein-nanoparticle cocktail can provide broad protection against SARS-CoV-2-like and SARS-CoV-1-like sarbecoviruses.

Defining Distinct RNA-Protein Interactomes of SARS-CoV-2 Genomic and Subgenomic RNAs.

Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. Individual interactomes indicated viral associations with cell response pathways, including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We tested the significance of three protein interactors in these pathways (APOBEC3F, PPP1CC, and MSI2) using siRNA knockdowns, with several knockdowns affecting viral gene expression, most consistently PPP1CC. This study describes a new technology for high-resolution studies of SARS-CoV-2 RNA regulation and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.

Neutralizing Immunity Against Antigenically Advanced Omicron BA.5 in Children After SARS-CoV-2 Infection.

We assessed serum neutralization of Omicron BA.5 in children following SARS-CoV-2 infection during the Delta or Omicron BA.1/BA.2 variant period. Convalescent BA.5 titers were higher following infections during the Omicron BA.1/BA.2 vs Delta variant period, and in vaccinated vs unvaccinated children. Titers against BA.5 did not differ by age group.

Comparative Analysis of SARS-CoV-2 Antigenicity across Assays and in Human and Animal Model Sera.

The antigenic evolution of SARS-CoV-2 requires ongoing monitoring to judge the immune escape of newly arising variants. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal sera. We compared 18 datasets generated using human, hamster, and mouse sera, and six different neutralization assays. Titer magnitude was lowest in human, intermediate in hamster, and highest in mouse sera. Fold change, immunodominance patterns and antigenic maps were similar among sera. Most assays yielded similar results, except for differences in fold change in cytopathic effect assays. Not enough data was available for conclusively judging mouse sera, but hamster sera were a consistent surrogate for human first-infection sera.

Airway surveillance and lung viral control by memory T cells induced by COVID-19 mRNA vaccine.

Although SARS-CoV-2 evolution seeds a continuous stream of antibody-evasive viral variants, COVID-19 mRNA vaccines provide robust protection against severe disease and hospitalization. Here, we asked whether mRNA vaccine-induced memory T cells limit lung SARS-CoV-2 replication and severe disease. We show that mice and humans receiving booster BioNTech mRNA vaccine developed potent CD8 T cell responses and showed similar kinetics of expansion and contraction of granzyme B/perforin-expressing effector CD8 T cells. Both monovalent and bivalent mRNA vaccines elicited strong expansion of a heterogeneous pool of terminal effectors and memory precursor effector CD8 T cells in spleen, inguinal and mediastinal lymph nodes, pulmonary vasculature, and most surprisingly in the airways, suggestive of systemic and regional surveillance. Furthermore, we document that: (a) CD8 T cell memory persists in multiple tissues for > 200 days; (b) following challenge with pathogenic SARS-CoV-2, circulating memory CD8 T cells rapidly extravasate to the lungs and promote expeditious viral clearance, by mechanisms that require CD4 T cell help; and (c) adoptively transferred splenic memory CD8 T cells traffic to the airways and promote lung SARS-CoV-2 clearance. These findings provide insights into the critical role of memory T cells in preventing severe lung disease following breakthrough infections with antibody-evasive SARS-CoV-2 variants.

Mapping SARS-CoV-2 antigenic relationships and serological responses.

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, multiple variants escaping preexisting immunity emerged, causing reinfections of previously exposed individuals. Here, we used antigenic cartography to analyze patterns of cross-reactivity among 21 variants and 15 groups of human sera obtained after primary infection with 10 different variants or after messenger RNA (mRNA)-1273 or mRNA-1273.351 vaccination. We found antigenic differences among pre-Omicron variants caused by substitutions at spike-protein positions 417, 452, 484, and 501. Quantifying changes in response breadth over time and with additional vaccine doses, our results show the largest increase between 4 weeks and >3 months after a second dose. We found changes in immunodominance of different spike regions, depending on the variant an individual was first exposed to, with implications for variant risk assessment and vaccine-strain selection.

Host range, transmissibility and antigenicity of a pangolin coronavirus.

The pathogenic and cross-species transmission potential of SARS-CoV-2-related coronaviruses (CoVs) remain poorly characterized. Here we recovered a wild-type pangolin (Pg) CoV GD strain including derivatives encoding reporter genes using reverse genetics. In primary human cells, PgCoV replicated efficiently but with reduced fitness and showed less efficient transmission via airborne route compared with SARS-CoV-2 in hamsters. PgCoV was potently inhibited by US Food and Drug Administration approved drugs, and neutralized by COVID-19 patient sera and SARS-CoV-2 therapeutic antibodies in vitro. A pan-Sarbecovirus antibody and SARS-CoV-2 S2P recombinant protein vaccine protected BALB/c mice from PgCoV infection. In K18-hACE2 mice, PgCoV infection caused severe clinical disease, but mice were protected by a SARS-CoV-2 human antibody. Efficient PgCoV replication in primary human cells and hACE2 mice, coupled with a capacity for airborne spread, highlights an emergence potential. However, low competitive fitness, pre-immune humans and the benefit of COVID-19 countermeasures should impede its ability to spread globally in human populations.

Evolution of a globally unique SARS-CoV-2 Spike E484T monoclonal antibody escape mutation in a persistently infected, immunocompromised individual.

Prolonged infections in immunocompromised individuals may be a source for novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants, particularly when both the immune system and antiviral therapy fail to clear the infection and enable within-host evolution. Here we describe a 486-day case of SARS-CoV-2 infection in an immunocompromised individual. Following monotherapy with the monoclonal antibody Bamlanivimab, the individual's virus acquired resistance, likely via the earliest known occurrence of Spike amino acid variant E484T. Recently, E484T has arisen again as a derivative of E484A in the Omicron Variant of Concern, supporting the hypothesis that prolonged infections can give rise to novel variants long before they become prevalent in the human population.

Ebola Virus Infection Induces HCAR2 Expression Leading to Cell Death.

Ebola virus (EBOV) induces cell death not only in infected permissive cells but also in nonpermissive, bystander cells by employing different mechanisms. Hydroxycarboxylic acid receptor 2 (HCAR2) has been reported to be involved in apoptotic cell death. We previously reported an increase in the expression of HCAR2-specific mRNA in EBOV-infected individuals with fatal outcomes. Here, we report that infection with an EBOV lacking the VP30 gene (EBOVΔVP30) results in the upregulation of HCAR2 mRNA expression in human hepatocyte Huh7.0 cells stably expressing VP30. Transient overexpression of HCAR2 reduced the viability of Huh7.0 cells and human embryonic kidney cells. Phosphatidylserine externalization and cell membrane permeabilization by HCAR2 overexpression was also observed. Interestingly, coexpression of HCAR2 with EBOV VP40 further reduced cell viability in transfected cells compared to HCAR2 coexpression with other viral proteins. Our data suggest that HCAR2 may contribute to EBOV-induced cell death.

An Antiviral Role for TRIM14 in Ebola Virus Infection.

Ebola virus (EBOV) is a highly pathogenic virus that encodes 7 multifunctional structural proteins. Multiple host factors have been reported to interact with the EBOV proteins. Here, we found that tripartite motif-containing 14 (TRIM14), an interferon-stimulated gene that mediates cellular signaling pathways associated with type I interferon and inflammatory cytokine production, interacts with EBOV nucleoprotein to enhance interferon-ß (IFN-ß) and nuclear factor-κB (NF-κB) promotor activation. Moreover, TRIM14 overexpression reduced viral replication in an infectious but biologically contained EBOVΔVP30 system by approximately 10-fold without affecting viral protein expression. Furthermore, TRM14-deficient mice were more susceptible to mouse-adapted EBOV infection than wild-type mice. Our data suggest that TRIM14 is a host factor with anti-EBOV activity that limits EBOV pathogenesis.

Recombinant spike protein vaccines coupled with adjuvants that have different modes of action induce protective immunity against SARS-CoV-2.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a glycoprotein, expressed on the virion surface, that mediates infection of host cells by directly interacting with host receptors. As such, it is a reasonable target to neutralize the infectivity of the virus. Here we found that a recombinant S protein vaccine adjuvanted with Alhydrogel or the QS-21-like adjuvant Quil-A effectively induced anti-S receptor binding domain (RBD) serum IgG and neutralizing antibody titers in the Syrian hamster model, resulting in significantly low SARS-CoV-2 replication in respiratory organs and reduced body weight loss upon virus challenge. Severe lung inflammation upon virus challenge was also strongly suppressed by vaccination. We also found that the S protein vaccine adjuvanted with Alhydrogel, Quil-A, or an AS03-like adjuvant elicited significantly higher neutralizing antibody titers in mice than did unadjuvanted vaccine. Although the neutralizing antibody titers against the variant viruses B.1.351 and B.1.617.2 declined markedly in mice immunized with wild-type S protein, the binding antibody levels against the variant S proteins were equivalent to those against wild-type S. When splenocytes from the immunized mice were re-stimulated with the S protein in vitro, the induced Th1 or Th2 cytokine levels were not significantly different upon re-stimulation with wild-type S or variant S, suggesting that the T-cell responses against the variants were the same as those against the wild-type virus. Upon Omicron XBB-challenge in hamsters, wild-type S-vaccination with Alhydrogel or AS03 reduced lung virus titers on Day 3, and the Quil-A adjuvanted group showed less body weight loss, although serum neutralizing antibody titers against XBB were barely detected in vitro. Collectively, recombinant vaccines coupled with different adjuvants may be promising modalities to combat new variant viruses by inducing various arms of the immune response.

Broad Protection Against Clade 1 Sarbecoviruses After a Single Immunization with Cocktail Spike-Protein-Nanoparticle Vaccine.

The 2002 SARS outbreak, the 2019 emergence of COVID-19, and the continuing evolution of immune-evading SARS-CoV-2 variants together highlight the need for a broadly protective vaccine against ACE2-utilizing sarbecoviruses. While updated variant-matched formulations such as Pfizer-BioNTech's bivalent vaccine are a step in the right direction, protection needs to extend beyond SARS-CoV-2 and its variants to include SARS-like viruses. Here, we introduce bivalent and trivalent vaccine formulations using our spike protein nanoparticle platform that completely protected hamsters against BA.5 and XBB.1 challenges with no detectable virus in the lungs. The trivalent cocktails elicited highly neutralizing responses against all tested Omicron variants and the bat sarbecoviruses SHC014 and WIV1. Finally, our 614D/SHC014/XBB trivalent spike formulation completely protected human ACE2-transgenic hamsters against challenges with WIV1 and SHC014 with no detectable virus in the lungs. Collectively, these results illustrate that our trivalent protein-nanoparticle cocktail can provide broad protection against SARS-CoV-2-like and SARS-CoV-1-like sarbecoviruses.

Defining distinct RNA-protein interactomes of SARS-CoV-2 genomic and subgenomic RNAs.

Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral defense mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA at either of two time points. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. The interactomes indicated viral associations with cell response pathways including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We validated the significance of five protein interactors predicted to exhibit antiviral activity (APOBEC3F, TRIM71, PPP1CC, LIN28B, and MSI2) using siRNA knockdowns, with each knockdown yielding increases in viral production. This study describes new technology for studying SARS-CoV-2 and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.