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Summary paragraphSmall animal models have been a challenge for the study of SARS-CoV-2 transmission, with most investigators using golden hamsters or ferrets 1,2. Mice have the advantages of low cost, wide availability, less regulatory and husbandry challenges, and the existence of a versatile reagent and genetic toolbox. However, adult mice do not transmit SARS-CoV-2 3. Here we establish a model based on neonatal mice that allows for transmission of clinical SARS-CoV-2 isolates. We characterize tropism, respiratory tract replication and transmission of ancestral WA-1 compared to variants alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2) and omicron (B.1.1.529). We identify inter-variant differences in timing and magnitude of infectious particle shedding from index mice, both of which shape transmission to contact mice. Furthermore, we characterize two recombinant SARS-CoV-2 lacking either the ORF6 or ORF8 host antagonists. The removal of ORF8 shifts viral replication towards the lower respiratory tract, resulting in significantly delayed and reduced transmission. Our results demonstrate the potential of our neonatal mouse model to characterize viral and host determinants of SARS-CoV-2 transmission, while revealing for the first time a role for an accessory protein this context.
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Due to the recrudescence of SARS-CoV-2 infections worldwide, mainly caused by Omicron BA.1 and BA.2 variants of concern, several jurisdictions are administering a mRNA vaccine boost. Here, we analyzed humoral responses induced after the second and third doses of mRNA vaccine in naive and previously-infected donors who received their second dose with an extended 16-week interval. We observed that the extended interval elicited robust humoral responses against VOCs, but this response was significantly diminished 4 months after the second dose. Administering a boost to these individuals brought back the humoral responses to the same levels obtained after the extended second dose. Interestingly, we observed that administering a boost to individuals that initially received a short 3-4 weeks regimen elicited humoral responses similar to those elicited in the long interval regimen. Nevertheless, humoral responses elicited by the boost in naive individuals did not reach those present in previously-infected vaccinated individuals.
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BackgroundThe emergence of recombinant viruses is a threat to public health. Recombination of viral variants may combine variant-specific features that together catalyze viral escape from treatment or immunity. The selective advantages of recombinant SARS-CoV-2 isolates over their parental lineages remain unknown. MethodsMulti-method amplicon and metagenomic sequencing of a clinical swab and the in vitro grown virus allowed for high-confidence detection of a novel recombinant variant. Mutational, phylogeographic, and structural analyses determined features of the recombinant genome and spike protein. Neutralization assays using infectious as well as pseudotyped viruses and point mutants thereof defined the recombinants sensitivity to a panel of monoclonal antibodies and sera from vaccinated and/or convalescent individuals. ResultsA novel Delta-Omicron SARS-CoV-2 recombinant was identified in an unvaccinated, immunosuppressed kidney transplant recipient treated with monoclonal antibody Sotrovimab. The recombination breakpoint is located in the spike N-terminal domain, adjacent to the Sotrovimab quaternary binding site, and results in a 5-Delta AY.45 and a 3-Omicron BA.1 mosaic spike protein. Delta and BA.1 are sensitive to Sotrovimab neutralization, whereas the Delta-Omicron recombinant is highly resistant to Sotrovimab, both with and without the RBD resistance mutation E340D. ConclusionsRecombination between circulating SARS-CoV-2 variants can functionally contribute to immune escape. It is critical to validate phenotypes of mosaic viruses and monitor immunosuppressed COVID-19 patients treated with monoclonal antibodies for the selection of recombinant and immune escape variants. (Funded by NYU, the National Institutes of Health, and others)
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In 2021, Delta has become the predominant SARS-CoV-2 variant worldwide. While vaccines effectively prevent COVID-19 hospitalization and death, vaccine breakthrough infections increasingly occur. The precise role of clinical and genomic determinants in Delta infections is not known, and whether they contribute to increased rates of breakthrough infections compared to unvaccinated controls. Here, we show a steep and near complete replacement of circulating variants with Delta between May and August 2021 in metropolitan New York. We observed an increase of the Delta sublineage AY.25, its spike mutation S112L, and nsp12 mutation F192V in breakthroughs. Delta infections were associated with younger age and lower hospitalization rates than Alpha. Delta breakthroughs increased significantly with time since vaccination, and, after adjusting for confounders, they rose at similar rates as in unvaccinated individuals. Our data indicate a limited impact of vaccine escape in favor of Deltas increased epidemic growth in times of waning vaccine protection.
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While the standard regimen of the BNT162b2 mRNA vaccine includes two doses administered three weeks apart, some public health authorities decided to space them, raising concerns about vaccine efficacy. Here, we analyzed longitudinal humoral responses including antibody binding, Fc-mediated effector functions and neutralizing activity against the D614G strain but also variants of concern and SARS-CoV-1 in a cohort of SARS-CoV-2 naive and previously infected individuals, with an interval of sixteen weeks between the two doses. While the administration of a second dose to previously infected individuals did not significantly improve humoral responses, we observed a significant increase of humoral responses in naive individuals after the 16-weeks delayed second shot, achieving similar levels as in previously infected individuals. We compared these responses to those elicited in individuals receiving a short (4-weeks) dose interval. For the naive donors, these responses were superior to those elicited by the short dose interval.
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The efficacy of COVID-19 mRNA vaccines is high, but breakthrough infections still occur. We compared the SARS-CoV-2 genomes of 76 breakthrough cases after full vaccination with BNT162b2 (Pfizer/BioNTech), mRNA-1273 (Moderna), or JNJ-78436735 (Janssen) to unvaccinated controls (February-April 2021) in metropolitan New York, including their phylogenetic relationship, distribution of variants, and full spike mutation profiles. Their median age was 48 years; seven required hospitalization and one died. Most breakthrough infections (57/76) occurred with B.1.1.7 (Alpha) or B.1.526 (Iota). Among the 7 hospitalized cases, 4 were infected with B.1.1.7, including 1 death. Both unmatched and matched statistical analyses considering age, sex, vaccine type, and study month as covariates supported the null hypothesis of equal variant distributions between vaccinated and unvaccinated in chi-squared and McNemar tests (p>0.1) highlighting a high vaccine efficacy against B.1.1.7 and B.1.526. There was no clear association among breakthroughs between type of vaccine received and variant. In the vaccinated group, spike mutations in the N-terminal domain and receptor-binding domain that have been associated with immune evasion were overrepresented. The evolving dynamic of SARS-CoV-2 variants requires broad genomic analyses of breakthrough infections to provide real-life information on immune escape mediated by circulating variants and their spike mutations.
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The standard dosing of the Pfizer/BioNTech BNT162b2 mRNA vaccine validated in clinical trials includes two doses administered three weeks apart. While the decision by some public health authorities to space the doses because of limiting supply has raised concerns about vaccine efficacy, data indicate that a single dose is up to 90% effective starting 14 days after its administration. We analyzed humoral and T cells responses three weeks after a single dose of this mRNA vaccine. Despite the proven efficacy of the vaccine at this time point, no neutralizing activity were elicited in SARS-CoV-2 naive individuals. However, we detected strong anti-receptor binding domain (RBD) and Spike antibodies with Fc-mediated effector functions and cellular responses dominated by the CD4+ T cell component. A single dose of this mRNA vaccine to individuals previously infected by SARS-CoV-2 boosted all humoral and T cell responses measured, with strong correlations between T helper and antibody immunity. Neutralizing responses were increased in both potency and breadth, with distinctive capacity to neutralize emerging variant strains. Our results highlight the importance of vaccinating uninfected and previously-infected individuals and shed new light into the potential role of Fc-mediated effector functions and T cell responses in vaccine efficacy. They also provide support to spacing the doses of two-vaccine regimens to vaccinate a larger pool of the population in the context of vaccine scarcity against SARS-CoV-2.
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Mortality among patients with COVID-19 and respiratory failure is high and there are no known lower airway biomarkers that predict clinical outcome. We investigated whether bacterial respiratory infections and viral load were associated with poor clinical outcome and host immune tone. We obtained bacterial and fungal culture data from 589 critically ill subjects with COVID-19 requiring mechanical ventilation. On a subset of the subjects that underwent bronchoscopy, we also quantified SARS-CoV-2 viral load, analyzed the microbiome of the lower airways by metagenome and metatranscriptome analyses and profiled the host immune response. We found that isolation of a hospital-acquired respiratory pathogen was not associated with fatal outcome. However, poor clinical outcome was associated with enrichment of the lower airway microbiota with an oral commensal (Mycoplasma salivarium), while high SARS-CoV-2 viral burden, poor anti-SARS-CoV-2 antibody response, together with a unique host transcriptome profile of the lower airways were most predictive of mortality. Collectively, these data support the hypothesis that 1) the extent of viral infectivity drives mortality in severe COVID-19, and therefore 2) clinical management strategies targeting viral replication and host responses to SARS-CoV-2 should be prioritized.
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Functional and lasting immune responses to the novel coronavirus (SARS-CoV-2) are currently under intense investigation as antibody titers in plasma have been shown to decline during convalescence. Since the absence of antibodies does not equate to absence of immune memory, we sought to determine the presence of SARS-CoV-2-specific memory B cells in COVID-19 convalescent patients. In this study, we report on the evolution of the overall humoral immune responses on 101 blood samples obtained from 32 COVID-19 convalescent patients between 16 and 233 days post-symptom onset. Our observations indicate that anti-Spike and anti-RBD IgM in plasma decay rapidly, whereas the reduction of IgG is less prominent. Neutralizing activity in convalescent plasma declines rapidly compared to Fc-effector functions. Concomitantly, the frequencies of RBD-specific IgM+ B cells wane significantly when compared to RBD-specific IgG+ B cells, which increase over time, and the number of IgG+ memory B cells which remain stable thereafter for up to 8 months after symptoms onset. With the recent approval of highly effective vaccines for COVID-19, data on the persistence of immune responses are of central importance. Even though overall circulating SARS-CoV-2 Spike-specific antibodies contract over time during convalescence, we demonstrate that RBD-specific B cells increase and persist up to 8 months post symptom onset. We also observe modest increases in RBD-specific IgG+ memory B cells and importantly, detectable IgG and sustained Fc-effector activity in plasma over the 8-month period. Our results add to the current understanding of immune memory following SARS-CoV-2 infection, which is critical for the prevention of secondary infections, vaccine efficacy and herd immunity against COVID-19.
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COVID-19 is a respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and declared by the World Health Organization a global public health emergency. Among the severe outbreaks across South America, Uruguay has become known for curtailing SARS-CoV-2 exceptionally well. To understand the SARS-CoV-2 introductions, local transmissions, and associations with genomic and clinical parameters in Uruguay, we sequenced the viral genomes of 44 outpatients and inpatients in a private healthcare system in its capital, Montevideo, from March to May 2020. We performed a phylogeographic analysis using sequences from our cohort and other studies that indicate a minimum of 23 independent introductions into Uruguay, resulting in five major transmission clusters. Our data suggest that most introductions resulting in chains of transmission originate from other South American countries, with the earliest seeding of the virus in late February 2020, weeks before the borders were closed to all non-citizens and a partial lockdown implemented. Genetic analyses suggest a dominance of S and G clades (G, GH, GR) that make up >90% of the viral strains in our study. In our cohort, lethal outcome of SARS-CoV-2 infection significantly correlated with arterial hypertension, kidney failure, and ICU admission (FDR < 0.01), but not with any mutation in a structural or non-structural protein, such as the spike D614G mutation. Our study contributes genetic, phylodynamic, and clinical correlation data about the exceptionally well-curbed SARS-CoV-2 outbreak in Uruguay, which furthers the understanding of disease patterns and regional aspects of the pandemic in Latin America.
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The SARS-CoV-2 virus is responsible for the current worldwide coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The Spike glycoprotein of SARS-CoV-2 mediates viral entry and is the main target for neutralizing antibodies. Understanding the antibody response directed against SARS-CoV-2 is crucial for the development of vaccine, therapeutic and public health interventions. Here we performed a cross-sectional study on 106 SARS-CoV-2-infected individuals to evaluate humoral responses against the SARS-CoV-2 Spike. The vast majority of infected individuals elicited anti-Spike antibodies within 2 weeks after the onset of symptoms. The levels of receptor-binding domain (RBD)-specific IgG persisted overtime, while the levels of anti-RBD IgM decreased after symptoms resolution. Some of the elicited antibodies cross-reacted with other human coronaviruses in a genus-restrictive manner. While most of individuals developed neutralizing antibodies within the first two weeks of infection, the level of neutralizing activity was significantly decreased over time. Our results highlight the importance of studying the persistence of neutralizing activity upon natural SARS-CoV-2 infection.
