SARS-CoV-2 Omicron BA.1 and BA.2 are attenuated in rhesus macaques as compared to Delta

Since the emergence of SARS-CoV-2, five different variants of concern (VOCs) have been identified: Alpha, Beta, Gamma, Delta, and Omicron. Due to confounding factors in the human population, such as pre-existing immunity, comparing severity of disease caused by different VOCs is challenging. Here, we investigate disease progression in the rhesus macaque model upon inoculation with the Delta, Omicron BA.1, and Omicron BA.2 VOCs. Disease severity in rhesus macaques inoculated with Omicron BA.1 or BA.2 was lower than those inoculated with Delta and resulted in significantly lower viral loads in nasal swabs, bronchial cytology brush samples, and lung tissue in rhesus macaques. Cytokines and chemokines were upregulated in nasosorption samples of Delta animals compared to Omicron BA.1 and BA.2 animals. Overall, these data suggests that in rhesus macaques, Omicron replicates to lower levels than the Delta VOC, resulting in reduced clinical disease.

Mild SARS-CoV-2 infection in rhesus macaques is associated with viral control prior to antigen-specific T cell responses in tissues

SARS-CoV-2 primarily replicates in mucosal sites, and more information is needed about immune responses in infected tissues. We used rhesus macaques to model protective primary immune responses in tissues during mild COVID-19. Viral RNA levels were highest on days 1-2 post-infection and fell precipitously thereafter. 18F-fluorodeoxyglucose (FDG)-avid lung abnormalities and interferon (IFN)-activated myeloid cells in the bronchoalveolar lavage (BAL) were found on days [~]3-4. Virus-specific effector CD8 and CD4 T cells were detectable in the BAL and lung tissue on days [~]7-10, after viral RNA, lung inflammation, and IFN-activated myeloid cells had declined. Notably, SARS-CoV-2-specific T cells were not detectable in the nasal turbinates, salivary glands, and tonsils on day 10 post-infection. Thus, SARS-CoV-2 replication wanes in the lungs prior to T cell responses, and in the nasal and oral mucosa despite the apparent lack of Ag-specific T cells, suggesting that innate immunity efficiently restricts viral replication during mild COVID-19. ONE SENTENCE SUMMARYSARS-CoV-2 infection leads to mild, focal lung inflammation, and type I IFN activated myeloid cells that mostly resolve prior to the influx of virus-specific effector T cells or antibody responses in rhesus macaques.

Year-long COVID-19 infection reveals within-host evolution of SARS-CoV-2 in a patient with B cell depletion

BackgroundB-cell depleting therapies may lead to protracted disease and prolonged viral shedding in individuals infected with SARS-CoV-2. Viral persistence in the setting of immunosuppression raises concern for viral evolution. MethodsAmplification of sub-genomic transcripts for the E gene (sgE) was done on nasopharyngeal samples over the course of 355 days in a patient infected with SARS-CoV-2 who had previously undergone CAR T cell therapy and had persistently positive SARS-CoV-2 nasopharyngeal swabs. Whole genome sequencing was performed on samples from the patients original presentation and 10 months later. ResultsOver the course of almost a year, the virus accumulated a unique in-frame deletion in the amino-terminal domain of the spike protein, and complete deletion of ORF7b and ORF8, the first report of its kind in an immunocompromised patient. Also, minority variants that were identified in the early samples--reflecting the heterogeneity of the initial infection--were found to be fixed late in the infection. Remdesivir and high-titer convalescent plasma treatment were given, and the infection was eventually cleared after 335 days of infection. ConclusionsThe unique viral mutations found in this study highlight the importance of analyzing viral evolution in protracted SARS-CoV-2 infection, especially in immunosuppressed hosts, and the implication of these mutations in the emergence of viral variants. SummaryWe report an immunocompromised patient with persistent symptomatic SARS-CoV-2 infection for 335 days. During this time, the virus accumulated a unique in-frame deletion in the spike, and a complete deletion of ORF7b and ORF8 which is the first report of its kind in an immunocompromised patient.

Age-related differences in immune dynamics during SARS-CoV-2 infection in rhesus macaques

Advanced age is a key predictor of severe COVID-19. To gain insight into this relationship, particularly with respect to immune responses, we utilized the rhesus macaque model of SARS-CoV-2 infection. Two cohorts of eight older (16-23 years) and eight younger (3-5 years) rhesus macaques were inoculated with SARS-CoV-2. Animals were evaluated using viral RNA quantification, clinical observations, thoracic radiographs, single-cell transcriptomics, multiparameter flow cytometry, multiplex immunohistochemistry, cytokine detection, and lipidomics analysis at pre-defined timepoints in various tissues. Differences in clinical signs, pulmonary infiltrates, and virus replication dynamics were limited between age cohorts. Transcriptional signatures of inflammation-associated genes in cells isolated from bronchoalveolar lavage fluid at 3 dpi revealed efficient mounting of innate immune defenses in both younger and older animals. These findings suggested that age did not substantially skew major facets of acute disease in this model. However, age-specific divergence of immune responses emerged during the post-acute phase of infection (7-21 dpi). Older animals exhibited sustained local inflammatory innate responses while local effector T-cell responses were induced earlier in the younger animals. Circulating lipid mediator and cytokine levels highlighted increased repair-associated signals in the younger animals, in contrast to persistent pro-inflammatory responses in the older animals. In summary, despite similar disease outcomes, multi-omics profiling in SARS-CoV-2-infected rhesus macaques suggests that age may delay or impair the induction of anti-viral cellular immune responses and delay efficient return to immune homeostasis following acute infection.

Subtle differences in the pathogenicity of SARS-CoV-2 variants of concern B.1.1.7 and B.1.351 in rhesus macaques

The emergence of several SARS-CoV-2 variants has caused global concerns about increased transmissibility, increased pathogenicity, and decreased efficacy of medical countermeasures. Animal models can be used to assess phenotypical changes in the absence of confounding factors that affect observed pathogenicity and transmissibility data in the human population. Here, we studied the pathogenicity of variants of concern (VOC) B.1.1.7 and B.1.351 in rhesus macaques and compared it to a recent clade B.1 SARS-CoV-2 isolate containing the D614G substitution in the spike protein. The B.1.1.7 VOC behaved similarly to the D614G with respect to clinical disease, virus shedding and virus replication in the respiratory tract. Inoculation with the B.1.351 isolate resulted in lower clinical scores in rhesus macaques that correlated with lower virus titers in the lungs, less severe histologic lung lesions and less viral antigen detected in the lungs. We observed differences in the local innate immune response to infection. In bronchoalveolar lavages, cytokines and chemokines were upregulated on day 4 in animals inoculated with D614G and B.1.1.7 but not in those inoculated with B.1.351. In nasal samples, we did not detect upregulation of cytokines and chemokines in D614G or B.1.351-inoculated animals. However, cytokines and chemokines were upregulated in the noses of B.1.1.7-inoculated animals. Taken together, our comparative pathogenicity study suggests that ongoing circulation under diverse evolutionary pressure favors transmissibility and immune evasion rather than an increase in intrinsic pathogenicity.

Prior aerosol infection with lineage A SARS-CoV-2 variant protects hamsters from disease, but not reinfection with B.1.351 SARS-CoV-2 variant

The circulation of SARS-CoV-2 has resulted in the emergence of variants of concern (VOCs). It is currently unclear whether previous infection with SARS-CoV-2 provides protection against reinfection with VOCs. Here, we show that low dose aerosol exposure to hCoV-19/human/USA/WA-CDC-WA1/2020 (WA1, lineage A), resulted in a productive mild infection. In contrast, low dose of SARS-CoV-2 via fomites did not result in productive infection in the majority of exposed hamsters and these animals remained non-seroconverted. After recovery, hamsters were re-exposed to hCoV-19/South African/KRISP-K005325/2020 (VOC B.1.351) via an intranasal challenge. Seroconverted rechallenged animals did not lose weight and shed virus for 3 days. They had little infectious virus and no pathology in the lungs. In contrast, shedding, weight loss and extensive pulmonary pathology caused by B.1.351 replication was observed in the non-seroconverted animals. The rechallenged seroconverted animals did not transmit virus to naive sentinels via direct contact transmission, in contrast to the non-seroconverted animals. Reinfection with B.1.351 triggered an anamnestic response that boosted not only neutralizing titers against lineage A, but also titers against B.1.351. Our results confirm that aerosol exposure is a more efficient infection route than fomite exposure. Furthermore, initial infection with SARS-CoV-2 lineage A does not prevent heterologous reinfection with B.1.351 but prevents disease and onward transmission. These data suggest that previous SARS-CoV-2 exposure induces partial protective immunity. The reinfection generated a broadly neutralizing humoral response capable of effectively neutralizing B.1.351 while maintaining its ability to neutralize the virus to which the initial response was directed against.

ChAdOx1 nCoV-19 (AZD1222) protects against SARS-CoV-2 B.1.351 and B.1.1.7

We investigated ChAdOx1 nCoV-19 (AZD1222) vaccine efficacy against SARS-CoV-2 variants of concern (VOCs) B.1.1.7 and B.1.351 in Syrian hamsters. We previously showed protection against SARS-CoV-2 disease and pneumonia in hamsters vaccinated with a single dose of ChAdOx1 nCoV-19. Here, we observed a 9.5-fold reduction of virus neutralizing antibody titer in vaccinated hamster sera against B.1.351 compared to B.1.1.7. Vaccinated hamsters challenged with B.1.1.7 or B.1.351 did not lose weight compared to control animals. In contrast to control animals, the lungs of vaccinated animals did not show any gross lesions. Minimal to no viral subgenomic RNA (sgRNA) and no infectious virus was detected in lungs of vaccinated animals. Histopathological evaluation showed extensive pulmonary pathology caused by B.1.1.7 or B.1.351 replication in the control animals, but none in the vaccinated animals. These data demonstrate the effectiveness of the ChAdOx1 nCoV-19 vaccine against clinical disease caused by B.1.1.7 or B.1.351 VOCs.

Microbial signatures in the lower airways of mechanically ventilated COVID19 patients associated with poor clinical outcome

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.

A betacoronavirus multiplex microsphere immunoassay detects early SARS-CoV-2 seroconversion and controls for pre-existing seasonal human coronavirus antibody cross-reactivity

With growing concern of persistent or multiple waves of SARS-CoV-2 in the United States, sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 surveillance. Here, we describe the development and application of a multiplex microsphere-based immunoassay (MMIA) for COVD-19 antibody studies, utilizing serum samples from non-human primate SARS-CoV-2 infection models, an archived human sera bank and subjects enrolled at five U.S. military hospitals. The MMIA incorporates prefusion stabilized spike glycoprotein trimers of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and the seasonal human coronaviruses HCoV-HKU1 and HCoV-OC43, into a multiplexing system that enables simultaneous measurement of off-target pre-existing cross-reactive antibodies. We report the sensitivity and specificity performances for this assay strategy at 98% sensitivity and 100% specificity for subject samples collected as early as 10 days after the onset of symptoms. In archival sera collected prior to 2019 and serum samples from subjects PCR negative for SARS-CoV-2, we detected seroprevalence of 72% and 98% for HCoV-HKU1 and HCoV-0C43, respectively. Requiring only 1.25 {micro}L of sera, this approach permitted the simultaneous identification of SARS-CoV-2 seroconversion and polyclonal SARS-CoV-2 IgG antibody responses to SARS-CoV-1 and MERS-CoV, further demonstrating the presence of conserved epitopes in the spike glycoprotein of zoonotic betacoronaviruses. Application of this serology assay in observational studies with serum samples collected from subjects before and after SARS-CoV-2 infection will permit an investigation of the influences of HCoV-induced antibodies on COVID-19 clinical outcomes.

SARS-CoV-2 infection dynamics in lungs of African green monkeys

Detailed knowledge about the dynamics of SARS-CoV-2 infection is important for unraveling the viral and host factors that contribute to COVID-19 pathogenesis. Old-World nonhuman primates recapitulate mild-moderate COVID-19 cases, thereby serving as important pathogenesis models. We compared African green monkeys inoculated with SARS-CoV-2 or inactivated virus to study the dynamics of virus replication throughout the respiratory tract. RNA sequencing of single cells from the lungs and mediastinal lymph nodes allowed a high-resolution analysis of virus replication and host responses over time. Viral replication was mainly localized to the lower respiratory tract, with evidence of replication in the pneumocytes. Macrophages were found to play a role in initiating a pro-inflammatory state in the lungs, while also interacting with infected pneumocytes. Our dataset provides a detailed view of changes in host and virus replication dynamics over the course of mild COVID-19 and serves as a valuable resource to identify therapeutic targets.

K18-hACE2 mice develop respiratory disease resembling severe COVID-19

SARS-CoV-2 emerged in late 2019 and resulted in the ongoing COVID-19 pandemic. Several animal models have been rapidly developed that recapitulate the asymptomatic to moderate disease spectrum. Now, there is a direct need for additional small animal models to study the pathogenesis of severe COVID-19 and for fast-tracked medical countermeasure development. Here, we show that transgenic mice expressing the human SARS-CoV-2 receptor (angiotensin-converting enzyme 2 [hACE2]) under a cytokeratin 18 promoter (K18) are susceptible to SARS-CoV-2 and that infection resulted in a dose-dependent lethal disease course. After inoculation with either 104 TCID50 or 105 TCID50, the SARS-CoV-2 infection resulted in rapid weight loss in both groups and uniform lethality in the 105 TCID50 group. High levels of viral RNA shedding were observed from the upper and lower respiratory tract and intermittent shedding was observed from the intestinal tract. Inoculation with SARS-CoV-2 resulted in upper and lower respiratory tract infection with high infectious virus titers in nasal turbinates, trachea and lungs. The observed interstitial pneumonia and pulmonary pathology, with SARS-CoV-2 replication evident in pneumocytes, were similar to that reported in severe cases of COVID-19. SARS-CoV-2 infection resulted in macrophage and lymphocyte infiltration in the lungs and upregulation of Th1 and proinflammatory cytokines/chemokines. Extrapulmonary replication of SARS-CoV-2 was observed in the cerebral cortex and hippocampus of several animals at 7 DPI but not at 3 DPI. The rapid inflammatory response and observed pathology bears resemblance to COVID-19. Taken together, this suggests that this mouse model can be useful for studies of pathogenesis and medical countermeasure development. Authors SummaryThe disease manifestation of COVID-19 in humans range from asymptomatic to severe. While several mild to moderate disease models have been developed, there is still a need for animal models that recapitulate the severe and fatal progression observed in a subset of patients. Here, we show that humanized transgenic mice developed dose-dependent disease when inoculated with SARS-CoV-2, the etiological agent of COVID-19. The mice developed upper and lower respiratory tract infection, with virus replication also in the brain after day 3 post inoculation. The pathological and immunological diseases manifestation observed in these mice bears resemblance to human COVID-19, suggesting increased usefulness of this model for elucidating COVID-19 pathogenesis further and testing of countermeasures, both of which are urgently needed.

Hydroxychloroquine Proves Ineffective in Hamsters and Macaques Infected with SARS-CoV-2

We remain largely without effective prophylactic/therapeutic interventions for COVID-19. Although many human clinical trials are ongoing, there remains a deficiency of supportive preclinical drug efficacy studies. Here we assessed the prophylactic/therapeutic efficacy of hydroxychloroquine (HCQ), a drug of interest for COVID-19 management, in two animal models. When used for prophylaxis or treatment neither the standard human malaria dose (6.5 mg/kg) nor a high dose (50 mg/kg) of HCQ had any beneficial effect on clinical disease or SARS-CoV-2 kinetics (replication/shedding) in the Syrian hamster disease model. Similarly, HCQ prophylaxis/treatment (6.5 mg/kg) did not significantly benefit clinical outcome nor reduce SARS-CoV-2 replication/shedding in the upper and lower respiratory tract in the rhesus macaque disease model. In conclusion, our preclinical animal studies do not support the use of HCQ in prophylaxis/treatment of COVID-19.One Sentence Summary Hydroxychloroquine prophylaxis/treatment showed no beneficial effect in SARS-CoV-2 hamster and macaque disease models.Competing Interest StatementThe authors have declared no competing interest.View Full Text

ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-2 pneumonia in rhesus macaques

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in December 20191,2 and is responsible for the COVID-19 pandemic3. Vaccines are an essential countermeasure urgently needed to control the pandemic4. Here, we show that the adenovirus-vectored vaccine ChAdOx1 nCoV-19, encoding the spike protein of SARS-CoV-2, is immunogenic in mice, eliciting a robust humoral and cell-mediated response. This response was not Th2 dominated, as demonstrated by IgG subclass and cytokine expression profiling. A single vaccination with ChAdOx1 nCoV-19 induced a humoral and cellular immune response in rhesus macaques. We observed a significantly reduced viral load in bronchoalveolar lavage fluid and respiratory tract tissue of vaccinated animals challenged with SARS-CoV-2 compared with control animals, and no pneumonia was observed in vaccinated rhesus macaques. Importantly, no evidence of immune-enhanced disease following viral challenge in vaccinated animals was observed. ChAdOx1 nCoV-19 is currently under investigation in a phase I clinical trial. Safety, immunogenicity and efficacy against symptomatic PCR-positive COVID-19 disease will now be assessed in randomised controlled human clinical trials.

Assessment of N95 respirator decontamination and re-use for SARS-CoV-2

The unprecedented pandemic of SARS-CoV-2 has created worldwide shortages of personal protective equipment, in particular respiratory protection such as N95 respirators. SARS-CoV-2 transmission is frequently occurring in hospital settings, with numerous reported cases of nosocomial transmission highlighting the vulnerability of healthcare workers. In general, N95 respirators are designed for single use prior to disposal. Here, we have analyzed four readily available and often used decontamination methods: UV, 70% ethanol, 70C heat and vaporized hydrogen peroxide for inactivation of SARS-CoV-2 on N95 respirators. Equally important we assessed the function of the N95 respirators after multiple wear and decontamination sessions.

Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2

BackgroundEffective therapeutics to treat COVID-19 are urgently needed. Remdesivir is a nucleotide prodrug with in vitro and in vivo efficacy against coronaviruses. Here, we tested the efficacy of remdesivir treatment in a rhesus macaque model of SARS-CoV-2 infection. MethodsTo evaluate the effect of remdesivir treatment on SARS-CoV-2 disease outcome, we used the recently established rhesus macaque model of SARS-CoV-2 infection that results in transient lower respiratory tract disease. Two groups of six rhesus macaques were infected with SARS-CoV-2 and treated with intravenous remdesivir or an equal volume of vehicle solution once daily. Clinical, virological and histological parameters were assessed regularly during the study and at necropsy to determine treatment efficacy. ResultsIn contrast to vehicle-treated animals, animals treated with remdesivir did not show signs of respiratory disease and had reduced pulmonary infiltrates on radiographs. Virus titers in bronchoalveolar lavages were significantly reduced as early as 12hrs after the first treatment was administered. At necropsy on day 7 after inoculation, lung viral loads of remdesivir-treated animals were significantly lower and there was a clear reduction in damage to the lung tissue. ConclusionsTherapeutic remdesivir treatment initiated early during infection has a clear clinical benefit in SARS-CoV-2-infected rhesus macaques. These data support early remdesivir treatment initiation in COVID-19 patients to prevent progression to severe pneumonia.

Aerosol and surface stability of HCoV-19 (SARS-CoV-2) compared to SARS-CoV-1

To the EditorA novel human coronavirus, now named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, referred to as HCoV-19 here) that emerged in Wuhan, China in late 2019 is now causing a pandemic1. Here, we analyze the aerosol and surface stability of HCoV-19 and compare it with SARS-CoV-1, the most closely related human coronavirus.2 We evaluated the stability of HCoV-19 and SARS-CoV-1 in aerosols and on different surfaces and estimated their decay rates using a Bayesian regression model (see Supplementary Appendix). All experimental measurements are reported as mean across 3 replicates.

Respiratory disease and virus shedding in rhesus macaques inoculated with SARS-CoV-2

An outbreak of a novel coronavirus, now named SARS-CoV-2, causing respiratory disease and a [~]2% case fatality rate started in Wuhan, China in December 2019. Following unprecedented rapid global spread, the World Health Organization declared COVID-19 a pandemic on March 11, 2020. Although data on disease in humans are emerging at a steady pace, certain aspects of the pathogenesis of SARS-CoV-2 can only be studied in detail in animal models, where repeated sampling and tissue collection is possible. Here, we show that SARS-CoV-2 causes respiratory disease in infected rhesus macaques, with disease lasting 8-16 days. Pulmonary infiltrates, a hallmark of human disease, were visible in lung radiographs of all animals. High viral loads were detected in swabs from the nose and throat of all animals as well as in bronchoalveolar lavages; in one animal we observed prolonged rectal shedding. Taken together, the rhesus macaque recapitulates moderate disease observed in the majority of human cases. The establishment of the rhesus macaque as a model of COVID-19 will increase our understanding of the pathogenesis of this disease and will aid development and testing of medical countermeasures.