SARS-CoV-2 Aerosol and Intranasal Exposure Models in Ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the worldwide COVID-19 pandemic. Animal models are extremely helpful for testing vaccines and therapeutics and for dissecting the viral and host factors that contribute to disease severity and transmissibility. Here, we report the assessment and comparison of intranasal and small particle (~3 µm) aerosol SARS-CoV-2 exposure in ferrets. The primary endpoints for analysis were clinical signs of disease, recovery of the virus in the upper respiratory tract, and the severity of damage within the respiratory tract. This work demonstrated that ferrets were productively infected with SARS-CoV-2 following either intranasal or small particle aerosol exposure. SARS-CoV-2 infection of ferrets resulted in an asymptomatic disease course following either intranasal or small particle aerosol exposure, with no clinical signs, significant weight loss, or fever. In both aerosol and intranasal ferret models, SARS-CoV-2 replication, viral genomes, and viral antigens were detected within the upper respiratory tract, with little to no viral material detected in the lungs. The ferrets exhibited a specific IgG immune response to the SARS-CoV-2 full spike protein. Mild pathological findings included inflammation, necrosis, and edema within nasal turbinates, which correlated to positive immunohistochemical staining for the SARS-CoV-2 virus. Environmental sampling was performed following intranasal exposure of ferrets, and SARS-CoV-2 genomic material was detected on the feeders and nesting areas from days 2-10 post-exposure. We conclude that both intranasal and small particle aerosol ferret models displayed measurable parameters that could be utilized for future studies, including transmission studies and testing SARS-CoV-2 vaccines and therapeutics.

Seroprevalence as an Indicator of Undercounting of COVID-19 Cases in a Large Well-Described Cohort.

Introduction: Reported confirmed cases represent a small portion of overall true cases for many infectious diseases. The undercounting of true cases can be considerable when a significant portion of infected individuals are asymptomatic or minimally symptomatic, as is the case with COVID-19. Seroprevalence studies are an efficient way to assess the extent to which true cases are undercounted during a large-scale outbreak and can inform efforts to improve case identification and reporting. Methods: A longitudinal seroprevalence study of active duty U.S. military members was conducted from May 2020 through June 2021. A random selection of service member serum samples submitted to the Department of Defense Serum Repository was analyzed for the presence of antibodies reactive to SARS-CoV-2. The monthly seroprevalence rates were compared with those of cumulative confirmed cases reported during the study period. Results: Seroprevalence was 2.3% in May 2020 and increased to 74.0% by June 2021. The estimated true case count based on seroprevalence was 9.3 times greater than monthly reported cases at the beginning of the study period and fell to 1.7 by the end of the study. Conclusions: In our sample, confirmed case counts significantly underestimated true cases of COVID-19. The increased availability of testing over the study period and enhanced efforts to detect asymptomatic and minimally symptomatic cases likely contributed to the fall in the seroprevalence to reported case ratio.

Antibody responses to recombinant vesicular stomatitis virus-Zaire Ebolavirus vaccination for Ebola virus disease across doses and continents: 5-year durability.

OBJECTIVES: To report 5-year persistence and avidity of antibodies produced by the live-attenuated recombinant vesicular stomatitis virus (rVSV) expressing the Zaire Ebolavirus (ZEBOV) glycoprotein (GP), known as rVSV-ZEBOV (Ervebo®). METHODS: Healthy adults vaccinated with 300,000 or 10-50 million plaque-forming units of rVSV-ZEBOV in the WHO-coordinated trials of 2014-2015 were followed for up to 4 (Lambaréné, Gabon) and 5 (Geneva, Switzerland) years. We report seropositivity rates, geometric mean titres (GMTs), and population distribution of ZEBOV-GP ELISA IgG antibodies, neutralizing antibodies (pseudovirus and live-virus neutralization) and antibody avidity; the primary outcome was ZEBOV-GP ELISA IgG GMTs at 4 or 5 years compared with 1 year (Y1) after immunization. RESULTS: Among the 168 eligible vaccinees (Geneva: 97 and Lambaréné: 71) enrolled 1 year post-immunization, 146 (87%) remained enrolled at 4 years (Geneva: n = 88, Lambaréné: n = 58), and 84 (87%, Geneva) at 5 years post-vaccination. ZEBOV-GP ELISA IgG GMTs plateaued, with no declining trend from 1 year through the last time point assessed (1147.8 [95% CI 874.3-1507.0] at Y1 versus 1548.1 [95% CI 1136.6-2108.5] at Y5 in Geneva volunteers receiving ≥10 million plaque-forming units of rVSV-ZEBOV), their avidity matching that of ZEBOV convalescents. Live-virus neutralizing antibodies were detected for shorter periods and in fewer vaccinees (53/95 [56%] at Y1 versus 35/84 [42%] at Y5 in Geneva volunteers, all dose levels). DISCUSSION: Titres at Y1 emerged as a correlate of antibody persistence at Y5. The findings of persistent ZEBOV-GP ELISA IgG titres yet shorter-lasting, lower titres of live-virus neutralizing antibodies suggest the contribution of antibody-mediated protective mechanisms other than neutralization. Long-term clinical efficacy of rVSV-ZEBOV, however, requires further study.

Induced protection from a CCHFV-M DNA vaccine requires CD8+ T cells.

Crimean-Congo hemorrhagic fever (CCHF) is a World Health Organization prioritized disease because its broad distribution and severity of disease make it a global health threat. Despite advancements in preclinical vaccine development for CCHF virus (CCHFV), including multiple platforms targeting multiple antigens, a clear definition of the adaptive immune correlates of protection is lacking. Levels of neutralizing antibodies in vaccinated animal models do not necessarily correlate with protection, suggesting that cellular immunity, such as CD8+ T cells, might have an important role in protection in this model. Using a well-established IFN-I antibody blockade mouse model (IS) and a DNA-based vaccine encoding the CCHFV M-segment glycoprotein precursor, we investigated the role of humoral and T cell immunity in vaccine-mediated protection in mice genetically devoid of these immune compartments. We found that in the absence of the B-cell compartment (µMT knockout mice), protection provided by the vaccine was not reduced. In contrast, in the absence of CD8+ T cells (CD8+ knockout mice) the vaccine-mediated protection was significantly diminished. Importantly, humoral responses to the vaccine in CD8+ T-cell knockout mice were equivalent to wild-type mice. These findings indicated that CD8+ T-cell responses are necessary and sufficient to promote protection in mice vaccinated with the M-segment DNA vaccine. Identifying a crucial role of the cellular immunity to protect against CCHFV should help guide the development of CCHFV-targeting vaccines.

A SARS-CoV-2 Spike Ferritin Nanoparticle Vaccine Is Protective and Promotes a Strong Immunological Response in the Cynomolgus Macaque Coronavirus Disease 2019 (COVID-19) Model.

The COVID-19 pandemic has had a staggering impact on social, economic, and public health systems worldwide. Vaccine development and mobilization against SARS-CoV-2 (the etiologic agent of COVID-19) has been rapid. However, novel strategies are still necessary to slow the pandemic, and this includes new approaches to vaccine development and/or delivery that will improve vaccination compliance and demonstrate efficacy against emerging variants. Here, we report on the immunogenicity and efficacy of a SARS-CoV-2 vaccine comprising stabilized, pre-fusion spike protein trimers displayed on a ferritin nanoparticle (SpFN) adjuvanted with either conventional aluminum hydroxide or the Army Liposomal Formulation QS-21 (ALFQ) in a cynomolgus macaque COVID-19 model. Vaccination resulted in robust cell-mediated and humoral responses and a significant reduction in lung lesions following SARS-CoV-2 infection. The strength of the immune response suggests that dose sparing through reduced or single dosing in primates may be possible with this vaccine. Overall, the data support further evaluation of SpFN as a SARS-CoV-2 protein-based vaccine candidate with attention to fractional dosing and schedule optimization.

Exposure Route Influences Disease Severity in the COVID-19 Cynomolgus Macaque Model.

The emergence of SARS-CoV-2 and the subsequent pandemic has highlighted the need for animal models that faithfully replicate the salient features of COVID-19 disease in humans. These models are necessary for the rapid selection, testing, and evaluation of potential medical countermeasures. Here, we performed a direct comparison of two distinct routes of SARS-CoV-2 exposure-combined intratracheal/intranasal and small particle aerosol-in two nonhuman primate species, rhesus and cynomolgus macaques. While all four experimental groups displayed very few outward clinical signs, evidence of mild to moderate respiratory disease was present on radiographs and at necropsy. Cynomolgus macaques exposed via the aerosol route also developed the most consistent fever responses and had the most severe respiratory disease and pathology. This study demonstrates that while all four models produced suitable representations of mild COVID-like illness, aerosol exposure of cynomolgus macaques to SARS-CoV-2 produced the most severe disease, which may provide additional clinical endpoints for evaluating therapeutics and vaccines.

Development of a coronavirus disease 2019 nonhuman primate model using airborne exposure.

Airborne transmission is predicted to be a prevalent route of human exposure with SARS-CoV-2. Aside from African green monkeys, nonhuman primate models that replicate airborne transmission of SARS-CoV-2 have not been investigated. A comparative evaluation of COVID-19 in African green monkeys, rhesus macaques, and cynomolgus macaques following airborne exposure to SARS-CoV-2 was performed to determine critical disease parameters associated with disease progression, and establish correlations between primate and human COVID-19. Respiratory abnormalities and viral shedding were noted for all animals, indicating successful infection. Cynomolgus macaques developed fever, and thrombocytopenia was measured for African green monkeys and rhesus macaques. Type II pneumocyte hyperplasia and alveolar fibrosis were more frequently observed in lung tissue from cynomolgus macaques and African green monkeys. The data indicate that, in addition to African green monkeys, macaques can be successfully infected by airborne SARS-CoV-2, providing viable macaque natural transmission models for medical countermeasure evaluation.

Chikungunya and O'nyong-nyong Viruses in Uganda: Implications for Diagnostics.

BACKGROUND: A serosurvey of healthy blood donors provided evidence of hemorrhagic fever and arthropod-borne virus infections in Uganda. METHODS: Antibody prevalence to arthropod-borne and hemorrhagic fever viruses in human sera was determined using enzyme-linked immunosorbent assay (ELISA) and plaque reduction neutralization test (PRNT). RESULTS: The greatest antibody prevalence determined by ELISA was to chikungunya virus (CHIKV) followed in descending order by West Nile virus (WNV), Crimean-Congo hemorrhagic fever virus (CCHFV), Ebola virus (EBOV), dengue virus (DEN), yellow fever virus (YFV), Rift Valley fever virus (RVFV), Marburg virus (MARV), and Lassa virus (LASV). Further investigation of CHIKV-positive sera demonstrated that the majority of antibody responses may likely be the result of exposure to the closely related alphavirus o'nyong-nyong virus (ONNV). CONCLUSIONS: As the use of highly specific and sensitive polymerase chain reaction-based assays becomes the diagnostic standard without the corresponding use of the less sensitive but more broadly reactive immunological-based assays, emerging and re-emerging outbreaks will be initially missed, illustrating the need for an orthogonal system for the detection and identification of viruses causing disease.

Isolation of west nile and sindbis viruses from mosquitoes collected in the Nile Valley of Egypt during an outbreak of Rift Valley fever.

As part of an evaluation of potential vectors of arboviruses during a Rift Valley fever (RVF) outbreak in the Nile Valley of Egypt in August 1993, we collected mosquitoes in villages with known RVF viral activity. Mosquitoes were sorted to species, pooled, and processed for virus isolation both by intracerebral inoculation into suckling mice and by inoculation into cell culture. A total of 33 virus isolates was made from 36,024 mosquitoes. Viruses were initially identified by indirect fluorescent antibody testing and consisted of 30 flaviviruses (all members of the Japanese encephalitis complex, most probably West Nile [WN] virus) and three alphaviruses (all members of western equine encephalitis complex, most probably Sindbis). The identity of selected viruses was confirmed by reverse transcriptase-polymerase chain reaction and sequencing. Culex antennatus (Becker) and Culex perexiguus Theobald accounted for five (17%) and 23 (77%) of the WN virus isolations, respectively. Despite isolation of viruses from 32 pools of mosquitoes (both WN and Sindbis viruses were isolated from a single pool), RVF virus was not isolated from these mosquitoes, even though most of them are known competent vectors collected during an ongoing RVF outbreak. Thus, it should be remembered, that even during a known arbovirus outbreak, other arboviruses may still be circulating and causing disease.