Molecular engineering of a cryptic epitope in Spike RBD improves manufacturability and neutralizing breadth against SARS-CoV-2 variants

There is a continued need for sarbecovirus vaccines that can be manufactured and distributed in low- and middle-income countries (LMICs). Subunit protein vaccines are manufactured at large scales at low costs, have less stringent temperature requirements for distribution in LMICs, and several candidates have shown protection against SARS-CoV-2. We previously reported an engineered variant of the SARS-CoV-2 Spike protein receptor binding domain antigen (RBD-L452K-F490W; RBD-J) with enhanced manufacturability and immunogenicity compared to the ancestral RBD. Here, we report a second-generation engineered RBD antigen (RBD-J6) with two additional mutations to a hydrophobic cryptic epitope in the RBD core, S383D and L518D, that further improved expression titers and biophysical stability. RBD-J6 retained binding affinity to human convalescent sera and to all tested neutralizing antibodies except antibodies that target the class IV epitope on the RBD core. K18-hACE2 transgenic mice immunized with three doses of a Beta variant of RBD-J6 displayed on a virus-like particle (VLP) generated neutralizing antibodies (nAb) to nine SARS-CoV-2 variants of concern at similar levels as two doses of Comirnaty. The vaccinated mice were also protected from challenge with Alpha or Beta SARS-CoV-2. This engineered antigen could be useful for modular RBD-based subunit vaccines to enhance manufacturability and global access, or for further development of variant-specific or broadly acting booster vaccines.

Diet induced obesity and type 2 diabetes drives exacerbated sex-associated disease profiles in K18-hACE2-mice challenged with SARS-CoV-2

SARS-CoV-2 infection results in wide-ranging disease manifestation from asymptomatic to potentially lethal. Infection poses an increased threat of severity to at-risk populations including those with hypertension, diabetes, and obesity. Type 2 Diabetes (T2DM), is characterized, in part, by insulin insensitivity and impaired glucose regulation. T2DM patients have increased disease severity and poorer outcomes with COVID-19. We utilized the diet-induced obesity (DIO) model of Type 2 Diabetes in SARS-CoV-2-susceptible K18-hACE2 transgenic mice to better understand the obesity co-morbidity. Female DIO, but not male DIO mice challenged with SARS-CoV-2 were observed to have shortened time to morbidity compared to normal diet mice. Increase in susceptibility to SARS-CoV2 in female DIO was associated with increased total viral RNA burden compared to male mice. RNAseq analysis was performed on the lungs of non-challenged, challenged, females, males, of either normal diet or DIO cohorts to determine the disease specific transcriptional profiles. DIO female mice had more total activated genes than normal diet mice after challenge; however, male mice experienced a decrease. GO term analysis revealed the DIO condition increased interferon response signatures and interferon gamma production following challenge. Male challenged mice had robust expression of antibody-related genes suggesting antibody producing cell localization in the lung. DIO reduced antibody gene expression in challenged males. Collectively this study establishes a preclinical T2DM/obesity co-morbidity model of COVID-19 in mice where we observed sex and diet specific responses that begin to explain the effects of obesity and diabetes on COVID-19 disease.

SARS-CoV-2 Delta variant induces enhanced pathology and inflammatory responses in K18-hACE2 mice

The COVID-19 pandemic has been fueled by novel variants of concern (VOC) that have increased transmissibility, receptor binding affinity, and other properties that enhance disease. The goal of this study is to characterize unique pathogenesis of the Delta VOC strain in the K18-hACE2-mouse challenge model. Challenge studies suggested that the lethal dose of Delta was higher than Alpha or Beta strains. To characterize the differences in the Delta strains pathogenesis, a time-course experiment was performed to evaluate the overall host response to Alpha or Delta variant challenge. qRT-PCR analysis of Alpha- or Delta- challenged mice revealed no significant difference between viral RNA burden in the lung, nasal wash or brain. However, histopathological analysis revealed high lung tissue inflammation and cell infiltration following Delta- but not Alpha-challenge at day 6. Additionally, pro-inflammatory cytokines were highest at day 6 in Delta-challenged mice suggesting enhanced pneumonia. Total RNA-sequencing analysis of lungs comparing infected to uninfected mice revealed that Alpha-challenged mice have more total genes differentially activated, conversely, Delta-challenged mice have a higher magnitude of differential gene expression. Delta-challenged mice have increased interferon-dependent gene expression and IFN-{gamma} production compared to Alpha. Analysis of TCR clonotypes suggested that Delta challenged mice have increased T-cell infiltration compared to Alpha challenged. Our data suggest that Delta has evolved to engage interferon responses in a manner that may enhance pathogenesis. The in vivo and in silico observations of this study underscore the need to conduct experiments with VOC strains to best model COVID-19 when evaluating therapeutics and vaccines. ImportanceThe Delta variant of SARS-CoV-2 is known to be more transmissible and cause severe disease in human hosts due to mutations in its genome that are divergent from previous variants of concern (VOC). Our study evaluates the pathogenesis of Delta in the K18-hACE2 mouse model compared to the Alpha VOC. We observed that relative to Alpha, Delta challenge results in enhanced inflammation and tissue damage with stronger antiviral responses. These observations provide insight into Deltas unique pathogenesis.

Serological survey of SARS-CoV-2 incidence conducted at a rural West Virginia hospital

The SARS-CoV-2 pandemic has affected all types of global communities. Differences in urban and rural environments have led to varying levels of transmission within these subsets of the population. To fully understand the prevalence and impact of SARS-CoV-2 it is critical to survey both types of community. This study establishes the prevalence of SARS-CoV-2 in a rural community: Montgomery, West Virginia. Approximately 10% of participants exhibited serological or PCR-based results indicating exposure to SARS-CoV-2 within 6 months of the sampling date. Quantitative analysis of IgG levels against SARS-CoV-2 receptor binding domain (RBD) was used to stratify individuals based on antibody response to SARS-CoV-2. A significant negative correlation between date of exposure and degree of anti-SARS-CoV-2 RBD IgG (R2 = 0.9006) was discovered in addition to a correlation between neutralizing anti-SARS-CoV-2 antibodies (R2 = 0.8880) and days post exposure. Participants were confirmed to have normal immunogenic profiles by determining serum reactivity B. pertussis antigens commonly used in standardized vaccines. No significant associations were determined between anti-SARS-CoV-2 RBD IgG and age or biological sex. Reporting of viral-like illness symptoms was similar in SARS-CoV-2 exposed participants greater than 30 years old (100% reporting symptoms 30-60 years old, 75% reporting symptoms >60 years old) in contrast to participants under 30 years old (25% reporting symptoms). Overall, this axnalysis of a rural population provides important information about the SARS-CoV-2 pandemic in small rural communities. The study also underscores the fact that prior infection with SARS-CoV-2 results in antibody responses that wane over time which highlights the need for vaccine mediated protection in the absence of lasting protection.

SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern induce lethal disease in K18-hACE2 transgenic mice despite convalescent plasma therapy

SARS-CoV-2 variants of concern (VoCs) are impacting responses to the COVID-19 pandemic. Here we present a comparison of the SARS-CoV-2 USA-WA1/2020 (WA-1) strain with B.1.1.7 and B.1.351 VoCs and identify significant differences in viral propagation in vitro and pathogenicity in vivo using K18-hACE2 transgenic mice. Passive immunization with plasma from an early pandemic SARS-CoV-2 patient resulted in significant differences in the outcome of VoC-infected mice. WA-1-infected mice were protected by plasma, B.1.1.7-infected mice were partially protected, and B.1.351-infected mice were not protected. Serological correlates of disease were different between VoC-infected mice, with B.1.351 triggering significantly altered cytokine profiles than other strains. In this study, we defined infectivity and immune responses triggered by VoCs and observed that early 2020 SARS-CoV-2 human immune plasma was insufficient to protect against challenge with B.1.1.7 and B.1.351 in the mouse model.

Interplay of antibody and cytokine production reveals CXCL-13 as a potential novel biomarker of lethal SARS-CoV-2 infection

The SARS-CoV-2 pandemic is continuing to impact the global population. This study was designed to assess the interplay of antibodies with the systemic cytokine response in SARS-CoV-2 patients. We demonstrate that significant anti-SARS-CoV-2 antibody production to Receptor Binding Domain (RBD), Nucleocapsid (N), and Spike S1 subunit (S1) of SARS-CoV-2 develops over the first 10 to 20 days of infection. The majority of patients produced antibodies against all three antigens (219/255 SARS-CoV-2 positive patient specimens, 86%) suggesting a broad response to viral proteins. Patient mortality, sex, blood type, and age were all associated with differences in antibody production to SARS-CoV-2 antigens which may help explain variation in immunity between these populations. To better understand the systemic immune response, we analyzed the production of 20 cytokines by SARS-CoV-2 patients over the course of infection. Cytokine analysis of SARS-CoV-2 positive patients exhibited increases in proinflammatory markers (IL-6, IL-8, IL-18) and chemotactic markers (IP-10, SDF-1, MIP-1{beta}, MCP-1, and eotaxin) relative to healthy individuals. Patients who succumbed to infection produced decreased IL-2, IL-4, IL-12, IL-13, RANTES, TNF-, GRO-, and MIP-1 relative to patients who survived infection. We also observed that the chemokine CXCL13 was particularly elevated in patients that succumbed to infection. CXCL13 is involved in B cell activation, germinal center development, and antibody maturation, and we observed that CXCL13 levels in blood trended with anti-SARS-CoV-2 antibody production. Furthermore, patients that succumbed to infection produced high CXCL13 and also tended to have high ratio of nucleocapsid to RBD antibodies. This study provides insights into SARS-CoV-2 immunity implicating the magnitude and specificity of response in relation to patient outcomes.