Animal Pre-Clinical Study of an Inactivated SARS-CoV-2 Vaccine candidate (Osvid-19 ): Immunogenicity, Protective, and Safety Aspects

Background: This study aimed to evaluate the outcomes of preclinical studies on the safety and immunogenicity of an inactivated COVID-19 vaccine candidate to warrant further clinical evaluation. Method(s): SARS-CoV-2 positive nasopharyngeal swab specimens were confirmed by real-time polymerase chain reaction and next-generation sequencing. The safety and immunogenicity tests of the COVID-19 vaccine were carried out in rats and Rhesus monkeys, and Balb/C mice and Rhesus monkeys, respectively. Result(s): The candidate vaccine was well tolerated and induced promising levels of SARS-CoV-2- specific IgG1, IgG2a, and Granzyme B in Balb/C mice, and anti-SARS-CoV-2 spike IgG and neutralizing antibodies in Rhesus monkeys. Based on cVNT results, the inactivated vaccine in 0.5 and 1 microg/100 microL doses was able to induce a neutralizing effect against the SARS-CoV-2 virus up to a dilution of 1:512 and 1:1000. The protective efficacy of the vaccine candidate was challenged with 2 x108 PFU of live viruses and confirmed by lung CT scan and histopathological evaluations compared to the control group. Repeated intramuscular injection of the candidate vaccine was generally well-tolerated in Rats and Rhesuses. No significant side effects were observed in rats injected with ten full human doses and in the Rhesus monkeys with three full human doses. Conclusion(s): Based on the findings presented in this study, it is recommended that this vaccine be moved into human testing commencing with a phase I clinical trial.Copyright © 2021 Muslim OT et al.

PROTECTION AGAINST HETEROLOGOUS CHALLENGE 1 YEAR AFTER INFANT SARS-CoV-2 IMMUNIZATION

Background: Although mRNA SARS-CoV-2 vaccines have received emergencyuse- authorization for infants age 6 months and older, vaccine uptake is slow, stressing that questions of safety and durability of vaccine efficacy remain prominent. Method(s): Infant rhesus macaques (RMs) (n=8/group) at 2 months of age, comparable to human toddler age, were immunized intramuscularly at weeks 0 and 4 with 30mug stabilized prefusion SARS-CoV-2 S-2P spike (S) protein (Washington strain) encoded by mRNA encapsulated in lipid nanoparticles (mRNA-LNP) or 15mug S protein mixed with 3M-052 in stable emulsion (Protein). At 1 year, vaccinated and age-matched unvaccinated RM (n=8) were challenged intranasally (106pfu) and intratracheally (2x106pfu) with B.1.617.2. Lung radiographs and pathology were blindly assessed, viral N gene RNA (vRNA) copies were measured by qPCR in pharyngeal swabs and lung, and neutralizing antibody and peripheral blood T cell responses were measured. Result(s): At 1 year, D614G-specific neutralizing antibody (nAb) titers were still detectable in the Protein (ID50=755;range: 359-1,949) and mRNA-LNP groups (ID50=73;range: 41-240). Both vaccines also induced cross-neutralizing antibodies to B.1.617.2. Peripheral blood CD4+ T cell responses to the ancestral spike protein at week 52 did not differ between the groups. However, median CD8+ T cell responses were higher (p=0.002, Mann Whitney) in the mRNA-LNP group (2.8%;range: 0.9%-7.1%) compared to the Protein group (0.8%;range: 0.1%-1.6%). Control RMs had significantly higher median vRNA copies/ml (1.4+/-2.7x108) in day 4 pharyngeal swabs compared to Protein (3.8+/-6.8x103) or mRNA-LNP (4.4+/-9.7x105) vaccinated RMs. Severe lung pathology was observed in 7 of 8 controls compared to 1 of 8 or 0 of 8 RMs in the mRNA-LNP or Protein group respectively. Protection against lung inflammation was associated with nAb titers (r=-0.592, p=0.003) (Figure 1). Conclusion(s): These results demonstrate that despite lower vaccine doses compared to adults, both protein and mRNA vaccines were safe, induced durable immune responses and provided comparable protective efficacy against infection with a heterologous SARS-CoV-2 variant in infants, implying that early life vaccination of human infants may lead to durable immunity. Neutralizing ID50 antibody titers are a correlate of protection in infant RMs challenged with SARS-CoV-2.

PROLONGED SARS-CoV-2 VIRAL BURDEN IN SIV-SARS-CoV-2 COINFECTED MACAQUES

Background: Individuals living with HIV are at increased risk of morbidity and mortality from COVID-19. Furthermore, SARS-CoV-2 infection in immunocompromised HIV infected individuals poses a risk to prolonged infection and viral shedding and the emergence of new variants of concern (VOCs). Using the SIV macaque model for AIDS, we are investigating the hypothesis that immune dysfunction during HIV infection will prolong SARSCoV- 2 viral infection, promote enhanced COVID-19 disease, and accelerate viral evolution. Here, we report the impact of SIV-CoV-2 co-infection on immune responses and pathogenesis. Method(s): Eight female rhesus macaques (aged 7-15 years, 5.5-9.9kg) were infected with SIVmac251 via low dose intravaginal challenge and then inoculated with 6.5x105 TCID50/mL SARS-CoV-2 (WA-1) at 17-34 weeks post-SIV infection via combined intranasal and intratracheal routes. Blood, bronchoalveolar lavage (BAL), stool, and nasal, oral, and rectal swabs were collected pre-infection through 14 days post-infection (DPI) to measure immune responses and viremia. ELISAs, ELISPOT, qRT-PCR, lung pathology, cytokine multiplex, and virus neutralization assays were performed to measure viral loads, pathogenesis, and immune responses. Result(s): Three days post-SARS-CoV-2 infection, we observed a transient decrease in CD4 counts, but there were no changes in clinical symptoms or plasma SIV viral loads. However, SARS-CoV-2 replication persisted in the upper respiratory tract, but not the lower respiratory tract. In addition, SARS-CoV-2 IgG seroconversion was delayed and antigen-specific T-cell responses were dampened. Notably, viral RNA levels in nasal swabs were significantly higher 7-14 DPI in SIV+ compared to previously published results using the same SARS-CoV-2 challenge virus in SIV- rhesus (PMCID: PMC8462335, PMC8829873). In addition, SIV/CoV-2 co-infected animals exhibited elevated levels of myeloperoxidase (MPO), a marker of neutrophil activation and increased lung inflammation. Conclusion(s): Here we provide evidence for the utility of the rhesus macaque in modeling human HIV-SARS-CoV-2 co-infection. Our results suggest that immunosuppression during SIV infection impairs de novo generation of anti-SARS-CoV-2 immunity, that may contribute to prolonged SARS-CoV-2 viral shedding, increased transmission windows, altered disease pathogenesis, and lower protection against subsequent SARS-CoV-2 exposures. Studies in progress will determine if SARS-CoV-2 viral evolution is accelerated in SIV-infected macaques.

HIGH FAT AND SUGAR DIET INDUCES GUT LEUKOCYTE DISRUPTIONS EXACERBATED BY SARS-CoV-2

Background: The disparity in COVID-19 disease burden between European, Asian, and African countries is notable, with considerably higher morbidity and mortality in many European countries as well as the U.S. Dietary differences between regions could play a role in differential COVID-19 pathogenesis, as Western diets high in fat and sugar have been implicated in enhancing gut damage and pathogenesis during viral infections. Here we investigate the effect of diet on gut immunity and SARS-CoV-2 infection. Method(s): Six pigtail macaques were fed a commercial monkey chow diet, then transitioned to a high fat and sugar chow diet (HFD) for approximately two months prior to infection with Delta strain SARS-CoV-2. Animals were sampled prior to HFD initiation, during HFD administration but prior to infection, and for approximately one month post-infection. HFD was maintained following infection and animals were euthanized at the study conclusion. Result(s): Viral RNA was detected for up to 28 days post-infection in nose swabs, with peak viral load at day 2 at a mean of 8.2x109 copies/mL of swab fluid. Subgenomic RNA (sgRNA, indicating viral replication) decayed more rapidly, with all animals having undetectable sgRNA by day 21, and a lower peak of 2.6x109 copies/mL swab fluid on day 2. Viral RNA load was approximately 3.5 logs greater and sgRNA load approximately 3 logs higher at day 2 than in rhesus macaques infected with WA2020 SARS-CoV-2 and fed standard monkey chow. Mucosal rectal biopsies indicated significantly lower B cell frequencies from baseline to approximately two months following HFD administration (p=0.04, Dunn's), and frequencies had not recovered approximately one month postinfection. GI tract-resident IgG+ B cells were nearly absent at necropsy, with mean frequency 0.03% of total B cells. B cell loss was coupled with modest T cell expansion during HFD administration, though frequencies declined following infection. Furthermore, NK cell frequencies tended to decline from baseline throughout HFD administration, and were further depleted at necropsy one month post-infection. Conclusion(s): SARS-CoV-2 infection can induce lymphopenia, and our sampling of gut mucosal tissue indicates B cell depletion and NK cell loss with a HFD that is further exacerbated by SARS-CoV-2 infection. Excess dietary fat and sugar may disrupt gut barrier integrity and immunity, in turn predisposing the tissue to pathology of viral infection.

Translational Model-Based Meta-Analysis -a Powerful Quantitative Tool in Developing Vaccines against Sars-Cov- 2 Variants

BACKGROUND: Quantitative models leveraging non-clinical data to predict clinical vaccine efficacy provide a translational framework to rapidly develop vaccines/ boosters against new strains of SARS-CoV- 2. METHOD(S): Previously, based on a systematic literature review, we performed a translational Model-Based Meta-Analysis (MBMA)1 integrating data of wild-type (WT) SARS-CoV- 2 from 13 rhesus macaques (RM) studies and eight clinical trials. The model is here updated with data from 32 additional RM studies including newer strains of SARS-CoV- 2 (e.g., omicron). Non-linear mixed-effects modeling was used to quantify the relationship in RM between serum neutralizing (SN) titers after vaccination and peak viral load (VL) post challenge in relevant tissue matrices. RESULT(S): The plot2 shows the model describes the relationship between SN titers and peak VL across all specimens well. The overlap between the confidence intervals across virus strains suggests that the model can be leveraged to describe RM data across viral SARS-CoV- 2 strains. CONCLUSION(S): The previous work1 demonstrated that RM VL is quantitatively predictive of clinical efficacy, and so this update provides a framework to predict clinical vaccine efficacy against newer variants using only RM data (Figure Presented).

PROTECTIVE EFFICACY of A GASTROINTESTINAL SARS-CoV-2 VACCINE

Background: SARS-CoV-2 remains a global threat, despite the rapid deployment but limited coverage of multiple vaccines. Alternative vaccine strategies that have favorable manufacturing timelines, greater ease of distribution and improved coverage may offer significant public health benefits, especially in resource-limited settings. Live oral vaccines have the potential to address some of these limitations;however no studies have yet been conducted to assess the immunogenicity and protective efficacy of a live oral vaccine against SARS-CoV-2. Thus far, we assessed whether oral administration of live SARS-CoV-2 in non-human primates might offer prophylactic benefits. Methods: In this study, we assessed the immunogenicity of gastrointestinal (GI) delivery of SARS-CoV-2 and the protective efficacy against intranasal and intratracheal SARS-CoV-2 challenge in rhesus macaques. Esophagogastroduodenoscopy (EGD) administration of 106 50% Tissue Culture Infectious Dose (TCID50) of SARS-CoV-2 elicited low levels of serum neutralizing antibodies (NAb), which correlated with modestly diminished viral loads in nasal swabs (NS) and Bronchoalveolar Lavage (BAL) post-challenge. In addition, mucosal NAb titers from the rectal swabs (RS), NS, and BAL and Spike-specific T-cell responses appear to be below the limit of detection post-vaccination. Replicating virus was only observed in 44% of macaques and on limited number of dates post vaccination, suggesting limited, if any, productive infection in the GI tract. Results: We demonstrate that GI delivery of live 1x106 TCID50 SARS-CoV-2 elicited modest immune responses and provided partial protection against intranasal and intratracheal challenge with SARS-CoV-2. Moreover, serum neutralizing antibody titers correlated with protective efficacy. Conclusion: These data provide proof-of-concept that an orally administered vaccine can protect against respiratory SARS-CoV-2 challenge, but the limited immunogenicity and protective efficacy observed here suggests that the oral vaccine approach will require optimization.

EVIDENCE of BRAIN HYPOXIA in NEUROPATHOLOGY of SARS-CoV-2-INFECTED NONHUMAN PRIMATES

Background: Neurological manifestations are a major complication of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and likely contribute to symptoms of "long COVID". Elucidating the mechanisms that underlie neuropathogenesis in infection is critical for identifying or developing viable therapeutic strategies. While neurological injury in infection is varied, cerebrovascular disease is seen at a high frequency among patients over 50 years of age. Additionally, microhemorrhages and hypoxic-ischemic injury are often described in brain autopsy series of human subjects who died from COVID-19. Here, we report neuropathology in aged SARS-CoV-2 infected non-human primates (NHPs) is consistent with that observed in aged human subjects and provide insight into the underlying cause. Methods: Four adult Rhesus macaques and four African green monkeys were inoculated with the 2019-nCoV/USA-WA1/2020strain of SARS-CoV-2 via a multi-route mucosal or aerosol challenge. Two of each species were included as age-matched controls. Frontal, parietal, occipital, and temporal lobes, basal ganglia, cerebellum, and brainstem were interrogated through histopathological and immunohistochemical techniques to identify and characterize the observed pathology. Results: Like humans, pathology was variable but included wide-spread inflammation with nodular lesions, neuronal injury, and microhemorrhages. Neuronal degeneration and apoptosis were confirmed with FluoroJade C and cleaved caspase 3 IHC, which showed foci of positivity, particularly among cerebellar Purkinje cells. This was seen even among infected animals that did not develop severe respiratory disease but was not seen in age-matched controls. Significant upregulation of the alpha subunit of hypoxia inducible factor 1 (HIF1-α), indicative of tissue hypoxia, was observed in brain of all infected animals, regardless of disease severity. Sparse virus was detected in brain endothelial cells but did not associate with the severity of CNS injury. Conclusion: SARS-CoV-2 infected NHPs are a viable animal model for advancing our current understanding of infection-associated neuropathogenesis. Upregulation of HIF1-α in brain of infected animals suggests cerebral hypoxia may underlie or contribute to neuroinflammation and neuronal injury/death and may provide some insight into neurological manifestations observed among asymptomatic patients or those only suffering mild disease.

SPIKE PLUS NUCLEOCAPSID VACCINE PROTECTS AGAINST SARS-CoV-2 DELTA INFECTION in NHPs

Background: SARS-CoV-2 vaccines capable of inducing broad and cross-reactive humoral and T cell responses help to fight against emerging variants. In this study we compared the immunogenicity and efficacy of modified vaccinia Ankara (MVA) based SARS-CoV-2 vaccine expressing furin-cleavage site inactivated stabilized spike (SdFCS) and nucleocapsid (N) delivered via intramuscular (IM), buccal or sublingual (SL) routes in rhesus macaques (RMs). Methods: Three groups (n=5/group) of RMs were immunized with MVA/SdFCS-N vaccine on weeks 0 and 4, via IM, buccal, or SL route. An additional group (control) received non-recombinant MVA via IM. IM vaccinations were delivered using needle and SL and buccal vaccinations were delivered using a needle-free injection device. All RMs were challenged with B.1.617.2 strain (Delta) of SARS-CoV-2 at week 8 via intratracheal and intranasal routes simultaneously. Various humoral and cellular immune parameters were determined post vaccination and challenge. SARS-CoV-2 subgenomic RNA (sgRNA) was measured to monitor virus replication in the upper (nose) and lower (lung) respiratory tract. Results: IM vaccination induced strong RBD-specific IgG antibody in serum, nose, throat, lung, and rectum. The serum antibody showed strong live virus neutralizing activity against WA-1/2020 (median of 415) and B.1.617.2 strains (median of 317). Serum from IM vaccinated animals also demonstrated strong non-neutralizing effector functions such as ADCD, ADCP and ADNKA. In addition, IM vaccination induced strong CD4 and CD8 T cell response in the blood that was directed against both S and N. In contrast, the SL and buccal vaccination-induced antibody showed lower neutralization titer against WA-1/2020 (143 and 302, respectively), and showed 4.5-fold lower cross reactivity neutralization titer against B.1.617.2 compared to WA-1/2020. Following challenge with B.1.617.2, the IM group RMs showed superior protection with 3 of the 5 animals being negative in upper and lower respiratory airways at Day 2. In contrast, no significant protection was observed in the SL group. Vaccine induced neutralizing and non-neutralizing antibody effector functions showed direct association with protection. Conclusion: Our findings showed that IM vaccination with improved MVA-based SARS-CoV-2 vaccine elicits cross-reactive antibody and T cell responses and protect against heterologous SARS-CoV-2 Delta challenge in RMs. They also showed IM vaccinations are superior to oral vaccinations.

PULMONARY CMV REACTIVATION FOLLOWING SARS-CoV-2: IMPLICATIONS for IMMUNOPATHOLOGY

Background: SARS-CoV-2 infection results in a spectrum of disease severity attributable to the magnitude of the underlying inflammatory response. Aged individuals with co-morbidities are most vulnerable and severely affected, but the mechanisms driving aberrant immune responses fueling SARS-CoV-2 immunopathology in this high-risk population are not fully elucidated. We hypothesized that asymptomatic CMV infection might exacerbate SARS-CoV-2 pathogenesis since its replication is both a cause and consequence of inflammation and appears to worsen oxygenation in critically ill patients (Limaye, JAMA, 2017). CMV-seropositivity was associated with increased hospitalization among people with SARS-CoV-2 infection (Shrock, Science, 2020). To begin to address this hypothesis, we utilized the rhesus macaque model of natural rhesus (Rh)CMV infection to investigate the extent to which SARS-CoV-2 induces CMV reactivation in the anatomic sites of SARS-CoV-2 pathology. Methods: To assess CMV reactivation, eight aged, type 2 diabetic RhCMV-seropositive rhesus macaques (sera anti-CMV IgG: 300-1400 ng/ml) were infected with high-dose SARS-CoV-2 (2.5x10 6 PFU) and monitored for 7 days prior to euthanasia. Samples from the respiratory tract, intestinal tract, and blood were collected to assess viral and inflammatory dynamics in distinct tissue compartments. Results: Following infection, SARS-CoV-2 replication was observed throughout the respiratory tract, which was associated with local and systemic inflammation and immune activation. Lung histopathological assessments revealed development of interstitial pneumonia with colocalization of SARS nucleocapsid protein within pneumocytes. qPCR assays targeting RhCMV gB showed CMV DNA within the caudal lung lobe (up to 103 CMV DNA copies/mg of tissue) in all animals at day 7, and the animal with the highest CMV DNA presented with the most profound clinical symptoms. Strikingly, CMV DNA copies strongly correlated with CD4 and CD8 T cell activation indices in blood and spleen (r = 0.96, p< 0.001). Additionally, we found RhCMV reactivation in the ileum, where high levels of ACE2 are reported. Conclusion: SARS-CoV-2 infection of RhCMV-seropositive macaques results in CMV reactivation in the anatomic sites where SARS-CoV-2 causes pathology. Future experimental studies should address whether CMV reactivation exacerbates SARS-CoV-2 pathogenesis.

TYPE-I INTERFERON MODULATION in VIVO BLOCKS SARS-CoV-2 REPLICATION and INFLAMMATION

Background: Systemic and local inflammation following SARS-CoV-2 infection has been widely described and predictive of disease severity and death. However, the exact immune mediators driving inflammation contributing to SARS-CoV-2 host defense vs. those driving immune-mediated pathology in humans have not been fully elucidated. Deficiencies in type-I interferon (IFN-I) responses, including inborn errors to genes in the IFN-I pathway, neutralizing auto-antibodies against all subtypes of IFN-I, or the lack of production of IFN-I, are associated with severe COVID-19 in otherwise healthy individuals. Conversely, sustained IFN-I responses have been shown to contribute to severe COVID-19 by exacerbating inflammation, and prolonged IFN-I signaling has been shown to interfere with lung repair following viral infection and to increase susceptibility to bacterial infections. Thus, it is critical to understand the roles of IFN-I signaling in COVID-19 to design therapeutic strategies. Methods: Here, we modulated IFN-I signaling in rhesus macaques (Macaca mulatta;RMs) from day-1 through day 2 post SARS-CoV-2 infection (dpi) using an IFN-I antagonist (IFNant). Eighteen RMs (9 control and 9 IFNant treated) were infected with SARS-CoV-2 on day 0, with 6 RMs sacrificed at 2, 4, and 7dpi. Nasal and throat swabs were collected for viral load;blood and bronchoalveolar lavage fluid (BAL) for flow cytometry and RNAseq. Results: IFNant treatment prior to infection resulted in a highly significant and consistent reduction in SARS-CoV-2 viral load in the lower airways (>3-log difference;2dpi BAL) and upper airways (nasal and throat swabs). Treatment with IFNant initiated also potently reduced: (i) soluble markers of inflammation in BAL, (ii) expansion of inflammatory monocytes (CD14+CD16+), and (iii) pathogenesis in the lung. Furthermore, Siglec-1 expression, which has been shown to enhance SARS-CoV-2 infection, was rapidly downregulated in the lung and in monocytes of IFNant-treated RMs. Remarkably, RNAseq analysis showed a robust reduction in pathways associated with inflammation and decreased levels of interferon-stimulated genes post-infection in treated RMs. Thus, IFNant treatment prior to infection resulted in limited viral replication, inflammation, and pathogenesis in SARS-CoV-2-infected RMs. Conclusion: These data indicate a vital, early role of IFN-I in regulating COVID-19 progression and emphasize the importance of understanding IFN-I pathways in COVID-19 for the development of targeted therapeutic strategies.

DURABILITY and BOOSTABILITY of Ad26.COV2.S in RHESUS MACAQUES

Background: Ad26.COV2.S is a single-shot vaccine that has demonstrated clinical efficacy against symptomatic COVID-19. In this study, we report the durability of immune responses in 20 rhesus macaques received single-shot Ad26.COV2.S and the immunogenicity of a booster shot at 8-10 months following the initial immunization. Methods: Animals were immunized by intramuscular route with 1011 vp (N=10) or 5x1010 vp (N=10) Ad26.COV2.S and were followed for either 230 or 315 days. Animals were then boosted with 5x1010 vp Ad26.COV2.S (N=10). Humoral immune responses including RBD-specific Ig ELISA and pseudovirus-based virus neutralization response were monitored. Circulating RBD-specific memory B cells and bone marrow plasma cells were assessed by multiparameter flow cytometry. Results: Ad26.COV2.S elicited robust and comparable RBD-specific binding and neutralizing antibody responses in animals that received the 1011 vp and 5x1010 vp doses, which peaked on days 28-56, and then showed a biphasic decay. All animals showed binding antibody responses for the duration of follow-up, and 17 of 20 animals showed neutralizing antibody responses by day 230-315. RBD-specific memory B cell response peaked on day 14-28 followed by a gradual decline, and remained detectable in 17 of 20 animals by day 230-315. On day 315 following vaccination, bone marrow RBD-specific PCs were detected in the majority of vaccinated macaques, including in all animals that received the 1011 vp dose. Following Ad26.COV2.S boost immunization, RBD-specific binding antibody responses increased 31-69 fold compared with pre-boost levels against the ancestral (WA1/2020), alpha (B.1.1.7), beta (B.1.351), kappa (B.1.617.1), and delta (B.1.617.2) SARS-CoV-2 variants. Neutralizing antibody responses increased 23-43 fold compared with pre-boost levels against the ancestral, alpha, beta, gamma (P.1), kappa, and delta SARS-CoV-2 variants. Antigen-specific memory B cell response also increased 8 fold following the boost immunization. Conclusion: Ad26.COV2.S elicited durable antibody and B cell responses, and a late boost with Ad26.COV2.S resulted in a dramatic increase in humoral immunity that were highly cross-reactive across multiple SARS-CoV-2 variants in rhesus macaques. These data contribute to our understanding of Ad26.COV2.S durability and boostability, and provide important data to inform COVID-19 vaccine boosting strategies in humans.

SARS-CoV-2-INFECTED MACAQUES EXHIBIT MICROBIAL TRANSLOCATION ACROSS INTESTINAL MUCOSA

Background: Gastrointestinal symptoms and viral RNA (vRNA) in stool have been described in human SARS-CoV-2 infections. However, intestinal pathology and related inflammation have not been extensively described in humans or animal models. Here we investigate the effect of SARS-CoV-2 infection on the gut mucosa and inflammation in rhesus macaques (RM) and humans. Methods: Fourteen adult RM were infected with US/WA-1/2020 SARS-CoV-2 instilled intranasally and intratracheally. Animal clinical features (mass, temperature, etc.) and samples (nasal swabs, throat swabs, blood, stool, etc.) were collected at baseline and up to day 10 post-infection at necropsy. RNA was extracted from swab and stool samples and vRNA measured by qRT-PCR. Plasma samples were assessed for inflammatory biomarkers by ELISA. Tissues collected at necropsy were fixed and evaluated for microbial translocation through immunohistochemical (IHC) staining of bacterial products;H&E staining was also performed. Tissues were additionally collected from uninfected RM and processed in the same manner. Human plasma samples from individuals with moderate COVID-19 were collected at early infection and recovery time points and assessed for inflammatory biomarkers. Results: SARS-CoV-2 infection of RM did not induce fever nor weight loss over five percent. vRNA was detected in all animals in nasal and throat swabs. vRNA, including subgenomic RNA indicative of viral replication, was also detected in stool samples. Scores for translocating bacteria in colon sections stained by IHC for bacterial products were higher for SARS-CoV-2 infected RM than uninfected controls. Additionally, follicles made up a higher percentage of total mesenteric lymph node area in SARS-CoV-2 infected animals than control RM. Furthermore, soluble CD14 in plasma increased significantly from baseline to day 10 of SARS-CoV-2 infection (p=0.0006) and decreased significantly in humans from early infection to recovery time points (p=0.0295). Conclusion: Thus, adult RM experienced mild to moderate SARS-CoV-2 infections yet demonstrated evidence of microbial translocation. Humans similarly demonstrated evidence of microbial translocation that decreased upon recovery from COVID-19. These data suggest gut pathology in SARS-CoV-2 infection may be contributing to systemic inflammation in COVID-19.

Cross-Species Translation of Correlates of Protection for COVID-19 Vaccine Candidates Using Quantitative Tools

Background. Several COVID-19 vaccines have been authorized, and the need for rapid, further modification is anticipated. This work uses a Model-Based Meta-Analysis (MBMA) to relate, across species, immunogenicity to peak viral load (VL) after challenge and to clinical efficacy. Together with non-clinical and/or early clinical immunogenicity data (ECID), this enables prediction of a candidate vaccine's clinical efficacy. The goal of this work was to enable the accelerated development of vaccine candidates by supporting Go/No-Go and study design decisions, and the resulting MBMA can be instrumental in decisions not to progress candidates to late stage development. Methods. A literature review with pre-specified inclusion/exclusion criteria enabled creation of a database including nonclinical serum neutralizing titers (SN), peak VL after challenge with SARS-CoV-2 (VL), along with data from several clinical vaccine candidates. Rhesus Macaque (RM) and golden hamster (GH) were selected (due to availability and consistency of data) for MBMA modeling. For both RM and GH, peak post-challenge VL in lung and nasal tissues were used as surrogates for clinical disease and were related to pre-challenge SN via the MBMA. The VL predictions from the RM MBMA were scaled to incidence rates in humans, with a scaling factor between RM and human SN estimated using early Phase 3 efficacy data. This enabled clinical efficacy predictions based on ECID. To qualify the model's predictive power, efficacies of COVID-19 vaccine candidates were compared to those predicted from the MBMA and their respective Ph1/2 SN data. More recently available clinical data enable building a clinical MBMA;comparing this to the RM MBMA further supports SN as predictive. Results. The MBMA analyses identified a sigmoidal decrease in VL (increasing protection) with increase in SN in all three species, with more SN needed (in both RM and GH) for protection in nasal swabs than in BAL (see figure). The comparison between predicted and reported clinical efficacies demonstrated the model's predictive power across vaccine platforms. RM and GH MBMA Protection Models and Translational Prediction with Observed Efficacies Sizes of circles indicate relative weight of the data in the respective quantitative model. Model and data visualizations have been harmonized (across tissue-types) separately for each of RM and GH using VACHER (Lommerse, et al., CPT:PSP, in press). Conclusion. By quantifying adjustments needed between species and assays, translational MBMA can inform development decisions by using nonclinical SN and VL, and ECID to predict protection from COVID-19.

Translational PET imaging evaluation of [18F]Favipiravir by positron emission tomography

Objectives: With over 90 million cases reported in the globe, the COVID-19 pandemic caused by SARS-CoV-2 has been a serious public health crisis. Development of novel and specific antiviral drugs against the SARS-CoV-2 has been an urgent demand. One such drug is Favipiravir, initially developed as an antiviral drug against influenza. Now Favipiravir has received approvals for emergency use against SARS-CoV-2 in many countries. A better understanding of Favipiravir’s biodistribution and pharmacokinetics in vivo will facilitate the clinical development of antiviral drugs against the SARS-CoV-2. Herein, we reported the evaluation of [18 F]Favipiravir with PET in cross-species studies to demonstrate the drug’s biodistribu-tion and pharmacokinetics and investigate the potentially increased risk of neurodegenerative diseases and/or neuroinflammation to COVID-19. Methods: The radiosynthesis of [18 F]Favipiravir was via labeling a commercially available precursor, methyl-5-chloroisoxazolo[4,5-b] pyrazine-3-carboxylate with K[18F]F/K222 and K2CO3 in DMSO at 130°C for 10 min, followed by hydrolysis with NaOH (aq.) at 110°C for 15 min.1 The whole body distribution on CD-1 mice was performed at four time points (5, 15, 30, 60 min). PET studies were carried out in CD-1 mice and AD mice (5XFAD) and naïve rhesus monkeys. We also performed the radiometabolite analysis of [18 F]Favipiravir in plasma and brain of CD-1 mice at 30 min post-injection. Results: [18 F]Favipiravir was obtained in 29% isolated radiochemi-cal yield (decay corrected). The radiochemical purity of the tracer was greater than 99%. No sign of radiolysis was observed for [18F] Favipiravir up to 120 min after formulation with 10% EtOH/saline. High radioactivity accumulation was observed in blood, lung, liver, kidney, and bone (around or more than 5% ID/g, injected dose per gram of wet tissue). The radioactivity level reached a plateau in small intestine, kidney and liver at 30,15 and 5 min, respectively, followed by slow washout, indicating that [18F]Favipiravir was possibly eliminated via the hepatobiliary and urinary pathway. For the radio-metabolic analysis of [18F]Favipiravir, average 41% and 89% of the radioactivity was parent fraction in the mice brain and plasma at 30 min post-injection (n=2), respectively. In PET imaging of CD-1 mice, the standard uptake value (SUV) of [18F]Favipiravir in brain reached its max value of 0.5 at 10 min and slowly reduced to 0.4 at 60 min. The results of PET imaging of AD mice with [18 F]Favipiravir were similar with that of CD-1 mice. In PET imaging of Rhesus monkeys, the brain uptake of [18 F]Favipiravir reached the max value of 0.5 SUV at 5 min and subsequently decreased to 50-60% of the maximum at 60 min. Conclusion: The evaluation of [18F]Favipiravir has demonstrated with bio-distribution and PET in mice and NHPs. Further evaluation of pharmacokinetics of [18F]Favipiravir in whole body monkey scans and LPS-induced neuroinflammation models is underway.

Evaluation of the Relationship between Clonal Hematopoiesis and Severe COVID-19 Disease in Rhesus Macaques

Clinical manifestations of infection with the novel SARS-CoV-2 in humans are widely varied, ranging from asymptomatic to COVID-19 respiratory failure and multiorgan damage. Profound inflammation is the hallmark of severe COVID-19 disease, and commonly does not occur until the second week of infection. Although risk factors for this late hyperinflammatory disease have been identified, most notably age and pre-existing co-morbidities, even within high-risk groups the specific factors leading to severe COVID-19 illness remain elusive. Acquired somatic mutations in hematopoietic stem and progenitor cells (HSPCs), termed clonal hematopoiesis (CH), are associated with advanced age, and loss of function (LOF) mutations in certain genes, most commonly DNMT3A and TET2, have been linked to a marked hyperinflammatory phenotype as well as clonal expansion of mutant HSPCs. Given the similar age range of frequent CH and severe COVID-19 disease, the presence of CH could impact the risk of severe COVID-19. Several human cohort studies have suggested this relationship may exist, but results to date are conflicting. Rhesus macaques (RM) have been established as a model for SARS-CoV infection and are being utilized to test therapies and vaccine development, but up to now, macaques have not been reported to develop late hyperinflammatory COVID-19 disease. We have created a robust RM model of CH by introducing LOF TET2 mutations into young adult HSPC via CRISPR/Cas9 followed by autologous transplantation, recapitulating the clonal expansion and hyperinflammatory phenotype. Thus, we hypothesized that macaques with CH could develop severe late COVID-19 disease and be utilized as a model to study disease pathophysiology or test therapeutic approaches. Macaques with either engineered (n=2) or natural CH (n=1) along with age-matched transplanted controls (n=3) were inoculated with SARS-CoV-2 and monitored clinically and via laboratory studies until 12 days post-inoculation (dpi). Macaques normally clear infection and symptoms within 3-5 days of infection. No significant differences in clinical symptoms and blood counts were noted, however, an aged animal with natural DNMT3A CH died on 10 dpi. IL-6 levels were somewhat higher in sera of the CH animals until 12 dpi, and in BAL, mean concentrations of MCP-1, IL-6, IL-8 and MIP-1b were consistently higher in CH macaques compared to controls. Interestingly, we found the median copy number of subgenomic SARS-CoV-2 RNA was higher at every timepoint in the CH group as compared with the control group, in both upper and lower respiratory samples. Lung sections from euthanasia at 10 or 12 dpi showed evidence of mild inflammation in all animals. However, in the immunohistochemical analysis, the viral antigen was detected in the lung tissues of all three animals in the CH group even at the time of autopsy, whereas only one animal of three controls had detectable viral antigen. Although the striking inflammation and serious disease have not been observed, data so far provide evidence of potential pathophysiological differences with or without CH upon SARS-CoV-2 infection. We continue to expand sample size and conduct further analyses to draw a solid conclusion, but we believe this model may be of benefit to understand the relationship between COVID-19 disease and CH. Disclosures: No relevant conflicts of interest to declare.

Previously published ethno-pharmacological reports reveal the potentiality of plants and plant-derived products used as traditional home remedies by Bangladeshi COVID-19 patients to combat SARS-CoV-2.

Several plants have traditionally been used since antiquity to treat various gastroenteritis and respiratory symptoms similar to COVID-19 outcomes. The common symptoms of COVID-19 include fever or chills, cold, cough, flu, headache, diarrhoea, tiredness/fatigue, sore throat, loss of taste or smell, asthma, shortness of breath, or difficulty breathing, etc. This study aims to find out the plants and plant-derived products which are being used by the COVID-19 infected patients in Bangladesh and how those plants are being used for the management of COVID-19 symptoms. In this study, online and partially in-person survey interviews were carried out among Bangladeshi respondents. We selected Bangladeshi COVID-19 patients who were detected Coronavirus positive (+) by RT-PCR nucleic acid test and later recovered. Furthermore, identified plant species from the surveys were thoroughly investigated for safety and efficacy based on the previous ethnomedicinal usage reports. Based on the published data, they were also reviewed for their significant potentialities as antiviral, anti-inflammatory, and immunomodulatory agents. We explored comprehensive information about a total of 26 plant species, belonging to 23 genera and 17 different botanical families, used in COVID-19 treatment as home remedies by the respondents. Most of the plants and plant-derived products were collected directly from the local marketplace. According to our survey results, greatly top 5 cited plant species measured as per the highest RFC value are Camellia sinensis (1.0) > Allium sativum (0.984) > Azadirachta indica (0.966) > Zingiber officinale (0.966) > Syzygium aromaticum (0.943). Previously published ethnomedicinal usage reports, antiviral, anti-inflammatory, and immunomodulatory activity of the concerned plant species also support our results. Thus, the survey and review analysis simultaneously reveals that these reported plants and plant-derived products might be promising candidates for the treatment of COVID-19. Moreover, this study clarifies the reported plants for their safety during COVID-19 management and thereby supporting them to include in any future pre-clinical and clinical investigation for developing herbal COVID-19 therapeutics.

CACO-2 cells: A continuous cell line with sensitive and broad-spectrum utility for respiratory virus culture.

BACKGROUND: Primary rhesus monkey kidney cells (RhMK) can be used for the detection of respiratory viruses, including influenza and parainfluenza. The human colon adeno-carcinoma cell line, CACO-2, has been previously used for the growth of multiple influenza viruses, including seasonal, novel and avian lineages. OBJECTIVE: We compared CACO-2, Madin-Darby Canine Kidney (MDCK), and RhMK cells for the isolation of viruses from patients presenting with influenza like-illness (ILI). STUDY DESIGN: Nasopharyngeal specimens from patients with ILI in primary care settings were processed for conventional viral culture in MDCK, RhMK, and CACO-2. Cells were examined microscopically for cytopathic effect (CPE) and confirmatory testing included immunofluorescent antigen (IFA) detection and real-time RT-PCR. Additionally, 16 specimens positive for respiratory syncytial virus (RSV) by PCR were inoculated on CACO-2 cells. Statistical analysis was done using Chi-square test with IBM Statistical Program. RESULTS: Of 1031 respiratory specimens inoculated, viruses were isolated and confirmed from 331 (32.1 %) in MDCK cells, 304 (29.5 %) in RhMk cells, and 433 (42.0 %) in CACO-2 cells. These included influenza A/(H1N1)pdm09, influenza A(H3N2), influenza B, parainfluenza virus (PIV) types 1, 2, and 3, human coronavirus 229E (CoV-229E), human adenovirus (HAdV), herpes simplex virus 1 (HSV 1), and enterovirus (EV). Influenza A viruses grew best in the CACO-2 cell line. Time to observation of CPE was similar for all three cell types but unlike RhMK and MDCK cells, virus-specific morphological changes were indistinguishable in CACO-2 cells. None of the 16 specimens positive for RSV by PCR grew on CACO-2 cells. CONCLUSIONS: The overall respiratory virus culture isolation rate in CACO-2 cells was significantly higher than that in RhMK or MDCK cells (p < 0.05). CACO-2 cells also supported the growth of some viruses that did not grow in either RhMK or MDCK cells. Except for RSV, CACO-2 cells provide a worthwhile addition to culture algorithms for respiratory specimens.

The Dynamic Expression of Potential Mediators of Severe Acute Respiratory Syndrome Coronavirus 2 Cellular Entry in Fetal, Neonatal, and Adult Rhesus Monkeys.

The coronavirus disease 2019 (COVID-19) pandemic, induced by the pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread rapidly all over the world. There is considerable variability among neonates, children, and adults in the incidence of infection and severe disease following exposure to SARS-CoV-2. In our study, we analyzed the transcriptome data of primate animal model of Rhesus monkeys to evaluate the expression levels of possible SARS-CoV-2 receptors and proteases and immunologic features in the lungs, colons, livers, and brains at different developmental stages. Our results revealed that ACE2 and TMPRSS2 were highly expressed in neonates compared with other populations, which imply the high incidence of infection. Other potential receptors and Type II transmembrane serine proteases (TTSPs) and cathepsin of endosomal proteases also exhibited dynamic and differential expression patterns. The expression of receptors (ACE2, BSG, and DPP4) and proteases (TMPRSS2, TMPRSS9, CTSL, and CTSB) were highly correlated during lung development, suggesting the high susceptibility of the lungs. TMPRSS9 was specifically highly expressed in the lungs and reached the highest level in neonates, similar to TMPRSS2. Moreover, the immune cell infiltration analysis revealed immunity immaturity in neonates, implying the association with the mild or moderate type of COVID-19. The results might help researchers design protective and therapeutic strategies for COVID-19 in populations at different ages.