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

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.

Recovery from acute SARS-CoV-2 infection and development of anamnestic immune responses in T cell-depleted rhesus macaques (preprint)

Severe COVID-19 has been associated with T cell lymphopenia 1,2, but no causal effect of T cell deficiency on disease severity has been established. To investigate the specific role of T cells in recovery from SARS-CoV-2 infections we studied rhesus macaques that were depleted of either CD4+, CD8+ or both T cell subsets prior to infection. Peak virus loads were similar in all groups, but the resolution of virus in the T cell-depleted animals was slightly delayed compared to controls. The T cell-depleted groups developed virus-neutralizing antibody responses and also class-switched to IgG. When re-infected six weeks later, the T cell-depleted animals showed anamnestic immune responses characterized by rapid induction of high-titer virus-neutralizing antibodies, faster control of virus loads and reduced clinical signs. These results indicate that while T cells play a role in the recovery of rhesus macaques from acute SARS-CoV-2 infections, their depletion does not induce severe disease, and T cells do not account for the natural resistance of rhesus macaques to severe COVID-19. Neither primed CD4+ or CD8+ T cells appeared critical for immunoglobulin class switching, the development of immunological memory or protection from a second infection.

Intranasal ChAdOx1 nCoV-19/AZD1222 vaccination reduces shedding of SARS-CoV-2 D614G in rhesus macaques (preprint)

Intramuscular vaccination with ChAdOx1 nCoV-19/AZD1222 protected rhesus macaques against pneumonia but did not reduce shedding of SARS-CoV-2. Here we investigate whether intranasally administered ChAdOx1 nCoV-19 reduces shedding, using a SARS-CoV-2 virus with the D614G mutation in the spike protein. Viral load in swabs obtained from intranasally vaccinated hamsters was significantly decreased compared to controls and no viral RNA or infectious virus was found in lung tissue, both in a direct challenge and a transmission model. Intranasal vaccination of rhesus macaques resulted in reduced shedding and a reduction in viral load in bronchoalveolar lavage and lower respiratory tract tissue. In conclusion, intranasal vaccination reduced shedding in two different SARS-CoV-2 animal models, justifying further investigation as a potential vaccination route for COVID-19 vaccines.

Impact of South African 501.V2 Variant on SARS-CoV-2 Spike Infectivity and Neutralization: A Structure-based Computational Assessment (preprint)

Motivation: The SARS-CoV-2 variants emerging from South Africa (501.V2) and the UK (B.1.1.7) necessitate rapid assessment of the effects of the corresponding amino acid substitutions in the spike (S) receptor-binding domain (RBD) of the variants on the interactions with the human ACE2 receptor and monoclonal antibodies (mAbs) reported earlier to neutralize the spike. Results: Molecular modeling and simulations reveal that N501Y, shared by both variants, increases ACE2 binding affinity, and may impact the collective dynamics of the ACE2-RBD complex, occupying a central hinge site that modulates the overall dynamics of the complex. In contrast, the substitutions K417N and E484K in the South African variant 501.V2 would reduce the ACE2-binding affinity by abolishing two interfacial salt bridges that facilitate RBD binding to ACE2, K417(S)-D30(ACE2) and E484 (S)-K31(ACE2). These two mutations may thus be more than compensating the attractive effect induced by N501Y, overall resulting in an ACE2-binding affinity comparable to that of the wildtype RBD. Further analysis of the impact of these mutations on the interactions with mAbs targeting the spike indicate that the substitutions K417N and E484K may also abolish the salt bridges between the spike and selected mAbs, such as REGN10933, BD23, H11_H4, and C105, thus reducing the binding affinity and effectiveness of these mAbs.

Microsecond simulation unravel the structural dynamics of SARS-CoV-2 Spike-C-terminal cytoplasmic tail (residues 1242-1273) (preprint)

Spike protein of human coronaviruses has been a vital drug and vaccine target. The multifunctionality of this protein including host receptor binding and apoptosis has been proved in several coronaviruses. It also interacts with other viral proteins such as membrane (M) protein through its C-terminal domain. The specific dibasic motif signal present in cytosolic region at C-terminal of spike protein helps it to localize within the endoplasmic reticulum (ER). However, the structural conformation of cytosolic region is not known in SARS-CoV-2 using which it interacts with other proteins and transporting vesicles. Therefore, we have demonstrated the conformation of cytosolic region and its dynamics through computer simulations up to microsecond timescale using OPLS and CHARMM forcefields. The simulations have revealed the unstructured conformation of cytosolic region (residues 1242-1273). Also, in temperature dependent replica-exchange molecular dynamics simulations it has shown to form secondary structures. We believe that our findings will surely help us understand the structure-function relationship of the spike protein's cytosolic region.

The hyperlipidaemic drug fenofibrate significantly reduces infection by SARS-CoV-2 in cell culture models (preprint)

The SARS-CoV-2 pandemic has caused a significant number of fatalities and worldwide disruption. To identify drugs to repurpose to treat SARS-CoV-2 infections, we established a screen to measure dimerization of ACE2, the primary receptor for the virus. This screen identified fenofibric acid, the active metabolite of fenofibrate. Fenofibric acid also destabilized the receptor binding domain (RBD) of the viral spike protein and inhibited RBD binding to ACE2 in ELISA and whole cell binding assays. Fenofibrate and fenofibric acid were tested by two independent laboratories measuring infection of cultured Vero cells using two different SARS-CoV-2 isolates. In both settings at drug concentrations which are clinically achievable, fenofibrate and fenofibric acid reduced viral infection by up to 70%. Together with its extensive history of clinical use and its relatively good safety profile, these studies identify fenofibrate as a potential therapeutic agent requiring urgent clinical evaluation to treat SARS-CoV-2 infection.

Validation and Application of a Bench Top Cell Sorter in a BSL-3 Containment Setting (preprint)

Rigorous assessment of the cellular and molecular changes during infection typically requires isolation of specific immune cell subsets for downstream application. While there are numerous options for enrichment/isolation of cells from tissues, fluorescent activated cell sorting (FACS) is accepted as a method that results in superior purification of a wide variety of cell types. Flow cytometry requires extensive fluidics and aerosol droplets can be generated during collection of target cells. Pathogens such as Francisella tularensis , Mycobacterium tuberculosis , Yersinia pestis , and SARS-CoV-2 require manipulation at biosafety level-3 (BSL-3). Due to the concern of potential aerosolization of these pathogens, use of flow cytometric-based cell sorting in these laboratory settings requires placement of the equipment in dedicated biosafety cabinets within the BSL-3. For many researchers, this is often not possible due to expense, space, or expertise available. Here we describe the safety validation and utility of a completely closed cell sorter that results in gentle, rapid, high purity, and safe sorting of cells on the benchtop at BSL-3. We also provide data demonstrating the need for cell sorting versus bead purification and the applicability of this technology for BSL-3 and potentially BSL-4 related infectious disease projects. Adoption of this technology will significantly expand our ability to uncover important features of the most dangerous infectious diseases leading to faster development of novel vaccines and therapeutics.