Rapid recall and de novo T cell responses during SARS-CoV-2 breakthrough infection (preprint)

While the protective role of neutralising antibodies against COVID-19 is well-established, questions remain about the relative importance of cellular immunity. Using 6 pMHC-multimers in a cohort with early and frequent sampling we define the phenotype and kinetics of recalled and primary T cell responses following Delta or Omicron breakthrough infection. Recall of spike-specific CD4+ T cells was rapid, with cellular proliferation and extensive activation evident as early as 1 day post-symptom onset. Similarly, spike-specific CD8+ T cells were rapidly activated but showed variable levels of expansion. Strikingly, high levels of SARS-CoV-2-specific CD8+ T cell activation at baseline and peak were strongly correlated with reduced peak SARS-CoV-2 RNA levels in nasal swabs and accelerated clearance of virus. Our study demonstrates rapid and extensive recall of memory T cell populations occurs early after breakthrough infection and suggests that CD8+ T cells contribute to the control of viral replication in breakthrough SARS-CoV-2 infections.

Robust SARS-CoV-2 antibody and T cell immunity following three COVID-19 vaccine doses in inflammatory bowel disease patients receiving anti-TNF or alternative treatments (preprint)

BACKGROUND AND AIMS: Vaccine-mediated immune responses in patients with inflammatory bowel disease (IBD) may be influenced by IBD therapies. We investigated in-depth humoral and T-cell responses to SARS-CoV-2 vaccination in IBD patients following three COVID-19 vaccine doses. METHODS: Immune responses of 100 SARS-CoV-2-uninfected IBD patients on varying treatments were compared to healthy controls (n=35). Anti-S1/2 and anti-RBD SARS-CoV-2-specific antibodies, CD4+ and CD8+ T-cell responses were measured at baseline and at five time-points after COVID-19 vaccination. RESULTS: Anti-S1/2 and anti-RBD antibody concentrations at ~1 month after second dose vaccination were significantly lower in anti-TNF-treated patients compared to non-TNF IBD patients and healthy controls (126.4 vs 262.1 and 295.5, p<0.0001). Anti-S1/2 antibodies remained reduced in anti-TNF treated patients before and after the third dose (285.7 vs 365.3, p=0.03), although anti-RBD antibodies reached comparable titres to non-TNF patients. Anti-RBD antibodies were higher in the vedolizumab group than controls after second dose (4.2 vs 3.6, p=0.003). Anti-TNF monotherapy was associated with increased CD4+ and CD8+ T-cell activation compared to combination anti-TNF patients after second dose, but comparable after third dose. Overall, IBD patients demonstrated similar CD4+/CD8+ T-cell responses compared to healthy controls regardless of treatment regimen. CONCLUSIONS: Anti-TNFs impaired antibody concentrations when compared to non-TNF patients and controls after two vaccine doses. These differences were not observed after the third vaccine dose. However, vaccine induced SARS-CoV-2-specific T cell responses are robust in anti-TNF-treated patients. Our study supports the need for timely booster vaccination particularly in anti-TNF treated patients to minimise the risk of severe SARS-CoV-2 infection.

Broad immunity to SARS-CoV-2 variants of concern mediated by a SARS-CoV-2 receptor-binding domain protein vaccine (preprint)

The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines. Here, we report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain. These were tested in conjunction with three different adjuvants: a TLR2 agonist R4-Pam2Cys, an NKT cell agonist glycolipid alpha-Galactosylceramide, or MF59 squalene oil-in-water adjuvant. Each formulation drove strong neutralising antibody (nAb) responses and provided durable and highly protective immunity against lower and upper airway infection in mouse models of COVID-19. We have also developed an RBD-human IgG1 Fc vaccine with an RBD sequence of the highly immunoevasive beta variant (N501Y, E484K, K417N). This beta variant RBD vaccine, combined with MF59 adjuvant, induced strong protection in mice against the beta strain as well as the ancestral strain. Furthermore, when used as a third dose booster vaccine following priming with whole spike vaccine, anti-sera from beta-RBD-Fc immunised mice increased titres of nAb against other variants including alpha, delta, delta+, gamma, lambda, mu, and omicron BA.1 and BA.2. These results demonstrated that an RBD-Fc protein subunit/MF59 adjuvanted vaccine can induce high levels of broad nAbs, including when used as a booster following prior immunisation of mice with whole ancestral-strain Spike vaccines. This vaccine platform offers a potential approach to augment some of the currently approved vaccines in the face of emerging variants of concern, and it has now entered a phase I clinical trial.

Virology and immune dynamics reveal high household transmission of ancestral SARS-CoV-2 strain (preprint)

Background: Household studies are crucial for understanding the transmission of SARS-CoV-2 infection, which may be underestimated from PCR testing of respiratory samples alone. We aim to combine assessment of household mitigation measures; nasopharyngeal, saliva and stool PCR testing; along with mucosal and systemic SARS-CoV-2 specific antibodies, to comprehensively characterise SARS-CoV-2 infection and transmission in households. Methods: Between March and September 2020, we obtained samples from 92 participants in 26 households in Melbourne, Australia, in a 4-week period following onset of infection with ancestral SARS-CoV-2 variants. Results: The secondary attack rate was 36% (24/66) when using nasopharyngeal swab (NPS) PCR positivity alone. However, when respiratory and non-respiratory samples were combined with antibody responses in blood and saliva, the secondary attack rate was 76% (50/66). SARS-CoV-2 viral load of the index case and household isolation measures were key factors that determine secondary transmission. In 27% (7/26) of households, all family members tested positive by NPS for SARS-CoV-2 and were characterised by lower respiratory Ct-values than low transmission families (Median 22.62 vs 32.91; IQR 17.06 to 28.67 vs 30.37 to 34.24). High transmission families were associated with enhanced plasma antibody responses to multiple SARS-CoV-2 antigens and the presence of neutralising antibodies. Three distinguishing saliva SARS-CoV-2 antibody features were identified according to age (IgA1 to Spike 1, IgA1 to nucleocapsid protein (NP), suggesting that adults and children generate distinct mucosal antibody responses during the acute phase of infection. Conclusion: Utilising respiratory and non-respiratory PCR testing, along with measurement of SARS-CoV-2 specific local and systemic antibodies, provides a more accurate assessment of infection within households and highlights some of the immunological differences in response between children and adults.

Anti-PEG Antibodies Boosted in Humans by SARS-CoV-2 Lipid Nanoparticle mRNA Vaccine (preprint)

ABSTRACT Humans commonly have low level antibodies to poly(ethylene) glycol (PEG) due to environmental exposure. Lipid nanoparticle (LNP) mRNA vaccines for SARS-CoV-2 contain small amounts of PEG but it is not known whether PEG antibodies are enhanced by vaccination and what their impact is on particle–immune cell interactions in human blood. We studied plasma from 130 adults receiving either the BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) mRNA vaccines, or no SARS-CoV-2 vaccine for PEG-specific antibodies. Anti-PEG IgG was commonly detected prior to vaccination and was significantly boosted a mean of 13.1-fold (range 1.0 to 70.9) following mRNA-1273 vaccination and a mean of 1.78-fold (range 0.68 to 16.6) following BNT162b2 vaccination. Anti-PEG IgM increased 68.5-fold (range 0.9 to 377.1) and 2.64-fold (0.76 to 12.84) following mRNA-1273 and BNT162b2 vaccination, respectively. The rise in PEG-specific antibodies following mRNA-1273 vaccination was associated with a significant increase in the association of clinically relevant PEGylated LNPs with blood phagocytes ex vivo . PEG antibodies did not impact the SARS-CoV-2 specific neutralizing antibody response to vaccination. However, the elevated levels of vaccine-induced anti-PEG antibodies correlated with increased systemic reactogenicity following two doses of vaccination. We conclude that PEG-specific antibodies can be boosted by LNP mRNA-vaccination and that the rise in PEG-specific antibodies is associated with systemic reactogenicity and an increase of PEG particle–leukocyte association in human blood. The longer-term clinical impact of the increase in PEG-specific antibodies induced by lipid nanoparticle mRNA-vaccines should be monitored.

Integrated immune networks in SARS-CoV-2 infected pregnant women reveal differential NK cell and unconventional T cell activation (preprint)

Although pregnancy poses a greater risk for severe COVID-19, the underlying immunological changes associated with SARS-CoV-2 infection during pregnancy are poorly understood. We defined immune responses to SARS-CoV-2 in pregnant and non-pregnant women during acute and convalescent COVID-19 up to 258 days post symptom onset, quantifying 217 immunological parameters. Additionally, matched maternal and cord blood were collected from COVID-19 convalescent pregnancies. Although serological responses to SARS-CoV-2 were similar in pregnant and non-pregnant women, cellular immune analyses revealed marked differences in key NK cell and unconventional T cell responses during COVID-19 in pregnant women. While NK, γδ T cells and MAIT cells displayed pre-activated phenotypes in healthy pregnant women when compared to non-pregnant age-matched women, activation profiles of these pre-activated NK and unconventional T cells remained unchanged at acute and convalescent COVID-19 in pregnancy. Conversely, activation dynamics of NK and unconventional T cells were prototypical in non-pregnant women in COVID-19. In contrast, activation of αβ CD4 + and CD8 + T cells, T follicular helper cells and antibody-secreting cells was similar in pregnant and non-pregnant women with COVID-19. Elevated levels of IL-1β, IFN-γ, IL-8, IL-18 and IL-33 were also found in pregnant women in their healthy state, and these cytokine levels remained elevated during acute and convalescent COVID-19. Collectively, our study provides the first comprehensive map of longitudinal immunological responses to SARS-CoV-2 infection in pregnant women, providing insights into patient management and education during COVID-19 pregnancy.

SARS-CoV-2 mRNA vaccination elicits robust and persistent T follicular helper cell response in humans (preprint)

SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we show that frequency of TFH correlates with that of S-binding germinal center B cells. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLADPB1* 04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.

Immune responses in COVID-19 respiratory tract and blood reveal mechanisms of disease severity (preprint)

Although the respiratory tract is the primary site of SARS-CoV-2 infection and the ensuing immunopathology, respiratory immune responses are understudied and urgently needed to understand mechanisms underlying COVID-19 disease pathogenesis. We collected paired longitudinal blood and respiratory tract samples (endotracheal aspirate, sputum or pleural fluid) from hospitalized COVID-19 patients and non-COVID-19 controls. Cellular, humoral and cytokine responses were analysed and correlated with clinical data. SARS-CoV-2-specific IgM, IgG and IgA antibodies were detected using ELISA and multiplex assay in both the respiratory tract and blood of COVID-19 patients, although a higher receptor binding domain (RBD)-specific IgM and IgG seroconversion level was found in respiratory specimens. SARS-CoV-2 neutralization activity in respiratory samples was detected only when high levels of RBD-specific antibodies were present. Strikingly, cytokine/chemokine levels and profiles greatly differed between respiratory samples and plasma, indicating that inflammation needs to be assessed in respiratory specimens for the accurate assessment of SARS-CoV-2 immunopathology. Diverse immune cell subsets were detected in respiratory samples, albeit dominated by neutrophils. Importantly, we also showed that dexamethasone and/or remdesivir treatment did not affect humoral responses in blood of COVID-19 patients. Overall, our study unveils stark differences in innate and adaptive immune responses between respiratory samples and blood and provides important insights into effect of drug therapy on immune responses in COVID-19 patients.

Immune responses in COVID-19 respiratory tract and blood reveal mechanisms of disease severity (preprint)

Although the respiratory tract is the primary site of SARS-CoV-2 infection and the ensuing immunopathology, respiratory immune responses are understudied and urgently needed to understand mechanisms underlying COVID-19 disease pathogenesis. We collected paired longitudinal blood and respiratory tract samples (endotracheal aspirate, sputum or pleural fluid) from hospitalized COVID-19 patients and non-COVID-19 controls. Cellular, humoral and cytokine responses were analysed and correlated with clinical data. SARS-CoV-2-specific IgM, IgG and IgA antibodies were detected using ELISA and multiplex assay in both the respiratory tract and blood of COVID-19 patients, although a higher receptor binding domain (RBD)-specific IgM and IgG seroconversion level was found in respiratory specimens. SARS-CoV-2 neutralization activity in respiratory samples was detected only when high levels of RBD-specific antibodies were present. Strikingly, cytokine/chemokine levels and profiles greatly differed between respiratory samples and plasma, indicating that inflammation needs to be assessed in respiratory specimens for the accurate assessment of SARS-CoV-2 immunopathology. Diverse immune cell subsets were detected in respiratory samples, albeit dominated by neutrophils. Importantly, we also showed that dexamethasone and/or remdesivir treatment did not affect humoral responses in blood of COVID-19 patients. Overall, our study unveils stark differences in innate and adaptive immune responses between respiratory samples and blood and provides important insights into effect of drug therapy on immune responses in COVID-19 patients.

Integrated immune networks in SARS-CoV-2 infected pregnant women reveal differential NK cell and unconventional T cell activation (preprint)

Although pregnancy poses a greater risk for severe COVID-19, the underlying immunological changes associated with SARS-CoV-2 infection during pregnancy are poorly understood. We defined immune responses to SARS-CoV-2 in pregnant and non-pregnant women during acute and convalescent COVID-19 up to 258 days post symptom onset, quantifying 217 immunological parameters. Additionally, matched maternal and cord blood were collected from COVID-19 convalescent pregnancies. Although serological responses to SARS-CoV-2 were similar in pregnant and non-pregnant women, cellular immune analyses revealed marked differences in key NK cell and unconventional T cell responses during COVID-19 in pregnant women. While NK cells, {gamma}{delta} T cells and MAIT cells displayed pre-activated phenotypes in healthy pregnant women when compared to non-pregnant age-matched women, activation profiles of these pre-activated NK and unconventional T cells remained unchanged at acute and convalescent COVID-19 in pregnancy. Conversely, activation dynamics of NK and unconventional T cells were prototypical in non-pregnant women in COVID-19. In contrast, activation of {beta} CD4+ and CD8+ T cells, T follicular helper cells and antibody-secreting cells was similar in pregnant and non-pregnant women with COVID-19. Elevated levels of IL-1{beta}, IFN-{gamma}, IL-8, IL-18 and IL-33 were also found in pregnant women in their healthy state, and these cytokine levels remained elevated during acute and convalescent COVID-19. Collectively, our study provides the first comprehensive map of longitudinal immunological responses to SARS-CoV-2 infection in pregnant women, providing insights into patient management and education during COVID-19 pregnancy.

A point-of-care lateral flow assay for neutralising antibodies against SARS-CoV-2 (preprint)

As vaccines against SARS-CoV-2 are now being rolled out, a better understanding of immunity to the virus; whether through infection, or passive or active immunisation, and the durability of this protection is required. This will benefit from the ability to measure SARS-CoV-2 immunity, ideally with rapid turnaround and without the need for laboratory-based testing. Current rapid point-of-care (POC) tests measure antibodies (Ab) against the SARS-CoV-2 virus, however, these tests provide no information on whether the antibodies can neutralise virus infectivity and are potentially protective, especially against newly emerging variants of the virus. Neutralising Antibodies (NAb) are emerging as a strong correlate of protection, but most current NAb assays require many hours or days, samples of venous blood, and access to laboratory facilities, which is especially problematic in resource-limited settings. We have developed a lateral flow POC test that can measure levels of RBD-ACE2 neutralising antibodies from whole blood, with a result that can be determined by eye (semi-quantitative) or on a small instrument (quantitative), and results show high correlation with microneutralisation assays. This assay also provides a measure of total anti-RBD antibody, thereby providing evidence of exposure to SARS-CoV-2 or immunisation, regardless of whether NAb are present in the sample. By testing samples from immunised macaques, we demonstrate that this test is equally applicable for use with animal samples, and we show that this assay is readily adaptable to test for immunity to newly emerging SARS-CoV-2 variants. Lastly, using a cohort of vaccinated humans, we demonstrate that our whole-blood test correlates closely with microneutralisation assay data (R 2 =0.75, p<0.0001), and that fingerprick whole blood samples are sufficient for this test. Accordingly, the COVID-19 NAb-test™ device described here can provide a rapid readout of immunity to SARS-CoV-2 at the point of care.

Altered microRNA expression in COVID-19 patients enables identification of SARS-CoV-2 infection (preprint)

The host response to SARS-CoV-2 infection provide insights into both viral pathogenesis and patient management. The host-encoded microRNA (miRNA) response to SARS-CoV-2 infection, however, remains poorly defined. Here we profiled circulating miRNAs from ten COVID-19 patients sampled longitudinally and ten age and gender matched healthy donors. We observed 55 miRNAs that were altered in COVID-19 patients during early-stage disease, with the inflammatory miR-31-5p the most strongly upregulated. Supervised machine learning analysis revealed that a three-miRNA signature (miR-423-5p, miR-23a-3p and miR-195-5p) independently classified COVID-19 cases with an accuracy of 99.9%. In a ferret COVID-19 model, the three-miRNA signature again detected SARS-CoV-2 infection with 99.7% accuracy, and distinguished SARS-CoV-2 infection from influenza A (H1N1) infection and healthy controls with 95% accuracy. Distinct miRNA profiles were also observed in COVID-19 patients requiring oxygenation. This study demonstrates that SARS-CoV-2 infection induces a robust host miRNA response that could improve COVID-19 detection and patient management

High expression of CD38 and MHC class II on CD8+ T cells during severe influenza disease reflects bystander activation and trogocytosis (preprint)

Although co-expression of CD38 and HLA-DR on CD8 + T cells reflects activation during influenza, SARS-CoV-2, Dengue, Ebola and HIV-1 viral infections, high and prolonged CD38 + HLA-DR + expression can be associated with severe and fatal disease outcomes. As the expression of CD38 + HLA-DR + is poorly understood, we used mouse models of influenza A/H7N9, A/H3N2 and A/H1N1 infection to investigate the mechanisms underpinning CD38 + MHC-II + phenotype on CD8 + T-cells. Our analysis of influenza-specific immunodominant D b NP 366 +CD8 + T-cell responses showed that CD38 + MHC-II + co-expression was detected on both virus-specific and bystander CD8 + T-cells, with increased numbers of both CD38 + MHC-II + CD8 + T-cell populations observed in the respiratory tract during severe infection. To understand the mechanisms underlying CD38 and MHC-II expression, we also used adoptively-transferred transgenic OT-I CD8 + T-cells recognising the ovalbumin-derived K b SIINFEKL epitope and A/H1N1-SIINKEKL infection. Strikingly, we found that OT-I cells adoptively-transferred into MHC-II −/− mice did not display MHC-II after influenza virus infection, suggesting that MHC-II was acquired via trogocytosis in wild-type mice. Additionally, detection of CD19 on CD38 + MHC II + OT-I cells further supports that MHC-II was acquired by trogocytosis, at least partially, sourced from B-cells. Our results also revealed that co-expression of CD38 + MHC II + on CD8 + T-cells was needed for the optimal recall ability following secondary viral challenge. Overall, our study provides evidence that both virus-specific and bystander CD38 + MHC-II + CD8 + T-cells are recruited to the site of infection during severe disease, and that MHC-II expression occurs via trogocytosis from antigen-presenting cells. Our findings also highlight the importance of the CD38 + MHC II + phenotype for CD8 + T-cell memory establishment and recall. Summary Co-expression of CD38 and MHC-II on CD8 + T cells is recognized as a classical hallmark of activation during viral infections. High and prolonged CD38 + HLA-DR + expression, however, can be associated with severe disease outcomes and the mechanisms are unclear. Using our established influenza wild-type and transgenic mouse models, we determined how disease severity affected the activation of influenza-specific CD38 + MHC-II + CD8 + T cell responses in vivo and the antigenic determinants that drive their activation and expansion. Overall, our study provides evidence that both virus-specific and bystander CD38 + MHC-II + CD8 + T-cells are recruited to the site of infection during severe disease, and that MHC-II expression occurs, at least in part, via trogocytosis from antigen-presenting cells. Our findings also highlight the importance of the CD38 + MHC II + phenotype for CD8 + T-cell memory establishment and recall.

First Report of a Phase 1 Randomised Trial of Molecular Clamp-Stabilised Spike Protein-Based and MF59-Adjuvanted Vaccine for SARS-CoV-2 (preprint)

Background: We assessed the safety and immunogenicity of an MF59-adjuvanted subunit vaccine for COVID-19 based on recombinant SARS-CoV-2 spike glycoprotein stabilised in a prefusion conformation by a novel molecular clamp (Sclamp).Methods: Phase 1, double-blind, placebo-controlled trial conducted in Australia (July 2020–ongoing; ClinicalTrials.gov NCT04495933). Healthy adults (18-55 years) received two doses of placebo, 5-μg, 15-μg, or 45-μg SARS-CoV-2 Sclamp, or one 45-μg dose of SARS-CoV-2 Sclamp followed by placebo, 28 days apart (n=120; 24 per group). Safety, humoral immunogenicity (ELISA, microneutralisation, pseudovirus neutralisation), and cellular immunogenicity (antigen-specific CD4+/CD8+ T-cells, antibody-secreting cells) were assessed up to 56 days after the first dose.Findings: The SARS-CoV-2 Sclamp vaccine was very well tolerated with few systemic reactions. All two-dose regimens elicited robust, broadly neutralising humoral responses. Geometric mean titres were higher than in sera from convalescent COVID-19 patients and strongly neutralised spike variants of concern, including N501Y. Moreover, humoral and cellular responses were highly correlated. However, antibodies elicited to a peptide sequence used in the molecular clamp derived from human immunodeficiency virus-1 (HIV-1) gp41 cross-reacted weakly with some HIV diagnostic screening tests.Interpretation: These first-in-human results demonstrate that a subunit vaccine comprising mammalian cell culture-derived, molecular clamp-stabilised recombinant spike protein formulated in a squalene-in-oil adjuvant elicits strong immune responses with an excellent safety profile. However, the gp41 peptide induced diagnostic interference, creates a likely barrier to widespread use and highlights the criticality of potential off-target immunogenicity during vaccine development. Studies are ongoing with alternative molecular clamp trimerisation domains to ameliorate this response.Clinical Trial Registration: ClinicalTrials.gov (NCT04495933).Funding: Coalition for Epidemic Preparedness Innovations; National Health and Medical Research Council, Queensland Government, and philanthropic sources.Declaration of Interests: KJC and DW report grants from the Coalition for Epidemic Preparedness Innovations, the National Health and Medical Research Council of Australia, and the Queensland Government, during the conduct of the study; other from ViceBio Limited, outside the submitted work; and has patents pending (AU 2018241252; BR112019019813.0; CA 3057171; CH 201880022016.9; EP 18775234.0; IN 201917038666; ID P00201909145; IL 269534; JP 2019-553883; MX/a/2019/011599; NZ 757178; KR 0-2019-7031415; SG 11201908280S; US 16/498865). JB reports personal fees from CSL Limited, during the conduct of the study, and other from CSL Limited, outside the submitted work. WZ reports grants from the National Health and Medical Research Council of Australia, the Research Grants Council of the Hong Kong Special Administrative Region, China, and the Jack Ma Foundation, during the conduct of the study. SM-H reports grants from Canarian Foundation Doctor Manuel Morales, during the conduct of the study. KJS reports grants from the the Australian Medical Research Future Fund, during the conduct of the study. AWC reports grants from the Australian Medical Research Future Fund and a National Health and Medical Research Council of Australia Career Development Fellowship, during the conduct of the study. BDW reports grants from the National Health and Medical Research Council of Australia, the Australian Medical Research Future Fund, and the Victorian State Government, during the conduct of the study. PMH reports grants from the Australian Medical Research Future Fund, during the conduct of the study. DP reports grants from the National Health and Medical Research Council of Australia, the A2 Milk Foundation, and the Jack Ma Foundation, during the conduct of the study. CR reports grants from the Coalition for Epidemic Preparedness Innovations, during the conduct of the study. PRY reports grants from the Coalition for Epidemic Preparedness Innovations, the National Health and Medical Research Council of Australia, and the Queensland Government, during the conduct of the study; grants from ViceBio Limited, outside the submitted work; and a patent issued (US 2020/0040042). FLM, Zl, DKW, PE, JAL, STMC, NM, SA, CLH, KH, PG, LH, THON, MHT, PT, JB, PCR, SN, SC, TH, KK, KS, and TPM have nothing to disclose.Ethics Approval Statement: The protocol was approved by the Alfred Health Human Research Ethics Committee (2020001376/334/20).

Single-cell RNA Expression of SARS-CoV-2 Cell Entry Factors in Human Endometrium during Preconception (preprint)

We investigated potential SARS-CoV-2 tropism in human endometrium by single-cell RNA-sequencing of viral entry-associated genes in healthy women. Percentages of endometrial cells expressing ACE2, TMPRSS2, CTSB, or CTSL were <2%, 12%, 80%, and 80%, respectively, with 0.7% of cells expressing all four genes. Our findings imply low efficiency of SARS-CoV-2 infection in the endometrium before embryo implantation, providing information to assess preconception risk in asymptomatic carriers.

Ambroxol Hydrochloride Inhibits the Interaction between Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein's Receptor Binding Domain and Recombinant Human ACE2. (preprint)

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), enters the host cells through two main pathways, both involving key interactions between viral envelope-anchored spike glycoprotein of the novel coronavirus and the host receptor, angiotensin-converting enzyme 2 (ACE2). To date, SARS-CoV-2 has infected up to 26 million people worldwide; yet, there is no clinically approved drug or vaccine available. Therefore, a rapid and coordinated effort to re-purpose clinically approved drugs that prevent or disrupt these critical entry pathways of SARS-CoV-2 spike glycoprotein interaction with human ACE2, could potentially accelerate the identification and clinical advancement of prophylactic and/or treatment options against COVID-19, thus providing possible countermeasures against viral entry, pathogenesis and survival. Herein, we discovered that Ambroxol hydrochloride (AMB), and its progenitor, Bromhexine hydrochloride (BHH), both clinically approved drugs are potent effective modulators of the key interaction between the receptor binding domain (RBD) of SARS-CoV-2 spike protein and human ACE2. We also found that both compounds inhibited SARS-CoV-2 infection-induced cytopathic effect at micromolar concentrations. Therefore, in addition to the known TMPRSS2 activity of BHH; we report for the first time that the BHH and AMB pharmacophore has the capacity to target and modulate yet another key protein-protein interaction essential for the two known SARS-CoV-2 entry pathways into host cells. Altogether, the potent efficacy, excellent safety and pharmacologic profile of both drugs along with their affordability and availability, makes them promising candidates for drug repurposing as possible prophylactic and/or treatment options against SARS-CoV-2 infection.