SARS-CoV-2 Spike N-Terminal Domain Engages 9-O-Acetylated α2-8-Linked Sialic Acids.

SARS-CoV-2 viruses engage ACE2 as a functional receptor with their spike protein. The S1 domain of the spike protein contains a C-terminal receptor binding domain (RBD) and an N-terminal domain (NTD). The NTD of other coronaviruses includes a glycan binding cleft. However, for the SARS-CoV-2 NTD, protein-glycan binding was only observed weakly for sialic acids with highly sensitive methods. Amino acid changes in the NTD of variants of concern (VoC) show antigenic pressure, which can be an indication of NTD-mediated receptor binding. Trimeric NTD proteins of SARS-CoV-2, alpha, beta, delta, and omicron did not reveal a receptor binding capability. Unexpectedly, the SARS-CoV-2 beta subvariant strain (501Y.V2-1) NTD binding to Vero E6 cells was sensitive to sialidase pretreatment. Glycan microarray analyses identified a putative 9-O-acetylated sialic acid as a ligand, which was confirmed by catch-and-release ESI-MS, STD-NMR analyses, and a graphene-based electrochemical sensor. The beta (501Y.V2-1) variant attained an enhanced glycan binding modality in the NTD with specificity toward 9-O-acetylated structures, suggesting a dual-receptor functionality of the SARS-CoV-2 S1 domain, which was quickly selected against. These results indicate that SARS-CoV-2 can probe additional evolutionary space, allowing binding to glycan receptors on the surface of target cells.

Bispecific antibodies combine breadth, potency, and avidity of parental antibodies to neutralize sarbecoviruses.

SARS-CoV-2 variants evade current monoclonal antibody therapies. Bispecific antibodies (bsAbs) combine the specificities of two distinct antibodies taking advantage of the avidity and synergy provided by targeting different epitopes. Here we used controlled Fab-arm exchange to produce bsAbs that neutralize SARS-CoV and SARS-CoV-2 variants, including Omicron and its subvariants, by combining potent SARS-CoV-2-specific neutralizing antibodies with broader antibodies that also neutralize SARS-CoV. We demonstrated that the parental antibodies rely on avidity for neutralization using bsAbs containing one irrelevant Fab arm. Using mass photometry to measure the formation of antibody:spike complexes, we determined that bsAbs increase binding stoichiometry compared to corresponding cocktails, without a loss of binding affinity. The heterogeneous binding pattern of bsAbs to spike, observed by negative-stain electron microscopy and mass photometry provided evidence for both intra- and inter-spike crosslinking. This study highlights the utility of cross-neutralizing antibodies for designing bivalent agents to combat circulating and future SARS-like coronaviruses.

Different configurations of SARS-CoV-2 spike protein delivered by integrase-defective lentiviral vectors induce persistent functional immune responses, characterized by distinct immunogenicity profiles.

Several COVID-19 vaccine strategies utilizing new formulations for the induction of neutralizing antibodies (nAbs) and T cell immunity are still under evaluation in preclinical and clinical studies. Here we used Simian Immunodeficiency Virus (SIV)-based integrase defective lentiviral vector (IDLV) delivering different conformations of membrane-tethered Spike protein in the mouse immunogenicity model, with the aim of inducing persistent nAbs against multiple SARS-CoV-2 variants of concern (VoC). Spike modifications included prefusion-stabilizing double proline (2P) substitutions, mutations at the furin cleavage site (FCS), D614G mutation and truncation of the cytoplasmic tail (delta21) of ancestral and Beta (B.1.351) Spike, the latter mutation to markedly improve IDLV membrane-tethering. BALB/c mice were injected once with IDLV delivering the different forms of Spike or the recombinant trimeric Spike protein with 2P substitutions and FCS mutations in association with a squalene-based adjuvant. Anti-receptor binding domain (RBD) binding Abs, nAbs and T cell responses were detected up to six months from a single immunization with escalating doses of vaccines in all mice, but with different levels and kinetics. Results indicated that IDLV delivering the Spike protein with all the combined modifications, outperformed the other candidates in terms of T cell immunity and level of both binding Abs and nAbs soon after the single immunization and persistence over time, showing the best capacity to neutralize all formerly circulating VoC Alpha, Beta, Gamma and Delta. Although present, the lowest response was detected against Omicron variants (BA.1, BA.2 and BA.4/5), suggesting that the magnitude of immune evasion may be related to the higher genetic distance of Omicron as indicated by increased number of amino acid substitutions in Spike acquired during virus evolution.

Bispecific antibodies combine breadth, potency, and avidity of parental antibodies to neutralize sarbecoviruses

SARS-CoV-2 variants evade current monoclonal antibody therapies. Bispecific antibodies (bsAbs) combine the specificities of two distinct antibodies taking advantage of the avidity and synergy provided by targeting different epitopes. Here we used controlled Fab-arm exchange to produce bsAbs that neutralize SARS-CoV and SARS-CoV-2 variants, including Omicron and its subvariants, by combining potent SARS-CoV-2-specific neutralizing antibodies with broader antibodies that also neutralize SARS-CoV. We demonstrated that the parental antibodies rely on avidity for neutralization using bsAbs containing one irrelevant Fab arm. Using mass photometry to measure formation of antibody:spike complexes we determined that bsAbs increase binding stoichiometry compared to corresponding cocktails, without a loss of binding affinity. The heterogeneous binding pattern of bsAbs to spike, observed by negative-stain electron microscopy and mass photometry provided evidence for both intra- and inter-spike crosslinking. This study highlights the utility of cross-neutralizing antibodies for designing bivalent agents to combat circulating and future SARS-like coronaviruses. Graphical abstract

Antigen- and scaffold-specific antibody responses to protein nanoparticle immunogens.

Protein nanoparticle scaffolds are increasingly used in next-generation vaccine designs, and several have established records of clinical safety and efficacy. Yet the rules for how immune responses specific to nanoparticle scaffolds affect the immunogenicity of displayed antigens have not been established. Here we define relationships between anti-scaffold and antigen-specific antibody responses elicited by protein nanoparticle immunogens. We report that dampening anti-scaffold responses by physical masking does not enhance antigen-specific antibody responses. In a series of immunogens that all use the same nanoparticle scaffold but display four different antigens, only HIV-1 envelope glycoprotein (Env) is subdominant to the scaffold. However, we also demonstrate that scaffold-specific antibody responses can competitively inhibit antigen-specific responses when the scaffold is provided in excess. Overall, our results suggest that anti-scaffold antibody responses are unlikely to suppress antigen-specific antibody responses for protein nanoparticle immunogens in which the antigen is immunodominant over the scaffold.

Co-display of diverse spike proteins on nanoparticles broadens sarbecovirus neutralizing antibody responses.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses continuous challenges in combating the virus. Here, we describe vaccination strategies to broaden SARS-CoV-2 and sarbecovirus immunity by combining spike proteins based on different viruses or viral strains displayed on two-component protein nanoparticles. First, we combined spike proteins based on ancestral and Beta SARS-CoV-2 strains to broaden SARS-CoV-2 immune responses. Inclusion of Beta spike improved neutralizing antibody responses against SARS-CoV-2 Beta, Gamma, and Omicron BA.1 and BA.4/5. A third vaccination with ancestral SARS-CoV-2 spike also improved cross-neutralizing antibody responses against SARS-CoV-2 variants, in particular against the Omicron sublineages. Second, we combined SARS-CoV and SARS-CoV-2 spike proteins to broaden sarbecovirus immune responses. Adding SARS-CoV spike to a SARS-CoV-2 spike vaccine improved neutralizing responses against SARS-CoV and SARS-like bat sarbecoviruses SHC014 and WIV1. These results should inform the development of broadly active SARS-CoV-2 and pan-sarbecovirus vaccines and highlight the versatility of two-component nanoparticles for displaying diverse antigens.

Discriminating cross-reactivity in polyclonal IgG1 responses against SARS-CoV-2 variants of concern.

Existing assays to measure antibody cross-reactivity against different SARS-CoV-2 spike (S) protein variants lack the discriminatory power to provide insights at the level of individual clones. Using a mass spectrometry-based approach we are able to monitor individual donors' IgG1 clonal responses following a SARS-CoV-2 infection. We monitor the plasma clonal IgG1 profiles of 8 donors who had experienced an infection by either the wild type Wuhan Hu-1 virus or one of 3 VOCs (Alpha, Beta and Gamma). In these donors we chart the full plasma IgG1 repertoires as well as the IgG1 repertoires targeting the SARS-CoV-2 spike protein trimer VOC antigens. The plasma of each donor contains numerous anti-spike IgG1 antibodies, accounting for <0.1% up to almost 10% of all IgG1s. Some of these antibodies are VOC-specific whereas others do recognize multiple or even all VOCs. We show that in these polyclonal responses, each clone exhibits a distinct cross-reactivity and also distinct virus neutralization capacity. These observations support the need for a more personalized look at the antibody clonal responses to infectious diseases.

Antigenic cartography using sera from sequence-confirmed SARS-CoV-2 variants of concern infections reveals antigenic divergence of Omicron.

Large-scale vaccination campaigns have prevented countless hospitalizations and deaths due to COVID-19. However, the emergence of SARS-CoV-2 variants that escape from immunity challenges the effectiveness of current vaccines. Given this continuing evolution, an important question is when and how to update SARS-CoV-2 vaccines to antigenically match circulating variants, similarly to seasonal influenza viruses where antigenic drift necessitates periodic vaccine updates. Here, we studied SARS-CoV-2 antigenic drift by assessing neutralizing activity against variants of concern (VOCs) in a set of sera from patients infected with viral sequence-confirmed VOCs. Infections with D614G or Alpha strains induced the broadest immunity, whereas individuals infected with other VOCs had more strain-specific responses. Omicron BA.1 and BA.2 were substantially resistant to neutralization by sera elicited by all other variants. Antigenic cartography revealed that Omicron BA.1 and BA.2 were antigenically most distinct from D614G, associated with immune escape, and possibly will require vaccine updates to ensure vaccine effectiveness.

Recent infection with HCoV-OC43 may be associated with protection against SARS-CoV-2 infection.

Antibodies against seasonal human coronaviruses (HCoVs) are known to cross-react with SARS-CoV-2, but data on cross-protective effects of prior HCoV infections are conflicting. In a prospective cohort of healthcare workers (HCWs), we studied the association between seasonal HCoV (OC43, HKU1, 229E and NL63) nucleocapsid protein IgG and SARS-CoV-2 infection during the first pandemic wave in the Netherlands (March 2020 - June 2020), by 4-weekly serum sampling. HCW with HCoV-OC43 antibody levels in the highest quartile, were less likely to become SARS-CoV-2 seropositive when compared with those with lower levels (6/32, 18.8%, versus 42/97, 43.3%, respectively: p = 0.019; HR 0.37, 95% CI 0.16-0.88). We found no significant association with HCoV-OC43 spike protein IgG, or with antibodies against other HCoVs. Our results indicate that the high levels of HCoV-OC43-nucleocapsid antibodies, as an indicator of a recent infection, are associated with protection against SARS-CoV-2 infection; this supports and informs efforts to develop pancoronavirus vaccines.

A public antibody class recognizes an S2 epitope exposed on open conformations of SARS-CoV-2 spike.

Delineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigate the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We show that ∼82% of SARS-CoV-2 S-reactive B cells harbor a naive phenotype, which represents an unusually high fraction of total human naive B cells (∼0.1%). Approximately 10% of these naive S-reactive B cells share an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines.

Modelling the response to vaccine in non-human primates to define SARS-CoV-2 mechanistic correlates of protection.

The definition of correlates of protection is critical for the development of next-generation SARS-CoV-2 vaccine platforms. Here, we propose a model-based approach for identifying mechanistic correlates of protection based on mathematical modelling of viral dynamics and data mining of immunological markers. The application to three different studies in non-human primates evaluating SARS-CoV-2 vaccines based on CD40-targeting, two-component spike nanoparticle and mRNA 1273 identifies and quantifies two main mechanisms that are a decrease of rate of cell infection and an increase in clearance of infected cells. Inhibition of RBD binding to ACE2 appears to be a robust mechanistic correlate of protection across the three vaccine platforms although not capturing the whole biological vaccine effect. The model shows that RBD/ACE2 binding inhibition represents a strong mechanism of protection which required significant reduction in blocking potency to effectively compromise the control of viral replication.

Antibody responses against SARS-CoV-2 variants induced by four different SARS-CoV-2 vaccines in health care workers in the Netherlands: A prospective cohort study.

BACKGROUND: Emerging and future SARS-CoV-2 variants may jeopardize the effectiveness of vaccination campaigns. Therefore, it is important to know how the different vaccines perform against diverse SARS-CoV-2 variants. METHODS AND FINDINGS: In a prospective cohort of 165 SARS-CoV-2 naive health care workers in the Netherlands, vaccinated with either one of four vaccines (BNT162b2, mRNA-1273, AZD1222 or Ad26.COV2.S), we performed a head-to-head comparison of the ability of sera to recognize and neutralize SARS-CoV-2 variants of concern (VOCs; Alpha, Beta, Gamma, Delta and Omicron). Repeated serum sampling was performed 5 times during a year (from January 2021 till January 2022), including before and after booster vaccination with BNT162b2. Four weeks after completing the initial vaccination series, SARS-CoV-2 wild-type neutralizing antibody titers were highest in recipients of mRNA-1273, followed by recipients of BNT162b2 (geometric mean titers (GMT) of 358 [95% CI 231-556] and 214 [95% CI 153-299], respectively; p<0.05), and substantially lower in those vaccinated with the adenovirus vector-based vaccines AZD1222 and Ad26.COV2.S (GMT of 18 [95% CI 11-30] and 14 [95% CI 8-25] IU/ml, respectively; p<0.001). VOCs neutralization was reduced in all vaccine groups, with the greatest reduction in neutralization GMT observed against the Omicron variant (fold change 0.03 [95% CI 0.02-0.04], p<0.001). The booster BNT162b2 vaccination increased neutralizing antibody titers for all groups with substantial improvement against the VOCs including the Omicron variant. We used linear regression and linear mixed model analysis. All results were adjusted for possible confounding of age and sex. Study limitations include the lack of cellular immunity data. CONCLUSIONS: Overall, this study shows that the mRNA vaccines appear superior to adenovirus vector-based vaccines in inducing neutralizing antibodies against VOCs four weeks after initial vaccination and after booster vaccination, which implies the use of mRNA vaccines for both initial and booster vaccination.

Immunogenicity of the mRNA-1273 COVID-19 vaccine in adult patients with inborn errors of immunity.

BACKGROUND: Patients with inborn errors of immunity (IEI) are at increased risk of severe coronavirus disease-2019 (COVID-19). Effective vaccination against COVID-19 is therefore of great importance in this group, but little is known about the immunogenicity of COVID-19 vaccines in these patients. OBJECTIVES: We sought to study humoral and cellular immune responses after mRNA-1273 COVID-19 vaccination in adult patients with IEI. METHODS: In a prospective, controlled, multicenter study, 505 patients with IEI (common variable immunodeficiency [CVID], isolated or undefined antibody deficiencies, X-linked agammaglobulinemia, combined B- and T-cell immunodeficiency, phagocyte defects) and 192 controls were included. All participants received 2 doses of the mRNA-1273 COVID-19 vaccine. Levels of severe acute respiratory syndrome coronavirus-2-specific binding antibodies, neutralizing antibodies, and T-cell responses were assessed at baseline, 28 days after first vaccination, and 28 days after second vaccination. RESULTS: Seroconversion rates in patients with clinically mild antibody deficiencies and phagocyte defects were similar to those in healthy controls, but seroconversion rates in patients with more severe IEI, such as CVID and combined B- and T-cell immunodeficiency, were lower. Binding antibody titers correlated well to the presence of neutralizing antibodies. T-cell responses were comparable to those in controls in all IEI cohorts, with the exception of patients with CVID. The presence of noninfectious complications and the use of immunosuppressive drugs in patients with CVID were negatively correlated with the antibody response. CONCLUSIONS: COVID-19 vaccination with mRNA-1273 was immunogenic in mild antibody deficiencies and phagocyte defects and in most patients with combined B- and T-cell immunodeficiency and CVID. Lowest response was detected in patients with X-linked agammaglobulinemia and in patients with CVID with noninfectious complications. The assessment of longevity of immune responses in these vulnerable patient groups will guide decision making for additional vaccinations.

A SARS-CoV-2 Wuhan spike virosome vaccine induces superior neutralization breadth compared to one using the Beta spike.

Current SARS-CoV-2 vaccines are effective, but long-term protection is threatened by the emergence of virus variants. We generated a virosome vaccine containing the Beta spike protein and compared its immunogenicity in mice to a virosome vaccine containing the original Wuhan spike. Two administrations of the virosomes induced potent SARS-CoV-2 neutralizing antibodies in both vaccine groups. The level of autologous neutralization in Beta-vaccinated mice was similar to the level of autologous neutralization in Wuhan-vaccinated mice. However, heterologous neutralization to the Wuhan strain in Beta-vaccinated mice was 4.7-fold lower than autologous neutralization, whereas heterologous neutralization to the Beta strain in Wuhan-vaccinated mice was reduced by only 1.9-fold compared to autologous neutralization levels. In addition, neutralizing activity against the D614G, Alpha and Delta variants was also significantly lower after Beta spike vaccination than after Wuhan spike vaccination. Our results show that Beta spike vaccination induces inferior neutralization breadth. These results are informative for programs aimed to develop broadly active SARS-CoV-2 vaccines.

Computed tomography and [18F]-FDG PET imaging provide additional readouts for COVID-19 pathogenesis and therapies evaluation in non-human primates.

Non-human primates (NHPs) are particularly relevant as preclinical models for SARS-CoV-2 infection and nuclear imaging may represent a valuable tool for monitoring infection in this species. We investigated the benefit of computed X-ray tomography (CT) and [18F]-FDG positron emission tomography (PET) to monitor the early phase of the disease in a large cohort (n = 76) of SARS-CoV-2 infected macaques. Following infection, animals showed mild COVID-19 symptoms including typical lung lesions. CT scores at the acute phase reflect the heterogeneity of lung burden following infection. Moreover, [18F]-FDG PET revealed that FDG uptake was significantly higher in the lungs, nasal cavities, lung-draining lymph nodes, and spleen of NHPs by 5 days postinfection compared to pre-infection levels, indicating early local inflammation. The comparison of CT and PET data from previous COVID-19 treatments or vaccines we tested in NHP, to this large cohort of untreated animals demonstrated the value of in vivo imaging in preclinical trials.

Distinct spatial arrangements of ACE2 and TMPRSS2 expression in Syrian hamster lung lobes dictates SARS-CoV-2 infection patterns.

SARS-CoV-2 attaches to angiotensin-converting enzyme 2 (ACE2) to gain entry into cells after which the spike protein is cleaved by the transmembrane serine protease 2 (TMPRSS2) to facilitate viral-host membrane fusion. ACE2 and TMPRSS2 expression profiles have been analyzed at the genomic, transcriptomic, and single-cell RNAseq levels. However, transcriptomic data and actual protein validation convey conflicting information regarding the distribution of the biologically relevant protein receptor in whole tissues. To describe the organ-level architecture of receptor expression, related to the ability of ACE2 and TMPRSS2 to mediate infectivity, we performed a volumetric analysis of whole Syrian hamster lung lobes. Lung tissue of infected and control animals was stained using antibodies against ACE2 and TMPRSS2, combined with SARS-CoV-2 nucleoprotein staining. This was followed by light-sheet microscopy imaging to visualize their expression and related infection patterns. The data demonstrate that infection is restricted to sites containing both ACE2 and TMPRSS2, the latter is expressed in the primary and secondary bronchi whereas ACE2 is predominantly observed in the bronchioles and alveoli. Conversely, infection completely overlaps where ACE2 and TMPRSS2 co-localize in the tertiary bronchi, bronchioles, and alveoli.

High thermostability improves neutralizing antibody responses induced by native-like HIV-1 envelope trimers.

Soluble HIV-1 envelope glycoprotein (Env) immunogens are a prime constituent of candidate vaccines designed to induce broadly neutralizing antibodies. Several lines of evidence suggest that enhancing Env immunogen thermostability can improve neutralizing antibody (NAb) responses. Here, we generated BG505 SOSIP.v9 trimers, which displayed virtually no reactivity with non-neutralizing antibodies and showed increased global and epitope thermostability, compared to previous BG505 SOSIP versions. Chemical crosslinking of BG505 SOSIP.v9 further increased the melting temperature to 91.3 °C, which is almost 25 °C higher than that of the prototype SOSIP.664 trimer. Next, we compared the immunogenicity of a palette of BG505-based SOSIP trimers with a gradient of thermostabilities in rabbits. We also included SOSIP.v9 proteins in which a strain-specific immunodominant epitope was masked by glycans to redirect the NAb response to other subdominant epitopes. We found that increased trimer thermostability correlated with increased potency and consistency of the autologous NAb response. Furthermore, glycan masking steered the NAb response to subdominant epitopes without decreasing the potency of the autologous NAb response. In summary, SOSIP.v9 trimers and their glycan masked versions represent an improved platform for HIV-1 Env based vaccination strategies.

Quantitative analysis of mRNA-1273 COVID-19 vaccination response in immunocompromised adult hematology patients.

Vaccination guidelines for patients treated for hematological diseases are typically conservative. Given their high risk for severe COVID-19, it is important to identify those patients that benefit from vaccination. We prospectively quantified serum immunoglobulin G (IgG) antibodies to spike subunit 1 (S1) antigens during and after 2-dose mRNA-1273 (Spikevax/Moderna) vaccination in hematology patients. Obtaining S1 IgG ≥ 300 binding antibody units (BAUs)/mL was considered adequate as it represents the lower level of S1 IgG concentration obtained in healthy individuals, and it correlates with potent virus neutralization. Selected patients (n = 723) were severely immunocompromised owing to their disease or treatment thereof. Nevertheless, >50% of patients obtained S1 IgG ≥ 300 BAUs/mL after 2-dose mRNA-1273. All patients with sickle cell disease or chronic myeloid leukemia obtained adequate antibody concentrations. Around 70% of patients with chronic graft-versus-host disease (cGVHD), multiple myeloma, or untreated chronic lymphocytic leukemia (CLL) obtained S1 IgG ≥ 300 BAUs/mL. Ruxolitinib or hypomethylating therapy but not high-dose chemotherapy blunted responses in myeloid malignancies. Responses in patients with lymphoma, patients with CLL on ibrutinib, and chimeric antigen receptor T-cell recipients were low. The minimal time interval after autologous hematopoietic cell transplantation (HCT) to reach adequate concentrations was <2 months for multiple myeloma, 8 months for lymphoma, and 4 to 6 months after allogeneic HCT. Serum IgG4, absolute B- and natural killer-cell number, and number of immunosuppressants predicted S1 IgG ≥ 300 BAUs/mL. Hematology patients on chemotherapy, shortly after HCT, or with cGVHD should not be precluded from vaccination. This trial was registered at Netherlands Trial Register as #NL9553.

SARS-CoV-2 infection activates dendritic cells via cytosolic receptors rather than extracellular TLRs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), an infectious disease characterized by strong induction of inflammatory cytokines, progressive lung inflammation, and potentially multiorgan dysfunction. It remains unclear how SARS-CoV-2 infection leads to immune activation. The Spike (S) protein of SARS-CoV-2 has been suggested to trigger TLR4 and thereby activate immunity. Here, we have investigated the role of TLR4 in SARS-CoV-2 infection and immunity. Neither exposure of isolated S protein, SARS-CoV-2 pseudovirus nor primary SARS-CoV-2 isolate induced TLR4 activation in a TLR4-expressing cell line. Human monocyte-derived DCs express TLR4 but not angiotensin converting enzyme 2 (ACE2), and DCs were not infected by SARS-CoV-2. Notably, neither S protein nor SARS-CoV-2 induced DC maturation or cytokines, indicating that both S protein and SARS-CoV-2 virus particles do not trigger extracellular TLRs including TLR4. Ectopic expression of ACE2 in DCs led to efficient infection by SARS-CoV-2 and, strikingly, efficient type I IFN and cytokine responses. These data strongly suggest that not extracellular TLRs but intracellular viral sensors are key players in sensing SARS-CoV-2. These data imply that SARS-CoV-2 escapes direct sensing by TLRs, which might underlie the lack of efficient immunity to SARS-CoV-2 early during infection.

Immunization with synthetic SARS-CoV-2 S glycoprotein virus-like particles protects macaques from infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused an ongoing global health crisis. Here, we present as a vaccine candidate synthetic SARS-CoV-2 spike (S) glycoprotein-coated lipid vesicles that resemble virus-like particles. Soluble S glycoprotein trimer stabilization by formaldehyde cross-linking introduces two major inter-protomer cross-links that keep all receptor-binding domains in the "down" conformation. Immunization of cynomolgus macaques with S coated onto lipid vesicles (S-LVs) induces high antibody titers with potent neutralizing activity against the vaccine strain, Alpha, Beta, and Gamma variants as well as T helper (Th)1 CD4+-biased T cell responses. Although anti-receptor-binding domain (RBD)-specific antibody responses are initially predominant, the third immunization boosts significant non-RBD antibody titers. Challenging vaccinated animals with SARS-CoV-2 shows a complete protection through sterilizing immunity, which correlates with the presence of nasopharyngeal anti-S immunoglobulin G (IgG) and IgA titers. Thus, the S-LV approach is an efficient and safe vaccine candidate based on a proven classical approach for further development and clinical testing.