Safety, immunogenicity, and optimal dosing of a modified vaccinia Ankara-based vaccine against MERS-CoV in healthy adults: a phase 1b, double-blind, randomised placebo-controlled clinical trial.

BACKGROUND: MERS-CoV is a respiratory pathogen with a case-fatality rate of 36%, and for which no vaccines are currently licensed. MVA-MERS-S is a candidate vaccine based on recombinant modified vaccinia virus Ankara (MVA). In this study, the safety, immunogenicity, and optimal dose schedule of MVA-MERS-S was assessed in individuals with previous exposure to SARS-CoV-2 infections and vaccines. METHODS: We conducted a multicentre, double-blind, randomised controlled phase 1b clinical trial at two university medical centres in Germany and the Netherlands. Healthy volunteers aged 18-55 years were assigned by computer randomisation to receive three intramuscular injections of 107 or 108 plaque-forming units (PFU) of MVA-MERS-S, with two treatment groups each of either 28-day or 56-day intervals between the initial two doses, and one control arm that received only placebo, at a ratio of 2:2:2:2:1. The third dose was given after 224 days. The sponsor, clinical and laboratory staff, and participants were masked to both vaccine dose and dosing interval. The primary outcome was safety, assessed in the all participants who had received at least one injection; daily solicited vaccine reactions were recorded after each dose for 7 days, unsolicited adverse events for 28 days, and serious adverse events throughout the study. The secondary outcome was humoral immunogenicity, measured with vaccine-induced geometric mean antibody concentrations and seroconversion rates, analysed in all participants who received at least three allocated treatments. This study is registered at ClinicalTrials.gov (NCT04119440) and is completed. FINDINGS: Between 26 July, 2021, and 3 March, 2022, 244 volunteers were screened, 177 of whom were eligible and 140 were randomly assigned either to the 28-day 107 PFU group (n=32), 56-day 107 PFU group (n=31), 28-day 108 PFU group (n=31), 56-day 108 PFU group (n=30), or placebo group (n=16). In total, 178 doses were administered of 107 PFU of MVA-MERS-S, 174 of 108 PFU, and 164 doses of placebo, and 139 participants received at least one injection. 73 (53%) were female and 66 (48%) were male. No serious vaccine-related adverse events occurred. Solicited local reactions were mild in 288 (93%, 95% CI 90-96) of 309 reports and consisted primarily of pain or tenderness. Pain or tenderness (of any severity) occurred after 69 (39%, 32-46) of 178 107 PFU injections, 138 (79%; 73-85) of 174 108 PFU injections, and 18 (11%; 7-11) of 164 placebo injections. Of 595 reported solicited systemic reactions, 479 (81%, 77-83) were graded as mild. Systemic reactions of any grade occurred after 77 (43%; 36-51) 107 PFU injections, 102 (59%; 51-66) 108 PFU injections, and 67 (41%; 34-49) of 164 placebo injections. At 28 days after the second dose, MERS-CoV neutralising antibodies were highest for participants assigned to 56-day 108 PFU, with geometric mean ratios of 7·2 (95% CI 3·9-13·3) for the 56-day 108 PFU group versus the 28-day 108 PFU group (p<0·0001), 3·9 (2·1-7·2) for the 56-day 108 PFU group versus the 56-day 107 PFU group (p=0·0031), and 5·4 (2·9-10·0) for the 56-day 108 PFU group versus the 28-day 107 PFU group (p=0·0003). INTERPRETATION: MVA-MERS-S was safe and immunogenic in individuals with previous and concurrent SARS-CoV-2 exposure. The second vaccination with the 108 PFU dose of MVA-MERS-S elicited a stronger humoral immune response when administered 56 days after the first dose than a 28-day interval. Further studies are needed to verify these findings in groups at risk for MERS-CoV exposure, and at risk of severe disease, including older individuals and those with relevant comorbidities. FUNDING: Coalition for Epidemic Preparedness Innovations, the German Centre for Infection Research, and the German Research Foundation.

Dissecting humoral immune responses to an MVA-vectored MERS-CoV vaccine in humans using a systems serology approach.

Besides neutralizing antibodies, which are considered an important measure for vaccine immunogenicity, Fc-mediated antibody functions can contribute to antibody-mediated protection. They are strongly influenced by structural antibody properties such as subclass and Fc glycan composition. We here applied a systems serology approach to dissect humoral immune responses induced by MVA-MERS-S, an MVA-vectored vaccine against the Middle East respiratory syndrome coronavirus (MERS-CoV). Building on preceding studies reporting the safety and immunogenicity of MVA-MERS-S, our study highlights the potential of a late boost, administered one year after prime, to enhance both neutralizing and Fc-mediated antibody functionality compared to the primary vaccination series. Distinct characteristics were observed for antibodies specific to the MERS-CoV spike protein S1 and S2 subunits, regarding subclass and glycan compositions as well as Fc functionality. These findings highlight the benefit of a late homologous booster vaccination with MVA-MERS-S and may be of interest for the design of future coronavirus vaccines.

Advancing COVID-19 diagnostics: rapid detection of intact SARS-CoV-2 using viability RT-PCR assay.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Commonly used methods for both clinical diagnosis of SARS-CoV-2 infection and management of infected patients involve the detection of viral RNA, but the presence of infectious virus particles is unknown. Viability PCR (v-PCR) uses a photoreactive dye to bind non-infectious RNA, ideally resulting in the detection of RNA only from intact virions. This study aimed to develop and validate a rapid v-PCR assay for distinguishing intact and compromised SARS-CoV-2. Propidium monoazide (PMAxx) was used as a photoreactive dye. Mixtures with decreasing percentages of intact SARS-CoV-2 (from 100% to 0%) were prepared from SARS-CoV-2 virus stock and a clinical sample. Each sample was divided into a PMAxx-treated part and a non-PMAxx-treated part. Reverse transcription-PCR (RT-PCR) using an in-house developed SARS-CoV-2 viability assay was then applied to both sample sets. The difference in intact SARS-CoV-2 was determined by subtracting the cycle threshold (Ct) value of the PMAxx-treated sample from the non-PMAxx-treated sample. Mixtures with decreasing concentrations of intact SARS-CoV-2 showed increasingly lower delta Ct values as the percentage of intact SARS-CoV-2 decreased, as expected. This relationship was observed in both high and low viral load samples prepared from cultured SARS-CoV-2 virus stock, as well as for a clinical sample prepared directly from a SARS-CoV-2 positive nasopharyngeal swab. In this study, a rapid v-PCR assay has been validated that can distinguish intact from compromised SARS-CoV-2. The presence of intact virus particles, as determined by v-PCR, may indicate SARS-CoV-2 infectiousness. IMPORTANCE: This study developed a novel method that can help determine whether someone who has been diagnosed with coronavirus disease 2019 (COVID-19) is still capable of spreading the virus to others. Current tests only detect the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, but cannot tell whether the particles are still intact and can thus infect cells. The researchers used a dye that selectively blocks the detection of damaged virions and free RNA. They showed that this viability PCR reliably distinguishes intact SARS-CoV-2 capable of infecting from damaged SARS-CoV-2 or free RNA in both cultured virus samples and a clinical sample. Being able to quickly assess contagiousness has important implications for contact tracing and safely ending isolation precautions. This viability PCR technique provides a simple way to obtain valuable information, beyond just positive or negative test results, about the actual risk someone poses of transmitting SARS-CoV-2 through the air or surfaces they come into contact with.

Virus neutralization assays for human respiratory syncytial virus using airway organoids.

Neutralizing antibodies are considered a correlate of protection against severe human respiratory syncytial virus (HRSV) disease. Currently, HRSV neutralization assays are performed on immortalized cell lines like Vero or A549 cells. It is known that assays on these cell lines exclusively detect neutralizing antibodies (nAbs) directed to the fusion (F) protein. For the detection of nAbs directed to the glycoprotein (G), ciliated epithelial cells expressing the cellular receptor CX3CR1 are required, but generation of primary cell cultures is expensive and labor-intensive. Here, we developed a high-throughput neutralization assay based on the interaction between clinically relevant HRSV grown on primary cells with ciliated epithelial cells, and validated this assay using a panel of infant sera. To develop the high-throughput neutralization assay, we established a culture of differentiated apical-out airway organoids (Ap-O AO). CX3CR1 expression was confirmed, and both F- and G-specific monoclonal antibodies neutralized HRSV in the Ap-O AO. In a side-by-side neutralization assay on Vero cells and Ap-O AO, neutralizing antibody levels in sera from 125 infants correlated well, although titers on Ap-O AO were consistently lower. We speculate that these lower titers might be an actual reflection of the neutralizing antibody capacity in vivo. The organoid-based neutralization assay described here holds promise for further characterization of correlates of protection against HRSV disease.

Direct comparison of SARS-CoV-2 variant specific neutralizing antibodies in human and hamster sera.

Antigenic characterization of newly emerging SARS-CoV-2 variants is important to assess their immune escape and judge the need for future vaccine updates. To bridge data obtained from animal sera with human sera, we analyzed neutralizing antibody titers in human and hamster single infection sera in a highly controlled setting using the same authentic virus neutralization assay performed in one laboratory. Using a Bayesian framework, we found that titer fold changes in hamster sera corresponded well to human sera and that hamster sera generally exhibited higher reactivity.

Original COVID-19 priming regimen impacts the immunogenicity of bivalent BA.1 and BA.5 boosters.

Waning antibody responses after COVID-19 vaccination combined with the emergence of the SARS-CoV-2 Omicron lineage led to reduced vaccine effectiveness. As a countermeasure, bivalent mRNA-based booster vaccines encoding the ancestral spike protein in combination with that of Omicron BA.1 or BA.5 were introduced. Since then, different BA.2-descendent lineages have become dominant, such as XBB.1.5, JN.1, or EG.5.1. Here, we report post-hoc analyses of data from the SWITCH-ON study, assessing how different COVID-19 priming regimens affect the immunogenicity of bivalent booster vaccinations and breakthrough infections (NCT05471440). BA.1 and BA.5 bivalent vaccines boosted neutralizing antibodies and T-cells up to 3 months after boost; however, cross-neutralization of XBB.1.5 was poor. Interestingly, different combinations of prime-boost regimens induced divergent responses: participants primed with Ad26.COV2.S developed lower binding antibody levels after bivalent boost while neutralization and T-cell responses were similar to mRNA-based primed participants. In contrast, the breadth of neutralization was higher in mRNA-primed and bivalent BA.5 boosted participants. Combined, our data further support the current use of monovalent vaccines based on circulating strains when vaccinating risk groups, as recently recommended by the WHO. We emphasize the importance of the continuous assessment of immune responses targeting circulating variants to guide future COVID-19 vaccination policies.

Filamentous fungus-produced human monoclonal antibody provides protection against SARS-CoV-2 in hamster and non-human primate models.

Monoclonal antibodies are an increasingly important tool for prophylaxis and treatment of acute virus infections like SARS-CoV-2 infection. However, their use is often restricted due to the time required for development, variable yields and high production costs, as well as the need for adaptation to newly emerging virus variants. Here we use the genetically modified filamentous fungus expression system Thermothelomyces heterothallica (C1), which has a naturally high biosynthesis capacity for secretory enzymes and other proteins, to produce a human monoclonal IgG1 antibody (HuMab 87G7) that neutralises the SARS-CoV-2 variants of concern (VOCs) Alpha, Beta, Gamma, Delta, and Omicron. Both the mammalian cell and C1 produced HuMab 87G7 broadly neutralise SARS-CoV-2 VOCs in vitro and also provide protection against VOC Omicron in hamsters. The C1 produced HuMab 87G7 is also able to protect against the Delta VOC in non-human primates. In summary, these findings show that the C1 expression system is a promising technology platform for the development of HuMabs in preventive and therapeutic medicine.

Human metapneumovirus infection of organoid-derived human bronchial epithelium represents cell tropism and cytopathology as observed in in vivo models.

Human metapneumovirus (HMPV), a member of the Pneumoviridae family, causes upper and lower respiratory tract infections in humans. In vitro studies with HMPV have mostly been performed in monolayers of undifferentiated epithelial cells. In vivo studies in cynomolgus macaques and cotton rats have shown that ciliated epithelial cells are the main target of HMPV infection, but these observations cannot be studied in monolayer systems. Here, we established an organoid-derived bronchial culture model that allows physiologically relevant studies on HMPV. Inoculation with multiple prototype HMPV viruses and recent clinical virus isolates led to differences in replication among HMPV isolates. Prolific HMPV replication in this model caused damage to the ciliary layer, including cilia loss at advanced stages post-infection. These cytopathic effects correlated with those observed in previous in vivo studies with cynomolgus macaques. The assessment of the innate immune responses in three donors upon HMPV and RSV inoculation highlighted the importance of incorporating multiple donors to account for donor-dependent variation. In conclusion, these data indicate that the organoid-derived bronchial cell culture model resembles in vivo findings and is therefore a suitable and robust model for future HMPV studies. IMPORTANCE: Human metapneumovirus (HMPV) is one of the leading causative agents of respiratory disease in humans, with no treatment or vaccine available yet. The use of primary epithelial cultures that recapitulate the tissue morphology and biochemistry of the human airways could aid in defining more relevant targets to prevent HMPV infection. For this purpose, this study established the first primary organoid-derived bronchial culture model suitable for a broad range of HMPV isolates. These bronchial cultures were assessed for HMPV replication, cellular tropism, cytopathology, and innate immune responses, where the observations were linked to previous in vivo studies with HMPV. This study exposed an important gap in the HMPV field since extensively cell-passaged prototype HMPV B viruses did not replicate in the bronchial cultures, underpinning the need to use recently isolated viruses with a controlled passage history. These results were reproducible in three different donors, supporting this model to be suitable to study HMPV infection.

Human conjunctiva organoids to study ocular surface homeostasis and disease.

The conjunctival epithelium covering the eye contains two main cell types: mucus-producing goblet cells and water-secreting keratinocytes, which present mucins on their apical surface. Here, we describe long-term expanding organoids and air-liquid interface representing mouse and human conjunctiva. A single-cell RNA expression atlas of primary and cultured human conjunctiva reveals that keratinocytes express multiple antimicrobial peptides and identifies conjunctival tuft cells. IL-4/-13 exposure increases goblet and tuft cell differentiation and drastically modifies the conjunctiva secretome. Human NGFR+ basal cells are identified as bipotent conjunctiva stem cells. Conjunctival cultures can be infected by herpes simplex virus 1 (HSV1), human adenovirus 8 (hAdV8), and SARS-CoV-2. HSV1 infection was reversed by acyclovir addition, whereas hAdV8 infection, which lacks an approved drug therapy, was inhibited by cidofovir. We document transcriptional programs induced by HSV1 and hAdV8. Finally, conjunctival organoids can be transplanted. Together, human conjunctiva organoid cultures enable the study of conjunctival (patho)-physiology.

Comparative Pathogenesis of Severe Acute Respiratory Syndrome Coronaviruses.

Over the last two decades the world has witnessed the global spread of two genetically related highly pathogenic coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. However, the impact of these outbreaks differed significantly with respect to the hospitalizations and fatalities seen worldwide. While many studies have been performed recently on SARS-CoV-2, a comparative pathogenesis analysis with SARS-CoV may further provide critical insights into the mechanisms of disease that drive coronavirus-induced respiratory disease. In this review, we comprehensively describe clinical and experimental observations related to transmission and pathogenesis of SARS-CoV-2 in comparison with SARS-CoV, focusing on human, animal, and in vitro studies. By deciphering the similarities and disparities of SARS-CoV and SARS-CoV-2, in terms of transmission and pathogenesis mechanisms, we offer insights into the divergent characteristics of these two viruses. This information may also be relevant to assessing potential novel introductions of genetically related highly pathogenic coronaviruses.

Transient Autoreactive PF4 and Antiphospholipid Antibodies in COVID-19 Vaccine Recipients.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare autoimmune condition associated with recombinant adenovirus (rAV)-based COVID-19 vaccines. It is thought to arise from autoantibodies targeting platelet factor 4 (aPF4), triggered by vaccine-induced inflammation and the formation of neo-antigenic complexes between PF4 and the rAV vector. To investigate the specific induction of aPF4 by rAV-based vaccines, we examined sera from rAV vaccine recipients (AZD1222, AD26.COV2.S) and messenger RNA (mRNA) based (mRNA-1273, BNT162b2) COVID-19 vaccine recipients. We compared the antibody fold change (FC) for aPF4 and for antiphospholipid antibodies (aPL) of rAV to mRNA vaccine recipients. We combined two biobanks of Dutch healthcare workers and matched rAV-vaccinated individuals to mRNA-vaccinated controls, based on age, sex and prior history of COVID-19 (AZD1222: 37, Ad26.COV2.S: 35, mRNA-1273: 47, BNT162b2: 26). We found no significant differences in aPF4 FCs after the first (0.99 vs. 1.08, mean difference (MD) = -0.11 (95% CI -0.23 to 0.057)) and second doses of AZD1222 (0.99 vs. 1.10, MD = -0.11 (95% CI -0.31 to 0.10)) and after a single dose of Ad26.COV2.S compared to mRNA-based vaccines (1.01 vs. 0.99, MD = 0.026 (95% CI -0.13 to 0.18)). The mean FCs for the aPL in rAV-based vaccine recipients were similar to those in mRNA-based vaccines. No correlation was observed between post-vaccination aPF4 levels and vaccine type (mean aPF difference -0.070 (95% CI -0.14 to 0.002) mRNA vs. rAV). In summary, our study indicates that rAV and mRNA-based COVID-19 vaccines do not substantially elevate aPF4 levels in healthy individuals.

Fangchinoline inhibits SARS-CoV-2 and MERS-CoV entry.

The COVID-19 pandemic caused by SARS-CoV-2, lead to mild to severe respiratory illness and resulted in 6.9 million deaths worldwide. Although vaccines are effective in preventing COVID-19, they may not be sufficient to protect immunocompromised individuals from this respiratory illness. Moreover, novel emerging variants of SARS-CoV-2 pose a risk of new COVID-19 waves. Therefore, identification of effective antivirals is critical in controlling SARS and other coronaviruses, such as MERS-CoV. We show that Fangchinoline (Fcn), a bisbenzylisoquinoline alkaloid, inhibits replication of SARS-CoV, SARS-CoV-2, and MERS-CoV in a range of in vitro assays, by blocking entry. Therapeutic use of Fcn inhibited viral loads in the lungs, and suppressed associated airway inflammation in hACE2. Tg mice and Syrian hamster infected with SARS-CoV-2. Combination of Fcn with remdesivir (RDV) or an anti-leprosy drug, Clofazimine, exhibited synergistic antiviral activity. Compared to Fcn, its synthetic derivative, MK-04-003, more effectively inhibited SARS-CoV-2 and its variants B.1.617.2 and BA.5 in mice. Taken together these data demonstrate that Fcn is a pan beta coronavirus inhibitor, which possibly can be used to combat novel emerging coronavirus diseases.

The impact of BNT162b2 mRNA vaccine on adaptive and innate immune responses.

The mRNA-based BNT162b2 protects against severe disease and mortality caused by SARS-CoV-2 via induction of specific antibody and T-cell responses. Much less is known about its broad effects on immune responses against other pathogens. Here, we investigated the adaptive immune responses induced by BNT162b2 vaccination against various SARS-CoV-2 variants and its effects on the responsiveness of immune cells upon stimulation with heterologous stimuli. BNT162b2 vaccination induced effective humoral and cellular immunity against SARS-CoV-2 that started to wane after six months. We also observed long-term transcriptional changes in immune cells after vaccination. Additionally, vaccination with BNT162b2 modulated innate immune responses as measured by inflammatory cytokine production after stimulation - higher IL-1/IL-6 release and decreased IFN-α production. Altogether, these data expand our knowledge regarding the overall immunological effects of this new class of vaccines and underline the need for additional studies to elucidate their effects on both innate and adaptive immune responses.

Glycosylated extracellular mucin domains protect against SARS-CoV-2 infection at the respiratory surface.

Mucins play an essential role in protecting the respiratory tract against microbial infections while also acting as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance. Transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on epithelial entry of SARS-CoV-2. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of terminal mucin glycans sialic acid and fucose from the epithelial surface did not impact viral entry. In Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are located to the apical surface in close proximity and StcE treatment results in enhanced binding of purified spike protein. Both MUC1 and MUC16 are expressed on the surface of human organoid-derived air-liquid interface (ALI) differentiated airway cultures and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 replication. In these cultures, MUC1 was highly expressed in non-ciliated cells while MUC16 was enriched in goblet cells. In conclusion, the glycosylated extracellular domains of different transmembrane mucins might have similar protective functions in different respiratory cell types by restricting SARS-CoV-2 binding and entry.

SARS-CoV-2 Omicron entry is type II transmembrane serine protease-mediated in human airway and intestinal organoid models.

SARS-CoV-2 can enter cells after its spike protein is cleaved by either type II transmembrane serine proteases (TTSPs), like TMPRSS2, or cathepsins. It is now widely accepted that the Omicron variant uses TMPRSS2 less efficiently and instead enters cells via cathepsins, but these findings have yet to be verified in more relevant cell models. Although we could confirm efficient cathepsin-mediated entry for Omicron in a monkey kidney cell line, experiments with protease inhibitors showed that Omicron (BA.1 and XBB1.5) did not use cathepsins for entry into human airway organoids and instead utilized TTSPs. Likewise, CRISPR-edited intestinal organoids showed that entry of Omicron BA.1 relied on the expression of the serine protease TMPRSS2 but not cathepsin L or B. Together, these data force us to rethink the concept that Omicron has adapted to cathepsin-mediated entry and indicate that TTSP inhibitors should not be dismissed as prophylactic or therapeutic antiviral strategy against SARS-CoV-2. IMPORTANCE Coronavirus entry relies on host proteases that activate the viral fusion protein, spike. These proteases determine the viral entry route, tropism, host range, and can be attractive drug targets. Whereas earlier studies using cell lines suggested that the Omicron variant of SARS-CoV-2 has changed its protease usage, from cell surface type II transmembrane serine proteases (TTSPs) to endosomal cathepsins, we report that this is not the case in human airway and intestinal organoid models, suggesting that host TTSP inhibition is still a viable prophylactic or therapeutic antiviral strategy against current SARS-CoV-2 variants and highlighting the importance of relevant human in vitro cell models.

Antigenic evolution of SARS coronavirus 2.

SARS coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, emerged in China in December 2019. Vaccines developed were very effective initially, however, the virus has shown remarkable evolution with multiple variants spreading globally over the last three years. Nowadays, newly emerging Omicron lineages are gaining substitutions at a fast rate, resulting in escape from neutralization by antibodies that target the Spike protein. Tools to map the impact of substitutions on the further antigenic evolution of SARS-CoV-2, such as antigenic cartography, may be helpful to update SARS-CoV-2 vaccines. In this review, we focus on the antigenic evolution of SARS-CoV-2, highlighting the impact of Spike protein substitutions individually and in combination on immune escape.

Preclinical immunogenicity and protective efficacy of a SARS-CoV-2 RBD-based vaccine produced with the thermophilic filamentous fungal expression system Thermothelomyces heterothallica C1.

Introduction: The emergency use of vaccines has been the most efficient way to control the coronavirus disease 19 (COVID-19) pandemic. However, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern has reduced the efficacy of currently used vaccines. The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein is the main target for virus neutralizing (VN) antibodies. Methods: A SARS-CoV-2 RBD vaccine candidate was produced in the Thermothelomyces heterothallica (formerly, Myceliophthora thermophila) C1 protein expression system and coupled to a nanoparticle. Immunogenicity and efficacy of this vaccine candidate was tested using the Syrian golden hamster (Mesocricetus auratus) infection model. Results: One dose of 10-µg RBD vaccine based on SARS-CoV-2 Wuhan strain, coupled to a nanoparticle in combination with aluminum hydroxide as adjuvant, efficiently induced VN antibodies and reduced viral load and lung damage upon SARS-CoV-2 challenge infection. The VN antibodies neutralized SARS-CoV-2 variants of concern: D614G, Alpha, Beta, Gamma, and Delta. Discussion: Our results support the use of the Thermothelomyces heterothallica C1 protein expression system to produce recombinant vaccines against SARS-CoV-2 and other virus infections to help overcome limitations associated with the use of mammalian expression system.