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New platforms are urgently needed for the design of novel prophylactic vaccines and advanced immune therapies. Live-attenuated yellow fever vaccine YF17D serves as vector for several licensed vaccines and platform for novel vaccine candidates. Based on YF17D, we developed YF-S0 as exceptionally potent COVID-19 vaccine candidate. However, use of such live RNA virus vaccines raises safety concerns, i.e., adverse events linked to original YF17D (yellow fever vaccine-associated neurotropic; YEL-AND, and viscerotropic disease; YEL-AVD). In this study, we investigated the biodistribution and shedding of YF-S0 in hamsters. Likewise, we introduced hamsters deficient in STAT2 signaling as new preclinical model of YEL-AND/AVD. Compared to parental YF17D, YF-S0 showed an improved safety with limited dissemination to brain and visceral tissues, absent or low viremia, and no shedding of infectious virus. Considering yellow fever virus is transmitted by Aedes mosquitoes, any inadvertent exposure to the live recombinant vector via mosquito bites is to be excluded. The transmission risk of YF-S0 was hence evaluated in comparison to readily transmitting YFV-Asibi strain and non-transmitting YF17D vaccine, with no evidence for productive infection of vector mosquitoes. The overall favorable safety profile of YF-S0 is expected to translate to other novel vaccines that are based on the same YF17D platform.
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The emergence of SARS-CoV-2 variants of concern (VoCs) has exacerbated the COVID-19 pandemic. End of November 2021, a new SARS-CoV-2 variant namely the omicron (B.1.1.529) emerged. Since this omicron variant is heavily mutated in the spike protein, WHO classified this variant as the 5th variant of concern (VoC). We previously demonstrated that the other SARS-CoV-2 VoCs replicate efficiently in Syrian hamsters, alike also the ancestral strains. We here wanted to explore the infectivity of the omicron variant in comparison to the ancestral D614G strain. Strikingly, in hamsters that had been infected with the omicron variant, a 3 log10 lower viral RNA load was detected in the lungs as compared to animals infected with D614G and no infectious virus was detectable in this organ. Moreover, histopathological examination of the lungs from omicron-infecetd hamsters revealed no signs of peri-bronchial inflammation or bronchopneumonia. Further experiments are needed to determine whether the omicron VoC replicates possibly more efficiently in the upper respiratory tract of hamsters than in their lungs.
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SARS-CoV-2 B.1.1.529, designated omicron, was recently identified as a new variant of concern by WHO and is rapidly replacing SARS-CoV-2 delta as the most dominant variant in many countries. Unfortunately, because of the high number of mutations present in the spike of SARS-CoV-2 omicron, most monoclonal antibodies (mAbs) currently approved for treatment of COVID-19 lose their in vitro neutralizing activity against this variant. We recently described a panel of human anti-SARS-CoV-2 mAbs that potently neutralize SARS-CoV-2 Wuhan, D614G and variants alpha, beta, gamma and delta. In this work, we evaluated our mAb panel for potential in vitro activity against SARS-CoV-2 delta and omicron. Three mAbs from our panel retain neutralizing activity against both delta and omicron, with mAb 3B8 still resulting in complete neutralization at a concentration as low as 0.02 g/ml for both variants. Overall, our data indicate that mAb 3B8 may have the potential to become a game-changer in the fight against the continuously evolving SARS-CoV-2.
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Current first-generation COVID-19 vaccines are based on prototypic spike sequences from ancestral 2019 SARS-CoV-2 strains. However, the ongoing pandemic is fueled by variants of concern (VOC) that threaten to escape vaccine-mediated protection. Here we show in a stringent hamster model that immunization using prototypic spike expressed from a potent YF17D viral vector (1) provides vigorous protection against infection with ancestral virus (B lineage) and VOC Alpha (B.1.1.7), however, is insufficient to provide maximum protection against the Beta (B.1.351) variant. To improve vaccine efficacy, we created a revised vaccine candidate that carries an evolved spike antigen. Vaccination of hamsters with this updated vaccine candidate provides full protection against intranasal challenge with all four VOCs Alpha, Beta, Gamma (P.1) and Delta (B.1.617.2) resulting in complete elimination of infectious virus from the lungs and a marked improvement in lung pathology. Vaccinated hamsters did also no longer transmit the Delta variant to non-vaccinated sentinels. Hamsters immunized with our modified vaccine candidate also mounted marked neutralizing antibody responses against the recently emerged Omicron (B.1.1.529) variant, whereas the old vaccine employing prototypic spike failed to induce immunity to this antigenically distant virus. Overall, our data indicate that current first-generation COVID-19 vaccines need to be urgently updated to cover newly emerging VOCs to maintain vaccine efficacy and to impede virus spread at the community level. Significance StatementSARS-CoV-2 keeps mutating rapidly, and the ongoing COVID-19 pandemic is fueled by new variants escaping immunity induced by current first-generation vaccines. There is hence an urgent need for universal vaccines that cover variants of concern (VOC). In this paper we show that an adapted version of our vaccine candidate YF-S0* provides full protection from infection, virus transmission and disease by VOCs Alpha, Beta, Gamma and Delta, and also results in markedly increased levels of neutralizing antibodies against recently emerged Omicron VOC in a stringent hamster model. Our findings underline the necessity to update COVID-19 vaccines to curb the pandemic, providing experimental proof on how to maintain vaccine efficacy in view of an evolving SARS-CoV-2 diversity.
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We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and -2, such as VHH72, which binds to a unique highly conserved epitope in the viral receptor-binding domain (RBD) that is difficult to access for human antibodies. Here, we establish a protein engineering path for how a stable, long-acting drug candidate can be generated out of such a VHH building block. When fused to human IgG1-Fc, the prototype VHH72 molecule prophylactically protects hamsters from SARS-CoV-2. In addition, we demonstrate that both systemic and intranasal application protects hACE-2-transgenic mice from SARS-CoV-2 induced lethal disease progression. To boost potency of the lead, we used structure-guided molecular modeling combined with rapid yeast-based Fc-fusion prototyping, resulting in the affinity-matured VHH72_S56A-Fc, with subnanomolar SARS-CoV-1 and -2 neutralizing potency. Upon humanization, VHH72_S56A was fused to a human IgG1 Fc with optimized manufacturing homogeneity and silenced effector functions for enhanced safety, and its stability as well as lack of off-target binding was extensively characterized. Therapeutic systemic administration of a low dose of VHH72_S56A-Fc antibodies strongly restricted replication of both original and D614G mutant variants of SARS-CoV-2 virus in hamsters, and minimized the development of lung damage. This work led to the selection of XVR011 for clinical development, a highly stable anti-COVID-19 biologic with excellent manufacturability. Additionally, we show that XVR011 is unaffected in its neutralizing capacity of currently rapidly spreading SARS-CoV-2 variants, and demonstrate its unique, wide scope of binding across the Sarbecovirus clades.
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Within one year after its emergence, more than 108 million people contracted SARS-CoV-2 and almost 2.4 million succumbed to COVID-19. New SARS-CoV-2 variants of concern (VoC) are emerging all over the world, with the threat of being more readily transmitted, being more virulent, or escaping naturally acquired and vaccine-induced immunity. At least three major prototypic VoC have been identified, i.e. the UK (B.1.1.7), South African (B.1.351) and Brazilian (B.1.1.28.1), variants. These are replacing formerly dominant strains and sparking new COVID-19 epidemics and new spikes in excess mortality. We studied the effect of infection with prototypic VoC from both B.1.1.7 and B.1.351 lineages in Syrian golden hamsters to assess their relative infectivity and pathogenicity in direct comparison to two basal SARS-CoV-2 strains isolated in early 2020. A very efficient infection of the lower respiratory tract of hamsters by these VoC is observed. In line with clinical evidence from patients infected with these VoC, no major differences in disease outcome were observed as compared to the original strains as was quantified by (i) histological scoring, (ii) micro-computed tomography, and (iii) analysis of the expression profiles of selected antiviral and pro-inflammatory cytokine genes. Noteworthy however, in hamsters infected with VoC B.1.1.7, a particularly strong elevation of proinflammatory cytokines was detected. Overall, we established relevant preclinical infection models that will be pivotal to assess the efficacy of current and future vaccine(s) (candidates) as well as therapeutics (small molecules and antibodies) against two important SARS-CoV-2 VoC.
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In response to the ongoing COVID-19 pandemic, repurposing of drugs for the treatment of SARS-CoV-2 infections is being explored. The HIV protease inhibitor Nelfinavir, widely prescribed in combination with other HIV inhibitors, has been shown to inhibit in vitro SARS-CoV-2 replication. We here report on the effect of Nelfinavir in the Syrian hamster SARS-CoV-2 infection model. Although treatment of infected hamsters with either 15 or 50 mg/kg BID Nelfinavir [for four consecutive days, initiated on the day of infection] does not reduce viral RNA loads nor infectious virus titres in the lungs compared to the vehicle control, the drug reduced virus-induced lung pathology to nearly the baseline scores of healthy animals. A substantial interstitial infiltration of neutrophils is observed in the lungs of treated (both infected and uninfected) animals. The protective effect of Nelfinavir on SARS-CoV-2-induced lung pathology (at doses that are well tolerated and that result in exposures nearing those observed in HIV-infected patients) may lay the foundation for clinical studies with this widely used drug.
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Favipiravir and Molnupiravir, orally available antivirals, have been reported to exert antiviral activity against SARS-CoV2. In recent days preliminary efficacy data have been reported in COVID-19 patients. We here studied the combined antiviral effect of the drugs in the SARS-CoV2 hamster infection model. We first demonstrate that Molnupiravir can reduce infectious virus titers in lungs of infected animals in a dose-dependent manner by up to 3.5 log10 which is associated with a marked improvement of virus-induced lung pathology. When animals are treated with a combination of suboptimal doses of Molnupiravir and Favipiravir (that each alone result in respectively a 1.3 log10 and 1.1 log10 reduction of infectious virus titers in the lungs), a marked combined potency is observed. Infectious virus titers in the lungs of animals treated with the combo are on average reduced by 4.5 log10 and infectious virus are no longer detected in the lungs of 60% of treated infected animals. Both drugs result in an increased mutation frequency of the remaining viral RNA recovered from the lungs. In the combo-treated hamsters an increased frequency of C-to-T and G-to-A mutations in the viral RNA is observed as compared to the single treatment groups which may explain the pronounced antiviral potency of the combination. Our findings may lay the basis for the design of clinical studies to test the efficacy of the combination of Molnupiravir and Favipiravir in the treatment of COVID-19.
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The ongoing COVID-19 pandemic is responsible for worldwide economic damage and nearly one million deaths. Potent drugs for the treatment of severe SARS-CoV-2 infections are not yet available. To identify host factors that support coronavirus infection, we performed genome-wide functional genetic screens with SARS-CoV-2 and the common cold virus HCoV-229E in non-transgenic human cells. These screens identified PI3K type 3 as a potential drug target against multiple coronaviruses. We discovered that the lysosomal protein TMEM106B is an important host factor for SARS-CoV-2 infection. Furthermore, we show that TMEM106B is required for replication in multiple human cell lines derived from liver and lung and is expressed in relevant cell types in the human airways. Our results identify new coronavirus host factors that may potentially serve as drug targets against SARS-CoV-2 or to quickly combat future zoonotic coronavirus outbreaks.
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The explosively expanding COVID-19 pandemic urges the development of safe, efficacious and fast-acting vaccines to quench the unrestrained spread of SARS-CoV-2. Several promising vaccine platforms, developed in recent years, are leveraged for a rapid emergency response to COVID-191. We employed the live-attenuated yellow fever 17D (YF17D) vaccine as a vector to express the prefusion form of the SARS-CoV-2 Spike antigen. In mice, the vaccine candidate, tentatively named YF-S0, induces high levels of SARS-CoV-2 neutralizing antibodies and a favorable Th1 cell-mediated immune response. In a stringent hamster SARS-CoV-2 challenge model2, vaccine candidate YF-S0 prevents infection with SARS-CoV-2. Moreover, a single dose confers protection from lung disease in most vaccinated animals even within 10 days. These results warrant further development of YF-S0 as a potent SARS-CoV-2 vaccine candidate.
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SARS-CoV-2 rapidly spread around the globe after its emergence in Wuhan in December 2019. With no specific therapeutic and prophylactic options available, the virus was able to infect millions of people. To date, close to half a million patients succumbed to the viral disease, COVID-19. The high need for treatment options, together with the lack of small animal models of infection has led to clinical trials with repurposed drugs before any preclinical in vivo evidence attesting their efficacy was available. We used Syrian hamsters to establish a model to evaluate antiviral activity of small molecules in both an infection and a transmission setting. Upon intranasal infection, the animals developed high titers of SARS-CoV-2 in the lungs and pathology similar to that observed in mild COVID-19 patients. Treatment of SARS-CoV-2-infected hamsters with favipiravir or hydroxychloroquine (with and without azithromycin) resulted in respectively a mild or no reduction in viral RNA and infectious virus. Micro-CT scan analysis of the lungs showed no improvement compared to non-treated animals, which was confirmed by histopathology. In addition, both compounds did not prevent virus transmission through direct contact and thus failed as prophylactic treatments. By modelling the PK profile of hydroxychloroquine based on the trough plasma concentrations, we show that the total lung exposure to the drug was not the limiting factor. In conclusion, we here characterized a hamster infection and transmission model to be a robust model for studying in vivo efficacy of antiviral compounds. The information acquired using hydroxychloroquine and favipiravir in this model is of critical value to those designing (current and) future clinical trials. At this point, the data here presented on hydroxychloroquine either alone or combined with azithromycin (together with previously reported in vivo data in macaques and ferrets) provide no scientific basis for further use of the drug in humans.
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Introductory paragraphSince the emergence of SARS-CoV-2 causing COVID-19, the world is being shaken to its core with numerous hospitalizations and hundreds of thousands of deaths. In search for key targets of effective therapeutics, robust animal models mimicking COVID-19 in humans are urgently needed. Here, we show that productive SARS-CoV-2 infection in the lungs of mice is limited and restricted by early type I interferon responses. In contrast, we show that Syrian hamsters are highly permissive to SARS- CoV-2 and develop bronchopneumonia and a strong inflammatory response in the lungs with neutrophil infiltration and edema. Moreover, we identify an exuberant innate immune response as a key player in pathogenesis, in which STAT2 signaling plays a dual role, driving severe lung injury on the one hand, yet restricting systemic virus dissemination on the other. Finally, we assess SARS-CoV- 2-induced lung pathology in hamsters by micro-CT alike used in clinical practice. Our results reveal the importance of STAT2-dependent interferon responses in the pathogenesis and virus control during SARS-CoV-2 infection and may help rationalizing new strategies for the treatment of COVID-19 patients.
