Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis.

We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection both in vitro, and in the Syrian golden hamster model in vivo. In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants. ONE SENTENCE SUMMARYSARS-CoV-2 ORF6 subverts bidirectional nucleo-cytoplasmic trafficking to inhibit host gene expression and contribute to viral pathogenesis.

The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters

A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs.

Discovery of a SARS-CoV-2 Broadly-Acting Neutralizing Antibody with Activity against Omicron and Omicron + R346K Variants

The continual emergence of SARS-CoV-2 variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant, has rendered ineffective a number of previously EUA approved SARS-CoV-2 neutralizing antibody therapies. Furthermore, even those approved antibodies with neutralizing activity against Omicron are reportedly ineffective against the subset of Omicron variants that contain a R346K substitution, demonstrating the continued need for discovery and characterization of candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat newly diagnosed COVID-19 linked to recently emerged variants of concern. Following a campaign of antibody discovery based on the vaccination of Harbour H2L2 mice with defined SARS-CoV-2 spike domains, we have characterized the activity of a large collection of Spike-binding antibodies and identified a lead neutralizing human IgG1 LALA antibody, STI-9167. STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against the Omicron and Omicron + R346K variants in both pseudotype and live virus neutralization assays. Furthermore, STI-9167 nAb administered intranasally or intravenously provided protection against weight loss and reduced virus lung titers to levels below the limit of quantitation in Omicron-infected K18-hACE2 transgenic mice. With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for use in human clinical trials.

SARS-CoV-2 variants of concern have acquired mutations associated with an increased spike cleavage

For efficient cell entry and membrane fusion, SARS-CoV-2 spike (S) protein needs to be cleaved at two different sites, S1/S2 and S2 by different cellular proteases such as furin and TMPRSS2. Polymorphisms in the S protein can affect cleavage, viral transmission, and pathogenesis. Here, we investigated the role of arising S polymorphisms in vitro and in vivo to understand the emergence of SARS-CoV-2 variants. First, we showed that the S:655Y is selected after in vivo replication in the mink model. This mutation is present in the Gamma Variant Of Concern (VOC) but it also occurred sporadically in early SARS-CoV-2 human isolates. To better understand the impact of this polymorphism, we analyzed the in vitro properties of a panel of SARS-CoV-2 isolates containing S:655Y in different lineage backgrounds. Results demonstrated that this mutation enhances viral replication and spike protein cleavage. Viral competition experiments using hamsters infected with WA1 and WA1-655Y isolates showed that the variant with 655Y became dominant in both direct infected and direct contact animals. Finally, we investigated the cleavage efficiency and fusogenic properties of the spike protein of selected VOCs containing different mutations in their spike proteins. Results showed that all VOCs have evolved to acquire an increased spike cleavage and fusogenic capacity despite having different sets of mutations in the S protein. Our study demonstrates that the S:655Y is an important adaptative mutation that increases viral cell entry, transmission, and host susceptibility. Moreover, SARS-COV-2 VOCs showed a convergent evolution that promotes the S protein processing.

A Newcastle disease virus-vector expressing a prefusion-stabilized spike protein of SARS-CoV-2 induces protective immune responses against prototype virus and variants of concern in mice and hamsters

Rapid development of coronavirus disease 2019 (COVID-19) vaccines and expedited authorization for use and approval has been proven beneficial to mitigate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread and given hope in this desperate situation. It is believed that sufficient supplies and equitable allocations of vaccines are necessary to limit the global impact of the COVID-19 pandemic and the emergence of additional variants of concern. We have developed a COVID-19 vaccine based on Newcastle disease virus (NDV) that can be manufactured at high yields in embryonated eggs. Here we provide evidence that the NDV vector expressing an optimized spike antigen (NDV-HXP-S), upgraded from our previous construct, is a versatile vaccine that can be used live or inactivated to induce strong antibody responses and to also cross-neutralize variants of concern. The immunity conferred by NDV-HXP-S effectively counteracts SARS-CoV-2 infection in mice and hamsters. It is noteworthy that vaccine lots produced by existing egg-based influenza virus vaccine manufacturers in Vietnam, Thailand and Brazil exhibited excellent immunogenicity and efficacy in hamsters, demonstrating that NDV-HXP-S vaccines can be quickly produced at large-scale to meet global demands.

Prophylactic protection against respiratory viruses conferred by a prototype live attenuated influenza virus vaccine.

The influenza A non-structural protein 1 (NS1) is known for its ability to hinder the synthesis of type I interferon (IFN) during viral infection. Influenza viruses lacking NS1 ({Delta}NS1) are under clinical development as live attenuated human influenza virus vaccines and induce potent influenza virus-specific humoral and cellular adaptive immune responses. Attenuation of {Delta}NS1 influenza viruses is due to their high IFN inducing properties, that limit their replication in vivo. This study demonstrates that pre-treatment with a {Delta}NS1 virus results in an immediate antiviral state which prevents subsequent replication of homologous and heterologous viruses, preventing disease from virus respiratory pathogens, including SARS-CoV-2. Our studies suggest that {Delta}NS1 influenza viruses could be used for the prophylaxis of influenza, SARS-CoV-2 and other human respiratory viral infections, and that an influenza virus vaccine based on {Delta}NS1 live attenuated viruses would confer broad protection against influenza virus infection from the moment of administration, first by non-specific innate immune induction, followed by specific adaptive immunity.

A Combination Adjuvant for the Induction of Potent Antiviral Immune Responses for a Recombinant SARS-CoV-2 Protein Vaccine

Several SARS-CoV-2 vaccines have received EUAs, but many issues remain unresolved, including duration of conferred immunity and breadth of cross-protection. Adjuvants that enhance and shape adaptive immune responses that confer broad protection against SARS-CoV-2 variants will be pivotal for long-term protection. We developed an intranasal, rationally designed adjuvant integrating a nanoemulsion (NE) that activates TLRs and NLRP3 with an RNA agonist of RIG-I (IVT DI). The combination adjuvant with spike protein antigen elicited robust responses to SARS-CoV-2 in mice, with markedly enhanced TH1-biased cellular responses and high virus-neutralizing antibody titers towards both homologous SARS-CoV-2 and a variant harboring the N501Y mutation shared by B1.1.7, B.1.351 and P.1 variants. Furthermore, passive transfer of vaccination-induced antibodies protected naive mice against heterologous viral challenge. NE/IVT DI enables mucosal vaccination, and has the potential to improve the immune profile of a variety of SARS-CoV-2 vaccine candidates to provide effective cross-protection against future drift variants.

The E484K mutation in the SARS-CoV-2 spike protein reduces but does not abolish neutralizing activity of human convalescent and post-vaccination sera.

One year in the coronavirus disease 2019 (COVID-19) pandemic, the first vaccines are being rolled out under emergency use authorizations. It is of great concern that newly emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can escape antibody-mediated protection induced by previous infection or vaccination through mutations in the spike protein. The glutamate (E) to Lysine (K) substitution at position 484 (E484K) in the receptor binding domain (RBD) of the spike protein is present in the rapidly spreading variants of concern belonging to the B.1.351 and P.1 lineages. We performed in vitro microneutralization assays with both the USA-WA1/2020 virus and a recombinant (r)SARS-CoV-2 virus that is identical to USA-WA1/2020 except for the E484K mutation introduced in the spike RBD. We selected 34 sera from study participants based on their SARS-CoV-2 spike ELISA antibody titer (negative [N=4] versus weak [N=8], moderate [N=11] or strong positive [N=11]). In addition, we included sera from five individuals who received two doses of the Pfizer SARS-CoV-2 vaccine BNT162b2. Serum neutralization efficiency was lower against the E484K rSARS-CoV-2 (vaccination samples: 3.4 fold; convalescent low IgG: 2.4 fold, moderate IgG: 4.2 fold and high IgG: 2.6 fold) compared to USA-WA1/2020. For some of the convalescent donor sera with low or moderate IgG against the SARS-CoV-2 spike, the drop in neutralization efficiency resulted in neutralization ID50 values similar to negative control samples, with low or even absence of neutralization of the E484K rSARS-CoV-2. However, human sera with high neutralization titers against the USA-WA1/2020 strain were still able to neutralize the E484K rSARS-CoV-2. Therefore, it is important to aim for the highest titers possible induced by vaccination to enhance protection against newly emerging SARS-CoV-2 variants. Two vaccine doses may be needed for induction of high antibody titers against SARS-CoV-2. Postponing the second vaccination is suggested by some public health authorities in order to provide more individuals with a primer vaccination. Our data suggests that this may leave vaccinees less protected against newly emerging variants.

The N501Y mutation in SARS-CoV-2 spike leads to morbidity in obese and aged mice and is neutralized by convalescent and post-vaccination human sera

The current COVID-19 (coronavirus disease 19) pandemic, caused by SARS-CoV-2, disproportionally affects the elderly and people with comorbidities like obesity and associated type 2 diabetes mellitus. Small animal models are crucial for the successful development and validation of antiviral vaccines, therapies and to study the role that comorbidities have on the outcome of viral infections. The initially available SARS-CoV-2 isolates require adaptation in order to use the mouse angiotensin converting enzyme 2 (mACE-2) entry receptor and to productively infect the cells of the murine respiratory tract. We have "mouse-adapted" SARS-CoV-2 by serial passaging a clinical virus isolate in the lungs of mice. We then used low doses of this virus in mouse models for advanced age, diabetes and obesity. Similar to SARS-CoV-2 infection in humans, the outcome of infection with mouse-adapted SARS-CoV-2 resulted in enhanced morbidity in aged and diabetic obese mice. Mutations associated with mouse adaptation occurred in the S, M, N and ORF8 genes. Interestingly, one mutation in the receptor binding domain of the S protein results in the change of an asparagine to tyrosine residue at position 501 (N501Y). This mutation is also present in the newly emerging SARS-CoV-2 variant viruses reported in the U.K. (20B/501Y.V1, B1.1.7 lineage) that is epidemiologically associated with high human to human transmission. We show that human convalescent and post vaccination sera can neutralize the newly emerging N501Y virus variant with similar efficiency as that of the reference USA-WA1/2020 virus, suggesting that current SARS-CoV-2 vaccines will protect against the 20B/501Y.V1 strain.

Topoisomerase 1 inhibition therapy protects against SARS-CoV-2-induced inflammation and death in animal models.

The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro and in vivo analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans.

Scalable, effective, and rapid decontamination of SARS-CoV-2 contaminated N95 respirators using germicidal ultra-violet C (UVC) irradiation device

ImportanceParticulate respirators such as N95 masks are an essential component of personal protective equipment (PPE) for front-line workers. This study describes a rapid and effective UVC irradiation system that would facilitate the safe re-use of N95 respirators and provides supporting information for deploying UVC for decontamination of SARS-CoV-2 during the COVID19 pandemic. ObjectiveTo assess the inactivation potential of the proposed UVC germicidal device as a function of time by using 3M(R) 8211 - N95 particulate respirators inoculated with SARS-CoV-2. DesignA germicidal UVC device to deliver tailored UVC dose was developed and snippets (2.5cm2) of the 3M-N95 respirator were inoculated with 106 plaque-forming units (PFU) of SARS-CoV-2 and were UV irradiated. Different exposure times were tested (0-164 seconds) by fixing the distance between the lamp (10 cm) and the mask while providing an exposure of at least 5.43 mWcm-2. SettingThe current work is broadly applicable for healthcare-settings, particularly during a pandemic such as COVID-19. ParticipantsNot applicable. Main Outcome(s) and Measure(s)Primary measure of outcome was titration of infectious virus recovered from virus-inoculated respirator pieces after UVC exposure. Other measures included the method validation of the irradiation protocol, using lentiviruses (biosafety level-2 agent) and establishment of the germicidal UVC exposure protocol. ResultsAn average of 4.38x103 PFUml-1(SD 772.68) was recovered from untreated masks while 4.44x102 PFUml-1(SD 203.67), 4.00x102 PFUml-1(SD 115.47), 1.56x102 PFUml-1(SD 76.98) and 4.44x101 PFUml-1(SD 76.98) was recovered in exposures 2s,6s,18s and 54 seconds per side respectively. The germicidal device output and positioning was monitored and a minimum output of 5.43 mWcm-2 was maintained. Infectious SARS-CoV-2 was not detected by plaque assays (minimal level of detection is 67 PFUml-1) on N95 respirator snippets when irradiated for 120s per side or longer suggesting 3.5 log reduction in 240 seconds of irradiation. Conclusions and RelevanceA scalable germicidal UVC device to deliver tailored UVC dose for rapid decontamination of SARS-CoV-2 was developed. UVC germicidal irradiation of N95 snippets inoculated with SARS-CoV-2 for 120s per side resulted in 100% (3.5 log in total) reduction of virus. These data support the reuse of N95 particle-filtrate apparatus upon irradiation with UVC and supports use of UVC-based decontamination of SARS-CoV-2 virus during the COVID19 pandemic.

Sterilizing Immunity against SARS-CoV-2 Infection in Mice by a Single-Shot and Modified Imidazoquinoline TLR7/8 Agonist-Adjuvanted Recombinant Spike Protein Vaccine

The search for vaccines that protect from severe morbidity and mortality as a result of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Several vaccine candidates are currently being tested in the clinic. Inactivated virus and recombinant protein vaccines can be safe options but may require adjuvants to induce robust immune responses efficiently. In this work we describe the use of a novel amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile). This amphiphile is water soluble and exhibits massive translocation to lymph nodes upon local administration, likely through binding to albumin. IMDQ-PEG-CHOL is used to induce a protective immune response against SARS-CoV-2 after single vaccination with trimeric recombinant SARS-CoV-2 spike protein in the BALB/c mouse model. Inclusion of amphiphilic IMDQ-PEG-CHOL in the SARS-CoV-2 spike vaccine formulation resulted in enhanced immune cell recruitment and activation in the draining lymph node. IMDQ-PEG-CHOL has a better safety profile compared to native soluble IMDQ as the former induces a more localized immune response upon local injection, preventing systemic inflammation. Moreover, IMDQ-PEG-CHOL adjuvanted vaccine induced enhanced ELISA and in vitro microneutralization titers, and a more balanced IgG2a/IgG1 response. To correlate vaccine responses with control of virus replication in vivo, vaccinated mice were challenged with SARS-CoV-2 virus after being sensitized by intranasal adenovirus-mediated expression of the human angiotensin converting enzyme 2 (ACE2) gene. Animals vaccinated with trimeric recombinant spike protein vaccine without adjuvant had lung virus titers comparable to non-vaccinated control mice, whereas animals vaccinated with IMDQ-PEG-CHOL-adjuvanted vaccine controlled viral replication and infectious viruses could not be recovered from their lungs at day 4 post infection. In order to test whether IMDQ-PEG-CHOL could also be used to adjuvant vaccines currently licensed for use in humans, proof of concept was also provided by using the same IMDQ-PEG-CHOL to adjuvant human quadrivalent inactivated influenza virus split vaccine, which resulted in enhanced hemagglutination inhibition titers and a more balanced IgG2a/IgG1 antibody response. Enhanced influenza vaccine responses correlated with better virus control when mice were given a lethal influenza virus challenge. Our results underscore the potential use of IMDQ-PEG-CHOL as an adjuvant to achieve protection after single immunization with recombinant protein and inactivated vaccines against respiratory viruses, such as SARS-CoV-2 and influenza viruses.

Introduction of two prolines and removal of the polybasic cleavage site leads to optimal efficacy of a recombinant spike based SARS-CoV-2 vaccine in the mouse model

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the prime target for vaccine development. The spike protein mediates both binding to host cells and membrane fusion and is also so far the only known viral target of neutralizing antibodies. Coronavirus spike proteins are large trimers that are relatively instable, a feature that might be enhanced by the presence of a polybasic cleavage site in the SARS-CoV-2 spike. Exchange of K986 and V987 to prolines has been shown to stabilize the trimers of SARS-CoV-1 and the Middle Eastern respiratory syndrome coronavirus spikes. Here, we test multiple versions of a soluble spike protein for their immunogenicity and protective effect against SARS-CoV-2 challenge in a mouse model that transiently expresses human angiotensin converting enzyme 2 via adenovirus transduction. Variants tested include spike protein with a deleted polybasic cleavage site, the proline mutations, a combination thereof, as well as the wild type protein. While all versions of the protein were able to induce neutralizing antibodies, only the antigen with both a deleted cleavage site and the PP mutations completely protected from challenge in this mouse model. ImportanceA vaccine for SARS-CoV-2 is urgently needed. A better understanding of antigen design and attributes that vaccine candidates need to have to induce protective immunity is of high importance. The data presented here validates the choice of antigens that contain the PP mutation and suggests that deletion of the polybasic cleavage site could lead to a further optimized design.

Comparison of Transgenic and Adenovirus hACE2 Mouse Models for SARS-CoV-2 Infection

Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is currently causing a worldwide pandemic with high morbidity and mortality. Development of animal models that recapitulate important aspects of coronavirus disease 2019 (COVID-19) is critical for the evaluation of vaccines and antivirals, and understanding disease pathogenesis. SARS-CoV-2 has been shown to use the same entry receptor as SARS-CoV-1, human angiotensin-converting enzyme 2 (hACE2)(1-3). Due to amino acid differences between murine and hACE2, inbred mouse strains fail to support high titer viral replication of SARS-CoV-2 virus. Therefore, a number of transgenic and knock-in mouse models, as well as viral vector-mediated hACE2 delivery systems have been developed. Here we compared the K18-hACE2 transgenic model to adenovirus-mediated delivery of hACE2 to the mouse lung. We show that K18-hACE2 mice replicate virus to high titers in both the lung and brain leading to lethality. In contrast, adenovirus-mediated delivery results in viral replication to lower titers limited to the lung, and no clinical signs of infection with a challenge dose of 104 plaque forming units. The K18-hACE2 model provides a stringent model for testing the ability of vaccines and antivirals to protect against disease, whereas the adenovirus delivery system has the flexibility to be used across multiple genetic backgrounds and modified mouse strains.Competing Interest StatementThe authors have declared no competing interest.View Full Text