ACE2 mimetic antibody potently neutralizes all SARS-CoV-2 variants and fully protects in XBB.1.5 challenged monkeys (preprint)

The rapid evolution of SARS-CoV-2 to variants with improved transmission efficiency and reduced sensitivity to vaccine-induced humoral immunity has abolished the protective effect of licensed therapeutic human monoclonal antibodies (mAbs). To fill this unmet medical need and protect vulnerable patient populations, we isolated the P4J15 mAb from a previously infected, vaccinated donor, with <20 ng/ml neutralizing activity against all Omicron variants including the latest XBB.2.3 and EG.1 sub-lineages. Structural studies of P4J15 in complex with Omicron XBB.1 Spike show that the P4J15 epitope shares ~93% of its buried surface area with the ACE2 contact region, consistent with an ACE2 mimetic antibody. Although SARS-CoV-2 mutants escaping neutralization by P4J15 were selected in vitro, these displayed lower infectivity, poor binding to ACE2, and the corresponding "escape" mutations are accordingly rare in public sequence databases. Using a SARS-CoV-2 XBB.1.5 monkey challenge model, we show that P4J15 confers complete prophylactic protection. We conclude that the P4J15 mAb has potential as a broad-spectrum anti-SARS-CoV-2 drug.

High efficacy of VLA2001 vaccine against SARS-CoV-2 infection in non-human primates (preprint)

The fight against COVID-19 requires mass vaccination strategies, and vaccines inducing durable cross-protective responses are still needed. Inactivated vaccines have proven lasting efficacy against many pathogens and good safety records. They contain multiple protein antigens that may improve response breadth and can be easily adapted every year to maintain preparedness for future seasonally emerging variants. Here we report the immunogenicity and efficacy of VLA2001 in animal models, the first inactivated whole virus COVID-19 vaccine that has received standard marketing authorization by the European Medicines Agency. VLA2001 formulated with alum and the TLR9 agonist CpG 1018™ adjuvant generated a Th1-biased immune response and serum neutralizing antibodies in BALB/c mice. In non-human primates, two injections of VLA2001 were sufficient to induce specific and polyfunctional T cell responses, predominantly Th1-biased, and high levels of antibodies neutralizing SARS-CoV-2 infection in cell culture. These antibodies also inhibited the binding of the Spike protein to human ACE2 receptor of several variants of concern most resistant to neutralization. After exposure to a high dose of SARS-CoV-2, all vaccinated groups of cynomolgus macaques exhibited significant levels of protection from viral replication in the upper and lower respiratory tracts and from lung tissue inflammation as compared to controls.

Adaptive MHC-E restricted tissue-resident NK cells are associated with persistent low antigen load in alveolar macrophages after SARS-CoV-2 infection (preprint)

Natural killer (NK) cells are innate lymphocytes with potent activity against a wide range of viruses. In SARS-CoV-2 infection, NK cell activity might be of particular importance within lung tissues. Here, we investigated whether NK cells with activity against Spike+ cells are induced during SARS-CoV-2 infection and have a role in modulating viral persistence beyond primary clearance from nasopharyngeal and tracheal tissues. We performed an integrated analysis of NK cells and macrophages in blood and bronchoalveolar lavage fluids (BALF) of COVID-19 convalescent non-human primates in comparison to uninfected control animals. SARS-CoV-2 protein expression was detected for at least 9–18 months post-infection in alveolar macrophages. Convalescent animals segregated into two groups based on cellular phenotypes and viral persistence profiles in BALF. The animals with lower persistent antigen displayed macrophages with a regulatory phenotype and enhanced MHC-E restricted NK cell activity toward cells presenting peptides derived from the SARS-CoV-2 Spike protein leader sequence, while NK cell activity from the other convalescent animals, control animals and healthy humans were strongly inhibited by these Spike peptides. The adaptive NK cell activity was not detected in blood but in tissue-resident NK cells, and cross-reacted against MERS-CoV and SARS-CoV Spike-derived peptides.

Immunization with synthetic SARS-CoV-2 S glycoprotein virus-like particles protects Macaques from infection (preprint)

The SARS-CoV-2 pandemic causes an ongoing global health crisis, which requires efficient and safe vaccination programs. Here, we present synthetic SARS-CoV2 S glycoprotein-coated liposomes that resemble in size and surface structure virus-like particles. Soluble S glycoprotein trimers were stabilized by formaldehyde cross-linking and coated onto lipid vesicles (S-VLP). Immunization of cynomolgus macaques with S-VLPs induced high antibody titers and TH1 CD4+ biased T cell responses. Although antibody responses were initially dominated by RBD specificity, the third immunization boosted non-RBD antibody titers. Antibodies showed potent neutralization against the vaccine strain and the Alpha variant after two immunizations and robust neutralization of Beta and Gamma strains. Challenge of animals with SARS-CoV-2 protected all vaccinated animals by sterilizing immunity. Thus, the S-VLP approach is an efficient and safe vaccine candidate based on a proven classical approach for further development and clinical testing.

Targeting SARS-CoV-2 receptor-binding domain to cells expressing CD40 improves protection to infection in convalescent macaques (preprint)

Controlling the circulation of the recently emerged SARS-CoV-2 in the human populations requires massive vaccination campaigns. Achieving sufficient worldwide vaccination coverage will require additional approaches to first generation of approved viral vector and mRNA vaccines. Subunit vaccines have excellent safety and efficacy records and may have distinct advantages, in particular when immunizing individuals with vulnerabilities or when considering the vaccination of children and pregnant women.. We have developed a new generation of subunit vaccines with enhanced immunogenicity by the targeting of viral antigens to CD40-expressing antigen-presenting cells, thus harnessing their intrinsic immune-stimulant properties. Here, we demonstrate that targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to CD40 (αCD40.RBD) induces significant levels of specific T and B cells, with a long-term memory phenotype, in a humanized mouse model. In addition, we demonstrate that a single dose of the αCD40.RBD vaccine, injected without adjuvant, is sufficient to boost a rapid increase in neutralizing antibodies in convalescent non-human primates (NHPs) exposed six months previously to SARS-CoV-2. Such vaccination thus significantly improved protection against a new high-dose virulent challenge versus that in non-vaccinated convalescent animals. Viral dynamics modelling showed the high efficiency of the vaccine at controlling the viral dissemination.

COVA1-18 neutralizing antibody protects against SARS-CoV-2 in three preclinical models (preprint)

One year into the Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), effective treatments are still needed1–3. Monoclonal antibodies, given alone or as part of a therapeutic cocktail, have shown promising results in patients, raising the hope that they could play an important role in preventing clinical deterioration in severely ill or in exposed, high risk individuals4–6. Here, we evaluated the prophylactic and therapeutic effect of COVA1-18 in vivo, a neutralizing antibody isolated from a convalescent patient7 and highly potent against the B.1.1.7. isolate8,9. In both prophylactic and therapeutic settings, SARS-CoV-2 remained undetectable in the lungs of COVA1-18 treated hACE2 mice. Therapeutic treatment also caused a dramatic reduction in viral loads in the lungs of Syrian hamsters. When administered at 10 mg kg− 1 one day prior to a high dose SARS-CoV-2 challenge in cynomolgus macaques, COVA1-18 had a very strong antiviral activity in the upper respiratory compartments with an estimated reduction in viral infectivity of more than 95%, and prevented lymphopenia and extensive lung lesions. Modelling and experimental findings demonstrate that COVA1-18 has a strong antiviral activity in three different preclinical models and could be a valuable candidate for further clinical evaluation.

Two-Component Spike Nanoparticle Vaccine Protects Macaques from SARS-CoV-2 Infection (preprint)

The SARS-CoV-2 pandemic is continuing to disrupt personal lives, global healthcare systems and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits and cynomolgus macaques. The vaccine-induced immunity protected macaques against a high dose challenge, resulting in strongly reduced viral infection and replication in upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.Funding: This work was supported by a Netherlands Organization for Scientific Research (NWO) Vici grant (to R.W.S.); by the Bill & Melinda Gates Foundation through the Collaboration for AIDS Vaccine Discovery (CAVD) grants OPP1111923, OPP1132237, and INV-002022 (to R.W.S. and/or N.P.K.), INV-008352/OPP1153692 and OPP1196345/INV-008813 (to M.C.), and grant OPP1170236 (to A.B.W.); by the Fondation Dormeur, Vaduz (to R.W.S. and to M.J.v.G.) and Health Holland PPS-allowance LSHM20040 (to M.J.v.G.); the University of Southampton Coronavirus Response Fund (to M.C.); and by the Netherlands Organisation for Health Research and Development ZONMW (to B.L.H). M.J.v.G. is a recipient of an AMC Fellowship from Amsterdam UMC and a COVID-19 grant from the Amsterdam Institute for Infection and Immunity. R.W.S and M.J.v.G. are recipients of support from the University of Amsterdam Proof of Concept fund (contract no. 200421) as managed by Innovation Exchange Amsterdam (IXA). The Infectious Disease Models and Innovative Therapies (IDMIT) research infrastructure is supported by the ‘Programme Investissements d’Avenir, managed by the ANR under reference ANR-11-INBS-0008. The Fondation Bettencourt Schueller and the Region Ile-de-France contributed to the implementation of IDMIT’s facilities and imaging technologies. The NHP study received financial support from REACTing, the National Research Agency (ANR; AM-CoV-Path) and the European Infrastructure TRANSVAC2 (730964). Conflict of Interest: N.P.K. is a co-founder, shareholder, and chair of the scientific advisory board of Icosavax, Inc. All other authors declare no competing interests.Ethical Approval: The protocols were approved by the institutional ethical committee “Comité d’Ethique en Expérimentation Animale du Commissariat à l’Energie Atomique et aux Energies Alternatives” (CEtEA #44) under statement number A20-011. The study was authorized by the “Research, Innovation and Education Ministry” under registration number APAFIS#24434-2020030216532863v1.

Landscape analysis of escape variants identifies SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization (preprint)

Although neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of most COVID-19 vaccines and being developed as therapeutics, escape mutations could compromise such countermeasures. To define the immune-mediated mutational landscape in S protein, we used a VSV-eGFP-SARS-CoV-2-S chimeric virus and 19 neutralizing monoclonal antibodies (mAbs) against the receptor binding domain (RBD) to generate 48 escape mutants. These variants were mapped onto the RBD structure and evaluated for cross-resistance by convalescent human plasma. Although each mAb had unique resistance profiles, many shared residues within an epitope, as several variants were resistant to multiple mAbs. Remarkably, we identified mutants that escaped neutralization by convalescent human sera, suggesting that some humans induce a narrow repertoire of neutralizing antibodies. By comparing the antibody-mediated mutational landscape in S protein with sequence variation in circulating SARS-CoV-2 strains, we identified single amino acid substitutions that could attenuate neutralizing immune responses in some humans.

in vitro: Natural Compounds (Thymol, Carvacrol, Hesperidine, And Thymoquinone) Against SARS-CoV2 Strain Isolated From Egyptian Patients (preprint)

The current pandemic of the coronavirus disease-2019 (COVID-19) has badly affected our life during the year 2020. SARS-CoV-2 is the primary causative agent of the newly emerged pandemic. Natural flavonoids, Terpenoid and Thymoquinone are tested against different viral and host-cell protein targets. These natural compounds have a good history in treating Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV). Molecular docking combined with cytotoxicity and plaque reduction assay is used to test the natural compounds against different viral (Spike, RdRp, and Mpro) and host-cell (TMPRSS II, keap 1, and ACE2) targets. The results demonstrate the binding possibility of the natural compounds (Thymol, Carvacrol, Hesperidine, and Thymoquinone) to the viral main protease (Mpro). Some of these natural compounds were approved to start clinical trail from Egypt Center for Research and Regenerative Medicine ECRRM IRB (Certificate No.IRB00012517)

Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection (preprint)

The SARS-CoV-2 pandemic is continuing to disrupt personal lives, global healthcare systems and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits and cynomolgus macaques. The vaccine-induced immunity protected macaques against a high dose challenge, resulting in strongly reduced viral infection and replication in upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.

Viral dynamic modeling of SARS-CoV-2 in non-human primates (preprint)

Non-human primates infected with SARS-CoV-2 exhibit mild clinical signs. Here we used a mathematical model to characterize in detail the viral dynamics in 31 cynomolgus macaques infected with 106 pfu of SARS-CoV-2 for which nasopharyngeal and tracheal viral load were frequently assessed. We identified that infected cells had a large daily viral production (>104 virus) and a within-host reproductive basic number of 6 and 4 in nasopharyngeal and tracheal compartment, respectively. After peak viral load, infected cells were rapidly cleared with a half-life of 9 hours, with no significant association between cytokine elevation and clearance. Translating our model to the context of human-to-human infection, human mild infection may be characterized by a peak occurring 4 days after infection, a viral shedding of ~11 days and a generation time of 4 days. These results improve the understanding of SARS-CoV-2 viral replication and better understand the infection to SARS-CoV-2 in humans.

Hydroxychloroquine in the treatment and prophylaxis of SARS-CoV-2 infection in non-human primates (preprint)

COVID-19 has become a pandemic that has caused over 200,000 deaths worldwide, with no antiviral drug or vaccine yet available. Several clinical studies are ongoing to evaluate the efficacy of repurposed drugs that have demonstrated antiviral efficacy in vitro. Among these candidates, hydroxychloroquine (HCQ) has been given to thousands of individuals worldwide but definitive evidence for HCQ efficacy in treatment of COVID-19 is still missing.We evaluated the antiviral activity of HCQ both in vitro and in SARS-CoV-2-infected macaques. HCQ showed antiviral activity in monkey African green monkey kidney (VeroE6) cells but not in a model of reconstituted human airway epithelium. In macaques, we tested different treatment strategies in comparison to placebo, before and after peak viral load, alone or in combination with azithromycin (AZTH). Neither HCQ nor HCQ+AZTH showed a significant effect on the viral load levels in any of the tested compartments. When the drug was used as a pre-exposure prophylaxis (PrEP), HCQ did not confer protection against acquisition of infection.Our findings do not support the use of HCQ, either alone or in combination with AZTH, as an antiviral treatment for COVID-19 in humans.