ABSTRACT
Emergence of novel variants of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean neutralizing antibody titers of 14,000-21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within four days in 7 of 8 animals receiving 50 {micro}g RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only [~]2-fold relative to USA-WA1. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-like betacoronavirus vaccine development. Significance StatementThe emergence of SARS-CoV-2 variants of concern (VOC) that reduce the efficacy of current COVID-19 vaccines is a major threat to pandemic control. We evaluate a SARS-CoV-2 Spike receptor-binding domain ferritin nanoparticle protein vaccine (RFN) in a nonhuman primate challenge model that addresses the need for a next-generation, efficacious vaccine with increased pan-SARS breadth of coverage. RFN, adjuvanted with a liposomal-QS21 formulation (ALFQ), elicits humoral and cellular immune responses exceeding those of current vaccines in terms of breadth and potency and protects against high-dose respiratory tract challenge. Neutralization activity against the B.1.351 VOC within two-fold of wild-type virus and against SARS-CoV-1 indicate exceptional breadth. Our results support consideration of RFN for SARS-like betacoronavirus vaccine development.
Subject(s)
COVID-19ABSTRACT
Recently approved vaccines have shown remarkable protection in limiting SARS-CoV-2 associated disease. However, immunologic mechanism(s) of protection, and how boosting alters immunity to wildtype and newly emerging strains, remains incompletely understood. Here we profiled the humoral immune response in a cohort of non-human primates immunized with a recombinant SARS-CoV-2 spike (S) glycoprotein (NVX-CoV2373) at two dose levels, administered as a one or two-dose regimen with a saponin-based adjuvant Matrix-M™. While antigen dose had minimal effects, boosting significantly altered the humoral response, driving unique vaccine-induced antibody fingerprints. Differences in antibody effector functions and neutralization were associated with protection in the upper and lower respiratory tract, pointing to compartment-specific determinants of protective immunity against infection. Moreover, NVX-CoV2373 elicited antibodies targeting emerging SARS-CoV-2 variants. Collectively, the data presented here suggest that a single dose may prevent disease, but that two doses may be essential to block further transmission of SARS-CoV-2 and emerging variants.Funding: This work was funded by Operation Warp Speed. We thank Colin Mann and Kathryn Hastie for production of Spike antigens. We thank Nancy Zimmerman, Mark and Lisa Schwartz, an anonymous donor (financial support), Terry and Susan Ragon, and the SAMANA Kay MGH Research Scholars award for their support. We acknowledge support from the Ragon Institute of MGH, MIT and Harvard, the Massachusetts Consortium on Pathogen Readiness (Mass CPR), the NIH (3R37AI080289-11S1, R01AI146785, U19AI42790-01, U19AI135995-02, U19AI42790-01, 1U01CA260476 – 01, CIVIC75N93019C00052), National Science Foundation Graduate Research Fellowship Grant No. #1745302, the Gates foundation Global Health Vaccine Accelerator Platform funding (OPP1146996 and INV-001650), and the Musk Foundation.Conflict of Interest: NP, MGX, JHT, BZ, SM, AMG, MJM, ADP, GG, GS, and LE are current or past employees of Novavax, Inc. and have stock options in the company. GA is the founder of Serom Yx Systems, Inc. AZ is a current employee of Moderna, Inc. but conducted this work before employment.Any opinion, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. YG, RC, MJG, CA, KMP, CL, DY, KB, MEM, JL, DM, CM, SS, FA, FK, EOS, DL, and MBF declare no competing interest.Ethical Approval: The work was conducted in accordance with a protocol approved by Texas Biomed’s Institutional Animal Care and Use Committee. All subjects signed informed consent and safety oversight was monitored by a data monitoring board.
Subject(s)
Myotonic Dystrophy , Adenomatous Polyposis ColiABSTRACT
The emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019 (COVID-19). We developed and evaluated an adjuvanted SARS-CoV-2 Spike Ferritin Nanoparticle (SpFN) vaccine in nonhuman primates. High-dose (50 mcg) SpFN vaccine, given twice within a 28 day interval, induced a Th1-biased CD4 T cell helper response and a peak neutralizing antibody geometric mean titer of 52,773 against wild-type virus, with activity against SARS-CoV-1 and minimal decrement against variants of concern. Vaccinated animals mounted an anamnestic response on high-dose SARS-CoV-2 respiratory challenge that translated into rapid elimination of replicating virus in their upper and lower airways and lung parenchyma. The potent and broad immunogenicity profile of this vaccine and its resulting efficacy in NHPs supports its utility as a candidate platform for SARS-like betacoronaviruses.
Subject(s)
Coronavirus Infections , Severe Acute Respiratory Syndrome , COVID-19ABSTRACT
Monoclonal antibodies against SARS-CoV-2 are a clinically validated therapeutic option against COVID-19. As rapidly emerging virus mutants are becoming the next major concern in the fight against the global pandemic, it is imperative that these therapeutic treatments provide coverage against circulating variants and do not contribute to development of treatment emergent resistance. To this end, we investigated the sequence diversity of the spike protein and monitored emergence of minor virus variants in SARS-COV-2 isolates found in nature or identified from preclinical in vitro and in vivo studies and in the clinic. This study demonstrates that a combination of non-competing antibodies not only provides full coverage against currently circulating variants but also protects against emergence of new such variants and their potential seeding into the population in a clinical setting.
Subject(s)
COVID-19ABSTRACT
Recently approved vaccines have already shown remarkable protection in limiting SARS-CoV-2 associated disease. However, immunologic mechanism(s) of protection, as well as how boosting alters immunity to wildtype and newly emerging strains, remain incompletely understood. Here we deeply profiled the humoral immune response in a cohort of non-human primates immunized with a stable recombinant full-length SARS-CoV-2 spike (S) glycoprotein (NVX-CoV2373) at two dose levels, administered as a single or two-dose regimen with a saponin-based adjuvant Matrix-M. While antigen dose had some effect on Fc-effector profiles, both antigen dose and boosting significantly altered overall titers, neutralization and Fc-effector profiles, driving unique vaccine-induced antibody fingerprints. Combined differences in antibody effector functions and neutralization were strongly associated with distinct levels of protection in the upper and lower respiratory tract, pointing to the presence of combined, but distinct, compartment-specific neutralization and Fc-mechanisms as key determinants of protective immunity against infection. Moreover, NVX-CoV2373 elicited antibodies functionally target emerging SARS-CoV-2 variants, collectively pointing to the critical collaborative role for Fab and Fc in driving maximal protection against SARS-CoV-2. Collectively, the data presented here suggest that a single dose may prevent disease, but that two doses may be essential to block further transmission of SARS-CoV-2 and emerging variants.
ABSTRACT
Recently approved vaccines have already shown remarkable protection in limiting SARS-CoV-2 associated disease. However, immunologic mechanism(s) of protection, as well as how boosting alters immunity to wildtype and newly emerging strains, remain incompletely understood. Here we deeply profiled the humoral immune response in a cohort of non-human primates immunized with a stable recombinant full-length SARS-CoV-2 spike (S) glycoprotein (NVX-CoV2373) at two dose levels, administered as a single or two-dose regimen with a saponin-based adjuvant Matrix-M™. While antigen dose had some effect on Fc-effector profiles, both antigen dose and boosting significantly altered overall titers, neutralization and Fc-effector profiles, driving unique vaccine-induced antibody fingerprints. Combined differences in antibody effector functions and neutralization were strongly associated with distinct levels of protection in the upper and lower respiratory tract, pointing to the presence of combined, but distinct, compartment-specific neutralization and Fc-mechanisms as key determinants of protective immunity against infection. Moreover, NVX-CoV2373 elicited antibodies functionally target emerging SARS-CoV-2 variants, collectively pointing to the critical collaborative role for Fab and Fc in driving maximal protection against SARS-CoV-2. Collectively, the data presented here suggest that a single dose may prevent disease, but that two doses may be essential to block further transmission of SARS-CoV-2 and emerging variants.
ABSTRACT
The hallmark of severe COVID-19 disease has been an uncontrolled inflammatory response, resulting from poorly understood immunological dysfunction. We explored the hypothesis that perturbations in FoxP3+ T regulatory cells (Treg), key enforcers of immune homeostasis, contribute to COVID-19 pathology. Cytometric and transcriptomic profiling revealed a distinct Treg phenotype in severe COVID-19 patients, with an increase in both Treg proportions and intracellular levels of the lineage-defining transcription factor FoxP3, which correlated with poor outcomes. Accordingly, these Tregs over-expressed a range of suppressive effectors, but also pro-inflammatory molecules like IL32. Most strikingly, they acquired similarity to tumor-infiltrating Tregs, known to suppress local anti-tumor responses. These traits were most marked in acute patients with severe disease, but persisted somewhat in convalescent patients. These results suggest that Tregs may play nefarious roles in COVID-19, via suppressing anti-viral T cell responses during the severe phase of the disease, and/or via a direct pro-inflammatory role.
Subject(s)
Neoplasms , Immune System Diseases , COVID-19ABSTRACT
The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity and provided evidence of expression of ORF10. Heterogeneous allelic frequencies along the 20kb ORF1ab gene suggested the presence of a defective interfering viral RNA species subpopulation in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.
Subject(s)
COVID-19 , InfectionsABSTRACT
A safe and effective vaccine against COVID-19 is urgently needed in quantities sufficient to immunise large populations. We report the preclinical development of two BNT162b vaccine candidates, which contain lipid-nanoparticle (LNP) formulated nucleoside-modified mRNA encoding SARS-CoV-2 spike glycoprotein-derived immunogens. BNT162b1 encodes a soluble, secreted, trimerised receptor-binding domain (RBD-foldon). BNT162b2 encodes the full-length transmembrane spike glycoprotein, locked in its prefusion conformation (P2 S). The flexibly tethered RBDs of the RBD-foldon bind ACE2 with high avidity. Approximately 20% of the P 2S trimers are in the two-RBD down, one-RBD up state. In mice, one intramuscular dose of either candidate elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong TH1 CD4+ and IFN{gamma}+ CD8+ T-cell responses. Prime/boost vaccination of rhesus macaques with BNT162b candidates elicits SARS-CoV-2 neutralising geometric mean titres 8.2 to 18.2 times that of a SARS-CoV-2 convalescent human serum panel. The vaccine candidates protect macaques from SARS-CoV-2 challenge, with BNT162b2 protecting the lower respiratory tract from the presence of viral RNA and with no evidence of disease enhancement. Both candidates are being evaluated in phase 1 trials in Germany and the United States. BNT162b2 is being evaluated in an ongoing global, pivotal Phase 2/3 trial (NCT04380701, NCT04368728).
Subject(s)
COVID-19ABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF7a, the ortholog of SARS-CoV ORF7a, is a type I transmembrane protein and plays an important role in virus-host interactions. Deletion variants in ORF7a may influence virulence, but only a few such isolates have been reported. Here, we report 8 unique ORF7a deletion variants of 6 to 96 nucleotides in length identified from 2,726 clinical specimens collected in March of 2020.
Subject(s)
Coronavirus Infections , Severe Acute Respiratory SyndromeABSTRACT
To contain the coronavirus disease 2019 (COVID-19) pandemic, a safe and effective vaccine against the new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is urgently needed in quantities sufficient to immunise large populations. In this study, we report the design, preclinical development, immunogenicity and anti-viral protective effect in rhesus macaques of the BNT162b2 vaccine candidate. BNT162b2 contains an LNP-formulated nucleoside-modified mRNA that encodes the spike glycoprotein captured in its prefusion conformation. After expression of the BNT162b2 coding sequence in cells, approximately 20% of the spike molecules are in the one-RBD up, two-RBD down state. Immunisation of mice with a single dose of BNT162b2 induced dose level-dependent increases in pseudovirus neutralisation titers. Prime-boost vaccination of rhesus macaques elicited authentic SARS-CoV-2 neutralising geometric mean titers 10.2 to 18.0 times that of a SARS-CoV-2 convalescent human serum panel. BNT162b2 generated strong TH1 type CD4+ and IFNy+ CD8+ T-cell responses in mice and rhesus macaques. The BNT162b2 vaccine candidate fully protected the lungs of immunised rhesus macaques from infectious SARS-CoV-2 challenge. BNT162b2 is currently being evaluated in a global, pivotal Phase 2/3 trial (NCT04368728).
Subject(s)
Coronavirus Infections , COVID-19ABSTRACT
Objectives: SARS-CoV-2 infection is the cause of a worldwide pandemic, currently with limited therapeutic options. It is characterised by being highly contagious and nasal mucosa appears to be the primary site with subsequent spread to the lungs and elsewhere. BromAc (Bromelain & Acetylcysteine) has been described to disrupt glycoproteins by the synchronous breakage of glycosidic linkages and disulphide bonds. The spike protein of SARS-CoV-2 is an attractive target as it is essential for binding to the ACE2 receptor in host cells and is formed of glycoprotein and disulphide bridges for stabilisation. Hence, we sought to determine whether BromAc has activity on the spike and envelope protein specific to SARS-CoV-2 virus. Design: Gel electrophoresis analysis was carried out on recombinant spike and envelope proteins that were treated with a range of concentrations of single agents and BromAc. For UV analysis of disulfide bonds reduction, both spike and envelope protein were treated with Acetylcysteine with the determination of loss of disulfide bonds. Results: Recombinant spike and envelope SARS-CoV-2 protein were fragmented by BromAc whilst single agents had minimal effect. Spike and envelope proteins disulphide bonds were reduced by Acetylcysteine. Conclusion: BromAc disintegrates the spike and envelope protein from SARS-CoV-2 and may render it non-infective. In vitro tests on live virus have been encouraging and clinical testing through nasal administration in patients with early SARS-CoV-2 infection is imminent.
Subject(s)
COVID-19ABSTRACT
Convalescent plasma from SARS-CoV-2 infected individuals and monoclonal antibodies were shown to potently neutralize viral and pseudoviral particles carrying the S glycoprotein. However, a non-negligent proportion of plasma samples from infected individuals as well as S-specific monoclonal antibodies were reported to be non-neutralizing despite efficient interaction with the S glycoprotein in different biochemical assays using soluble recombinant forms of S or when expressed at the cell surface. How neutralization relates to binding of S glycoprotein in the context of viral particles remains to be established. Here we developed a pseudovirus capture assay (VCA) to measure the capacity of plasma samples or antibodies immobilized on ELISA plates to bind to membrane-bound S glycoproteins from SARS-CoV-2 expressed at the surface of lentiviral particles. By performing VCA and neutralization assays we observed a strong correlation between these two parameters. However, while we found that plasma samples unable to capture viral particles did not neutralize, capture did not guarantee neutralization, indicating that the capacity of antibodies to bind to the S glycoprotein at the surface of viral particles is required but not sufficient to mediate neutralization. Altogether, our results highlights the importance of better understanding the inactivation of S by plasma and neutralizing antibodies.
Subject(s)
Severe Acute Respiratory SyndromeABSTRACT
A novel severe acute respiratory (SARS)-like coronavirus (SARS-CoV-2) is responsible for the current global coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The viral entry of SARS-CoV-2 depends on an interaction between the receptor binding domain of its trimeric Spike glycoprotein and the human angiotensin converting enzyme 2 (ACE2) receptor. A better understanding of the Spike/ACE2 interaction is still required to design anti-SARS-CoV-2 therapeutics. Here, we investigated the degree of cooperativity of ACE2 within both the SARS-CoV-2 and the closely related SARS-CoV-1 membrane-bound S glycoproteins. We show that there exist differential inter-protomer conformational transitions between both Spike trimers. Interestingly, the SARS-CoV-2 spike exhibits a positive cooperativity for monomeric soluble ACE2 binding when compared to the SARS-CoV-1 spike, which might have more structural restrains. Our findings can be of importance in the development of therapeutics that block the Spike/ACE2 interaction.
Subject(s)
COVID-19ABSTRACT
The novel virus SARS-CoV-2 has infected more than 14 million people worldwide resulting in the Coronavirus disease 2019 (COVID-19). Limited information on the underlying immune mechanisms that drive disease or protection during COVID-19 severely hamper development of therapeutics and vaccines. Thus, the establishment of relevant animal models that mimic the pathobiology of the disease is urgent. Rhesus macaques infected with SARS-CoV-2 exhibit disease pathobiology similar to human COVID-19, thus serving as a relevant animal model. In the current study, we have characterized the transcriptional signatures induced in the lungs of juvenile and old rhesus macaques following SARS-CoV-2 infection. We show that genes associated with Interferon (IFN) signaling, neutrophil degranulation and innate immune pathways are significantly induced in macaque infected lungs, while pathways associated with collagen formation are downregulated. In COVID-19, increasing age is a significant risk factor for poor prognosis and increased mortality. We demonstrate that Type I IFN and Notch signaling pathways are significantly upregulated in lungs of juvenile infected macaques when compared with old infected macaques. These results are corroborated with increased peripheral neutrophil counts and neutrophil lymphocyte ratio in older individuals with COVID-19 disease. In contrast, pathways involving VEGF are downregulated in lungs of old infected macaques. Using samples from humans with SARS-CoV-2 infection and COVID-19, we validate a subset of our findings. Finally, neutrophil degranulation, innate immune system and IFN gamma signaling pathways are upregulated in both tuberculosis and COVID-19, two pulmonary diseases where neutrophils are associated with increased severity. Together, our transcriptomic studies have delineated disease pathways to improve our understanding of the immunopathogenesis of COVID-19 to facilitate the design of new therapeutics for COVID-19.
Subject(s)
Lung Diseases , Tuberculosis , COVID-19 , DiseaseABSTRACT
An urgent global quest for effective therapies to prevent and treat COVID-19 disease is ongoing. We previously described REGN-COV2, a cocktail of two potent neutralizing antibodies (REGN10987+REGN10933) targeting non-overlapping epitopes on the SARS-CoV-2 spike protein. In this report, we evaluate the in vivo efficacy of this antibody cocktail in both rhesus macaques and golden hamsters and demonstrate that REGN-COV-2 can greatly reduce virus load in lower and upper airway and decrease virus induced pathological sequalae when administered prophylactically or therapeutically. Our results provide evidence of the therapeutic potential of this antibody cocktail.
Subject(s)
COVID-19ABSTRACT
ABSTRACTVaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease currently lacks a validated small animal model. Here, we show that transgenic mice expressing human angiotensin converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2-transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2-transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 4. K18 hACE2-transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.
Subject(s)
COVID-19ABSTRACT
There are no known cures or vaccines for COVID-19, the defining pandemic of this era. Animal models are essential to fast track new interventions and nonhuman primate (NHP) models of other infectious diseases have proven extremely valuable. Here we compare SARS-CoV-2 infection in three species of experimentally infected NHPs (rhesus macaques, baboons, and marmosets). During the first 3 days, macaques developed clinical signatures of viral infection and systemic inflammation, coupled with early evidence of viral replication and mild-to-moderate interstitial and alveolar pneumonitis, as well as extra-pulmonary pathologies. Cone-beam CT scans showed evidence of moderate pneumonia, which progressed over 3 days. Longitudinal studies showed that while both young and old macaques developed early signs of COVID-19, both groups recovered within a two-week period. Recovery was characterized by low-levels of viral persistence in the lung, suggesting mechanisms by which individuals with compromised immune systems may be susceptible to prolonged and progressive COVID-19. The lung compartment contained a complex early inflammatory milieu with an influx of innate and adaptive immune cells, particularly interstitial macrophages, neutrophils and plasmacytoid dendritic cells, and a prominent Type I-interferon response. While macaques developed moderate disease, baboons exhibited prolonged shedding of virus and extensive pathology following infection; and marmosets demonstrated a milder form of infection. These results showcase in critical detail, the robust early cellular immune responses to SARS-CoV-2 infection, which are not sterilizing and likely impact development of antibody responses. Thus, various NHP genera recapitulate heterogeneous progression of COVID-19. Rhesus macaques and baboons develop different, quantifiable disease attributes making them immediately available essential models to test new vaccines and therapies.
Subject(s)
COVID-19ABSTRACT
Prolonged survival of SARS-CoV-2 on environmental surfaces and personal protective equipment (PPE) may lead to these surfaces transmitting disease to others. This article reports the effectiveness of a pulsed xenon ultraviolet (PX-UV) disinfection system in reducing the load of SARS-CoV-2 on hard surfaces and N95 respirators. Chamber slides and N95 respirator material were directly inoculated with SARS-CoV-2 and exposed to different durations of PX-UV disinfection. For hard surfaces, disinfection for 1, 2, and 5 minutes resulted in 3.53 Log10, >4.54 Log10, and >4.12 Log10 reductions in viral load, respectively. For N95 respirators, disinfection for 5 minutes resulted in >4.79 Log10 reduction in viral load. We found that PX-UV significantly reduces SARS-CoV-2 on hard surfaces and N95 respirators. With the potential to rapidly disinfectant environmental surfaces and N95 respirators, PX-UV devices are a promising technology for the reduction of environmental and PPE bioburden and to enhance both HCW and patient safety by reducing the risk of exposure to SARS-CoV-2.