ABSTRACT
With the continued transmission of SARS-CoV-2 across widely vaccinated populations, it remains important to develop new vaccines and vaccination strategies capable of providing protective immunity and limiting the spread of disease. Heterologous prime-boost vaccination based on the selection of different vaccine formulations and administration routes for priming and booster doses presents a promising strategy for inducing broader immune responses in key systemic and respiratory mucosal compartments. Intranasal vaccination can induce mucosal immune responses at the site of SARS-CoV-2 infection; however, the lack of clinically approved mucosal adjuvants makes it difficult to induce robust immune responses with protein subunit vaccines. Herein, we evaluated the immunogenicity of heterologous prime-boost regimens in mice and hamsters based on a parenteral vaccination of the antigen in combination with sulfated lactosylarchaeol (SLA) archaeosomes, a liposome adjuvant comprised of a single semisynthetic archaeal lipid, followed by an intranasally administered unadjuvanted SARS-CoV-2 spike antigen. Intranasal administration of unadjuvanted spike to mice and hamsters increased serum spike-specific IgG titers and spike-neutralizing activity compared with nonboosted animals. Spike-specific IgA responses were also detected in the bronchoalveolar lavage fluid in the lungs of mice that received an intranasal boost. In hamsters, the intranasal boost showed high efficacy against SARS-CoV-2 infection by protecting from body weight loss and reducing viral titers in the lungs and nasal turbinate. Overall, our heterologous intramuscular prime-intranasal boost with SLA-adjuvanted and unadjuvanted spike, respectively, demonstrated the potential of protein subunit formulations to promote antigen-specific systemic and mucosal immune responses.
Subject(s)
Administration, Intranasal , Antibodies, Viral , COVID-19 Vaccines , COVID-19 , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Vaccines, Subunit , Animals , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/administration & dosage , Mice , SARS-CoV-2/immunology , COVID-19 Vaccines/immunology , COVID-19 Vaccines/administration & dosage , Vaccines, Subunit/immunology , Vaccines, Subunit/administration & dosage , Cricetinae , COVID-19/prevention & control , COVID-19/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Female , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/blood , Immunization, Secondary , Adjuvants, Immunologic/administration & dosage , Mice, Inbred BALB C , Immunity, Mucosal/immunology , Humans , Vaccination/methodsABSTRACT
Follicular helper T (Tfh) cells promote, whereas follicular regulatory T (Tfr) cells restrain, germinal center (GC) reactions. However, the precise roles of these cells in the complex GC reaction remain poorly understood. Here, we perturb Tfh or Tfr cells after SARS-CoV-2 spike protein vaccination in mice. We find that Tfh cells promote the frequency and somatic hypermutation (SHM) of Spike-specific GC B cells and regulate clonal diversity. Tfr cells similarly control SHM and clonal diversity in the GC but do so by limiting clonal competition. In addition, deletion of Tfh or Tfr cells during primary vaccination results in changes in SHM after vaccine boosting. Aged mice, which have altered Tfh and Tfr cells, have lower GC responses, presenting a bimodal distribution of SHM. Together, these data demonstrate that GC responses to SARS-CoV-2 spike protein vaccines require a fine balance of positive and negative follicular T cell help to optimize humoral immunity.
Subject(s)
COVID-19/prevention & control , Germinal Center/immunology , Spike Glycoprotein, Coronavirus/administration & dosage , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Regulatory/immunology , Aging , Animals , Antibodies, Viral/blood , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , COVID-19/virology , Germinal Center/cytology , Germinal Center/metabolism , Immunity, Humoral , Mice , Mice, Inbred C57BL , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocytes, Helper-Inducer/cytology , T-Lymphocytes, Helper-Inducer/metabolism , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/metabolism , Vaccination , Vaccines, Subunit/immunologyABSTRACT
AIMS: Angiotensin-converting enzyme 2 (ACE2) is a key regulator of the renin-angiotensin system (RAS) recently identified as the membrane receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we aim to study whether two receptors from RAS, the angiotensin receptor type 1 (AT1R) and the bradykinin 2 receptor (B2R) modulate ACE2 internalization induced by a recombinant receptor binding domain (RBD) of SARS-CoV-2 spike protein. Also, we investigated the impact of ACE2 coexpression on AT1R and B2R functionality. MATERIALS AND METHODS: To study ACE2 internalization, we assessed the distribution of green fluorescent protein (GFP) signal in HEK293T cells coexpressing GFP-tagged ACE2 and AT1R, or B2R, or AT1R plus B2R in presence of RBD alone or in combination with AT1R or B2R ligands. To estimate ACE2 internalization, we classified GFP signal distribution as plasma membrane uniform GFP (PMU-GFP), plasma membrane clustered GFP (PMC-GFP) or internalized GFP and calculated its relative frequency. Additionally, we investigated the effect of ACE2 coexpression on AT1R and B2R inhibitory action on voltage-gated calcium channels (CaV2.2) currents by patch-clamp technique. KEY FINDINGS: RBD induced ACE2-GFP internalization in a time-dependent manner. RBD-induced ACE2-GFP internalization was increased by angiotensin II and reduced by telmisartan in cells coexpressing AT1R. RBD-induced ACE2-GFP internalization was strongly inhibited by B2R co-expression. This effect was mildly modified by bradykinin and rescued by angiotensin II in presence of AT1R. ACE2 coexpression impacted on B2R- and AT1R-mediated inhibition of CaV2.2 currents. SIGNIFICANCE: Our work contributes to understand the role of RAS modulators in the susceptibility to SARS-CoV-2 infection and severity of COVID-19.
Subject(s)
Angiotensin-Converting Enzyme 2/biosynthesis , Receptor, Angiotensin, Type 1/biosynthesis , Receptor, Bradykinin B2/biosynthesis , Spike Glycoprotein, Coronavirus/administration & dosage , Angiotensin II/pharmacology , Angiotensin II Type 1 Receptor Blockers/pharmacology , Angiotensin-Converting Enzyme 2/analysis , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/biosynthesis , HEK293 Cells , Humans , Receptor, Angiotensin, Type 1/analysis , Receptor, Bradykinin B2/analysis , Recombinant Proteins/administration & dosageABSTRACT
Mucosal immunity provides a potential for preventing initial infection and stopping subsequent transmission of SARS-CoV-2. Here, we examined the safety and immunogenicity of a replication-defective adenovirus type-5 vectored vaccine (Ad5-nCov) encoding SARS-CoV-2 spike protein delivered by nebulization inhalation in rhesus macaques. The vaccine-associated clinical pathology and toxicity were not observed in the NHP model. The extensive safety study indicated that Ad5-nCoV was mainly confined to the organs related to respiratory system and was rapidly cleared away from the system. Our results showed that Ad5-nCoV delivered by inhalation robustly elicited both systematic and mucosal immune responses against SARS-nCoV-2 and variants. Thus, Ad5-nCoV inhalation may provide an effective, safe and non-invasive vaccination strategy for the control of SARS-CoV-2.
Subject(s)
Adenoviridae/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , Genetic Vectors/immunology , Immunity, Mucosal , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adenoviridae/genetics , Administration, Inhalation , Animals , COVID-19/prevention & control , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/genetics , Disease Models, Animal , Genetic Vectors/genetics , Humans , Immunogenicity, Vaccine , Macaca mulatta , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/geneticsABSTRACT
The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (SpikeRBD) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHHMHCII). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHHMHCII-SpikeRBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHHMHCII-SpikeRBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy.
Subject(s)
COVID-19 Vaccines/immunology , COVID-19 Vaccines/pharmacology , COVID-19/immunology , COVID-19/prevention & control , Pandemics , SARS-CoV-2/immunology , Amino Acid Sequence , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , Antigen-Presenting Cells/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/epidemiology , COVID-19 Vaccines/administration & dosage , Camelids, New World/immunology , Female , Histocompatibility Antigens Class II/immunology , Humans , Immunity, Cellular , Immunity, Humoral , Immunization, Secondary , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Pandemics/prevention & control , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SARS-CoV-2/genetics , Single-Domain Antibodies/administration & dosage , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunologyABSTRACT
We previously reported that recombinant Newcastle disease virus LaSota (rLS) expressing infectious bronchitis virus (IBV) Arkansas (Ark)-type trimeric spike (S) ectodomain (Se; rLS/ArkSe) provides suboptimal protection against IBV challenge. We have now developed rLS expressing chicken granulocyte-macrophage colony-stimulating factor (GMCSF) and IBV Ark Se in an attempt to enhance vaccine effectiveness. In the current study, we first compared protection conferred by vaccination with rLS/ArkSe and rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with virulent Ark, and protection was determined by clinical signs, viral load, and tracheal histomorphometry. Results showed that coexpression of GMCSF and the Se from rLS significantly reduced tracheal viral load and tracheal lesions compared with chickens vaccinated with rLS/ArkSe. In a second experiment, we evaluated enhancement of cross-protection of a Massachusetts (Mass) attenuated vaccine by priming or boosting with rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with Ark, and protection was evaluated. Results show that priming or boosting with the recombinant virus significantly increased cross-protection conferred by Mass against Ark virulent challenge. Greater reductions of viral loads in both trachea and lachrymal fluids were observed in chickens primed with rLS/ArkSe.GMCSF and boosted with Mass. Consistently, Ark Se antibody levels measured with recombinant Ark Se protein-coated ELISA plates 14 days after boost were significantly higher in these chickens. Unexpectedly, the inverse vaccination scheme, that is, priming with Mass and boosting with the recombinant vaccine, proved somewhat less effective. We concluded that a prime and boost strategy by using rLS/ArkSe.GMCSF and the worldwide ubiquitous Mass attenuated vaccine provides enhanced cross-protection. Thus, rLS/GMCSF coexpressing the Se of regionally relevant IBV serotypes could be used in combination with live Mass to protect against regionally circulating IBV variant strains.
Protección incrementada por el virus recombinante de la enfermedad de Newcastle que expresa el ectodominio de la espícula del virus de la bronquitis infecciosa y el factor estimulante de colonias de granulocitos y macrófagos del pollo. Anteriormente se reportó que la cepa LaSota recombinante del virus de la enfermedad de Newcastle (rLS) que expresa el ectodominio de la espícula trimérica (S) de tipo Arkansas (Ark) del virus de la bronquitis infecciosa (IBV) (Se; rLS/ArkSe) proporciona una protección subóptima contra la exposición al virus de la bronquitis infecciosa. Ahora se ha desarrollado hemos desarrollado una cepa LaSota recombinante (rLS) que expresa el factor estimulante de colonias de granulocitos y macrófagos de pollo (GMCSF) y la espícula del virus de bronquitis Arkansas en un intento para mejorar la efectividad de la vacuna. En el estudio actual, primero se comparó la protección conferida por la vacunación con los virus rLS/ArkSe y rLS/ArkSe.GMCSF. Los pollos vacunados se desafiaron con un virus Arkansas virulento y la protección se determinó mediante los signos clínicos, la carga viral y la histomorfometría de la tráquea. Los resultados mostraron que la coexpresión del factor estimulante de colonias de granulocitos y macrófagos de pollo y la espícula de la cepa recombinante LaSota redujo significativamente la carga viral traqueal y las lesiones traqueales en comparación con los pollos vacunados con el virus rLS/ArkSe. En un segundo experimento, se evaluó el incremento en la protección cruzada por una vacuna atenuada de Massachusetts (Mass) mediante la primovacunación o la vacunación de refuerzo con rLS/ArkSe.GMCSF. Los pollos vacunados fueron desafiados con el virus Arkansas y se evaluó la protección. Los resultados mostraron que la primovacunación o la vacunación de refuerzo con el virus recombinante aumentó significativamente la protección cruzada conferida por el virus Massachusetts contra el desafío virulento con el virus Arkansas. Se observaron mayores reducciones de las cargas virales en los fluidos traqueales y lagrimales en pollos primovacunadoss con rLS/ArkSe.GMCSF y con vacunación de refuerzo con Massachusetts. De manera consistente, los niveles de anticuerpos Ark Se medidos con placas de ELISA recubiertas con proteína Ark Se recombinante a los 14 días después del refuerzo fueron significativamente más altos en estos pollos. De manera inesperada, el esquema de vacunación inverso, es decir, la primovacunación con Massachusetts y el refuerzo con la vacuna recombinante, resultó menos efectivo. Se concluye que una estrategia de primovacunación y refuerzo mediante el uso de rLS/ArkSe.GMCSF y la vacuna atenuada con Massachusetts usada en todo el mundo proporciona una protección cruzada aumentada. Por tanto, el virus rLS/GMCSF que coexpresa la proteína de la espícula de los serotipos regionales relevantes de bronquitis infecciosa podría usarse en combinación con una vacuna viva Massachusetts para proteger contra cepas variantes del virus de la bronquitis infecciosa que circulan regionalmente.
Subject(s)
Coronavirus Infections/veterinary , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Infectious bronchitis virus/immunology , Newcastle disease virus/genetics , Poultry Diseases/prevention & control , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Viral/immunology , Chickens/genetics , Chickens/immunology , Chickens/virology , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Cross Protection , Gene Expression , Granulocyte-Macrophage Colony-Stimulating Factor/administration & dosage , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Infectious bronchitis virus/chemistry , Infectious bronchitis virus/genetics , Infectious bronchitis virus/physiology , Newcastle disease virus/metabolism , Poultry Diseases/immunology , Poultry Diseases/virology , Protein Domains , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Trachea/immunology , Trachea/virology , Vaccination , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Viral LoadABSTRACT
A safe and effective vaccine is urgently needed to control the unprecedented COVID-19 pandemic. Four adenovirus-vectored vaccines expressing spike (S) protein have been approved for use. Here, we generated several recombinant chimpanzee adenovirus (AdC7) vaccines expressing S, receptor-binding domain (RBD), or tandem-repeat dimeric RBD (RBD-tr2). We found vaccination via either intramuscular or intranasal route was highly immunogenic in mice to elicit both humoral and cellular immune responses. AdC7-RBD-tr2 showed higher antibody responses compared to either AdC7-S or AdC7-RBD. Intranasal administration of AdC7-RBD-tr2 additionally induced mucosal immunity with neutralizing activity in bronchoalveolar lavage fluid. Either single-dose or two-dose mucosal administration of AdC7-RBD-tr2 protected mice against SARS-CoV-2 challenge, with undetectable subgenomic RNA in lung and relieved lung injury. AdC7-RBD-tr2-elicted sera preserved the neutralizing activity against the circulating variants, especially the Delta variant. These results support AdC7-RBD-tr2 as a promising COVID-19 vaccine candidate.
Subject(s)
Adenoviridae/genetics , Antibodies, Viral/blood , COVID-19 Vaccines/immunology , Spike Glycoprotein, Coronavirus/immunology , Administration, Intranasal , Animals , Antibodies, Neutralizing/blood , COVID-19 , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/genetics , Chlorocebus aethiops , Female , Genetic Vectors/genetics , HEK293 Cells , Humans , Immunogenicity, Vaccine , Injections, Intramuscular , Mice , Mice, Inbred BALB C , Pan troglodytes/virology , Protein Binding , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/genetics , Vaccination , Vero CellsABSTRACT
To curb the pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), multiple platforms have been employed toward a safe and highly effective vaccine. Here, we develop a novel cell-based vaccine candidate, namely K562-S, by utilizing human cell K562 as a cellular carrier to display Spike (S) protein of SARS-CoV-2 on the membrane. Analogous to the traditional inactivated vaccine, K562-S cells can be propagated to a large scale by culturing and completely lose their viability after exposure to X-ray irradiation or formalin. We in turn demonstrated high immunogenicity of formalin-inactivated K562-S vaccine in both mouse and non-human primates and its protective efficacy in mice. In mice, immunization with inactivated K562-S vaccines can elicit potent neutralizing antibody (nAb) responses persisting longer than 5 months. We consequently showed in a hACE2 mouse model of SARS-CoV-2 infection that a two-shot vaccination with adjuvanted K562-S rendered greater than 3 log reduction in viral lung load and concomitant ameliorated lung pathology. Of importance, the administration of the same regimen in non-human primates was able to induce a neutralizing antibody titer averaging three-fold higher relative to human convalescent serum. These results together support the promise of K562-based, S-protein-expressing vaccines as a novel vaccination approach against SARS-CoV-2. Importantly, with a powerful capacity to carry external genes for cell-based vectors, this platform could rapidly generate two- and multiple-valent vaccines by incorporating SARS-CoV-2 mutants, SARS-CoV, or MERS-CoV.
Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 Vaccines/immunology , COVID-19/prevention & control , Immunogenicity, Vaccine , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Animals , Animals, Genetically Modified , COVID-19 Vaccines/administration & dosage , Female , HEK293 Cells , Humans , K562 Cells , Macaca mulatta , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Primates , Specific Pathogen-Free Organisms , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vaccination/methods , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/immunologyABSTRACT
The newly emerged novel coronavirus, SARS-CoV-2, the causative agent of COVID-19 has proven to be a threat to the human race globally, thus, vaccine development against SARS-CoV-2 is an unmet need driving mass vaccination efforts. The receptor binding domain of the spike protein of this coronavirus has multiple neutralizing epitopes and is associated with viral entry. Here we have designed and characterized the SARS-CoV-2 spike protein fragment 330-526 as receptor binding domain 330-526 (RBD330-526) with two native glycosylation sites (N331 and N343); as a potential subunit vaccine candidate. We initially characterized RBD330-526 biochemically and investigated its thermal stability, humoral and T cell immune response of various RBD protein formulations (with or without adjuvant) to evaluate the inherent immunogenicity and immunomodulatory effect. Our result showed that the purified RBD immunogen is stable up to 72 h, without any apparent loss in affinity or specificity of interaction with the ACE2 receptor. Upon immunization in mice, RBD generates a high titer humoral response, elevated IFN-γ producing CD4+ cells, cytotoxic T cells, and robust neutralizing antibodies against live SARS-CoV-2 virus. Our results collectively support the potential of RBD330-526 as a promising vaccine candidate against SARS-CoV-2.
Subject(s)
Antibodies, Viral/blood , COVID-19 Vaccines/administration & dosage , Immunity, Humoral/drug effects , Immunogenicity, Vaccine , Peptide Fragments/administration & dosage , Spike Glycoprotein, Coronavirus/administration & dosage , Th1 Cells/drug effects , Adjuvants, Immunologic/administration & dosage , Animals , Biomarkers/blood , COVID-19 Vaccines/immunology , Chlorocebus aethiops , Drug Stability , Glycosylation , HEK293 Cells , Humans , Immunization , Interferon-gamma/blood , Male , Mice, Inbred C57BL , Peptide Fragments/immunology , Protein Interaction Domains and Motifs , Protein Stability , Spike Glycoprotein, Coronavirus/immunology , Th1 Cells/immunology , Th1 Cells/metabolism , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/immunology , Vero CellsABSTRACT
The inclusion of infants in the SARS-CoV-2 vaccine roll-out is important to prevent severe complications of pediatric SARS-CoV-2 infections and to limit transmission and could possibly be implemented via the global pediatric vaccine schedule. However, age-dependent differences in immune function require careful evaluation of novel vaccines in the pediatric population. Toward this goal, we assessed the safety and immunogenicity of two SARS-CoV-2 vaccines. Two groups of 8 infant rhesus macaques (RMs) were immunized intramuscularly at weeks 0 and 4 with stabilized prefusion SARS-CoV-2 S-2P spike (S) protein encoded by mRNA encapsulated in lipid nanoparticles (mRNA-LNP) or the purified S protein mixed with 3M-052, a synthetic TLR7/8 agonist in a squalene emulsion (Protein+3M-052-SE). Neither vaccine induced adverse effects. Both vaccines elicited high magnitude IgG binding to RBD, N terminus domain, S1, and S2, ACE2 blocking activity, and high neutralizing antibody titers, all peaking at week 6. S-specific memory B cells were detected by week 4 and S-specific T cell responses were dominated by the production of IL-17, IFN-γ, or TNF-α. Antibody and cellular responses were stable through week 22. The immune responses for the mRNA-LNP vaccine were of a similar magnitude to those elicited by the Moderna mRNA-1273 vaccine in adults. The S-2P mRNA-LNP and Protein-3M-052-SE vaccines were well-tolerated and highly immunogenic in infant RMs, providing proof-of concept for a pediatric SARS-CoV-2 vaccine with the potential for durable immunity that might decrease the transmission of SARS-CoV-2 and mitigate the ongoing health and socioeconomic impacts of COVID-19.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , Animals , Animals, Newborn , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/prevention & control , Macaca mulatta , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/immunologyABSTRACT
The COVID-19 pandemic continues to wreak havoc as worldwide SARS-CoV-2 infection, hospitalization, and death rates climb unabated. Effective vaccines remain the most promising approach to counter SARS-CoV-2. Yet, while promising results are emerging from COVID-19 vaccine trials, the need for multiple doses and the challenges associated with the widespread distribution and administration of vaccines remain concerns. Here, we engineered the coat protein of the MS2 bacteriophage and generated nanoparticles displaying multiple copies of the SARS-CoV-2 spike (S) protein. The use of these nanoparticles as vaccines generated high neutralizing antibody titers and protected Syrian hamsters from a challenge with SARS-CoV-2 after a single immunization with no infectious virus detected in the lungs. This nanoparticle-based vaccine platform thus provides protection after a single immunization and may be broadly applicable for protecting against SARS-CoV-2 and future pathogens with pandemic potential.
Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19/immunology , COVID-19/prevention & control , Pandemics , SARS-CoV-2 , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , Drug Delivery Systems , Female , Humans , Immunization/methods , Levivirus/genetics , Levivirus/immunology , Mesocricetus , Microscopy, Electron, Transmission , Models, Animal , Nanoparticles/administration & dosage , Nanoparticles/ultrastructure , Nanotechnology , Pandemics/prevention & control , Protein Engineering , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Combined/administration & dosage , Vaccines, Combined/genetics , Vaccines, Combined/immunology , Vaccines, Virus-Like Particle/administration & dosage , Vaccines, Virus-Like Particle/genetics , Vaccines, Virus-Like Particle/immunologyABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has initiated a global pandemic, and several vaccines have now received emergency use authorization. Using the reference strain SARS-CoV-2 USA-WA1/2020, we evaluated modes of transmission and the ability of prior infection or vaccine-induced immunity to protect against infection in ferrets. Ferrets were semipermissive to infection with the USA-WA1/2020 isolate. When transmission was assessed via the detection of viral RNA (vRNA) at multiple time points, direct contact transmission was efficient to 3/3 and 3/4 contact animals in 2 respective studies, while respiratory droplet transmission was poor to only 1/4 contact animals. To determine if previously infected ferrets were protected against reinfection, ferrets were rechallenged 28 or 56 days postinfection. Following viral challenge, no infectious virus was recovered in nasal wash samples. In addition, levels of vRNA in the nasal wash were several orders of magnitude lower than during primary infection, and vRNA was rapidly cleared. To determine if intramuscular vaccination protected ferrets, ferrets were vaccinated using a prime-boost strategy with the S protein receptor-binding domain formulated with an oil-in-water adjuvant. Upon viral challenge, none of the mock or vaccinated animals were protected against infection, and there were no significant differences in vRNA or infectious virus titers in the nasal wash. Combined, these studies demonstrate direct contact is the predominant mode of transmission of the USA-WA1/2020 isolate in ferrets and that immunity to SARS-CoV-2 is maintained for at least 56 days. Our studies also indicate protection of the upper respiratory tract against SARS-CoV-2 will require vaccine strategies that mimic natural infection or induce site-specific immunity. IMPORTANCE The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) USA-WA1/2020 strain is a CDC reference strain used by multiple research laboratories. Here, we show that the predominant mode of transmission of this isolate in ferrets is by direct contact. We further demonstrate ferrets are protected against reinfection for at least 56 days even when levels of neutralizing antibodies are low or undetectable. Last, we show that when ferrets were vaccinated by the intramuscular route to induce antibodies against SARS-CoV-2, ferrets remain susceptible to infection of the upper respiratory tract. Collectively, these studies suggest that protection of the upper respiratory tract will require vaccine approaches that mimic natural infection.
Subject(s)
COVID-19/transmission , Disease Models, Animal , Reinfection/prevention & control , SARS-CoV-2/physiology , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , COVID-19/immunology , COVID-19/prevention & control , COVID-19 Vaccines/administration & dosage , Ferrets , Injections, Intramuscular , Nose/virology , Reinfection/immunology , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/administration & dosage , Viral LoadABSTRACT
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed into a global pandemic since its first outbreak in the winter of 2019. An extensive investigation of SARS-CoV-2 is critical for disease control. Various recombinant monoclonal antibodies of human origin that neutralize SARS-CoV-2 infection have been isolated from convalescent patients and will be applied as therapies and prophylaxis. However, the need for dedicated monoclonal antibodies suitable for molecular pathology research is not fully addressed. Here, we produced six mouse anti-SARS-CoV-2 spike monoclonal antibodies that not only exhibit robust performance in immunoassays including western blotting, ELISA, immunofluorescence, and immunoprecipitation, but also demonstrate neutralizing activity against SARS-CoV-2 infection to VeroE6/TMPRSS2 cells. Due to their mouse origin, our monoclonal antibodies are compatible with the experimental immunoassay setups commonly used in basic molecular biology research laboratories, providing a useful tool for future research. Furthermore, in the hope of applying the antibodies of clinical setting, we determined the variable regions of the antibodies and used them to produce recombinant human/mouse chimeric antibodies.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , COVID-19/prevention & control , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/isolation & purification , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/isolation & purification , Antibodies, Viral/chemistry , Antibodies, Viral/isolation & purification , Binding Sites , COVID-19/immunology , COVID-19/virology , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Mice , Neutralization Tests , Protein Binding , Protein Interaction Domains and Motifs , Protein Subunits/administration & dosage , Protein Subunits/genetics , Protein Subunits/immunology , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/immunology , VaccinationABSTRACT
The COVID-19 pandemic continues to spread throughout the world with an urgent need for a safe and protective vaccine to effectuate herd protection and control the spread of SARS-CoV-2. Here, we report the development of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) from the full-length spike (S) protein that is stable in the prefusion conformation. NVX-CoV2373 S form 27.2-nm nanoparticles that are thermostable and bind with high affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. In mice, low-dose NVX-CoV2373 with saponin-based Matrix-M adjuvant elicit high titer anti-S IgG that blocks hACE2 receptor binding, neutralize virus, and protects against SARS-CoV-2 challenge with no evidence of vaccine-associated enhanced respiratory disease. NVX-CoV2373 also elicits multifunctional CD4+ and CD8+ T cells, CD4+ follicular helper T cells (Tfh), and antigen-specific germinal center (GC) B cells in the spleen. In baboons, low-dose levels of NVX-CoV2373 with Matrix-M was also highly immunogenic and elicited high titer anti-S antibodies and functional antibodies that block S-protein binding to hACE2 and neutralize virus infection and antigen-specific T cells. These results support the ongoing phase 1/2 clinical evaluation of the safety and immunogenicity of NVX-CoV2373 with Matrix-M (NCT04368988).
Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/genetics , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/genetics , Disease Models, Animal , Female , Male , Mice , Mice, Inbred BALB C , Papio , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/genetics , T-Lymphocytes/immunology , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/genetics , Vaccines, Subunit/immunologyABSTRACT
The ongoing COVID-19 vaccine rollout is critical for reducing SARS-CoV-2 infections, hospitalizations, and deaths worldwide. Unfortunately, massive disparities exist in getting vaccines to vulnerable populations, including people living with HIV. Preliminary studies indicate that COVID-19 mRNA vaccines are safe and immunogenic in people living with HIV that are virally suppressed with potent antiretroviral therapy but may be less efficacious in immunocompromised individuals. This raises the concern that COVID-19 vaccines may be less effective in resource poor settings with limited access to antiretroviral therapy. Here, we evaluated the immunogenicity of a single dose COVID-19 replicon RNA vaccine expressing Spike protein (A.1) from SARS-CoV-2 (repRNA-CoV2S) in immunocompromised, SIV infected and immune competent, naïve pigtail macaques. Moderate vaccine-specific cellular Th1 T-cell responses and binding and neutralizing antibodies were induced by repRNA-CoV2S in SIV infected animals and naïve animals. Furthermore, vaccine immunogenicity was elicited even among the animals with the highest SIV viral burden or lowest peripheral CD4 counts prior to immunization. This study provides evidence that a SARS-CoV-2 repRNA vaccine could be employed to induce strong immunity against COVID-19 in HIV infected and other immunocompromised individuals.
Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Immunity, Cellular/drug effects , Immunity, Humoral/drug effects , Immunogenicity, Vaccine , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/immunology , Spike Glycoprotein, Coronavirus/administration & dosage , Vaccine Efficacy , mRNA Vaccines/administration & dosage , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , Cells, Cultured , Disease Models, Animal , Host-Pathogen Interactions , Immunocompromised Host , Macaca nemestrina , Male , Simian Acquired Immunodeficiency Syndrome/blood , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Th1 Cells/drug effects , Th1 Cells/immunology , Th1 Cells/virology , Time Factors , Vaccination , mRNA Vaccines/genetics , mRNA Vaccines/immunologyABSTRACT
It is unclear whether severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019, can enter the brain. Severe acute respiratory syndrome coronavirus 2 binds to cells via the S1 subunit of its spike protein. We show that intravenously injected radioiodinated S1 (I-S1) readily crossed the blood-brain barrier in male mice, was taken up by brain regions and entered the parenchymal brain space. I-S1 was also taken up by the lung, spleen, kidney and liver. Intranasally administered I-S1 also entered the brain, although at levels roughly ten times lower than after intravenous administration. APOE genotype and sex did not affect whole-brain I-S1 uptake but had variable effects on uptake by the olfactory bulb, liver, spleen and kidney. I-S1 uptake in the hippocampus and olfactory bulb was reduced by lipopolysaccharide-induced inflammation. Mechanistic studies indicated that I-S1 crosses the blood-brain barrier by adsorptive transcytosis and that murine angiotensin-converting enzyme 2 is involved in brain and lung uptake, but not in kidney, liver or spleen uptake.
Subject(s)
Blood-Brain Barrier/metabolism , Spike Glycoprotein, Coronavirus/pharmacokinetics , Administration, Intranasal , Administration, Intravenous , Angiotensin-Converting Enzyme 2/metabolism , Animals , Apolipoproteins E/genetics , COVID-19 , Genotype , Hippocampus/metabolism , Humans , Inflammation/chemically induced , Inflammation/metabolism , Lipopolysaccharides/pharmacology , Male , Mice , Mice, Transgenic , Olfactory Bulb/metabolism , Sex Characteristics , Spike Glycoprotein, Coronavirus/administration & dosage , Tissue Distribution , TranscytosisABSTRACT
A novel STING agonist, CDGSF, ipsilaterally modified with phosphorothioate and fluorine, was synthesized. The phosphorothioate in CDGSF might be a site for covalent conjugation. Injection of CDGSF generated an immunogenic ("hot") tumor microenvironment to suppress melanoma, more efficiently than dithio CDG. In particular, immunization with SARS-CoV-2 spike protein using CDGSF as an adjuvant elicited an exceptionally high antibody titer and a robust T cell response, overcoming the drawbacks of aluminum hydroxide. These results highlighted the therapeutic potential of CDGSF for cancer immunotherapy and the adjuvant potential of the STING agonist in the SARS-CoV-2 vaccine for the first time.
Subject(s)
Adjuvants, Immunologic/administration & dosage , COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Melanoma, Experimental/drug therapy , Membrane Proteins/agonists , Nucleotides, Cyclic/administration & dosage , Skin Neoplasms/drug therapy , Adjuvants, Immunologic/chemical synthesis , Aluminum Hydroxide/administration & dosage , Aluminum Hydroxide/chemistry , Animals , Antibodies, Viral/biosynthesis , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , B-Lymphocytes/virology , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/chemistry , Enzyme-Linked Immunospot Assay , Humans , Immunotherapy/methods , Interferon-gamma/biosynthesis , Melanoma, Experimental/immunology , Melanoma, Experimental/mortality , Melanoma, Experimental/pathology , Membrane Proteins/genetics , Membrane Proteins/immunology , Mice , Nucleotides, Cyclic/chemical synthesis , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Skin Neoplasms/immunology , Skin Neoplasms/mortality , Skin Neoplasms/pathology , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Survival Analysis , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/virology , Tumor Burden/drug effects , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunology , Vaccination/methodsABSTRACT
The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and has spread worldwide, with millions of cases and more than 1 million deaths to date. The gravity of the situation mandates accelerated efforts to identify safe and effective vaccines. Here, we generated measles virus (MeV)-based vaccine candidates expressing the SARS-CoV-2 spike glycoprotein (S). Insertion of the full-length S protein gene in two different MeV genomic positions resulted in modulated S protein expression. The variant with lower S protein expression levels was genetically stable and induced high levels of effective Th1-biased antibody and T cell responses in mice after two immunizations. In addition to neutralizing IgG antibody responses in a protective range, multifunctional CD8+ and CD4+ T cell responses with S protein-specific killing activity were detected. Upon challenge using a mouse-adapted SARS-CoV-2, virus loads in vaccinated mice were significantly lower, while vaccinated Syrian hamsters revealed protection in a harsh challenge setup using an early-passage human patient isolate. These results are highly encouraging and support further development of MeV-based COVID-19 vaccines.
Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , Measles virus/immunology , SARS-CoV-2/immunology , Th1 Cells/immunology , Animals , Antibodies, Viral/immunology , COVID-19/epidemiology , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/genetics , Humans , Measles Vaccine/genetics , Measles Vaccine/immunology , Measles virus/genetics , Mice , Pandemics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocytes/immunologyABSTRACT
The current COVID-19 pandemic has claimed hundreds of thousands of lives and its causative agent, SARS-CoV-2, has infected millions, globally. The highly contagious nature of this respiratory virus has spurred massive global efforts to develop vaccines at record speeds. In addition to enhanced immunogen delivery, adjuvants may greatly impact protective efficacy of a SARS-CoV-2 vaccine. To investigate adjuvant suitability, we formulated protein subunit vaccines consisting of the recombinant S1 domain of SARS-CoV-2 Spike protein alone or in combination with either CoVaccine HT™ or Alhydrogel. CoVaccine HT™ induced high titres of antigen-binding IgG after a single dose, facilitated affinity maturation and class switching to a greater extent than Alhydrogel and elicited potent cell-mediated immunity as well as virus neutralizing antibody titres. Data presented here suggests that adjuvantation with CoVaccine HT™ can rapidly induce a comprehensive and protective immune response to SARS-CoV-2.
Subject(s)
Adjuvants, Immunologic/administration & dosage , COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Aluminum Hydroxide/administration & dosage , Aluminum Hydroxide/immunology , Animals , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , Female , Humans , Immunity, Cellular , Immunity, Humoral , Immunization , Immunoglobulin G/immunology , Male , Mice , Mice, Inbred BALB C , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/genetics , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunologyABSTRACT
The coronavirus disease 2019 (COVID-19) pandemic is still ongoing and has become an important public health threat. This disease is caused by a new coronavirus named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, and so far, little is known about this virus. In this study, by using plaque purification, we purified two SARS-CoV-2 virus strains from the same specimen, one named F8 containing a 12-bp deletion in the E gene and the other named 8X containing the wild-type E gene. There was no significant difference in the viral titer and infectivity of these two strains. The S protein content of the F8 viral culture was 0.39â µg/ml, much higher than that of 8X. An inactivated vaccine made from the F8 strain could trigger high levels of the IgG titer and neutralizing antibody titer, which could last for at least 6 weeks and were significantly higher than those from the 8X strain at 1 and 3 weeks post vaccination, respectively. In conclusion, we reported that both the E gene mutant and wild-type SARS-CoV-2 strains were isolated from the same clinical sample by plaque purification. A 12-bp deletion in the E gene was important for SARS-CoV-2 replication and immunogenicity.