RESUMEN
The diversity of SARS-CoV-2 mutations raises the possibility of reinfection of individuals previously infected with earlier variants, and this risk is further increased by the emergence of the B.1.1.529 Omicron variant. In this study, we used an in vivo, hamster infection model to assess the potential for individuals previously infected with SARS-CoV-2 to be reinfected with Omicron variant and we also investigated the pathology associated with such infections. Initially, Syrian hamsters were inoculated with a lineage A, B.1.1.7, B.1.351, B.1.617.2 or a subvariant of Omicron, BA.1 strain and then reinfected with the BA.1 strain 5 weeks later. Subsequently, the impact of reinfection with Omicron subvariants (BA.1 and BA.2) in individuals previously infected with the BA.1 strain was examined. Although viral infection and replication were suppressed in both the upper and lower airways, following reinfection, virus-associated RNA was detected in the airways of most hamsters. Viral replication was more strongly suppressed in the lower respiratory tract than in the upper respiratory tract. Consistent amino acid substitutions were observed in the upper respiratory tract of infected hamsters after primary infection with variant BA.1, whereas diverse mutations appeared in hamsters reinfected with the same variant. Histopathology showed no acute pneumonia or disease enhancement in any of the reinfection groups and, in addition, the expression of inflammatory cytokines and chemokines in the airways of reinfected animals was only mildly elevated. These findings are important for understanding the risk of reinfection with new variants of SARS-CoV-2. IMPORTANCE The emergence of SARS-CoV-2 variants and the widespread use of COVID-19 vaccines has resulted in individual differences in immune status against SARS-CoV-2. A decay in immunity over time and the emergence of variants that partially evade the immune response can also lead to reinfection. In this study, we demonstrated that, in hamsters, immunity acquired following primary infection with previous SARS-CoV-2 variants was effective in preventing the onset of pneumonia after reinfection with the Omicron variant. However, viral infection and multiplication in the upper respiratory tract were still observed after reinfection. We also showed that more diverse nonsynonymous mutations appeared in the upper respiratory tract of reinfected hamsters that had acquired immunity from primary infection. This hamster model reveals the within-host evolution of SARS-CoV-2 and its pathology after reinfection, and provides important information for countermeasures against diversifying SARS-CoV-2 variants.
Asunto(s)
COVID-19 , Reinfección , Animales , Cricetinae , Mesocricetus , ARN Viral , SARS-CoV-2/genéticaRESUMEN
SARS-CoV-2 nucleocapsid protein (NP) is the main target for COVID-19-diagnostic PCR and antigen rapid diagnostic tests (Ag-RDTs). Ag-RDTs are more convenient than PCR tests for point-of-care testing or self-testing to identify the SARS-CoV-2 antigen. The sensitivity and specificity of this method depends mainly on the affinity and specificity of NP-binding antibodies; therefore, antigen-antibody binding is key elements for the Ag-RDTs. Here, we applied the high-throughput antibody isolation platform that has been utilized to isolate therapeutic antibodies against rare epitopes. Two NP antibodies were identified to recognize non-overlapping epitopes with high affinity. One antibody specifically binds to SARS-CoV-2 NP, and the other rapidly and tightly binds to SARS-CoV-2 NP with cross-reactivity to SARS-CoV NP. Furthermore, these antibodies were compatible with a sandwich enzyme-linked immunosorbent assay that exhibited enhanced sensitivity for NP detection compared to the previously isolated NP antibodies. Thus, the NP antibody pair is applicable to more sensitive and specific Ag-RDTs, highlighting the utility of a high-throughput antibody isolation platform for diagnostics development.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/diagnóstico , Nucleocápside , Anticuerpos Antivirales , Epítopos , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Vaccination against mpox (formerly known as monkeypox), an infectious disease caused by the monkeypox virus (MPXV), is needed to prevent outbreaks and consequent public health concerns. The LC16m8 vaccine, a dried cell-cultured proliferative live attenuated vaccinia virusbased vaccine, was approved in Japan against smallpox and mpox. However, its immunogenicity and efficacy against MPXV have not been fully assessed. We assessed the safety and immunogenicity of LC16m8 against MPXV in healthy adults. METHODS: We conducted a single-arm study that included 50 participants who were followed up for 168 days postvaccination. The primary end point was the neutralizing antibody seroconversion rate against MPXVs, including the Zr599 and Liberia strains, on day 28. The secondary end points included the vaccine "take" (major cutaneous reaction) rate, neutralizing titer kinetics against MPXV and vaccinia virus (LC16m8) strains, and safety outcomes. RESULTS: Seroconversion rates on day 28 were 72% (36 of 50), 70% (35 of 50), and 88% (44 of 50) against the Zr599 strain, the Liberia strain, and LC16m8, respectively. On day 168, seroconversion rates decreased to 30% (15 of 50) against the Zr599 and Liberia strains and to 76% (38 of 50) against LC16m8. The vaccine "take" (broad definition) rate on day 14 was 94% (46 of 49). Adverse events (AEs), including common solicited cutaneous reactions, occurred in 98% (45 of 48) of participants; grade 3 severity AEs occurred in 16% (8 of 50). No deaths, serious AEs, or mpox onset incidences were observed up to day 168. CONCLUSIONS: The LC16m8 vaccine generated neutralizing antibody responses against MPXV in healthy adults. No serious safety concerns occurred with LC16m8 use. (Funded by the Ministry of Health, Labour and Welfare of Japan; Japan Registry of Clinical Trials number, jRCTs031220171.)
Asunto(s)
Mpox , Vacuna contra Viruela , Vacunas , Adulto , Humanos , Anticuerpos Neutralizantes , Antígenos ViralesRESUMEN
BACKGROUND: In early 2020, developing vaccines was an urgent need for preventing COVID-19 from a contingency perspective. METHODS: S-268019-a is a recombinant protein-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising a modified recombinant spike protein antigen adjuvanted with agatolimod sodium, a Toll-like receptor-9 agonist. In the preclinical phase, it was administered intramuscularly twice at a 2-week interval in 7-week-old mice. Immunogenicity was assessed, and the mice were challenged intranasally with mouse-adapted SARS-CoV-2 at 2 and 8 weeks, respectively, after the second immunization. After confirming the preclinical effect, a Phase 1/2, randomized, parallel-group clinical study was conducted in healthy adults (aged 20-64 years). All participants received 2 intramuscular injections at various combinations of the antigen and the adjuvant (S-910823/agatolimod sodium, in µg: 12.5/250, 25/250, 50/250, 25/500, 50/500, 100/500, 10/500, 100/100, 200/1000) or placebo (saline) in an equivalent volume at a 3-week interval and were followed up until Day 50 in this interim analysis. RESULTS: In the preclinical studies, S-268019-a was safe and elicited robust immunoglobulin G (IgG) and neutralizing antibody responses in mice. When challenged with SARS-CoV-2, all S-268019-a-treated mice survived and maintained weight until 10 days, whereas all placebo- or adjuvant-treated (without antigen) mice died within 6 days. In the Phase 1/2 trial, although S-268019-a was well tolerated in adult participants, was safe up to Day 50, and elicited robust anti-spike protein IgG antibodies, it did not elicit sufficient neutralizing antibody levels. CONCLUSIONS: The S-268019-a vaccine was not sufficiently immunogenic in Japanese adults despite robust immunogenicity and efficacy in mice. Our results exemplify the innate challenges in translating preclinical data in animals to clinical trials, and highlight the need for continued research to overcome such barriers. (jRCT2051200092).
Asunto(s)
Vacunas contra la COVID-19 , COVID-19 , Inmunogenicidad Vacunal , Animales , Humanos , Ratones , Adyuvantes Inmunológicos , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19/prevención & control , Vacunas contra la COVID-19/inmunología , Método Doble Ciego , Pueblos del Este de Asia , Inmunoglobulina G , SARS-CoV-2 , Sodio , Vacunas Sintéticas/inmunologíaRESUMEN
Evaluating the serum cross-neutralization responses after breakthrough infection with various SARS-CoV-2 variants provides valuable insight for developing variant-proof COVID-19 booster vaccines. However, fairly comparing the impact of breakthrough infections with distinct epidemic timing on cross-neutralization responses, influenced by the exposure interval between vaccination and infection, is challenging. To compare the impact of pre-Omicron to Omicron breakthrough infection, we estimated the effects on cross-neutralizing responses by the exposure interval using Bayesian hierarchical modeling. The saturation time required to generate saturated cross-neutralization responses differed by variant, with variants more antigenically distant from the ancestral strain requiring longer intervals of 2-4 months. The breadths of saturated cross-neutralization responses to Omicron lineages were comparable in pre-Omicron and Omicron breakthrough infections. Our results highlight the importance of vaccine dosage intervals of 4 months or longer, regardless of the antigenicity of the exposed antigen, to maximize the breadth of serum cross-neutralization covering SARS-CoV-2 Omicron lineages.
RESUMEN
In cultured cells, SARS-CoV-2 infects cells via multiple pathways using different host proteases. Recent studies have shown that the furin and TMPRSS2 (furin/TMPRSS2)-dependent pathway plays a minor role in infection of the Omicron variant. Here, we confirm that Omicron uses the furin/TMPRSS2-dependent pathway inefficiently and enters cells mainly using the cathepsin-dependent endocytosis pathway in TMPRSS2-expressing VeroE6/TMPRSS2 and Calu-3 cells. This is the case despite efficient cleavage of the spike protein of Omicron. However, in the airways of TMPRSS2-knockout mice, Omicron infection is significantly reduced. We furthermore show that propagation of the mouse-adapted SARS-CoV-2 QHmusX strain and human clinical isolates of Beta and Gamma is reduced in TMPRSS2-knockout mice. Therefore, the Omicron variant isn't an exception in using TMPRSS2 in vivo, and analysis with TMPRSS2-knockout mice is important when evaluating SARS-CoV-2 variants. In conclusion, this study shows that TMPRSS2 is critically important for SARS-CoV-2 infection of murine airways, including the Omicron variant.
Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Humanos , Ratones , Catepsinas , Furina/genética , Furina/metabolismo , Ratones Noqueados , Péptido Hidrolasas , Serina Endopeptidasas/genética , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del VirusRESUMEN
The vaccine S-268019-b is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)-protein vaccine consisting of full-length recombinant SARS-CoV-2 S-protein (S-910823) as antigen, mixed with the squalene-based adjuvant A-910823. The current study evaluated the immunogenicity of S-268019-b using various doses of S-910823 and its vaccine efficacy against SARS-CoV-2 challenge in cynomolgus monkeys. The different doses of S-910823 combined with A-910823 were intramuscularly administered twice at a 3-week interval. Two weeks after the second dosing, dose-dependent humoral immune responses were observed with neutralizing antibody titers being comparable to that of human convalescent plasma. Pseudoviruses harboring S proteins from Beta and Gamma SARS-CoV-2 variants displayed approximately 3- to 4-fold reduced sensitivity to neutralizing antibodies induced after two vaccine doses compared with that against ancestral viruses, whereas neutralizing antibody titers were reduced >14-fold against the Omicron variant. Cellular immunity was also induced with a relative Th1 polarized response. No adverse clinical signs or weight loss associated with the vaccine were observed, suggesting safety of the vaccine in cynomolgus monkeys. Immunization with 10 µg of S-910823 with A-910823 demonstrated protective efficacy against SARS-CoV-2 challenge according to genomic and subgenomic viral RNA transcript levels in nasopharyngeal, throat, and rectal swab specimens. Pathological analysis revealed no detectable vaccine-dependent enhancement of disease in the lungs of challenged vaccinated monkeys. The current findings provide fundamental information regarding vaccine doses for human trials and support the development of S-268019-b as a safe and effective vaccine for controlling the current pandemic, as well as general protection against SARS-CoV-2 moving forward.
Asunto(s)
COVID-19 , Vacunas Virales , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19/prevención & control , COVID-19/terapia , Inmunización Pasiva , Inmunogenicidad Vacunal , Macaca fascicularis , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Sueroterapia para COVID-19RESUMEN
To control the coronavirus disease 2019 (COVID-19) pandemic, there is a need to develop vaccines to prevent infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. One candidate is a nasal vaccine capable of inducing secretory IgA antibodies in the mucosa of the upper respiratory tract, the initial site of infection. However, regarding the development of COVID-19 vaccines, there is concern about the potential risk of inducing lung eosinophilic immunopathology as a vaccine-associated enhanced respiratory disease as a result of the T helper 2 (Th2)-dominant adaptive immune response. In this study, we investigated the protective effect against virus infection induced by intranasal vaccination of recombinant trimeric spike protein derived from SARS-CoV-2 adjuvanted with CpG oligonucleotides, ODN2006, in mouse model. The intranasal vaccine combined with ODN2006 successfully induced not only systemic spike-specific IgG antibodies, but also secretory IgA antibodies in the nasal mucosa. Secretory IgA antibodies showed high protective ability against SARS-CoV-2 variants (Alpha, Beta and Gamma variants) compared to IgG antibodies in the serum. The nasal vaccine of this formulation induced a high number of IFN-γ-secreting cells in the draining cervical lymph nodes and a lower spike-specific IgG1/IgG2a ratio compared to that of subcutaneous vaccination with alum as a typical Th2 adjuvant. These features are consistent with the induction of the Th1 adaptive immune response. In addition, mice intranasally vaccinated with ODN2006 showed less lung eosinophilic immunopathology after viral challenge than mice subcutaneously vaccinated with alum adjuvant. Our findings indicate that intranasal vaccine adjuvanted with ODN2006 could be a candidate that can prevent the infection of antigenically different variant viruses, reducing the risk of vaccine-associated enhanced respiratory disease.
Asunto(s)
COVID-19 , SARS-CoV-2 , Adyuvantes Inmunológicos , Administración Intranasal , Compuestos de Alumbre , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19/prevención & control , Vacunas contra la COVID-19 , Humanos , Inmunoglobulina A Secretora , Inmunoglobulina G , Pulmón , Ratones , Oligonucleótidos , Glicoproteína de la Espiga del Coronavirus , VacunaciónRESUMEN
Vaccines that efficiently target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent for coronavirus disease (COVID-19), are the best means for controlling viral spread. This study evaluated the efficacy of the COVID-19 vaccine S-268019-b, which comprises the recombinant full-length SARS-CoV-2 spike protein S-910823 (antigen) and A-910823 (adjuvant). In addition to eliciting both Th1-type and Th2-type cellular immune responses, two doses of S-910823 plus A-910823 induced anti-spike protein IgG antibodies and neutralizing antibodies against SARS-CoV-2. In a SARS-CoV-2 challenge test, S-910823 plus A-910823 mitigated SARS-CoV-2 infection-induced weight loss and death and inhibited viral replication in mouse lungs. S-910823 plus A-910823 promoted cytokine and chemokine at the injection site and immune cell accumulation in the draining lymph nodes. This led to the formation of germinal centers and the induction of memory B cells, antibody-secreting cells, and memory T cells. These findings provide fundamental property of S-268019-b, especially importance of A-910823 to elicit humoral and cellular immune responses.
Asunto(s)
COVID-19 , Vacunas , Ratones , Animales , Humanos , Glicoproteína de la Espiga del Coronavirus/genética , SARS-CoV-2 , Vacunas contra la COVID-19 , COVID-19/prevención & control , Anticuerpos Neutralizantes , InmunidadRESUMEN
Monoclonal antibody therapy is a promising option for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and a cocktail of antibodies (REGN-COV) has been administered to infected patients with a favorable outcome. However, it is necessary to continue generating novel sets of monoclonal antibodies with neutralizing activity because viral variants can emerge that show resistance to the currently utilized antibodies. Here, we isolated a new cocktail of antibodies, EV053273 and EV053286, from peripheral blood mononuclear cells derived from convalescent patients infected with wild-type SARS-CoV-2. EV053273 exerted potent antiviral activity against the Wuhan wild-type virus as well as the Alpha and Delta variants in vitro, whereas the antiviral activity of EV053286 was moderate, but it had a wide-range of suppressive activity on the wild-type virus as well as the Alpha, Beta, Delta, Kappa, Omicron BA.1, and BA.2 variants. With the combined use of EV053273 and EV053286, we observed similar inhibitory effects on viral replication as with REGN-COV in vitro. We further assessed their activity in vivo by using a mouse model infected with a recently established viral strain with adopted infectious activity in mice. Independent experiments revealed that the combined use of EV053273 and EV053286 or the single use of each monoclonal antibody efficiently blocked infection in vivo. Together with data showing that these two monoclonal antibodies could neutralize REGN-COV escape variants and the Omicron variant, our findings suggest that the EV053273 and EV053286 monoclonal antibody cocktail is a novel clinically applicable therapeutic candidate for SARS-CoV-2 infection.
Asunto(s)
Antineoplásicos Inmunológicos , Tratamiento Farmacológico de COVID-19 , Anticuerpos Monoclonales , Anticuerpos Monoclonales Humanizados , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Antivirales/farmacología , Antivirales/uso terapéutico , Combinación de Medicamentos , Humanos , Leucocitos Mononucleares , SARS-CoV-2 , Glicoproteína de la Espiga del CoronavirusRESUMEN
Background: The immune profile against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has dramatically diversified due to a complex combination of exposure to vaccines and infection by various lineages/variants, likely generating a heterogeneity in protective immunity in a given population. To further complicate this, the Omicron variant, with numerous spike mutations, has emerged. These circumstances have created the need to assess the potential of immune evasion by Omicron in individuals with various immune histories. Methods: The neutralization susceptibility of the variants, including Omicron and their ancestors, was comparably assessed using a panel of plasma/serum derived from individuals with divergent immune histories. Blood samples were collected from either mRNA vaccinees or from those who suffered from breakthrough infections of Alpha/Delta with multiple time intervals following vaccination. Findings: Omicron was highly resistant to neutralization in fully vaccinated individuals without a history of breakthrough infections. In contrast, robust cross-neutralization against Omicron was induced in vaccinees that experienced breakthrough infections. The time interval between vaccination and infection, rather than the variant types of infection, was significantly correlated with the magnitude and potency of Omicron-neutralizing antibodies. Conclusions: Immune histories with breakthrough infections can overcome the resistance to infection by Omicron, with the vaccination-infection interval being the key determinant of the magnitude and breadth of neutralization. The diverse exposure history in each individual warrants a tailored and cautious approach to understanding population immunity against Omicron and future variants. Funding: This study was supported by grants from the Japan Agency for Medical Research and Development (AMED).