RESUMO
BACKGROUND: The association between low-frequency human immunodeficiency virus type 1 (HIV-1) drug resistance mutations (DRMs) and treatment failure (TF) is controversial. We explore this association using next-generation sequencing (NGS) methods that accurately sample low-frequency DRMs. METHODS: We enrolled women with HIV-1 in Malawi who were either antiretroviral therapy (ART) naive (cohort A), had ART failure (cohort B), or had discontinued ART (cohort C). At entry, cohorts A and C began a nonnucleoside reverse transcriptase inhibitor-based regimen and cohort B started a protease inhibitor-based regimen. We used Primer ID MiSeq to identify regimen-relevant DRMs in entry and TF plasma samples, and a Cox proportional hazards model to calculate hazard ratios (HRs) for entry DRMs. Low-frequency DRMs were defined as ≤20%. RESULTS: We sequenced 360 participants. Cohort B and C participants were more likely to have TF than cohort A participants. The presence of K103N at entry significantly increased TF risk among A and C participants at both high and low frequency, with HRs of 3.12 (95% confidence interval [CI], 1.58-6.18) and 2.38 (95% CI, 1.00-5.67), respectively. At TF, 45% of participants showed selection of DRMs while in the remaining participants there was an apparent lack of selective pressure from ART. CONCLUSIONS: Using accurate NGS for DRM detection may benefit an additional 10% of patients by identifying low-frequency K103N mutations.
Assuntos
Farmacorresistência Viral , Infecções por HIV , HIV-1 , Mutação , Falha de Tratamento , Humanos , HIV-1/genética , HIV-1/efeitos dos fármacos , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Farmacorresistência Viral/genética , Adulto , Malaui , Fármacos Anti-HIV/uso terapêutico , Sequenciamento de Nucleotídeos em Larga Escala , Estudos de Coortes , Adulto Jovem , Resultado do TratamentoRESUMO
Antibodies perform both neutralizing and non-neutralizing effector functions that protect against certain pathogen-induced diseases. A human antibody directed at the SARS-CoV-2 Spike N-terminal domain (NTD), DH1052, was recently shown to be non-neutralizing, yet it protected mice and cynomolgus macaques from severe disease. The mechanisms of NTD non-neutralizing antibody-mediated protection are unknown. Here we show that Fc effector functions mediate NTD non-neutralizing antibody (non-nAb) protection against SARS-CoV-2 MA10 viral challenge in mice. Though non-nAb prophylactic infusion did not suppress infectious viral titers in the lung as potently as neutralizing antibody (nAb) infusion, disease markers including gross lung discoloration were similar in nAb and non-nAb groups. Fc functional knockout substitutions abolished non-nAb protection and increased viral titers in the nAb group. Fc enhancement increased non-nAb protection relative to WT, supporting a positive association between Fc functionality and degree of protection from SARS-CoV-2 infection. For therapeutic administration of antibodies, non-nAb effector functions contributed to virus suppression and lessening of lung discoloration, but the presence of neutralization was required for optimal protection from disease. This study demonstrates that non-nAbs can utilize Fc-mediated mechanisms to lower viral load and prevent lung damage due to coronavirus infection.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , Fragmentos Fc das Imunoglobulinas , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , SARS-CoV-2/imunologia , Camundongos , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Anticorpos Antivirais/imunologia , Anticorpos Neutralizantes/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Humanos , Feminino , Domínios Proteicos/imunologia , Carga Viral , Pulmão/virologia , Pulmão/imunologia , Pulmão/patologiaRESUMO
Despite the wide availability of several safe and effective vaccines that prevent severe COVID-19, the persistent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that can evade vaccine-elicited immunity remains a global health concern. In addition, the emergence of SARS-CoV-2 VOCs that can evade therapeutic monoclonal antibodies underscores the need for additional, variant-resistant treatment strategies. Here, we characterize the antiviral activity of GS-5245, obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved viral RNA-dependent RNA polymerase (RdRp). We show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, SARS-CoV, SARS-CoV-related bat-CoV RsSHC014, Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant. Moreover, in mouse models of SARS-CoV, SARS-CoV-2 (WA/1 and Omicron B1.1.529), MERS-CoV, and bat-CoV RsSHC014 pathogenesis, we observed a dose-dependent reduction in viral replication, body weight loss, acute lung injury, and pulmonary function with GS-5245 therapy. Last, we demonstrate that a combination of GS-5245 and main protease (Mpro) inhibitor nirmatrelvir improved outcomes in vivo against SARS-CoV-2 compared with the single agents. Together, our data support the clinical evaluation of GS-5245 against coronaviruses that cause or have the potential to cause human disease.
Assuntos
Antivirais , Pró-Fármacos , SARS-CoV-2 , Animais , SARS-CoV-2/efeitos dos fármacos , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Humanos , Camundongos , Administração Oral , Chlorocebus aethiops , Células Vero , Tratamento Farmacológico da COVID-19 , COVID-19/virologia , Replicação Viral/efeitos dos fármacos , Nucleosídeos/farmacologia , Nucleosídeos/uso terapêutico , Nucleosídeos/química , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Feminino , Modelos Animais de DoençasRESUMO
Whole virus-based inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide have been critical to the COVID-19 pandemic response. Although these vaccines are protective against homologous coronavirus infection, the emergence of novel variants and the presence of large zoonotic reservoirs harboring novel heterologous coronaviruses provide significant opportunities for vaccine breakthrough, which raises the risk of adverse outcomes like vaccine-associated enhanced respiratory disease. Here, we use a female mouse model of coronavirus disease to evaluate inactivated vaccine performance against either homologous challenge with SARS-CoV-2 or heterologous challenge with a bat-derived coronavirus that represents a potential emerging disease threat. We show that inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide can cause enhanced respiratory disease during heterologous infection, while use of an alternative adjuvant does not drive disease and promotes heterologous viral clearance. In this work, we highlight the impact of adjuvant selection on inactivated vaccine safety and efficacy against heterologous coronavirus infection.
Assuntos
Hidróxido de Alumínio , Vacinas contra COVID-19 , COVID-19 , SARS-CoV-2 , Vacinas de Produtos Inativados , Animais , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Feminino , COVID-19/prevenção & controle , COVID-19/imunologia , COVID-19/virologia , Camundongos , Vacinas de Produtos Inativados/imunologia , SARS-CoV-2/imunologia , Hidróxido de Alumínio/administração & dosagem , Modelos Animais de Doenças , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes de Vacinas , Anticorpos Antivirais/imunologia , Camundongos Endogâmicos BALB C , Humanos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologiaRESUMO
Pulmonary anthrax caused by exposure to inhaled Bacillus anthracis, the most lethal form of anthrax disease, is a continued military and public health concern for the United States. The vaccine AV7909, consisting of the licensed anthrax drug substance AVA adjuvanted with CpG7909, induces high levels of toxin neutralizing antibodies in healthy adults using fewer doses than AVA. This study compares the ability of one- or two-dose regimens of AV7909 to induce a protective immune response in guinea pigs challenged with a lethal dose of aerosolized B. anthracis spores 6 weeks after the last vaccine dose. The results indicated that AV7909 was less effective when delivered as a single dose compared to the two-dose regimen that resulted in dose-dependent protection against death. The toxin neutralizing assay (TNA) titer and anti-PA IgG responses were proportional to the protective efficacy, with a 50% TNA neutralizing factor (NF50) greater than 0.1 associated with survival in animals receiving two doses of vaccine. The strong protection at relatively low TNA NF50 titers in this guinea pig model supports the exploration of lower doses in clinical trials to determine if these protective levels of neutralizing antibodies can be achieved in humans; however, protection with a single dose may not be feasible.
Assuntos
Vacinas contra Antraz , Antraz , Bacillus anthracis , Adulto , Humanos , Animais , Cobaias , Antraz/prevenção & controle , Anticorpos Antibacterianos , Anticorpos Neutralizantes , Antígenos de BactériasRESUMO
Inactivated whole virus SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide (Alum) are among the most widely used COVID-19 vaccines globally and have been critical to the COVID-19 pandemic response. Although these vaccines are protective against homologous virus infection in healthy recipients, the emergence of novel SARS-CoV-2 variants and the presence of large zoonotic reservoirs provide significant opportunities for vaccine breakthrough, which raises the risk of adverse outcomes including vaccine-associated enhanced respiratory disease (VAERD). To evaluate this possibility, we tested the performance of an inactivated SARS-CoV-2 vaccine (iCoV2) in combination with Alum against either homologous or heterologous coronavirus challenge in a mouse model of coronavirus-induced pulmonary disease. Consistent with human results, iCoV2 + Alum protected against homologous challenge. However, challenge with a heterologous SARS-related coronavirus, Rs-SHC014-CoV (SHC014), up to at least 10 months post-vaccination, resulted in VAERD in iCoV2 + Alum-vaccinated animals, characterized by pulmonary eosinophilic infiltrates, enhanced pulmonary pathology, delayed viral clearance, and decreased pulmonary function. In contrast, vaccination with iCoV2 in combination with an alternative adjuvant (RIBI) did not induce VAERD and promoted enhanced SHC014 clearance. Further characterization of iCoV2 + Alum-induced immunity suggested that CD4+ T cells were a major driver of VAERD, and these responses were partially reversed by re-boosting with recombinant Spike protein + RIBI adjuvant. These results highlight potential risks associated with vaccine breakthrough in recipients of Alum-adjuvanted inactivated vaccines and provide important insights into factors affecting both the safety and efficacy of coronavirus vaccines in the face of heterologous virus infections.
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The pathogenic and cross-species transmission potential of SARS-CoV-2-related coronaviruses (CoVs) remain poorly characterized. Here we recovered a wild-type pangolin (Pg) CoV GD strain including derivatives encoding reporter genes using reverse genetics. In primary human cells, PgCoV replicated efficiently but with reduced fitness and showed less efficient transmission via airborne route compared with SARS-CoV-2 in hamsters. PgCoV was potently inhibited by US Food and Drug Administration approved drugs, and neutralized by COVID-19 patient sera and SARS-CoV-2 therapeutic antibodies in vitro. A pan-Sarbecovirus antibody and SARS-CoV-2 S2P recombinant protein vaccine protected BALB/c mice from PgCoV infection. In K18-hACE2 mice, PgCoV infection caused severe clinical disease, but mice were protected by a SARS-CoV-2 human antibody. Efficient PgCoV replication in primary human cells and hACE2 mice, coupled with a capacity for airborne spread, highlights an emergence potential. However, low competitive fitness, pre-immune humans and the benefit of COVID-19 countermeasures should impede its ability to spread globally in human populations.
Assuntos
COVID-19 , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Cricetinae , Humanos , Animais , Camundongos , Especificidade de Hospedeiro , Pangolins , SARS-CoV-2/genética , COVID-19/prevenção & controle , Anticorpos Antivirais , Vacinas contra COVID-19 , Camundongos Endogâmicos BALB CRESUMO
Antibodies perform both neutralizing and non-neutralizing effector functions that protect against certain pathogen-induced diseases. A human antibody directed at the SARS-CoV-2 Spike N-terminal domain (NTD), DH1052, was recently shown to be non-neutralizing yet it protected mice and cynomolgus macaques from severe disease. The mechanisms of this non-neutralizing antibody-mediated protection are unknown. Here we show that Fc effector functions mediate non-neutralizing antibody (non-nAb) protection against SARS-CoV-2 MA10 viral challenge in mice. Though non-nAb infusion did not suppress infectious viral titers in the lung as potently as NTD neutralizing antibody (nAb) infusion, disease markers including gross lung discoloration were similar in nAb and non-nAb groups. Fc functional knockout substitutions abolished non-nAb protection and increased viral titers in the nAb group. Finally, Fc enhancement increased non-nAb protection relative to WT, supporting a positive association between Fc functionality and degree of protection in SARS-CoV-2 infection. This study demonstrates that non-nAbs can utilize Fc-mediated mechanisms to lower viral load and prevent lung damage due to coronavirus infection.
RESUMO
Despite the wide availability of several safe and effective vaccines that can prevent severe COVID-19 disease, the emergence of SARS-CoV-2 variants of concern (VOC) that can partially evade vaccine immunity remains a global health concern. In addition, the emergence of highly mutated and neutralization-resistant SARS-CoV-2 VOCs such as BA.1 and BA.5 that can partially or fully evade (1) many therapeutic monoclonal antibodies in clinical use underlines the need for additional effective treatment strategies. Here, we characterize the antiviral activity of GS-5245, Obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved RNA-dependent viral RNA polymerase (RdRp). Importantly, we show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-related Bat-CoV RsSHC014, Middle East Respiratory Syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant in vitro and highly effective as antiviral therapy in mouse models of SARS-CoV, SARS-CoV-2 (WA/1), MERS-CoV and Bat-CoV RsSHC014 pathogenesis. In all these models of divergent coronaviruses, we observed protection and/or significant reduction of disease metrics such as weight loss, lung viral replication, acute lung injury, and degradation in pulmonary function in GS-5245-treated mice compared to vehicle controls. Finally, we demonstrate that GS-5245 in combination with the main protease (Mpro) inhibitor nirmatrelvir had increased efficacy in vivo against SARS-CoV-2 compared to each single agent. Altogether, our data supports the continuing clinical evaluation of GS-5245 in humans infected with COVID-19, including as part of a combination antiviral therapy, especially in populations with the most urgent need for more efficacious and durable interventions.
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The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that evade immunity elicited by vaccination has placed an imperative on the development of countermeasures that provide broad protection against SARS-CoV-2 and related sarbecoviruses. Here, we identified extremely potent monoclonal antibodies (mAbs) that neutralized multiple sarbecoviruses from macaques vaccinated with AS03-adjuvanted monovalent subunit vaccines. Longitudinal analysis revealed progressive accumulation of somatic mutation in the immunoglobulin genes of antigen-specific memory B cells (MBCs) for at least 1 year after primary vaccination. Antibodies generated from these antigen-specific MBCs at 5 to 12 months after vaccination displayed greater potency and breadth relative to those identified at 1.4 months. Fifteen of the 338 (about 4.4%) antibodies isolated at 1.4 to 6 months after the primary vaccination showed potency against SARS-CoV-2 BA.1, despite the absence of serum BA.1 neutralization. 25F9 and 20A7 neutralized authentic clade 1 sarbecoviruses (SARS-CoV, WIV-1, SHC014, SARS-CoV-2 D614G, BA.1, and Pangolin-GD) and vesicular stomatitis virus-pseudotyped clade 3 sarbecoviruses (BtKY72 and PRD-0038). 20A7 and 27A12 showed potent neutralization against all SARS-CoV-2 variants and multiple Omicron sublineages, including BA.1, BA.2, BA.3, BA.4/5, BQ.1, BQ.1.1, and XBB. Crystallography studies revealed the molecular basis of broad and potent neutralization through targeting conserved sites within the RBD. Prophylactic protection of 25F9, 20A7, and 27A12 was confirmed in mice, and administration of 25F9 particularly provided complete protection against SARS-CoV-2, BA.1, SARS-CoV, and SHC014 challenge. These data underscore the extremely potent and broad activity of these mAbs against sarbecoviruses.
Assuntos
COVID-19 , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Animais , Humanos , Camundongos , Anticorpos Amplamente Neutralizantes , Vacinas contra COVID-19 , Macaca , SARS-CoV-2 , COVID-19/prevenção & controle , Imunização , Vacinação , Anticorpos Monoclonais , Anticorpos Antivirais , Anticorpos NeutralizantesRESUMO
Group 2B ß-coronaviruses (sarbecoviruses) have caused regional and global epidemics in modern history. Here, we evaluate the mechanisms of cross-sarbecovirus protective immunity, currently less clear yet important for pan-sarbecovirus vaccine development, using a panel of alphavirus-vectored vaccines covering bat to human strains. While vaccination does not prevent virus replication, it protects against lethal heterologous disease outcomes in both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and clade 2 bat sarbecovirus challenge models. The spike vaccines tested primarily elicit a highly S1-specific homologous neutralizing antibody response with no detectable cross-virus neutralization. Rather, non-neutralizing antibody functions, mechanistically linked to FcgR4 and spike S2, mediate cross-protection in wild-type mice. Protection is lost in FcR knockout mice, further supporting a model for non-neutralizing, protective antibodies. These data highlight the importance of FcR-mediated cross-protective immune responses in universal pan-sarbecovirus vaccine designs.
Assuntos
Alphavirus , COVID-19 , Quirópteros , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Vacinas Virais , Humanos , Animais , Camundongos , Anticorpos Antivirais , SARS-CoV-2 , COVID-19/prevenção & controle , Anticorpos Neutralizantes , VacinaçãoRESUMO
The rapid emergence of SARS-CoV-2 variants that evade immunity to vaccination has placed a global health imperative on the development of therapeutic countermeasures that provide broad protection against SARS-CoV-2 and related sarbecoviruses. Here, we identified extremely potent pan-sarbecovirus antibodies from non-human primates vaccinated with an AS03 adjuvanted subunit vaccine against SARS-CoV-2 that recognize conserved epitopes in the receptor binding domain (RBD) with femtomolar affinities. Longitudinal analysis revealed progressive accumulation of somatic mutation in the immunoglobulin genes of antigen-specific memory B cells for at least one year following primary vaccination. 514 monoclonal antibodies (mAbs) were generated from antigen-specific memory B cells. Antibodies isolated at 5 to 12 months following vaccination displayed greater potency and breadth, relative to those identified at 1.4 months. Notably, 15 out of 338 (â¼4.4%) antibodies isolated at 1.4â¼6 months after the primary vaccination showed extraordinary neutralization potency against SARS-CoV-2 omicron BA.1, despite the absence of BA.1 neutralization in serum. Two of them, 25F9 and 20A7, neutralized authentic clade Ia sarbecoviruses (SARS-CoV, WIV-1, SHC014) and clade Ib sarbecoviruses (SARS-CoV-2 D614G, SARS-CoV-2 BA.1, Pangolin-GD) with half-maximal inhibition concentrations of (0.85 ng/ml, 3 ng/ml, 6 ng/ml, 6 ng/ml, 42 ng/ml, 6 ng/ml) and (13 ng/ml, 2 ng/ml, 18 ng/ml, 9 ng/ml, 6 ng/ml, 345 ng/ml), respectively. Furthermore, 20A7 and 27A12 showed potent neutralization against all SARS-CoV-2 variants of concern and multiple Omicron sublineages, including BA.1, BA.2, BA.3, BA.4/5, BQ.1, BQ.1.1 and XBB variants. X-ray crystallography studies revealed the molecular basis of broad and potent neutralization through targeting conserved RBD sites. In vivo prophylactic protection of 25F9, 20A7 and 27A12 was confirmed in aged Balb/c mice. Notably, administration of 25F9 provided complete protection against SARS-CoV-2, SARS-CoV-2 BA.1, SARS-CoV, and SHC014 challenge, underscoring that these mAbs are promising pan-sarbecovirus therapeutic antibodies. One Sentence Summary: Extremely potent pan-sarbecovirus neutralizing antibodies.
RESUMO
Two group 2B ß-coronaviruses (sarbecoviruses) have caused regional and global epidemics in modern history. The mechanisms of cross protection driven by the sarbecovirus spike, a dominant immunogen, are less clear yet critically important for pan-sarbecovirus vaccine development. We evaluated the mechanisms of cross-sarbecovirus protective immunity using a panel of alphavirus-vectored vaccines covering bat to human strains. While vaccination did not prevent virus replication, it protected against lethal heterologous disease outcomes in both SARS-CoV-2 and clade 2 bat sarbecovirus HKU3-SRBD challenge models. The spike vaccines tested primarily elicited a highly S1-specific homologous neutralizing antibody response with no detectable cross-virus neutralization. We found non-neutralizing antibody functions that mediated cross protection in wild-type mice were mechanistically linked to FcgR4 and spike S2-binding antibodies. Protection was lost in FcR knockout mice, further supporting a model for non-neutralizing, protective antibodies. These data highlight the importance of FcR-mediated cross-protective immune responses in universal pan-sarbecovirus vaccine designs.
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Maturation of dengue viruses (DENVs) alters the structure, immunity, and infectivity of the virion and highly mature particles represent the dominant form in vivo. The production of highly mature virions principally relies on the structure and function of the viral premature membrane protein (prM) and its cleavage by the host protease furin. We redeveloped a reliable clonal cell line (VF1) which produces single-round mature DENVs without the need for DENV reverse genetics. More importantly, using protein engineering and directed evolution of the prM cleavage site, we engineered genetically stable mature DENVs in all serotypes independent of cell or host, usually with minimal impact on viral yield. Using these complementary strategies to regulate maturation, we demonstrate that the resulting mature DENVs are antigenically distinct from their isogenic partially mature forms. Given the clinical importance of mature DENVs in immunity, our study provides reliable strategies and reagents for the production of stable, high-titer mature DENVs for DENV antibody neutralization and vaccination immunity studies. Biologically, our data from directed evolution across host species reveals distinct maturation-dependent selective pressures between mammalian and insect cells, verifying the substrate preference between mammalian and insect furin, while hinting at an evolutionary equilibrium of DENV prM cleavage site between its host and vector in nature. IMPORTANCE Mature DENVs represent the dominant form in vivo and are the target for vaccine development. Here, we used multiple strategies, including protein engineering and natural and directed evolution to generate DENV1, -2, -3, and -4 variants that are highly mature without compromising replication efficiency compared to the parental strains. Given the clinical importance of mature DENVs in immunity, this work provides a roadmap for engineering highly mature DENV that could apply to future vaccine development. Our directed-evolution data also shed light on the divergent evolutionary relationship of DENVs between its host and vector.
Assuntos
Vírus da Dengue , Dengue , Animais , Anticorpos Antivirais , Vírus da Dengue/fisiologia , Furina/genética , Mamíferos , Sorogrupo , Proteínas do Envelope Viral/genética , VírionRESUMO
Zoonotic transmission of coronaviruses poses an ongoing threat to human populations. Endemic outbreaks of swine acute diarrhea syndrome coronavirus (SADS-CoV) have caused severe economic losses in the pig industry and have the potential to cause human outbreaks. Currently, there are no vaccines or specific antivirals against SADS-CoV, and our limited understanding of SADS-CoV host entry factors could hinder prompt responses to a potential human outbreak. Using a genomewide CRISPR knockout screen, we identified placenta-associated 8 protein (PLAC8) as an essential host factor for SADS-CoV infection. Knockout of PLAC8 abolished SADS-CoV infection, which was restored by complementing PLAC8 from multiple species, including human, rhesus macaques, mouse, pig, pangolin, and bat, suggesting a conserved infection pathway and susceptibility of SADS-CoV among mammals. Mechanistically, PLAC8 knockout does not affect viral entry; rather, knockout cells displayed a delay and reduction in viral subgenomic RNA expression. In a swine primary intestinal epithelial culture (IEC) infection model, differentiated cultures have high levels of PLAC8 expression and support SADS-CoV replication. In contrast, expanding IECs have low levels of PLAC8 expression and are resistant to SADS-CoV infection. PLAC8 expression patterns translate in vivo; the immunohistochemistry of swine ileal tissue revealed high levels of PLAC8 protein in neonatal compared to adult tissue, mirroring the known SADS-CoV pathogenesis in neonatal piglets. Overall, PLAC8 is an essential factor for SADS-CoV infection and may serve as a promising target for antiviral development for potential pandemic SADS-CoV.
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Alphacoronavirus , Infecções por Coronavirus , Doenças dos Suínos , Alphacoronavirus/genética , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Infecções por Coronavirus/epidemiologia , SuínosRESUMO
Understanding vaccine-mediated protection against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is critical to overcoming the global coronavirus disease 2019 (COVID-19) pandemic. We investigate mRNA-vaccine-induced antibody responses against the reference strain, seven variants, and seasonal coronaviruses in 168 healthy individuals at three time points: before vaccination, after the first dose, and after the second dose. Following complete vaccination, both naive and previously infected individuals developed comparably robust SARS-CoV-2 spike antibodies and variable levels of cross-reactive antibodies to seasonal coronaviruses. However, the strength and frequency of SARS-CoV-2 neutralizing antibodies in naive individuals were lower than in previously infected individuals. After the first vaccine dose, one-third of previously infected individuals lacked neutralizing antibodies; this was improved to one-fifth after the second dose. In all individuals, neutralizing antibody responses against the Alpha and Delta variants were weaker than against the reference strain. Our findings support future tailored vaccination strategies against emerging SARS-CoV-2 variants as mRNA-vaccine-induced neutralizing antibodies are highly variable among individuals.
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Anticorpos Neutralizantes/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , Reações Cruzadas , Imunoglobulina G/imunologia , SARS-CoV-2/imunologia , Anticorpos Antivirais/imunologia , Formação de Anticorpos , COVID-19/prevenção & controle , Vacinas contra COVID-19/administração & dosagem , Coronavirus/imunologia , Humanos , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas de mRNA/administração & dosagem , Vacinas de mRNA/imunologiaRESUMO
The angiotensin-converting enzyme 2 (ACE2) receptor is a major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) host range determinant, and understanding SARS-CoV-2-ACE2 interactions will provide important insights into COVID-19 pathogenesis and animal model development. SARS-CoV-2 cannot infect mice due to incompatibility between its receptor binding domain and the murine ACE2 receptor. Through molecular modeling and empirical in vitro validation, we identified 5 key amino acid differences between murine and human ACE2 that mediate SARS-CoV-2 infection, generating a chimeric humanized murine ACE2. Additionally, we examined the ability of the humanized murine ACE2 receptor to permit infection by an additional preemergent group 2B coronavirus, WIV-1, providing evidence for the potential pan-virus capabilities of this chimeric receptor. Finally, we predicted the ability of these determinants to inform host range identification of preemergent coronaviruses by evaluating hot spot contacts between SARS-CoV-2 and additional potential host receptors. Our results identify residue determinants that mediate coronavirus receptor usage and host range for application in SARS-CoV-2 and emerging coronavirus animal model development.IMPORTANCE SARS-CoV-2 (the causative agent of COVID-19) is a major public health threat and one of two related coronaviruses that have caused epidemics in modern history. A method of screening potential infectible hosts for preemergent and future emergent coronaviruses would aid in mounting rapid response and intervention strategies during future emergence events. Here, we evaluated determinants of SARS-CoV-2 receptor interactions, identifying key changes that enable or prevent infection. The analysis detailed in this study will aid in the development of model systems to screen emergent coronaviruses as well as treatments to counteract infections.
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Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Betacoronavirus/fisiologia , Replicação Viral , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/genética , Animais , Betacoronavirus/metabolismo , Sítios de Ligação , COVID-19/virologia , Linhagem Celular , Infecções por Coronavirus/virologia , Especificidade de Hospedeiro , Humanos , Camundongos , Modelos Moleculares , Mutação , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Coronaviruses are prone to transmission to new host species, as recently demonstrated by the spread to humans of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic1. Small animal models that recapitulate SARS-CoV-2 disease are needed urgently for rapid evaluation of medical countermeasures2,3. SARS-CoV-2 cannot infect wild-type laboratory mice owing to inefficient interactions between the viral spike protein and the mouse orthologue of the human receptor, angiotensin-converting enzyme 2 (ACE2)4. Here we used reverse genetics5 to remodel the interaction between SARS-CoV-2 spike protein and mouse ACE2 and designed mouse-adapted SARS-CoV-2 (SARS-CoV-2 MA), a recombinant virus that can use mouse ACE2 for entry into cells. SARS-CoV-2 MA was able to replicate in the upper and lower airways of both young adult and aged BALB/c mice. SARS-CoV-2 MA caused more severe disease in aged mice, and exhibited more clinically relevant phenotypes than those seen in Hfh4-ACE2 transgenic mice, which express human ACE2 under the control of the Hfh4 (also known as Foxj1) promoter. We demonstrate the utility of this model using vaccine-challenge studies in immune-competent mice with native expression of mouse ACE2. Finally, we show that the clinical candidate interferon-λ1a (IFN-λ1a) potently inhibits SARS-CoV-2 replication in primary human airway epithelial cells in vitro-both prophylactic and therapeutic administration of IFN-λ1a diminished SARS-CoV-2 replication in mice. In summary, the mouse-adapted SARS-CoV-2 MA model demonstrates age-related disease pathogenesis and supports the clinical use of pegylated IFN-λ1a as a treatment for human COVID-196.
Assuntos
Betacoronavirus , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , Modelos Animais de Doenças , Interferons/farmacologia , Interferons/uso terapêutico , Interleucinas/farmacologia , Interleucinas/uso terapêutico , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Vacinas Virais/imunologia , Envelhecimento/imunologia , Enzima de Conversão de Angiotensina 2 , Animais , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , COVID-19 , Vacinas contra COVID-19 , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Feminino , Fatores de Transcrição Forkhead/genética , Humanos , Interferon-alfa/administração & dosagem , Interferon-alfa/farmacologia , Interferon-alfa/uso terapêutico , Interferons/administração & dosagem , Interleucinas/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Modelos Moleculares , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/genética , Pneumonia Viral/imunologia , Receptores Virais/genética , Receptores Virais/metabolismo , SARS-CoV-2RESUMO
The head of Salmonella virus SPN3US is composed of ~50 different proteins and is unusual because within its packaged genome there is a mass (>40 MDa) of ejection or E proteins that enter the Salmonella cell. The assembly mechanisms of this complex structure are poorly understood. Previous studies showed that eight proteins in the mature SPN3US head had been cleaved by the prohead protease. In this study, we present the characterization of SPN3US prohead protease mutants using transmission electron microscopy and mass spectrometry. In the absence of the prohead protease, SPN3US head formation was severely impeded and proheads accumulated on the Salmonella inner membrane. This impediment is indicative of proteolysis being necessary for the release and subsequent DNA packaging of proheads in the wild-type phage. Proteomic analyses of gp245- proheads that the normal proteolytic processing of head proteins had not occurred. Assays of a recombinant, truncated form of the protease found it was active, leading us to hypothesize that the C-terminal propeptide has a role in targeting the protease into the prohead core. Our findings provide new evidence regarding the essential role of proteolysis for correct head assembly in this remarkable parasite.