RESUMO
BACKGROUND: BBV152 (Covaxin™) is a whole-virion inactivated SARS-CoV-2 vaccine mixed with an immune adjuvant. We aimed to compare immune responses after booster vaccination with heterologous BBV152 versus homologous mRNA vaccine. METHODS: We conducted a randomized, participant-blinded, controlled trial. Fifty mRNA-vaccinated participants were enrolled and randomized to receive an mRNA booster (n = 26) or BBV152 (n = 24). Blood samples were collected pre-vaccination, and at Day 7, 28, 180 and 360 post-booster for analysis of humoral and cellular immune responses. Primary end point was the SARS-CoV-2 anti-spike antibody titer at day 28. RESULTS: Recruitment began in January 2022 and was terminated early due to the BBV152 group meeting pre-specified criteria for futility. At Day 28 post-boost, mean SARS-CoV-2 spike antibody titers were lower with BBV152 (2004 IU/mL; 95 % confidence interval [CI], 1132-3548) vs mRNA (26,669 IU/mL; 95 % CI, 21,330-33,266; p < 0.0001), but comparable levels of spike-specific CD4 and cytotoxic T-cells were observed. Anti-spike antibody titers remained significantly different at Day 180: BBV152 4467 IU/mL (95 % CI, 1959-10,186) vs mRNA 20,749 IU/mL (95 % CI, 12,303-35,075; p = 0.0017). Levels of surrogate virus neutralizing antibodies against ancestral and Omicron subvariants BA.1 and BA.2 were significantly higher among mRNA recipients at Day 180, including after adjusting for intercurrent infection. By Day 360, anti-spike antibody titers and neutralizing antibody levels against Omicron subvariants became similar between vaccine groups. By the end of the study, 16 in each arm (mRNA 64 % and BBV152 69.6 %) had breakthrough infections and time to COVID-19 infection between vaccine groups were similar (p = 0.63). CONCLUSIONS: Wild-type SARS-CoV-2 anti-spike antibody titer and surrogate virus neutralizing test levels against wild-type SARS-CoV-2 and Omicron subvariants BA.1/BA.2/BA.5 were significantly higher at Day 28 and 180 in individuals who received booster vaccination with an mRNA vaccine compared with BBV152. CLINICAL TRIAL REGISTRATION NUMBER: NCT05142319.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , Imunização Secundária , Imunogenicidade da Vacina , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Humanos , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Feminino , Masculino , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , COVID-19/prevenção & controle , COVID-19/imunologia , Adulto , Imunização Secundária/métodos , Pessoa de Meia-Idade , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de mRNA/imunologia , Adulto Jovem , Imunidade Humoral , Imunidade Celular , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/administração & dosagemRESUMO
The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered global difficulties for both individuals and economies, with new variants continuing to emerge. The Delta variant of SARS-CoV-2 remains most prevalent worldwide, and it affects the efficacy of coronavirus disease 2019 (COVID-19) vaccination. Expedited testing to detect the Delta variant of SARS-CoV-2 and monitor viral transmission is necessary. This study aimed to develop and evaluate a colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) technique targeting the L452R mutation in the S gene for the specific detection of the Delta variant. In the test, positivity was indicated as a color change from purple to yellow. The assay's 95% limit of detection was 57 copies per reaction for the L452R (U1355G)-specific standard plasmid. Using 126 clinical samples, our assay displayed 100% specificity, 97.06% sensitivity, and 98.41% accuracy in identifying the Delta variant of SARS-CoV-2 compared to real-time RT-PCR. To our knowledge, this is the first colorimetric RT-LAMP assay that can differentiate the Delta variant from its generic SARS-CoV-2, enabling it as an approach for studying COVID-19 demography and facilitating proper effective control measure establishment to fight against the reemerging variants of SARS-CoV-2 in the future.
Assuntos
COVID-19 , Colorimetria , Mutação , Técnicas de Amplificação de Ácido Nucleico , SARS-CoV-2 , SARS-CoV-2/genética , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Colorimetria/métodos , COVID-19/virologia , COVID-19/diagnóstico , COVID-19/genética , Sensibilidade e Especificidade , Técnicas de Diagnóstico Molecular/métodos , Glicoproteína da Espícula de Coronavírus/genética , RNA Viral/genética , Teste de Ácido Nucleico para COVID-19/métodosRESUMO
In post-COVID-19 syndrome, clinical presentation of the nerve fiber dysfunction plays an important role. The possibility of autoantigen cross-mimicry of human coronaviruses and the peripheral nervous system needs to be investigated. The bioinformatic analysis was applied to search for possible common protein sequences located in the immunoreactive epitopes. Among the autoantigens of the human nervous system, fibroblast growth factor receptor protein 3, myelin protein P0, myelin protein P2, sodium channel protein type 9, alpha protein subunit, plexin-D1 protein and ubiquitin-carboxyl-terminal hydrolase protein of the L1 isoenzyme were selected. The original "Alignmentaj" analytical program was created. The UniProt database, Protein Data Bank, and AlphaFold databases were used. The analysis of protein sequence similarities of spike glycoproteins in human coronaviruses revealed common pentapeptides of the MERS-CoV-2 virus with the fibroblast growth factor receptor 3 and myelin protein P2. Among seasonal coronaviruses, common peptide sequences were identified in HCoV-HKU-1 virus with sodium channel protein type 9 subunit alpha and Plexin-D1, HCoV-OC43 with Plexin-D1, as well as HCoV-NL63 with Plexin-D1 and Ubiquitin carboxyl-terminal hydrolase isozyme L1. Some shared peptides belong to immunoreactive epitopes. The most important targets for the molecular similarities are the sodium channel subunits and fibroblast growth factor receptor 3, both for seasonal and highly pathogenic coronaviruses. The data obtained make it possible to identify new potential targets for the development of autoimmune reactions that may occur against the background of the infections with highly pathogenic as well as seasonal coronaviruses.
Assuntos
Autoantígenos , Mimetismo Molecular , Humanos , Autoantígenos/imunologia , COVID-19/imunologia , COVID-19/virologia , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Antígenos Virais/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Epitopos/imunologia , Biologia Computacional , Coronavirus/imunologia , Coronavirus/genética , Sequência de AminoácidosRESUMO
A mucosal route of vaccination could prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication at the site of infection and limit transmission. We compared protection against heterologous XBB.1.16 challenge in nonhuman primates (NHPs) ~5 months following intramuscular boosting with bivalent mRNA encoding WA1 and BA.5 spike proteins or mucosal boosting with a WA1-BA.5 bivalent chimpanzee adenoviral-vectored vaccine delivered by intranasal or aerosol device. NHPs boosted by either mucosal route had minimal virus replication in the nose and lungs, respectively. By contrast, protection by intramuscular mRNA was limited to the lower airways. The mucosally delivered vaccine elicited durable airway IgG and IgA responses and, unlike the intramuscular mRNA vaccine, induced spike-specific B cells in the lungs. IgG, IgA and T cell responses correlated with protection in the lungs, whereas mucosal IgA alone correlated with upper airway protection. This study highlights differential mucosal and serum correlates of protection and how mucosal vaccines can durably prevent infection against SARS-CoV-2.
Assuntos
Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , Imunização Secundária , Imunoglobulina A , SARS-CoV-2 , Animais , Imunoglobulina A/imunologia , SARS-CoV-2/imunologia , COVID-19/prevenção & controle , COVID-19/imunologia , COVID-19/virologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Macaca mulatta , Adenoviridae/imunologia , Adenoviridae/genética , Imunidade nas Mucosas , Vacinas contra Adenovirus/imunologia , Vacinas contra Adenovirus/administração & dosagem , Feminino , Pulmão/virologia , Pulmão/imunologia , Linfócitos B/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Administração Intranasal , Vacinação/métodos , HumanosRESUMO
MoMo30 is an antiviral protein isolated from aqueous extracts of Momordica balsamina L. (Senegalese bitter melon). Previously, we demonstrated MoMo30's antiviral activity against HIV-1. Here, we explore whether MoMo30 has antiviral activity against the COVID-19 virus, SARS-CoV-2. MLV particles pseudotyped with the SARS-CoV-2 Spike glycoprotein and a Luciferase reporter gene (SARS2-PsV) were developed from a three-way co-transfection of HEK293-T17 cells. MoMo30's inhibition of SARS2-PsV infection was measured using a luciferase assay and its cytotoxicity using an XTT assay. Additionally, MoMo30's interactions with the variants and domains of Spike were determined by ELISA. We show that MoMo30 inhibits SARS2-PsV infection. We also report evidence of the direct interaction of MoMo30 and SARS-CoV-2 Spike from WH-1, Alpha, Delta, and Omicron variants. Furthermore, MoMo30 interacts with both the S1 and S2 domains of Spike but not the receptor binding domain (RBD), suggesting that MoMo30 inhibits SARS-CoV-2 infection by inhibiting fusion of the virus and the host cell via interactions with Spike.
Assuntos
Antivirais , Ligação Proteica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/química , Humanos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Células HEK293 , Antivirais/farmacologia , COVID-19/virologia , Internalização do Vírus/efeitos dos fármacos , Pseudotipagem ViralRESUMO
The most recent wave of SARS-CoV-2 Omicron variants descending from BA.2 and BA.2.86 exhibited improved viral growth and fitness due to convergent evolution of functional hotspots. These hotspots operate in tandem to optimize both receptor binding for effective infection and immune evasion efficiency, thereby maintaining overall viral fitness. The lack of molecular details on structure, dynamics and binding energetics of the latest FLiRT and FLuQE variants with the ACE2 receptor and antibodies provides a considerable challenge that is explored in this study. We combined AlphaFold2-based atomistic predictions of structures and conformational ensembles of the SARS-CoV-2 spike complexes with the host receptor ACE2 for the most dominant Omicron variants JN.1, KP.1, KP.2 and KP.3 to examine the mechanisms underlying the role of convergent evolution hotspots in balancing ACE2 binding and antibody evasion. Using the ensemble-based mutational scanning of the spike protein residues and computations of binding affinities, we identified binding energy hotspots and characterized the molecular basis underlying epistatic couplings between convergent mutational hotspots. The results suggested the existence of epistatic interactions between convergent mutational sites at L455, F456, Q493 positions that protect and restore ACE2-binding affinity while conferring beneficial immune escape. To examine immune escape mechanisms, we performed structure-based mutational profiling of the spike protein binding with several classes of antibodies that displayed impaired neutralization against BA.2.86, JN.1, KP.2 and KP.3. The results confirmed the experimental data that JN.1, KP.2 and KP.3 harboring the L455S and F456L mutations can significantly impair the neutralizing activity of class 1 monoclonal antibodies, while the epistatic effects mediated by F456L can facilitate the subsequent convergence of Q493E changes to rescue ACE2 binding. Structural and energetic analysis provided a rationale to the experimental results showing that BD55-5840 and BD55-5514 antibodies that bind to different binding epitopes can retain neutralizing efficacy against all examined variants BA.2.86, JN.1, KP.2 and KP.3. The results support the notion that evolution of Omicron variants may favor emergence of lineages with beneficial combinations of mutations involving mediators of epistatic couplings that control balance of high ACE2 affinity and immune evasion.
Assuntos
Enzima de Conversão de Angiotensina 2 , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Humanos , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , COVID-19/virologia , COVID-19/genética , Epistasia Genética , Evasão da Resposta Imune , Anticorpos Antivirais/imunologia , Conformação ProteicaRESUMO
Infectious Bronchitis Virus (IBV) is a major threat to the poultry industry worldwide, causing significant economic losses. While the virus's genetic structure is well understood, the specific strains circulating in Bolivia have remained uncharacterized until now. This study aimed to identify and characterize new IBV strains in Bolivia. Tissue samples from broilers exhibiting clinical signs of Infectious Bronchitis were screened to detect IBV using real-time RT-PCR (RT-qPCR). Positive samples with low cycle threshold (Ct) values were selected for sequencing the full S1 gene. Of the 12 samples analyzed, 10 were determined to be positive for IBV. However, only four samples yielded sufficient genetic material for sequencing and subsequent phylogenetic analysis. The results revealed the presence of GI-1 and GI-23 lineages, both belonging to genotype I (GI). The GI-1 lineage showed >99% sequence identity to the H120 and Massachusetts vaccine strains, suggesting a close relationship. In contrast, the GI-23 lineage clustered with other IBV strains, showing a distinct subclade that is genetically distant from Brazilian strains. No evidence of recombination was found. Furthermore, amino acid substitution analysis identified specific mutations in the S1 subunit, particularly in the hypervariable regions 1, 2, and 3. These mutations could potentially alter the virus's antigenicity, leading to reduced vaccine efficacy. The findings of this study highlight the importance of continued and broad genomic surveillance of circulating IBV strains and the need to improve vaccination strategies in Bolivia.
Assuntos
Galinhas , Infecções por Coronavirus , Genótipo , Vírus da Bronquite Infecciosa , Filogenia , Doenças das Aves Domésticas , Animais , Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/isolamento & purificação , Vírus da Bronquite Infecciosa/classificação , Galinhas/virologia , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/epidemiologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Infecções por Coronavirus/epidemiologia , Bolívia/epidemiologia , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
The COVID-19 pandemic was characterized by the emergence and succession of SARS-CoV-2 variants able to evade the antibody response induced by natural infection and vaccination. To evaluate the IgG reactivity and neutralizing capacity of the serum of individuals vaccinated with Sputnik V (105 volunteers vaccinated) against different viral variants. IgG reactivity to the Spike protein (S) was evaluated by ELISA. A plaque reduction neutralization test was performed using different viral variant isolates. At 42 days post-vaccination, the frequency of recognition and reactivity to the S protein of the Omicron variant was lower compared to that of the other variants. In general, a higher average neutralization titer was seen against the ancestral variant compared to the variants, especially Omicron. However, some sera exhibited a higher neutralization titer to the Gamma variant compared to the ancestral variant, suggesting unapparent exposure during the clinical trial. Antibodies induced by Sputnik V can recognize, persist, and neutralize SARS-CoV-2 variants, with Omicron being the one that best evades this response. These results represent a unique report on the humoral response induced by a globally lesser-studied vaccine in terms of efficacy and immune escape, offering insights into developing vaccines targeting unknown coronaviruses.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , Imunoglobulina G , Testes de Neutralização , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Humanos , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , COVID-19/epidemiologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Venezuela/epidemiologia , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Adulto , Feminino , Masculino , Vacinação , Pessoa de Meia-IdadeRESUMO
Over the past three years, new SARS-CoV-2 variants have continuously emerged, evolving to a point where an immune response against the original vaccine no longer provided optimal protection against these new strains. During this time, high-throughput neutralization assays based on pseudoviruses have become a valuable tool for assessing the efficacy of new vaccines, screening updated vaccine candidates against emerging variants, and testing the efficacy of new therapeutics such as monoclonal antibodies. Lentiviral vectors derived from HIV-1 are popular for developing pseudo and chimeric viruses due to their ease of use, stability, and long-term transgene expression. However, the HIV-based platform has lower transduction rates for pseudotyping coronavirus spike proteins than other pseudovirus platforms, necessitating more optimized methods. As the SARS-CoV-2 virus evolved, we produced over 18 variants of the spike protein for pseudotyping with an HIV-based vector, optimizing experimental parameters for their production and transduction. In this article, we present key parameters that were assessed to improve such technology, including (a) the timing and method of collection of pseudovirus supernatant; (b) the timing of host cell transduction; (c) cell culture media replenishment after pseudovirus adsorption; and (d) the centrifugation (spinoculation) parameters of the host cell+ pseudovirus mix, towards improved transduction. Additionally, we found that, for some pseudoviruses, the addition of a cationic polymer (polybrene) to the culture medium improved the transduction process. These findings were applicable across variant spike pseudoviruses that include not only SARS-CoV-2 variants, but also SARS, MERS, Alpha Coronavirus (NL-63), and bat-like coronaviruses. In summary, we present improvements in transduction efficiency, which can broaden the dynamic range of the pseudovirus titration and neutralization assays.
Assuntos
HIV-1 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Transdução Genética , Humanos , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , HIV-1/genética , HIV-1/fisiologia , Células HEK293 , Pseudotipagem Viral , Vetores Genéticos/genética , COVID-19/virologia , Testes de NeutralizaçãoRESUMO
Porcine epidemic diarrhea virus (PEDV) causes diarrhea and vomiting in piglets, leading to a mortality rate of 100%. Due to the high frequency of mutation, it is important to monitor the evolution of PEDV and develop potential vaccine candidates. In this study, two PEDV strains (ZJ2022 and ZQ2022) were identified by PCR. These strains were subsequently isolated, and their genome sequences, growth characteristics, and pathogenicity were compared. Phylogenetic and recombination analyses revealed that both strains belonged to GIIa-subgroup, and ZQ2022 was identified as a recombinant strain derived from ZJ2022. Further sequence analysis showed that the ZJ2022 strain had a modified top region of the S1 protein due to a three amino acid insertion (T380_Y380insGGE) in the S1 gene. According to the virus growth curve, ZJ2022 exhibited better cellular adaptation than ZQ2022, with higher viral titers from 8 hpi to 24 hpi. Additionally, ZQ2022 exhibited a high level of pathogenicity, causing severe diarrhea in piglets at 36 hpi and a 100% mortality rate by 96 hpi. In contrast, ZJ2022 showed lower pathogenicity, inducing severe diarrhea in piglets at 60 hpi, with a mortality rate of 60% at 96 hpi and 100% at 120 hpi. In summary, our findings provided evidence of the undergoing mutations in Chinese PEDV strains. Furthermore, the S gene insertion strain ZJ2022 exhibited strong cellular adaptability and low pathogenicity, making it a potential candidate strain for vaccine development.
Assuntos
Animais Recém-Nascidos , Infecções por Coronavirus , Diarreia , Filogenia , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Animais , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/classificação , Suínos , Doenças dos Suínos/virologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Virulência , Diarreia/virologia , Diarreia/veterinária , Glicoproteína da Espícula de Coronavírus/genética , Genoma Viral , Mutagênese Insercional , China , Células VeroRESUMO
Influenza and coronavirus disease 2019 (COVID-19) represent two respiratory diseases that have significantly impacted global health, resulting in substantial disease burden and mortality. An optimal solution would be a combined vaccine capable of addressing both diseases, thereby obviating the need for multiple vaccinations. Previously, we conceived a chimeric protein subunit vaccine targeting both influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), utilizing the receptor binding domain of spike protein (S-RBD) and the stalk region of hemagglutinin protein (HA-stalk) components. By integrating the S-RBD from the SARS-CoV-2 Delta variant with the headless hemagglutinin (HA) from H1N1 influenza virus, we constructed stable trimeric structures that remain accessible to neutralizing antibodies. This vaccine has demonstrated its potential by conferring protection against a spectrum of strains in mouse models. In this study, we designed an mRNA vaccine candidate encoding the chimeric antigen. The resultant humoral and cellular immune responses were meticulously evaluated in mouse models. Furthermore, the protective efficacy of the vaccine was rigorously examined through challenges with either homologous or heterologous influenza viruses or SARS-CoV-2 strains. Our findings reveal that the mRNA vaccine exhibited robust immunogenicity, engendering high and sustained levels of neutralizing antibodies accompanied by robust and persistent cellular immunity. Notably, this vaccine effectively afforded complete protection to mice against H1N1 or heterosubtypic H5N8 subtypes, as well as the SARS-CoV-2 Delta and Omicron BA.2 variants. Additionally, our mRNA vaccine design can be easily adapted from Delta RBD to Omicron RBD antigens, providing protection against emerging variants. The development of two-in-one vaccine targeting both influenza and COVID-19, incorporating the mRNA platform, may provide a versatile approach to combating future pandemics.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Glicoproteínas de Hemaglutininação de Vírus da Influenza , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Vacinas de mRNA , Animais , Camundongos , SARS-CoV-2/imunologia , COVID-19/prevenção & controle , COVID-19/imunologia , Vacinas de mRNA/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Humanos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vacinas contra COVID-19/imunologia , Vacinas contra Influenza/imunologia , Anticorpos Antivirais/imunologia , Camundongos Endogâmicos BALB C , Feminino , Vírus da Influenza A Subtipo H1N1/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/imunologia , Vacinas Sintéticas/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/imunologia , Anticorpos Neutralizantes/imunologiaRESUMO
Porcine epidemic diarrhea virus (PEDV) has caused significant economic losses to the pig farming industry in various countries for a long time. Currently, there are no highly effective preventive or control measures available. Research into the pathogenic mechanism of PEDV has shown that it primarily causes infection by binding the S protein to the CD13 (APN) receptor on the membrane of porcine intestinal epithelial cells. The S1 region contains three neutralization epitopes and multiple receptor-binding domains, which are closely related to viral antigenicity and ad-sorption invasion. Nanobodies are a type of single-domain antibody that have been discovered in recent years. They can be expressed on a large scale through prokaryotic expression systems, which makes them cost-effective, stable, and less immunogenic. This study used a phage display library of nanobodies against the PEDV S1 protein. After three rounds of selection and enrichment, the DNA sequence of the highly specific nanobody S1Nb1 was successfully obtained. To obtain soluble nanobody S1Nb1, its DNA sequence was inserted into the vector Pcold and a solubility-enhancing SUMO tag was added. The resulting recombinant vector, Pcold-SUMO-S1Nb1, was then transformed into E. coli BL21(DE3) to determine the optimal expression conditions for the nanobody. Following purification using Ni-column affinity chromatography, Western blot analysis confirmed the successful purification of S1Nb1 carrying the solubility-enhancing tag. ELISA results demonstrated a strong affinity between the S1Nb1 nanobody and PEDV S1 protein.
Assuntos
Escherichia coli , Vírus da Diarreia Epidêmica Suína , Anticorpos de Domínio Único , Vírus da Diarreia Epidêmica Suína/imunologia , Vírus da Diarreia Epidêmica Suína/genética , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/isolamento & purificação , Anticorpos de Domínio Único/química , Escherichia coli/genética , Escherichia coli/metabolismo , Animais , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Suínos , Biblioteca de Peptídeos , Expressão GênicaRESUMO
The ongoing co-circulation of multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains necessitates advanced methods such as high-throughput multiplex pseudovirus systems for evaluating immune responses to different variants, crucial for developing updated vaccines and neutralizing antibodies (nAbs). We have developed a quadri-fluorescence (qFluo) pseudovirus platform by four fluorescent reporters with different spectra, allowing simultaneous measurement of the nAbs against four variants in a single test. qFluo shows high concordance with the classical single-reporter assay when testing monoclonal antibodies and human plasma. Utilizing qFluo, we assessed the immunogenicities of the spike of BA.5, BQ.1.1, XBB.1.5, and CH.1.1 in hamsters. An analysis of cross-neutralization against 51 variants demonstrated superior protective immunity from XBB.1.5, especially against prevalent strains such as "FLip" and JN.1, compared to BA.5. Our finding partially fills the knowledge gap concerning the immunogenic efficacy of the XBB.1.5 vaccine against current dominant variants, being instrumental in vaccine-strain decisions and insight into the evolutionary path of SARS-CoV-2.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , SARS-CoV-2 , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Animais , Humanos , COVID-19/imunologia , COVID-19/virologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Cricetinae , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Testes de Neutralização/métodos , Fluorescência , Células HEK293 , Antígenos Virais/imunologia , Anticorpos Monoclonais/imunologia , MesocricetusRESUMO
BACKGROUND: The hedgehogs have been recently identified as possible reservoir of Middle East respiratory syndrome coronavirus like (MERS-CoV-like). These viruses were classified as a distinct Betacoronavirus erinacei (BCoV-Eri) species within the MerBCoV-Eriirus subgenus. As coronaviruses are known for their ability to jump between different hosts, including humans, this can pose a particular threat to people in direct contact with hedgehogs, such as those working at animal asylums. Our previous studies have shown the presence of BCoV-Eri strains in animals collected in the wildlife rehabilitation centre. This study aimed to investigate the presence of CoV in subsequent hedgehogs collected from the urban area of Poland and their molecular characteristics. RESULTS: Monitoring for the presence of coronavirus infection in hedgehogs revealed five positive individuals. The presence of BCoV-Eri was found in a total of 20% of animals tested. Our analyses revealed no correlation between CoVs positivity and animal health conditions but a higher probability of such infection in juveniles and females. The whole genome of two Polish Hedgehog coronavirus 1 strains were sequenced and compared with available counterparts from European and Asian countries. Phylogenetic analysis showed that both CoV strains formed common cluster with other similar MerBCoV-Eriirus, but they were also found to be genetically variable and most changes in the S protein were identified. Our analysis revealed that some S protein sites of the Hedgehog coronavirus 1 strains evolved under positive selection pressure and of five such sites, three are in the S1 region while the other two in the S2 region of the Spike. CONCLUSIONS: BCoV-Eri is to some extent prevalent in wildlife asylums in Poland. Given that the S protein of BCoVs-Eri is highly variable and that some sites of this protein evolve under positive selection pressure, these strains could potentially acquire a favourable feature for cross-species transmission. Consequently, the threat to humans working in such asylums is particularly high. Adequate biosecurity safeguards, but also human awareness of such risks, are therefore essential.
Assuntos
Infecções por Coronavirus , Genoma Viral , Ouriços , Filogenia , Glicoproteína da Espícula de Coronavírus , Ouriços/virologia , Animais , Polônia/epidemiologia , Glicoproteína da Espícula de Coronavírus/genética , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Infecções por Coronavirus/epidemiologia , Feminino , Masculino , Coronavirus/genética , Coronavirus/classificação , Evolução MolecularRESUMO
Significant variations have been observed in viral copies generated during SARS-CoV-2 infections. However, the factors that impact viral copies and infection dynamics are not fully understood, and may be inherently dependent upon different viral and host factors. Here, we conducted virus whole genome sequencing and measured viral copies using RT-qPCR from 9,902 SARS-CoV-2 infections over a 2-year period to examine the impact of virus genetic variation on changes in viral copies adjusted for host age and vaccination status. Using a genome-wide association study (GWAS) approach, we identified multiple single-nucleotide polymorphisms (SNPs) corresponding to amino acid changes in the SARS-CoV-2 genome associated with variations in viral copies. We further applied a marginal epistasis test to detect interactions among SNPs and identified multiple pairs of substitutions located in the spike gene that have non-linear effects on viral copies. We also analyzed the temporal patterns and found that SNPs associated with increased viral copies were predominantly observed in Delta and Omicron BA.2/BA.4/BA.5/XBB infections, whereas those associated with decreased viral copies were only observed in infections with Omicron BA.1 variants. Our work showcases how GWAS can be a useful tool for probing phenotypes related to SNPs in viral genomes that are worth further exploration. We argue that this approach can be used more broadly across pathogens to characterize emerging variants and monitor therapeutic interventions.
Assuntos
COVID-19 , Genoma Viral , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único , SARS-CoV-2 , Polimorfismo de Nucleotídeo Único/genética , Humanos , SARS-CoV-2/genética , Estudo de Associação Genômica Ampla/métodos , COVID-19/genética , COVID-19/virologia , Genoma Viral/genética , Glicoproteína da Espícula de Coronavírus/genética , Pessoa de Meia-Idade , Adulto , Masculino , Feminino , Carga Viral/genética , Idoso , Sequenciamento Completo do Genoma/métodosRESUMO
Although the primary pandemic of SARS-CoV-2 is over, there are concerns about the resurgence of the next wave of related viruses, including a wide range of variant viruses. The soluble ACE2 (sACE2) inhibits the SARS-CoV-2 spike protein ACE2 interaction and has potential as a variant-independent therapeutic against SARS-CoV-2. Here, we introduce novel disulfide bonds in the wild-type sACE2-Fc by structure-guided mutagenesis, aiming to improve its stability. The stability of each mutant was assessed by a thermal shift assay to screen mutants with increased thermal stability. As a result, we identified a mutant sACE2-Fc with a significantly increased melting temperature. X-ray crystal structure determination of the sACE2 mutant confirmed the correct formation of the designed disulfide bond, and there were no significant structural disturbances. We also proved that the thermostable sACE2-Fc preserved the spike protein binding affinity comparable to the wild-type sACE2-Fc in both molecular and cellular environments, suggesting its therapeutic potential.
Assuntos
Enzima de Conversão de Angiotensina 2 , Dissulfetos , Estabilidade Proteica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Engenharia de Proteínas/métodos , Ligação Proteica , Cristalografia por Raios X , COVID-19/virologia , MutaçãoRESUMO
The N121 site on the spike protein of SARS-CoV-2 is associated with heme and its metabolite, biliverdin, which can affect antibody binding. Both N121T and N121S substitutions have been observed in natural conditions and in a hamster model of dual infection with SARS-CoV-2 and Influenza A virus. Serum pseudotype neutralization assays against HIV-1 particles carrying wild-type, N121T, and N121S spikes with immune mouse and human sera revealed that N121T and N121S mutations had a greater impact on serum neutralization than biliverdin treatment. Although N121T and N121S substitutions are not currently major SARS-CoV-2 variants of concern, this study could provide fundamental information to prepare for potential future mutations at the N121 site of SARS-CoV-2.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , Testes de Neutralização , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Humanos , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Camundongos , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/virologia , Substituição de Aminoácidos , MutaçãoRESUMO
SARS-CoV-2 cell-mediated immunity remains understudied during pregnancy in unvaccinated Black African women living with HIV (WLWH) from low- and middle-income countries. We investigated SARS-CoV-2-specific T-cell responses 1 month following infection in 24 HIV-uninfected women and 15 WLWH at any stage during pregnancy or postpartum. The full-length spike (FLS) glycoprotein and nucleocapsid (N) protein of wild-type (WT) SARS-CoV-2, as well as mutated spike protein regions found in the Omicron variant (B.1.1.529) were targeted by flow cytometry. WT-specific CD4+ and CD8+ T cells elicited similar FLS- and N-specific responses in HIV-uninfected women and WLWH. SARS-CoV-2-specific T-lymphocytes were predominantly TNF-α monofunctional in pregnant and postpartum women living with and without HIV, with fever cells producing either IFN-γ or IL-2. Furthermore, T-cell responses were unaffected by Omicron-specific spike mutations as similar responses between Omicron and the ancestral virus were detected for CD4+ and CD8+ T cells. Our results collectively demonstrate comparable T-cell responses between WLWH on antiretroviral therapy and HIV-uninfected pregnant and postpartum women who were naïve to Covid-19 vaccination. Additionally, we show that T cells from women infected with the ancestral virus, Beta variant (B.1.351), or Delta variant (B.1.617.2) can cross-recognize Omicron, suggesting an overall preservation of T-cell immunity.
Assuntos
COVID-19 , Infecções por HIV , Período Pós-Parto , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Humanos , Feminino , Gravidez , África do Sul/epidemiologia , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/virologia , Adulto , Infecções por HIV/imunologia , Infecções por HIV/virologia , Período Pós-Parto/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Linfócitos T CD8-Positivos/imunologia , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/virologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T/imunologiaRESUMO
The recently dominant SARS-CoV-2 Omicron JN.1 has evolved into multiple sublineages, with recurrent spike mutations R346T, F456L, and T572I, some of which exhibit growth advantages, such as KP.2 and KP.3. We investigated these mutations in JN.1, examining their individual and combined effects on immune evasion, ACE2 receptor affinity, and in vitro infectivity. F456L increased resistance to neutralization by human sera, including those after JN.1 breakthrough infections, and by RBD class-1 monoclonal antibodies, significantly altering JN.1 antigenicity. R346T enhanced ACE2-binding affinity and modestly boosted the infectivity of JN.1 pseudovirus, without a discernible effect on serum neutralization, while T572I slightly bolstered evasion of SD1-directed mAbs against JN.1's ancestor, BA.2, possibly by altering SD1 conformation. Importantly, expanding sublineages such as KP.2 containing R346T, F456L, and V1104L, showed similar neutralization resistance as JN.1 with R346T and F456L, suggesting V1104L does not appreciably affect antibody evasion. Furthermore, the hallmark mutation Q493E in KP.3 significantly reduced ACE2-binding affinity and viral infectivity, without noticeably impacting serum neutralization. Our findings illustrate how certain JN.1 mutations confer growth advantages in the population and could inform the design of the next COVID-19 vaccine booster.