RESUMEN
Porcine Rotavirus (PoRV) is a significant pathogen affecting swine-rearing regions globally, presenting a substantial threat to the economic development of the livestock sector. At present, no specific pharmaceuticals are available for this disease, and treatment options remain exceedingly limited. This study seeks to design a multi-epitope peptide vaccine for PoRV employing bioinformatics approaches to robustly activate T-cell and B-cell immune responses. Two antigenic proteins, VP7 and VP8*, were selected from PoRV, and potential immunogenic T-cell and B-cell epitopes were predicted using immunoinformatic tools. These epitopes were further screened according to non-toxicity, antigenicity, non-allergenicity, and immunogenicity criteria. The selected epitopes were linked with linkers to form a novel multi-epitope vaccine construct, with the PADRE sequence (AKFVAAWTLKAAA) and RS09 peptide attached at the N-terminus of the designed peptide chain to enhance the vaccine's antigenicity. Protein-protein docking of the vaccine constructs with toll-like receptors (TLR3 and TLR4) was conducted using computational methods, with the lowest energy docking results selected as the optimal predictive model. Subsequently, molecular dynamics (MD) simulation methods were employed to assess the stability of the protein vaccine constructs and TLR3 and TLR4 receptors. The results indicated that the vaccine-TLR3 and vaccine-TLR4 docking models remained stable throughout the simulation period. Additionally, the C-IMMSIM tool was utilized to determine the immunogenic triggering capability of the vaccine protein, demonstrating that the constructed vaccine protein could induce both cell-mediated and humoral immune responses, thereby playing a role in eliciting host immune responses. In conclusion, this study successfully constructed a multi-epitope vaccine against PoRV and validated the stability and efficacy of the vaccine through computational analysis. However, as the study is purely computational, experimental evaluation is required to validate the safety and immunogenicity of the newly constructed vaccine protein.
Asunto(s)
Antígenos Virales , Biología Computacional , Epítopos de Linfocito B , Epítopos de Linfocito T , Simulación de Dinámica Molecular , Infecciones por Rotavirus , Vacunas contra Rotavirus , Rotavirus , Vacunas de Subunidad , Animales , Porcinos , Rotavirus/inmunología , Rotavirus/genética , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/química , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/genética , Vacunas contra Rotavirus/inmunología , Vacunas contra Rotavirus/química , Vacunas contra Rotavirus/genética , Infecciones por Rotavirus/prevención & control , Infecciones por Rotavirus/inmunología , Infecciones por Rotavirus/virología , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/genética , Vacunas de Subunidad/química , Antígenos Virales/inmunología , Antígenos Virales/genética , Antígenos Virales/química , Simulación del Acoplamiento Molecular , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/virología , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/genética , Proteínas de la Cápside/química , Desarrollo de Vacunas , Inmunogenicidad VacunalRESUMEN
The available Epstein Barr virus vaccine has tirelessly harnessed the gp350 glycoprotein as its target epitope, but the result has not been preventive. Right here, we designed a global multi-epitope vaccine for EBV; with special attention to making sure all strains and preventive antigens are covered. Using a robust computational vaccine design approach, our proposed vaccine is armed with 6-16 mers linear B-cell epitopes, 4-9 mer CTL epitopes, and 8-15 mer HTL epitopes which are verified to induce interleukin 4, 10 & IFN-gamma. We employed deep computational mining coupled with expert intelligence in designing the vaccine, using human Beta defensin-3-which has been reported to induce the same TLRs as EBV-as the adjuvant. The tendency of the vaccine to cause autoimmune disorder is quenched by the assurance that the construct contains no EBNA-1 homolog. The protein vaccine construct exhibited excellent physicochemical attributes such as Aliphatic index 59.55 and GRAVY - 0.710; and a ProsaWeb Z score of - 3.04. Further computational analysis revealed the vaccine docked favorably with EBV indicted TLR 1, 2, 4 & 9 with satisfactory interaction patterns. With global coverage of 85.75% and the stable molecular dynamics result obtained for the best two interactions, we are optimistic that our nontoxic, non-allergenic multi-epitope vaccine will help to ameliorate the EBV-associated diseases-which include various malignancies, tumors, and cancers-preventively.
Asunto(s)
Proteínas de la Cápside , Herpesvirus Humano 4 , Herpesvirus Humano 4/inmunología , Humanos , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/química , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/prevención & control , Epítopos de Linfocito B/inmunología , Biología Computacional/métodos , Epítopos de Linfocito T/inmunología , Vacunas Virales/inmunología , Antígenos Virales/inmunología , Antígenos Virales/química , Modelos Moleculares , Simulación del Acoplamiento MolecularRESUMEN
The global spread of monkeypox has become a worldwide public healthcare issue. Therefore, there is an urgent need for accurate and sensitive detection methods to effectively control its spreading. Herein, we screened by phage display two peptides M4 (sequence: DPCGERICSIAL) and M6 (sequence: SCSSFLCSLKVG) with good affinity and specificity to monkeypox virus (MPXV) B21R protein. To simulate the state of the peptide in the phage and to avoid spatial obstacles of the peptide, GGGSK was added at the C terminus of M4 and named as M4a. Molecular docking shows that peptide M4a and peptide M6 are bound to different epitopes of B21R by hydrogen bonds and salt-bridge interactions, respectively. Then, peptide M4a was selected as the capture probe, phage M6 as the detection probe, and carbonized polymer dots (CPDs) as the fluorescent probe, and a colorimetric and fluorescent double-signal capture peptide/antigen/signal peptide-displayed phage sandwich ELISA triggered by horseradish peroxidase (HRP) through a simple internal filtration effect (IFE) was constructed. HRP catalyzes H2O2 to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to generate blue oxidized TMB, which can further quench the fluorescence of CPDs through IFE, enabling to detect MPXV B21R in colorimetric and fluorescent modes. The proposed simple immunoassay platform shows good sensitivity and reliability in MPXV B21R detection. The limit of detection for colorimetric and fluorescent modes was 27.8 and 9.14 pg/mL MPXV B21R, respectively. Thus, the established double-peptide sandwich-based dual-signal immunoassay provides guidance for the development of reliable and sensitive antigen detection capable of mutual confirmation, which also has great potential for exploring various analytical strategies for other respiratory virus surveillance.
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Ensayo de Inmunoadsorción Enzimática , Péptidos , Ensayo de Inmunoadsorción Enzimática/métodos , Péptidos/química , Antígenos Virales/inmunología , Antígenos Virales/análisis , Antígenos Virales/química , Simulación del Acoplamiento Molecular , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/metabolismo , Límite de Detección , Colorantes Fluorescentes/química , Biblioteca de Péptidos , Bencidinas/química , Colorimetría/métodosRESUMEN
African swine fever virus (ASFV) is a large double-stranded DNA virus with a complex structural architecture and encodes more than 150 proteins, where many are with unknown functions. E184L has been reported as one of the immunogenic ASFV proteins that may contribute to ASFV pathogenesis and immune evasion. However, the antigenic epitopes of E184L are not yet characterized. In this study, recombinant E184L protein was expressed in prokaryotic expression system and four monoclonal antibodies (mAbs), designated as 1A10, 2D2, 3H6, and 4C10 were generated. All four mAbs reacted specifically with ASFV infected cells. To identify the epitopes of the mAbs, a series of overlapped peptides of E184L were designed and expressed as maltose binding fusion proteins. Accordingly, the expressed fusion proteins were probed with each E184L mAb separately by using Western blot. Following a fine mapping, the minimal linear epitope recognized by mAb 1A10 was identified as 119IQRQGFL125, and mAbs 2D2, 3H6, and 4C10 recognized a region located between 153DPTEFF158. Alignment of amino acids of E184L revealed that the two linear epitopes are highly conserved among different ASFV isolates. Furthermore, the potential application of the two epitopes in ASFV diagnosis was assessed through epitope-based ELISA using 24 ASFV positive and 18 negative pig serum and the method were able to distinguish positive and negative samples, indicating the two epitopes are dominant antigenic sites. To our knowledge, this is the first study to characterize the B cell epitopes of the antigenic E184L protein of ASFV, offering valuable tools for future research, as well as laying a foundation for serological diagnosis and epitope-based marker vaccine development.
Asunto(s)
Virus de la Fiebre Porcina Africana , Anticuerpos Monoclonales , Anticuerpos Antivirales , Mapeo Epitopo , Epítopos de Linfocito B , Virus de la Fiebre Porcina Africana/inmunología , Virus de la Fiebre Porcina Africana/genética , Anticuerpos Monoclonales/inmunología , Epítopos de Linfocito B/inmunología , Animales , Anticuerpos Antivirales/inmunología , Porcinos , Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/virología , Ratones , Proteínas Virales/inmunología , Proteínas Virales/genética , Proteínas Virales/química , Antígenos Virales/inmunología , Antígenos Virales/genética , Antígenos Virales/química , Ratones Endogámicos BALB CRESUMEN
The ability to control protein conformations and dynamics through structure-based design has been useful in various scenarios, including engineering of viral antigens for vaccines. One effective design strategy is the substitution of residues to proline amino acids, which due to its unique cyclic side chain can favor and rigidify key backbone conformations. To provide the community with a means to readily identify and explore proline designs for target proteins of interest, we developed the Proscan web server. Proscan provides assessment of backbone angles, energetic and deep learning-based favorability scores, and other parameters for proline substitutions at each position of an input structure, along with interactive visualization of backbone angles and candidate substitution sites on structures. It identifies known favorable proline substitutions for viral antigens, and was benchmarked against datasets of proline substitution stability effects from deep mutational scanning and thermodynamic measurements. This tool can enable researchers to identify and prioritize designs for prospective vaccine antigen targets, or other designs to favor stability of key protein conformations. Proscan is available at: https://proscan.ibbr.umd.edu.
Asunto(s)
Internet , Prolina , Conformación Proteica , Programas Informáticos , Prolina/química , Sustitución de Aminoácidos , Termodinámica , Modelos Moleculares , Ingeniería de Proteínas/métodos , Antígenos Virales/química , Antígenos Virales/genética , Antígenos Virales/inmunología , Aprendizaje ProfundoRESUMEN
Influenza causes significant morbidity and mortality. As an alternative approach to current seasonal vaccines, the computationally optimized broadly reactive antigen (COBRA) platform has been previously applied to hemagglutinin (HA). This approach integrates wild-type HA sequences into a single immunogen to expand the breadth of accessible antibody epitopes. Adding to previous studies of H1, H3, and H5 COBRA HAs, we define the structural features of another H1 subtype COBRA, X6, that incorporates HA sequences from before and after the 2009 H1N1 influenza pandemic. We determined structures of this antigen alone and in complex with COBRA-specific as well as broadly reactive and functional antibodies, analyzing its antigenicity. We found that X6 possesses features representing both historic and recent H1 HA strains, enabling binding to both head- and stem-reactive antibodies. Overall, these data confirm the integrity of broadly reactive antibody epitopes of X6 and contribute to design efforts for a next-generation vaccine.
Asunto(s)
Anticuerpos Antivirales , Glicoproteínas Hemaglutininas del Virus de la Influenza , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Humanos , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/química , Modelos Moleculares , Antígenos Virales/inmunología , Antígenos Virales/química , Antígenos Virales/genética , Epítopos/inmunología , Epítopos/química , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/química , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/química , Gripe Humana/inmunología , Gripe Humana/virología , Cristalografía por Rayos X , Unión ProteicaRESUMEN
The signal sequence played a crucial role in the efficacy of mRNA vaccines against virus pandemic by influencing antigen translation. However, limited research had been conducted to compare and analyze the specific mechanisms involved. In this study, a novel approach was introduced by substituting the signal sequence of the mRNA antigen to enhance its immune response. Computational simulations demonstrated that various signal peptides differed in their binding capacities with the signal recognition particle (SRP) 54 M subunit, which positively correlated with antigen translation efficiency. Our data revealed that the signal sequences of tPA and IL-6-modified receptor binding domain (RBD) mRNA vaccines sequentially led to higher antigen expression and elicited more robust humoral and cellular immune protection against the SARS-CoV-2 compared to the original signal sequence. By highlighting the importance of the signal sequence, this research provided a foundational and safe approach for ongoing modifications in signal sequence-antigen design, aiming to optimize the efficacy of mRNA vaccines.
Asunto(s)
Señales de Clasificación de Proteína , SARS-CoV-2 , Vacunas de ARNm , Animales , Ratones , SARS-CoV-2/inmunología , COVID-19/prevención & control , COVID-19/inmunología , Ratones Endogámicos BALB C , ARN Mensajero/genética , Vacunas contra la COVID-19/inmunología , Femenino , Humanos , Antígenos Virales/inmunología , Antígenos Virales/genética , Antígenos Virales/química , Anticuerpos Antivirales/inmunología , Inmunidad Humoral , Vacunas Sintéticas/inmunología , Inmunidad CelularRESUMEN
Variants of coronavirus porcine epidemic diarrhea virus (PEDV) frequently emerge, causing an incomplete match between the vaccine and variant strains, which affects vaccine efficacy. Designing vaccines with rapidly replaceable antigens and high efficacy is a promising strategy for the prevention of infection with PEDV variant strains. In our study, three different types of self-assembled nanoparticles (nps) targeting receptor-binding N-terminal domain (NTD) and C-terminal domain (CTD) of S1 protein, named NTDnps, CTDnps, and NTD/CTDnps, were constructed and evaluated as vaccine candidates against PEDV. NTDnps and CTDnps vaccines mediated significantly higher neutralizing antibody (NAb) titers than NTD and CTD recombinant proteins in mice. The NTD/CTDnps in varying ratios elicited significantly higher NAb titers when compared with NTDnps and CTDnps alone. The NTD/CTDnps (3:1) elicited NAb with titers up to 92.92% of those induced by the commercial vaccine. Piglets immunized with NTD/CTDnps (3:1) achieved a passive immune protection rate of 83.33% of that induced by the commercial vaccine. NTD/CTDnps (3:1) enhanced the capacity of mononuclear macrophages and dendritic cells to take up and present antigens by activating major histocompatibility complex I and II molecules to stimulate humoral and cellular immunity. These data reveal that a combination of S1-NTD and S1-CTD antigens targeting double receptor-binding domains strengthens the protective immunity of nanoparticle vaccines against PEDV. Our findings will provide a promising vaccine candidate against PEDV.
Asunto(s)
Nanopartículas , Virus de la Diarrea Epidémica Porcina , Vacunas Virales , Virus de la Diarrea Epidémica Porcina/inmunología , Animales , Nanopartículas/química , Porcinos , Ratones , Vacunas Virales/inmunología , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/inmunología , Ratones Endogámicos BALB C , Antígenos Virales/inmunología , Antígenos Virales/química , Anticuerpos Neutralizantes/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Dominios Proteicos/inmunología , Femenino , NanovacunasRESUMEN
The rotavirus capsid protein VP6 forms the middle of three protein layers and is responsible for many critical steps in the viral life cycle. VP6 as a structural protein can be used in various applications including as a subunit vaccine component. The head domain of VP6 (VP6H) contains key sequences that allow the protein to trimerize and that represent epitopes that are recognized by human antibodies in the viral particle. The domain is rich in ß-sheet secondary structures. Here, VP6H was solubilised from bacterial inclusion bodies and purified using a single affinity chromatography step. Spectral (far-UV circular dichroism and intrinsic tryptophan fluorescence) analysis revealed that the purified domain had native-like secondary and tertiary structures. The domain could maintain structure up to 44°C during thermal denaturation following which structural changes result in an intermediate forming and finally irreversible aggregation and denaturation. The chemical denaturation with urea and guanidinium hydrochloride produces intermediates that represent a loss in the cooperativity. The VP6H domain is stable and can fold to produce its native structure in the absence of the VP6 base domain but cannot be defined as an independent folding unit.
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Antígenos Virales , Proteínas de la Cápside , Rotavirus , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Antígenos Virales/química , Antígenos Virales/genética , Rotavirus/química , Desnaturalización Proteica , Dominios Proteicos , Dicroismo Circular , Pliegue de Proteína , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The convergence strategies of antigenic subunits and synthetic nanoparticle scaffold platform improve the vaccine production efficiency and enhance vaccine-induced immunogenicity. Selecting the appropriate nanoparticle scaffold is crucial to controlling target antigens immunologically. Lumazine synthase (LS) is an attractive candidate for a vaccine display system due to its thermostability, modification tolerance, and morphological plasticity. Here, the first development of a multivalent thermostable scaffold, LS-SUMO (SUMO, small ubiquitin-likemodifier), and a divalent nanovaccine covalently conjugated with Chikungunya virus E2 and Zika virus EDIII antigens, is reported. Compared with antigen monomers, LS-SUMO nanoparticle vaccines elicit a higher humoral response and neutralizing antibodies against both antigen targets in mouse sera. Mice immunized with LS-SUMO conjugates produce CD4+ T cell-mediated Th2-biased responses and promote humoral immunity. Importantly, LS-SUMO conjugates possess equivalent humoral immunogenicity after heat treatment. Taken together, LS-SUMO is a powerful biotargeting nanoplatform with high-yield production, thermal stability and opens a new avenue for multivalent presentation of various antigens.
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Virus Chikungunya , Virus Zika , Animales , Ratones , Virus Chikungunya/inmunología , Virus Zika/inmunología , Nanopartículas/química , Vacunas Virales/inmunología , Vacunas Virales/química , Ratones Endogámicos BALB C , Femenino , Fiebre Chikungunya/inmunología , Fiebre Chikungunya/prevención & control , Inmunidad Humoral/efectos de los fármacos , Anticuerpos Neutralizantes/inmunología , Antígenos Virales/inmunología , Antígenos Virales/química , Nanovacunas , Complejos MultienzimáticosRESUMEN
A major challenge in creating universal influenza vaccines is to focus immune responses away from the immunodominant, variable head region of hemagglutinin (HA-head) and toward the evolutionarily conserved stem region (HA-stem). Here we introduce an approach to control antigen orientation via site-specific insertion of aspartate residues that facilitates antigen binding to alum. We demonstrate the generalizability of this approach with antigens from Ebola, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses and observe enhanced neutralizing antibody responses in all cases. We then reorient an H2 HA in an 'upside-down' configuration to increase the exposure and immunogenicity of HA-stem. The reoriented H2 HA (reoH2HA) on alum induced stem-directed antibodies that cross-react with both group 1 and group 2 influenza A subtypes. Electron microscopy polyclonal epitope mapping (EMPEM) revealed that reoH2HA (group 1) elicits cross-reactive antibodies targeting group 2 HA-stems. Our results highlight antigen reorientation as a generalizable approach for designing epitope-focused vaccines.
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Vacunas contra la Influenza , SARS-CoV-2 , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/química , Humanos , SARS-CoV-2/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Antígenos Virales/inmunología , Antígenos Virales/química , Reacciones Cruzadas/inmunología , Ratones , Epítopos/inmunología , Epítopos/química , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/virología , Ebolavirus/inmunología , Virus de la Influenza A/inmunología , Compuestos de Alumbre/química , Mapeo Epitopo , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/químicaRESUMEN
IMPORTANCE: AAVs are extensively studied as promising therapeutic gene delivery vectors. In order to circumvent pre-existing antibodies targeting primate-based AAV capsids, the AAAV capsid was evaluated as an alternative to primate-based therapeutic vectors. Despite the high sequence diversity, the AAAV capsid was found to bind to a common glycan receptor, terminal galactose, which is also utilized by other AAVs already being utilized in gene therapy trials. However, contrary to the initial hypothesis, AAAV was recognized by approximately 30% of human sera tested. Structural and sequence comparisons point to conserved epitopes in the fivefold region of the capsid as the reason determinant for the observed cross-reactivity.
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Antígenos Virales , Cápside , Parvovirinae , Animales , Humanos , Cápside/química , Proteínas de la Cápside/química , Dependovirus/química , Vectores Genéticos , Primates/genética , Antígenos Virales/química , Parvovirinae/químicaRESUMEN
BACKGROUND: We characterized and identified the genetic and antigenic variations of circulating rotavirus strains in comparison to used rotavirus vaccines. METHODS: Rotavirus-positive samples (n = 231) were collected and analyzed. The VP7 and VP4 genes were sequenced and analyzed against the rotavirus vaccine strains. Antigenic variations were illustrated on the three-dimensional models of surface proteins. RESULTS: In all, 59.7% of the hospitalized children were vaccinated, of which only 57.2% received two doses. There were no significant differences between the vaccinated and non-vaccinated groups in terms of clinical outcome. The G3 was the dominant genotype (40%) regardless of vaccination status. Several amino acid changes were identified in the VP7 and VP4 antigenic epitopes compared to the licensed vaccines. The highest variability was seen in the G3 (6 substitutions) and P[4] (11 substitutions) genotypes in comparison to RotaTeq®. In comparison to Rotarix®, G1 strains possessed three amino acid changes in 7-1a and 7-2 epitopes while P[8] strains possessed five amino acid changes in 8-1 and 8-3 epitopes. CONCLUSIONS: The current use of Rotarix® vaccine might not be effective in preventing the infection due to the higher numbers of G3-associated cases. The wide range of mutations in the antigenic epitopes compared to vaccine strains may compromise the vaccine's effectiveness. IMPACT: The reduced rotavirus vaccine effectiveness necessitate regular evaluation of the vaccine content to ensure optimal protection. We characterized and identified the genetic and antigenic variations of circulating rotavirus strains in comparison to the Rotarix vaccine strain that is used in Qatar. The study highlight the importance for regular monitoring of emerging rotavirus variants and their impact on vaccine effectiveness in young children.
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Infecciones por Rotavirus , Rotavirus , Humanos , Niño , Lactante , Preescolar , Rotavirus/genética , Infecciones por Rotavirus/epidemiología , Infecciones por Rotavirus/prevención & control , Qatar , Antígenos Virales/genética , Antígenos Virales/química , Proteínas de la Cápside/genética , Genotipo , Epítopos/genéticaRESUMEN
Human metapneumovirus (hMPV) is a leading cause of morbidity and hospitalization among children worldwide, however, no vaccines or therapeutics are currently available for hMPV disease prevention and treatment. The hMPV fusion (F) protein is the sole target of neutralizing antibodies. To map the immunodominant epitopes on the hMPV F protein, we isolated a panel of human monoclonal antibodies (mAbs), and the mAbs were assessed for binding avidity, neutralization potency, and epitope specificity. We found the majority of the mAbs target diverse epitopes on the hMPV F protein, and we discovered multiple mAb binding approaches for antigenic site III. The most potent mAb, MPV467, which had picomolar potency, was examined in prophylactic and therapeutic mouse challenge studies, and MPV467 limited virus replication in mouse lungs when administered 24 h before or 72 h after viral infection. We determined the structure of MPV467 in complex with the hMPV F protein using cryo-electron microscopy to a resolution of 3.3 Å, which revealed a complex novel prefusion-specific epitope overlapping antigenic sites II and V on a single protomer. Overall, our data reveal insights into the immunodominant antigenic epitopes on the hMPV F protein, identify a mAb therapy for hMPV F disease prevention and treatment, and provide the discovery of a prefusion-specific epitope on the hMPV F protein.
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Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Antígenos Virales , Metapneumovirus , Infecciones por Paramyxoviridae , Proteínas Virales de Fusión , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/química , Anticuerpos Antivirales/aislamiento & purificación , Anticuerpos Antivirales/uso terapéutico , Antígenos Virales/química , Antígenos Virales/inmunología , Microscopía por Crioelectrón , Epítopos/inmunología , Humanos , Metapneumovirus/inmunología , Ratones , Infecciones por Paramyxoviridae/prevención & control , Prevención Primaria , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/inmunologíaRESUMEN
Regulatory T cells (Tregs) are critical for regulating immunopathogenic responses in a variety of infections, including infection of mice with JHM strain of mouse hepatitis virus (JHMV), a neurotropic coronavirus that causes immune-mediated demyelinating disease. Although virus-specific Tregs are known to mitigate disease in this infection by suppressing pathogenic effector T cell responses of the same specificity, it is unclear whether these virus-specific Tregs form memory populations and persist similar to their conventional T cell counterparts of the same epitope specificity. Using congenically labeled JHMV-specific Tregs, we found that virus-specific Tregs persist long-term after murine infection, through at least 180 d postinfection and stably maintain Foxp3 expression. We additionally demonstrate that these cells are better able to proliferate and inhibit virus-specific T cell responses postinfection than naive Tregs of the same specificity, further suggesting that these cells differentiate into memory Tregs upon encountering cognate Ag. Taken together, these data suggest that virus-specific Tregs are able to persist long-term in the absence of viral Ag as memory Tregs.
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Infecciones por Coronavirus , Virus de la Hepatitis Murina , Animales , Antígenos Virales/química , Antígenos Virales/inmunología , Ratones , Linfocitos T ReguladoresRESUMEN
Nipah virus (NiV) and Hendra virus (HeV) are zoonotic henipaviruses (HNVs) responsible for outbreaks of encephalitis and respiratory illness. The entry of HNVs into host cells requires the attachment (G) and fusion (F) glycoproteins, which are the main targets of antibody responses. To understand viral infection and host immunity, we determined a cryo-electron microscopy structure of the NiV G homotetrameric ectodomain in complex with the nAH1.3 broadly neutralizing antibody Fab fragment. We show that a cocktail of two nonoverlapping G-specific antibodies neutralizes NiV and HeV synergistically and limits the emergence of escape mutants. Analysis of polyclonal serum antibody responses elicited by vaccination of macaques with NiV G indicates that the receptor binding head domain is immunodominant. These results pave the way for implementing multipronged therapeutic strategies against these deadly pathogens.
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Antígenos Virales , Glicoproteínas , Virus Nipah , Proteínas Virales , Acoplamiento Viral , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Antígenos Virales/química , Glicoproteínas/química , Glicoproteínas/inmunología , Humanos , Virus Nipah/genética , Virus Nipah/inmunología , Multimerización de Proteína , Proteínas Virales/química , Proteínas Virales/inmunología , Internalización del VirusRESUMEN
SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.
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Anticuerpos Antivirales/sangre , Antígenos Virales/química , COVID-19/sangre , COVID-19/diagnóstico , Proteínas de la Nucleocápside de Coronavirus/química , Inmunoglobulina G/sangre , Cuerpos de Inclusión/química , Replegamiento Proteico , SARS-CoV-2/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , COVID-19/virología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Presión Hidrostática , Inmunoglobulina G/inmunología , Fosfoproteínas/química , Fosfoproteínas/inmunología , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , SolubilidadRESUMEN
Coronavirus disease 2019, caused by SARS-CoV-2, remains an on-going pandemic, partly due to the emergence of variant viruses that can "break-through" the protection of the current vaccines and neutralizing antibodies (nAbs), highlighting the needs for broadly nAbs and next-generation vaccines. We report an antibody that exhibits breadth and potency in binding the receptor-binding domain (RBD) of the virus spike glycoprotein across SARS coronaviruses. Initially, a lead antibody was computationally discovered and crystallographically validated that binds to a highly conserved surface of the RBD of wild-type SARS-CoV-2. Subsequently, through experimental affinity enhancement and computational affinity maturation, it was further developed to bind the RBD of all concerning SARS-CoV-2 variants, SARS-CoV-1 and pangolin coronavirus with pico-molar binding affinities, consistently exhibited strong neutralization activity against wild-type SARS-CoV-2 and the Alpha and Delta variants. These results identify a vulnerable target site on coronaviruses for development of pan-sarbecovirus nAbs and vaccines.
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Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/metabolismo , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Antígenos Virales/química , Antígenos Virales/genética , Anticuerpos ampliamente neutralizantes/genética , Anticuerpos ampliamente neutralizantes/metabolismo , Cristalografía por Rayos X , Epítopos/química , Epítopos/inmunología , Humanos , Fragmentos de Inmunoglobulinas/inmunología , Simulación del Acoplamiento Molecular , Método de Montecarlo , Pruebas de Neutralización , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Dominios Proteicos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Adeno-associated viruses (AAV) serve as vectors for therapeutic gene delivery. AAV9 vectors have been FDA approved, as Zolgensma, for the treatment of spinal muscular atrophy and are being evaluated in clinical trials for the treatment of neurotropic and musculotropic diseases. A major hurdle for AAV-mediated gene delivery is the presence of preexisting neutralizing antibodies in 40 to 80% of the general population. These preexisting antibodies can reduce therapeutic efficacy through viral neutralization and the size of the patient cohort eligible for treatment. In this study, cryo-electron microscopy and image reconstruction were used to define the epitopes of five anti-AAV9 monoclonal antibodies (MAbs), ADK9, HL2368, HL2370, HL2372, and HL2374, on the capsid surface. Three of these, ADK9, HL2370, and HL2374, bound to or near the icosahedral 3-fold axes, HL2368 bound to the 2/5-fold wall, and HL2372 bound to the region surrounding the 5-fold axes. Pseudoatomic modeling enabled the mapping and identification of antibody contact amino acids on the capsid, including S454 and P659. These epitopes overlap previously defined parvovirus antigenic sites. Capsid amino acids critical for the interactions were confirmed by mutagenesis, followed by biochemical assays testing recombinant AAV9 (rAAV9) variants capable of escaping recognition and neutralization by the parental MAbs. These variants retained parental tropism and had similar or improved transduction efficiency compared to AAV9. These engineered rAAV9 variants could expand the patient cohort eligible for AAV9-mediated gene delivery by avoiding preexisting circulating neutralizing antibodies. IMPORTANCE The use of recombinant adeno-associated viruses (rAAVs) as delivery vectors for therapeutic genes is becoming increasingly popular, especially following the FDA approval of Luxturna and Zolgensma, based on serotypes AAV2 and AAV9, respectively. However, high-titer anti-AAV neutralizing antibodies in the general population exempt patients from treatment. The goal of this study is to circumvent this issue by creating AAV variant vectors not recognized by preexisting neutralizing antibodies. The mapping of the antigenic epitopes of five different monoclonal antibodies (MAbs) on AAV9, to recapitulate a polyclonal response, enabled the rational design of escape variants with minimal disruption to cell tropism and gene expression. This study, which included four newly developed and now commercially available MAbs, provides a platform for the engineering of rAAV9 vectors that can be used to deliver genes to patients with preexisting AAV antibodies.
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Antígenos Virales/química , Antígenos Virales/inmunología , Dependovirus/inmunología , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos/inmunología , Sitios de Unión , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Línea Celular , Microscopía por Crioelectrón , Dependovirus/ultraestructura , Mapeo Epitopo/métodos , Humanos , Modelos Moleculares , Pruebas de Neutralización , Unión Proteica , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a large number of mutations in its genome have been reported. Some of the mutations occur in noncoding regions without affecting the pathobiology of the virus, while mutations in coding regions are significant. One of the regions where a mutation can occur, affecting the function of the virus is at the receptor-binding domain (RBD) of the spike protein. RBD interacts with angiotensin-converting enzyme 2 (ACE2) and facilitates the entry of the virus into the host cells. There is a lot of focus on RBD mutations, especially the displacement of N501Y which is observed in the UK/Kent, South Africa, and Brazilian lineages of SARS-CoV-2. Our group utilizes computational biology approaches such as immunoinformatics, protein-protein interaction analysis, molecular dynamics, free energy computation, and tertiary structure analysis to disclose the consequences of N501Y mutation at the molecular level. Surprisingly, we discovered that this mutation reduces the immunogenicity of the spike protein; also, displacement of Asn with Tyr reduces protein compactness and significantly increases the stability of the spike protein and its affinity to ACE2. Moreover, following the N501Y mutation secondary structure and folding of the spike protein changed dramatically.