RESUMEN

Limited knowledge exists on the quality of polyclonal antibody responses generated following Marburg virus (MARV) infection and its evolution in survivors. In this study, we evaluate MARV proteome-wide antibody repertoire longitudinally in convalescent phase approximately every six months for five years following MARV infection in ten human survivors. Differential kinetics were observed for IgM vs IgG vs IgA epitope diversity, antibody binding, antibody affinity maturation and Fc-receptor interaction to MARV proteins. Durability of MARV-neutralizing antibodies is low in survivors. MARV infection induces a diverse epitope repertoire with predominance against GP, VP40, VP30 and VP24 that persisted up to 5 years post-exposure. However, the IgM and IgA repertoire declines over time. Within MARV-GP, IgG recognize antigenic sites predominantly in the amino-terminus, wing domain and GP2-heptad repeat. Interestingly, MARV infection generates robust durable FcɣRI, FcɣRIIA and FcɣRIIIA IgG-Fc receptor interactions. Immunization with immunodominant MARV epitopes reveals conserved wing region between GP1 and GP2, induces neutralizing antibodies against MARV. These findings demonstrate that MARV infection generates a diverse, long-lasting, non-neutralizing, IgG antibody repertoire that perturbs disease by FcɣR activity. This information, along with discovery of neutralizing immunogen in wing domain, could aid in development of effective therapeutics and vaccines against Marburg virus.

Asunto(s)

Anticuerpos Neutralizantes , Anticuerpos Antivirales , Enfermedad del Virus de Marburg , Marburgvirus , Proteoma , Marburgvirus/inmunología , Humanos , Enfermedad del Virus de Marburg/inmunología , Enfermedad del Virus de Marburg/virología , Anticuerpos Antivirales/inmunología , Anticuerpos Neutralizantes/inmunología , Proteoma/inmunología , Femenino , Vacunas Virales/inmunología , Inmunoglobulina G/inmunología , Masculino , Epítopos/inmunología , Adulto , Inmunoglobulina M/inmunología , Persona de Mediana Edad , Estudios Longitudinales , Inmunoglobulina A/inmunología , Desarrollo de Vacunas , Proteínas del Envoltorio Viral/inmunología

RESUMEN

The human respiratory syncytial virus (RSV) is a significant health concern, particularly for infants, young children, and the elderly. This virus is known to evolve continuously due to environmental factors and herd immunity. In light of this, our study aimed to analyze the genetic variability of the G protein in RSV-A and RSV-B genotypes in Kuwait from 2020 to 2022. Between January 2020 and September 2022, we collected 490 respiratory samples from hospitalized patients with acute respiratory tract infections. These samples were tested and confirmed positive for RSV using multiplex Real-Time PCR. Subsequently, the samples underwent nucleic acid sequencing using the advanced Nanopore sequencing technology to analyze the full-length G gene. Sequence analysis showed that 64 isolates (76%) were RSV-A, and 20 isolates (24%) were RSV-B. The G genes of RSV-A belonged to genotype GA2.3.5, while all the RSV-B genotypes belonged to GB5.0.5a. New lineages and sub-lineages of RSV-A and RSV-B were detected, indicating the circulation of new strains in Kuwait. Many unique and new amino acid changes, including insertions, were found in the G proteins of Kuwaiti isolates, with the highest variability in the second hypervariable region. An increased number of N and O-linked glycosylation sites were also identified in the G protein, which could speculate to alter the antigenicity of RSV. The identified changes in the G protein of RSV-A and RSV-B genotypes might result from immune pressure and could affect the antigenic characteristics of circulating strains in Kuwait. This could potentially lead to new RSV variants that can evade the immune response. Our in-depth analysis of the G proteins of both RSV-A and RSV-B could aid in the development of more potent treatments and vaccines.

Asunto(s)

Variación Genética , Genotipo , Filogenia , Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Humanos , Kuwait , Virus Sincitial Respiratorio Humano/genética , Virus Sincitial Respiratorio Humano/clasificación , Virus Sincitial Respiratorio Humano/aislamiento & purificación , Virus Sincitial Respiratorio Humano/inmunología , Infecciones por Virus Sincitial Respiratorio/virología , Glicosilación , Lactante , Preescolar , Femenino , Masculino , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunología , Adulto , Niño , Persona de Mediana Edad , Anciano , Adulto Joven , Adolescente , Infecciones del Sistema Respiratorio/virología , Proteínas del Envoltorio Viral/genética

RESUMEN

Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 Å resolution cryoelectron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses.

Asunto(s)

Citomegalovirus , Proteínas del Envoltorio Viral , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Citomegalovirus/inmunología , Humanos , Animales , Ratones , Microscopía por Crioelectrón , Conformación Proteica , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Internalización del Virus , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Estabilidad Proteica , Vacunas contra Citomegalovirus/inmunología , Sustitución de Aminoácidos , Modelos Moleculares

RESUMEN

Zika virus (ZIKV; family, Flaviviridae), which causes congenital Zika syndrome, Guillain-Barré Syndrome, and other severe diseases, is transmitted mainly by mosquitoes; however, the virus can be transmitted through other routes. Among the three structural and seven nonstructural proteins, the surface envelope (E) protein of ZIKV plays a critical role in viral entry and pathogenesis, making it a key target for the development of effective entry inhibitors. This review article describes the life cycle, genome, and encoded proteins of ZIKV, illustrates the structure and function of the ZIKV E protein, summarizes E protein-targeting entry inhibitors (with a focus on those based on natural products and small molecules), and highlights challenges that may potentially hinder the development of effective inhibitors of ZIKV infection. Overall, the article will provide useful guidance for further development of safe and potent ZIKV entry inhibitors targeting the viral E protein.

Asunto(s)

Antivirales , Proteínas del Envoltorio Viral , Internalización del Virus , Infección por el Virus Zika , Virus Zika , Virus Zika/efectos de los fármacos , Virus Zika/fisiología , Internalización del Virus/efectos de los fármacos , Humanos , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Infección por el Virus Zika/virología , Infección por el Virus Zika/tratamiento farmacológico , Antivirales/farmacología , Animales

RESUMEN

Human herpes viruses (HHV) are ubiquitous and have been implicated in numerous long-term health conditions. Since the association between viral exposure and long-term health impacts is partially influenced by variation in human leukocyte antigen (HLA) genes, we evaluated in silico the binding affinities of 9 HHV envelope glycoproteins with 127 common HLA Class I and Class II molecules. The findings show substantial variability in HHV binding affinity across viruses, HLA Class, HLA genes, and HLA alleles. Specific findings were as follows: (1) the predicted binding affinities of HHVs were characterized by four distinct groupings-[HHV1, HHV2], [HHV3, HHV4, HHV5], [HHV6A], [HHV6B, HHV7, HHV8]-with relatively lower binding affinities for HHV1, HHV2, and HHV6a compared to other HHVs; (2) significantly higher binding affinity was found for HLA Class I relative to Class II; (3) analyses within each class demonstrated that alleles of the C gene (for Class I) and DRB1 gene (for Class II) had the highest binding affinities; and (4) for each virus, predicted binding affinity to specific alleles varied, with HHV6a having the lowest affinity for HHV-HLA complexes, and HHV3, HHV4, and HHV5 having the highest. Since HLA-antigen binding is the first step in initiating an immune response to foreign antigens, these relative differences in HHV binding affinities are likely to influence long-term health impacts such that the cells infected with viruses associated with higher binding affinities across common HLA alleles may be more reduced in numbers, thereby lowering the potential for long-term sequelae of their infections.

Asunto(s)

Alelos , Proteínas del Envoltorio Viral , Humanos , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Herpesviridae/inmunología , Herpesviridae/genética , Antígenos HLA/genética , Antígenos HLA/inmunología , Unión Proteica , Inmunogenética , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología

RESUMEN

Multivalent live-attenuated or inactivated vaccines are often used to control the bovine viral diarrhea disease (BVD). Still, they retain inherent disadvantages and do not provide the expected protection. This study developed a new vaccine prototype, including the external segment of the E2 viral protein from five different subgenotypes selected after a massive screening. The E2 proteins of every subgenotype (1aE2, 1bE2, 1cE2, 1dE2, and 1eE2) were produced in mammalian cells and purified by IMAC. An equimolar mixture of E2 proteins formulated in an oil-in-water adjuvant made up the vaccine candidate, inducing a high humoral response at 50, 100, and 150 µg doses in sheep. A similar immune response was observed in bovines at 50 µg. The cellular response showed a significant increase in the transcript levels of relevant Th1 cytokines, while those corresponding to the Th2 cytokine IL-4 and the negative control were similar. High levels of neutralizing antibodies against the subgenotype BVDV1a demonstrated the effectiveness of our vaccine candidate, similar to that observed in the sera of animals vaccinated with the commercial vaccine. These results suggest that our vaccine prototype could become an effective recombinant vaccine against the BVD.

Asunto(s)

Anticuerpos Antivirales , Diarrea Mucosa Bovina Viral , Vacunas de Subunidad , Vacunas Sintéticas , Vacunas Virales , Animales , Bovinos , Vacunas Virales/inmunología , Vacunas de Subunidad/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Vacunas Sintéticas/inmunología , Diarrea Mucosa Bovina Viral/prevención & control , Diarrea Mucosa Bovina Viral/inmunología , Diarrea Mucosa Bovina Viral/virología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Ovinos , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/genética , Citocinas/metabolismo , Virus de la Diarrea Viral Bovina/inmunología , Virus de la Diarrea Viral Bovina/genética , Virus de la Diarrea Viral Bovina Tipo 1/inmunología , Virus de la Diarrea Viral Bovina Tipo 1/genética

RESUMEN

An efficient gene transfer and expression tool is lacking for shrimps and shrimp cells. To solve this, this study has developed a shrimp DNA virus-mediated gene transfer and expression system, consisting of insect Sf9 cells for viral packaging, the shrimp viral vector of pUC19-IHHNV-PH-GUS and the baculoviral vector of Bacmid or Bacmid-VP28 encoding the shrimp WSSV envelope protein VP28. The pUC19-IHHNV-PH-GUS vector was constructed by assembling the genomic DNA of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), which has shortened inverted terminal repeats, into a pUC19 backbone, and then an expression cassette of baculoviral polyhedron (PH) promoter-driven GUS (ß-glucuronidase) reporter gene was inserted immediately downstream of IHHNV for proof-of-concept. It was found that the viral vector of pUC19-IHHNV-PH-GUS could be successfully packaged into IHHNV-like infective virions in the Sf9 cells, and the gene transfer efficiency of this system was evaluated and verified in three systems of Sf9 cells, shrimp hemolymph cells and tissues of infected shrimps, but the GUS expression could only be detected in cases where the viral vector was co-transfected or co-infected with a baculovirus of Bacmid or Bacmid-VP28 due to the Bacmid-dependence of the PH promoter. Moreover, the packaging and infection efficiencies could be significantly improved when Bacmid-VP28 was used instead of Bacmid.

Asunto(s)

Técnicas de Transferencia de Gen , Vectores Genéticos , Penaeidae , Animales , Penaeidae/virología , Penaeidae/genética , Células Sf9 , Vectores Genéticos/genética , Baculoviridae/genética , Regiones Promotoras Genéticas , Spodoptera/virología , Densovirinae/genética , Expresión Génica , Virus del Síndrome de la Mancha Blanca 1/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Glucuronidasa/genética , Glucuronidasa/metabolismo

RESUMEN

Atypical porcine pestivirus (APPV) can cause congenital tremor type A-II in neonatal piglets, posing a significant threat to swine herd health globally. Our previous study demonstrated that the Mut domains, comprising 112 amino acids at the N-terminus, are the primary functional regions of the E2 protein of APPV. This study identified 14 host cellular proteins that exhibit potential interactions with the Mut domains of the E2 protein using yeast two-hybrid screening. Using bioinformatics analysis, we discovered that the Mut domains of the E2 protein might exert regulatory effects on apoptosis by modulating energy metabolism within the mitochondria. We also conducted co-immunoprecipitation, glutathione S-transferase pull-down, and immunofluorescence assays to confirm the interaction between the Mut domains of the E2 protein and cathepsin H and signal sequence receptor subunit 4 (SSR4). Ultimately, SSR4 enhanced APPV replication in vitro. In summary, our study successfully elucidated the interactions between the Mut domains of the E2 protein and host cell protein, predicted the potential pathways implicated in these interactions, and demonstrated SSR4 involvement in APPV infection. These significant findings contribute valuable knowledge toward a deeper understanding of APPV pathogenesis and the role of the Mut domains of the E2 protein in this intricate process.

Asunto(s)

Infecciones por Pestivirus , Pestivirus , Animales , Pestivirus/genética , Pestivirus/metabolismo , Porcinos , Infecciones por Pestivirus/veterinaria , Infecciones por Pestivirus/virología , Infecciones por Pestivirus/genética , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/genética , Enfermedades de los Porcinos/metabolismo , Interacciones Huésped-Patógeno/genética , Dominios Proteicos , Replicación Viral/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Humanos , Mapas de Interacción de Proteínas/genética

RESUMEN

BACKGROUND: Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonotic disease that presents with severe hemorrhagic manifestations and is associated with significant fatality rates. The causative agent, Crimean-Congo Hemorrhagic Fever Virus (CCHFV), is a high-priority pathogen identified by the World Health Organization with no approved vaccine or specific treatment available. In addition, there is a critical need for enhanced diagnostic tools to improve public health awareness, prevention measures, and disease control strategies. METHODS: We designed plasmids to enable the purification of soluble CCHFV glycoprotein Gc expressed in mammalian 293 F cells, followed by purification using affinity and size exclusion chromatography. The purified antigen was analyzed by SDS-PAGE and Western blotting to confirm its reactivity to antibodies from CCHF survivors. Additionally, an in-house indirect ELISA was developed using the purified Gc as a coating antigen. RESULTS: The optimized expression system successfully produced soluble and pure Gc antigen after affinity chromatography. The protein showed specific reactivity with CCHFV-positive serum antibodies in Western blot analysis. The indirect ELISA assay demonstrated high efficacy in distinguishing between CCHFV-positive and -negative serum samples, indicating its potential as a valuable diagnostic tool. Size exclusion chromatography further confirmed the presence of aggregates in our protein preparation. CONCLUSIONS: The purified Gc antigen shows promise for developing direct diagnostic assays for CCHFV. The antigen's suitability for subunit vaccine development and its application as bait for monoclonal antibody isolation from survivors could be investigated further. This work lays the foundation for future research into the development of rapid diagnostic tests for field deployment.

Asunto(s)

Virus de la Fiebre Hemorrágica de Crimea-Congo , Proteínas Recombinantes , Humanos , Virus de la Fiebre Hemorrágica de Crimea-Congo/inmunología , Virus de la Fiebre Hemorrágica de Crimea-Congo/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Células HEK293 , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/aislamiento & purificación , Fiebre Hemorrágica de Crimea/diagnóstico , Fiebre Hemorrágica de Crimea/virología , Ensayo de Inmunoadsorción Enzimática , Animales , Cromatografía de Afinidad/métodos , Cromatografía en Gel , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre

RESUMEN

Genetic editing of the germline using CRISPR/Cas9 technology has made it possible to alter livestock traits, including the creation of resistance to viral diseases. However, virus adaptability could present a major obstacle in this effort. Recently, chickens resistant to avian leukosis virus subgroup J (ALV-J) were developed by deleting a single amino acid, W38, within the ALV-J receptor NHE1 using CRISPR/Cas9 genome editing. This resistance was confirmed both in vitro and in vivo. In vitro resistance of W38-/- chicken embryonic fibroblasts to all tested ALV-J strains was shown. To investigate the capacity of ALV-J for further adaptation, we used a retrovirus reporter-based assay to select adapted ALV-J variants. We assumed that adaptive mutations overcoming the cellular resistance would occur within the envelope protein. In accordance with this assumption, we isolated and sequenced numerous adapted virus variants and found within their envelope genes eight independent single nucleotide substitutions. To confirm the adaptive capacity of these substitutions, we introduced them into the original retrovirus reporter. All eight variants replicated effectively in W38-/- chicken embryonic fibroblasts in vitro while in vivo, W38-/- chickens were sensitive to tumor induction by two of the variants. Importantly, receptor alleles with more extensive modifications have remained resistant to the virus. These results demonstrate an important strategy in livestock genome engineering towards antivirus resistance and illustrate that cellular resistance induced by minor receptor modifications can be overcome by adapted virus variants. We conclude that more complex editing will be necessary to attain robust resistance.

Asunto(s)

Virus de la Leucosis Aviar , Leucosis Aviar , Pollos , Animales , Virus de la Leucosis Aviar/genética , Virus de la Leucosis Aviar/fisiología , Pollos/virología , Leucosis Aviar/virología , Leucosis Aviar/genética , Enfermedades de las Aves de Corral/virología , Enfermedades de las Aves de Corral/genética , Resistencia a la Enfermedad/genética , Sistemas CRISPR-Cas , Edición Génica , Embrión de Pollo , Evolución Molecular , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Fibroblastos/virología , Fibroblastos/metabolismo

RESUMEN

Ebola disease is a lethal viral hemorrhagic fever caused by ebolaviruses within the Filoviridae family with mortality rates of up to 90%. Monoclonal antibody (mAb) based therapies have shown great potential for the treatment of EVD. However, the potential emerging ebolavirus isolates and the negative effect of decoy protein on the therapeutic efficacy of antibodies highlight the necessity of developing novel antibodies to counter the threat of Ebola. Here, 11 fully human mAbs were isolated from transgenic mice immunized with GP protein and recombinant vesicular stomatitis virus-bearing GP (rVSV-EBOV GP). These mAbs were divided into five groups according to their germline genes and exhibited differential binding activities and neutralization capabilities. In particular, mAbs 8G6, 2A4, and 5H4 were cross-reactive and bound at least three ebolavirus glycoproteins. mAb 4C1 not only exhibited neutralizing activity but no cross-reaction with sGP. mAb 7D8 exhibited the strongest neutralizing capacity. Further analysis on the critical residues for the bindings of 4C1 and 8G6 to GPs was conducted using antibodies complementarity-determining regions (CDRs) alanine scanning. It has been shown that light chain CDR3 played a crucial role in binding and neutralization and that any mutation in CDRs could not improve the binding of 4C1 to sGP. Importantly, mAbs 7D8, 8G6, and 4C1 provided complete protections against EBOV infection in a hamster lethal challenge model when administered 12 h post-infection. These results support mAbs 7D8, 8G6, and 4C1 as potent antibody candidates for further investigations and pave the way for further developments of therapies and vaccines.

Asunto(s)

Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Modelos Animales de Enfermedad , Ebolavirus , Fiebre Hemorrágica Ebola , Animales , Ebolavirus/inmunología , Ebolavirus/genética , Anticuerpos Monoclonales/inmunología , Fiebre Hemorrágica Ebola/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Fiebre Hemorrágica Ebola/virología , Anticuerpos Antivirales/inmunología , Cricetinae , Ratones , Anticuerpos Neutralizantes/inmunología , Humanos , Ratones Transgénicos , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/genética , Reacciones Cruzadas

RESUMEN

Nipah virus infection, one of the top priority diseases recognized by the World Health Organization, underscores the urgent need to develop effective countermeasures against potential epidemics and pandemics. Here, we identify a fully human single-domain antibody that targets a highly conserved cryptic epitope situated at the dimeric interface of the Nipah virus G protein (receptor binding protein, RBP), as elucidated through structures by high-resolution cryo-electron microscopy (cryo-EM). This unique binding mode disrupts the tetramerization of the G protein, consequently obstructing the activation of the F protein and inhibiting viral membrane fusion. Furthermore, our investigations reveal that this compact antibody displays enhanced permeability across the blood-brain barrier (BBB) and demonstrates superior efficacy in eliminating pseudovirus within the brain in a murine model of Nipah virus infection, particularly compared to the well-characterized antibody m102.4 in an IgG1 format. Consequently, this single-domain antibody holds promise as a therapeutic candidate to prevent Nipah virus infections and has potential implications for vaccine development.

Asunto(s)

Anticuerpos Antivirales , Microscopía por Crioelectrón , Epítopos , Infecciones por Henipavirus , Virus Nipah , Anticuerpos de Dominio Único , Virus Nipah/inmunología , Humanos , Animales , Infecciones por Henipavirus/inmunología , Infecciones por Henipavirus/prevención & control , Infecciones por Henipavirus/virología , Epítopos/inmunología , Ratones , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos Antivirales/inmunología , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/química , Femenino , Células HEK293

RESUMEN

Goatpox and sheeppox are highly contagious and economically important viral diseases of small ruminants. Due to the risk they pose to animal health, livestock production, and international trade, capripoxviruses are a considerable threat to the livestock economy. In this study, we expressed two core proteins (A4L and A12L) and one extracellular enveloped virion protein (A33R) of goatpox virus in a baculovirus expression vector system and evaluated their use as diagnostic antigens in ELISA. Full-length A4L, A12L, and A33R genes of the GTPV Uttarkashi strain were amplified, cloned into the pFastBac HT A donor vector, and introduced into DH10Bac cells containing a baculovirus shuttle vector plasmid to generate recombinant bacmids. The recombinant baculoviruses were produced in Sf-21 cells by transfection, and proteins were expressed in TN5 insect cells. The recombinant proteins were analysed by SDS-PAGE and confirmed by western blot, with expected sizes of ~30 kDa, ~31 kDa, and ~32 kDa for A4L, A12L, and A33R, respectively. The recombinant proteins were purified, and the immunoreactivity of the purified proteins was confirmed by western blot using anti-GTPV serum. The antigenic specificity of the expressed proteins as diagnostic antigens was evaluated by testing their reactivity with infected, vaccinated, and negative GTPV/SPPV serum in indirect ELISA, and the A33R-based indirect ELISA was optimized. The diagnostic sensitivity and specificity of the A33R-based indirect ELISA were found to be of 89% and 94% for goats and 98% and 91%, for sheep, respectively. No cross-reactivity was observed with other related viruses. The recombinant-A33R-based indirect ELISA developed in the present study shows that it has potential for the detection of antibodies in GTPV and SPPV infected/vaccinated animals.

Asunto(s)

Baculoviridae , Capripoxvirus , Ensayo de Inmunoadsorción Enzimática , Enfermedades de las Cabras , Cabras , Proteínas del Envoltorio Viral , Capripoxvirus/genética , Capripoxvirus/aislamiento & purificación , Baculoviridae/genética , Animales , Enfermedades de las Cabras/virología , Enfermedades de las Cabras/diagnóstico , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Cabras/virología , Ensayo de Inmunoadsorción Enzimática/métodos , Infecciones por Poxviridae/diagnóstico , Infecciones por Poxviridae/veterinaria , Infecciones por Poxviridae/virología , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/inmunología , Virión/genética , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Células Sf9 , Antígenos Virales/genética , Antígenos Virales/inmunología , Línea Celular , Expresión Génica

RESUMEN

Analysis of protein modifications is critical for quality control of therapeutic biologics. However, the identification and quantification of naturally occurring glycation of membrane proteins by mass spectrometry remain technically challenging. We used highly sensitive LC MS/MS analyses combined with multiple enzyme digestions to determine low abundance early-stage lysine glycation products of influenza vaccines derived from embryonated chicken eggs and cultured cells. Straightforward sequencing was enhanced by MS/MS fragmentation of small peptides. As a result, we determined a widespread distribution of lysine modifications attributed by the region-selectivity and site-specificity of glycation toward influenza matrix 1, hemagglutinin and neuraminidase. Topological analysis provides insights into the site-specific lysine glycation, localizing in the distinct structural regions of proteins surrounding the viral envelope membrane. Our finding highlights the proteome-wide discovery of lysine glycation of influenza membrane proteins and potential effects on the structural assembly, stability, receptor binding and enzyme activity, demonstrating that the impacts of accumulated glycation on the quality of products can be directly monitored by mass spectrometry-based structural proteomics analyses.

Asunto(s)

Espectrometría de Masas en Tándem , Glicosilación , Animales , Vacunas contra la Influenza/metabolismo , Neuraminidasa/metabolismo , Humanos , Lisina/metabolismo , Embrión de Pollo , Proteínas de la Matriz Viral/metabolismo , Proteínas de la Matriz Viral/química , Proteómica/métodos , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Cromatografía Liquida

RESUMEN

Zika virus (ZIKV) infection remains a global public health problem. After the "Public Health Emergencies of International Concern" declared in February 2016, the incidence of new infections by this pathogen has been decreasing in many areas. However, there is still a likely risk that ZIKV will spread to more countries. To date, there is no vaccine or antiviral drug available to prevent or treat Zika virus infection. In the Zika vaccine development, those based on protein subunits are attractive as a non-replicable platform due to their potentially enhanced safety profile to be used in all populations. However, these vaccines frequently require multiple doses and adjuvants to achieve protective immunity. In this study we show the immunological evaluation of new formulations of the recombinant protein ZEC, which combines regions of domain III of the envelope and the capsid from ZIKV. Two nucleotide-based adjuvants were used to enhance the immunity elicited by the vaccine candidate ZEC. ODN 39M or c-di-AMP was incorporated as immunomodulator into the formulations combined with aluminum hydroxide. Following immunizations in immunocompetent BALB/c mice, the formulations stimulated high IgG antibodies. Although the IgG subtypes suggested a predominantly Th1-biased immune response by the formulation including the ODN 39M, cellular immune responses measured by IFNγ secretion from spleen cells after in vitro stimulations were induced by both immunomodulators. These results demonstrate the capacity of both immunomodulators to enhance the immunogenicity of the recombinant subunit ZEC as a vaccine candidate against ZIKV.

Asunto(s)

Adyuvantes Inmunológicos , Anticuerpos Antivirales , Ratones Endogámicos BALB C , Vacunas de Subunidad , Vacunas Sintéticas , Infección por el Virus Zika , Virus Zika , Animales , Virus Zika/inmunología , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/administración & dosificación , Infección por el Virus Zika/prevención & control , Infección por el Virus Zika/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Ratones , Femenino , Adyuvantes Inmunológicos/administración & dosificación , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunogenicidad Vacunal , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Adyuvantes de Vacunas , Inmunidad Celular , Proteínas del Envoltorio Viral/inmunología , Proteínas de la Cápside/inmunología , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/inmunología

RESUMEN

mRNA platform holds promise for next-generation Varicella-zoster Virus (VZV) vaccine development due to its high potency at inducing strong T-cell response. Built upon the design of our 1st-generation VZV mRNA vaccine that encodes for full-length gE antigen, in this study we reported on a novel combinatorial strategy to further optimize the gE-encoding mRNA sequence through signal peptide replacement, C-terminal modification, and insertion of mRNA-stabilizing motif, which collectively contributed to significantly improved vaccine immunogenicity. In adult mice, aged mice, and immunocompromised mice, this optimized VZV mRNA vaccine showed strong superiority in multiple aspects including the induction of gE-specific antibodies, specific memory B-cell response, as well as Th1-type T-cell response.

Asunto(s)

Anticuerpos Antivirales , Herpesvirus Humano 3 , Proteínas del Envoltorio Viral , Animales , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/genética , Herpesvirus Humano 3/inmunología , Herpesvirus Humano 3/genética , Ratones , Anticuerpos Antivirales/inmunología , Humanos , Vacunas de ARNm , ARN Mensajero/genética , ARN Mensajero/inmunología , Femenino , Desarrollo de Vacunas , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Inmunogenicidad Vacunal , Vacuna contra la Varicela/inmunología , Vacuna contra la Varicela/administración & dosificación , Vacuna contra la Varicela/genética , Linfocitos B/inmunología , Ratones Endogámicos BALB C , Células TH1/inmunología

RESUMEN

Endogenous retroviruses (ERVs) occupy a significant part of the human genome, with some encoding proteins that influence the immune system or regulate cell-cell fusion in early extra-embryonic development. However, whether ERV-derived proteins regulate somatic development is unknown. Here, we report a somatic developmental function for the primate-specific ERVH48-1 (SUPYN/Suppressyn). ERVH48-1 encodes a fragment of a viral envelope that is expressed during early embryonic development. Loss of ERVH48-1 led to impaired mesoderm and cardiomyocyte commitment and diverted cells to an ectoderm-like fate. Mechanistically, ERVH48-1 is localized to sub-cellular membrane compartments through a functional N-terminal signal peptide and binds to the WNT antagonist SFRP2 to promote its polyubiquitination and degradation, thus limiting SFRP2 secretion and blocking repression of WNT/ß-catenin signaling. Knockdown of SFRP2 or expression of a chimeric SFRP2 with the ERVH48-1 signal peptide rescued cardiomyocyte differentiation. This study demonstrates how ERVH48-1 modulates WNT/ß-catenin signaling and cell type commitment in somatic development.

Asunto(s)

Diferenciación Celular , Retrovirus Endógenos , Proteínas de la Membrana , Miocitos Cardíacos , Vía de Señalización Wnt , Humanos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/citología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Retrovirus Endógenos/metabolismo , Retrovirus Endógenos/genética , Animales , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/genética , Primates , Células HEK293 , Mesodermo/metabolismo

RESUMEN

Systemic viral infection of insects typically begins with the primary infection of midgut epithelial cells (enterocytes) and subsequent transit of the progeny virus in an apical-to-basal orientation into the hemocoel. For insect-vectored viruses, an oppositely oriented process (basal-to-apical transit) occurs upon secondary infection of salivary glands and is necessary for virus transmission to non-insect hosts. To examine this inversely oriented virus transit in these polarized tissues, we assessed the intracellular trafficking of two model viral envelope proteins (baculovirus GP64 and vesicular stomatitis virus G) in the midgut and salivary gland cells of the model insect, Drosophila melanogaster. Using fly lines that inducibly express either GP64 or VSV G, we found that each protein, expressed alone, was trafficked basally in midgut enterocytes. In salivary gland cells, VSV G was trafficked apically in most but not all cells, whereas GP64 was consistently trafficked basally. We demonstrated that a YxxØ motif present in both proteins was critical for basal trafficking in midgut enterocytes but dispensable for trafficking in salivary gland cells. Using RNAi, we found that clathrin adaptor protein complexes AP-1 and AP-3, as well as seven Rab GTPases, were involved in polarized VSV G trafficking in midgut enterocytes. Our results indicate that these viral envelope proteins encode the requisite information and require no other viral factors for appropriately polarized trafficking. In addition, they exploit tissue-specific differences in protein trafficking pathways to facilitate virus egress in the appropriate orientation for establishing systemic infections and vectoring infection to other hosts. IMPORTANCE: Viruses that use insects as hosts must navigate specific routes through different insect tissues to complete their life cycles. The routes may differ substantially depending on the life cycle of the virus. Both insect pathogenic viruses and insect-vectored viruses must navigate through the polarized cells of the midgut epithelium to establish a systemic infection. In addition, insect-vectored viruses must also navigate through the polarized salivary gland epithelium for transmission. Thus, insect-vectored viruses appear to traffic in opposite directions in these two tissues. In this study, we asked whether two viral envelope proteins (VSV G and baculovirus GP64) alone encode the signals necessary for the polarized trafficking associated with their respective life cycles. Using Drosophila as a model to examine tissue-specific polarized trafficking of these viral envelope proteins, we identified one of the virus-encoded signals and several host proteins associated with regulating the polarized trafficking in the midgut epithelium.

Asunto(s)

Drosophila melanogaster , Transporte de Proteínas , Glándulas Salivales , Proteínas del Envoltorio Viral , Animales , Glándulas Salivales/virología , Glándulas Salivales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/genética , Drosophila melanogaster/virología , Drosophila melanogaster/metabolismo , Insectos Vectores/virología , Insectos Vectores/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Enterocitos/virología , Enterocitos/metabolismo , Tracto Gastrointestinal/virología , Tracto Gastrointestinal/metabolismo

RESUMEN

Pseudorabies virus (PRV) is one of the herpes viruses that can infect a wide range of animals including pigs, cattle, sheep, mice, and wild animals. PRV is a neurotropic alphaherpesvirus capable of infecting a variety of mammals. There is a rising interest in the targeted application of probiotic bacteria to prevent viral diseases, including PRV. In this study, the surface expression of enhanced green fluorescent protein (EGFP) on recombinant Lactiplantibacillus plantarum NC8 (rNC8) through the LP3065 LPxTG motif of Lactobacillus plantarum WCFS1 was generated. The surface expression was observed through confocal microscopy. Dendritic cell targeting peptides (DCpep) were also fused with LPxTG that help to bind with mouse DCs. The PRV-gD was cloned in LP3065 LPxTG, resulting in the generation of rNC8-LP3065-gD. Inactivated rNC8-LP3065-gD was administered intravenously in mice on days 1 and 7 at a dose of 200 µL (109 CFU/mouse) for monitoring immunogenicity. Subsequently, a challenge dose of PRV TJ (104 TCID50) was administered intramuscularly at 14 days post-immunization. The survival rate of the immunized mice reached 80% (4/5) with no significant signs of illness. A significant rise in anti-gD antibodies was detected in the immunized mice by ELISA. Quantitative PCR (qPCR) results showed decreased viral loading in different body tissues. Flow cytometry of lymphocytes derived from mice spleen indicated an increase in CD3+CD4+ T cells, but CD3+CD8+ T cells were not detected. Moreover, it offers a model to delineate immune correlates with rNC8-induced immunity against swine viral diseases.

Asunto(s)

Herpesvirus Suido 1 , Seudorrabia , Animales , Herpesvirus Suido 1/inmunología , Herpesvirus Suido 1/genética , Ratones , Seudorrabia/prevención & control , Seudorrabia/inmunología , Seudorrabia/virología , Femenino , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Lactobacillus plantarum/genética , Lactobacillus plantarum/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/genética , Ratones Endogámicos BALB C , Vacunas contra la Seudorrabia/inmunología , Porcinos , Proteínas Fluorescentes Verdes/genética , Técnicas de Visualización de Superficie Celular

RESUMEN

The white spot syndrome virus (WSSV), a rapidly replicating and highly lethal pathogen that targets Penaeid shrimp, has emerged as one of the most widespread viruses globally due to its high virulence. With effective chemotherapeutics still unavailable, the pursuit of novel and viable strategies against WSSV remains a crucial focus in the field of shrimp farming. The envelope proteins of WSSV are essential for virus entry, serving as excellent targets for the development of antiviral therapeutics. Novel strategies in the design of inhibitory peptides, especially those targeting envelope protein (VP28) located on the surface of the virus particle, play a critical role as a significant virulence factor during the early stages of inherent WSSV infection in shrimp. In this direction, the current computational study focused on identifying self-inhibitory peptides from the hydrophobic membrane regions of the VP28 protein, employing peptide docking and molecular dynamics simulation (MDS) approaches. Such inhibitory peptides could be useful building blocks for the rational engineering of inhibitory therapeutics since they imitate the mechanism of binding to homologous partners used by their origin domain to interact with other molecules. The N-terminal sequence of VP28 has been reported as the potential site for membrane interactions during the virus entry. Moreover, drug delivery systems mediated by chitosan and gold nanoparticles are being developed to enhance the therapeutic efficacy of anti-viral peptides. These systems can increase the solubility, stability, and selectivity of peptides, possessing better qualities than conventional delivery methods. This computational study on self-inhibitory peptides could be a valuable resource for further in vitro and in vivo studies on anti-viral therapeutics in the aquaculture industry.

Asunto(s)

Antivirales , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Penaeidae , Péptidos , Virus del Síndrome de la Mancha Blanca 1 , Virus del Síndrome de la Mancha Blanca 1/efectos de los fármacos , Virus del Síndrome de la Mancha Blanca 1/genética , Antivirales/farmacología , Animales , Péptidos/farmacología , Péptidos/química , Penaeidae/virología , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/química , Internalización del Virus/efectos de los fármacos