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SARS-CoV-2 vaccination is known to induce antibodies that recognize also variants of concerns (VoCs) of the virus. However, epidemiological and laboratory evidences indicate that these antibodies have a reduced neutralization ability against VoCs. We studied binding and neutralizing antibodies against the Spike protein domains and subunits of the Wuhan-Hu-1 virus and its alpha, beta, delta VoCs and of seasonal betacoronaviruses (HKU1 and OC43) in a cohort of 31 health care workers prospectively followed post-vaccination with BNT162b2-Comirnaty. The study of sequential samples collected up to 64 days post-vaccination showed that serological assays measuring IgG against Wuhan-Hu-1 antigens were a poor proxy for VoC neutralization. In addition, in subjects who had asymptomatic or mild COVID-19 prior to vaccination, the loss of nAbs following disease could be rapid and accompanied by post-vaccination antibody levels similar to those of naïve vaccinees. Interestingly, in health care workers naïve for SARS-CoV-2 infection, vaccination induced a rapid and transient reactivation of pre-existing seasonal coronaviruses IgG responses that was associated with a subsequent reduced ability to neutralize alpha and beta VoCs.
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COVID-19 , SARS-CoV-2 , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacina BNT162 , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Imunoglobulina G , Estações do Ano , VacinaçãoRESUMO
Monoclonal antibodies (mAb) targeting the SARS-CoV-2 Spike (S) glycoprotein have been exploited for the treatment of severe COVID-19. In this study, we evaluated the immune-regulatory features of two neutralizing anti-S mAbs (nAbs), named J08 and F05, with wild-type (WT) conformation or silenced Fc functions. In the presence of D614G SARS-CoV-2, WT nAbs enhance intracellular viral uptake in immune cells and amplify antiviral type I Interferon and inflammatory cytokine and chemokine production without viral replication, promoting the differentiation of CD16+ inflammatory monocytes and innate/adaptive PD-L1+ and PD-L1+CD80+ plasmacytoid Dendritic Cells. In spite of a reduced neutralizing property, WT J08 nAb still promotes the IL-6 production and differentiation of CD16+ monocytes once binding Omicron BA.1 variant. Fc-mediated regulation of antiviral and inflammatory responses, in the absence of viral replication, highlighted in this study, might positively tune immune response during SARS-CoV-2 infection and be exploited also in mAb-based therapeutic and prophylactic strategies against viral infections.
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Several COVID-19 vaccine strategies utilizing new formulations for the induction of neutralizing antibodies (nAbs) and T cell immunity are still under evaluation in preclinical and clinical studies. Here we used Simian Immunodeficiency Virus (SIV)-based integrase defective lentiviral vector (IDLV) delivering different conformations of membrane-tethered Spike protein in the mouse immunogenicity model, with the aim of inducing persistent nAbs against multiple SARS-CoV-2 variants of concern (VoC). Spike modifications included prefusion-stabilizing double proline (2P) substitutions, mutations at the furin cleavage site (FCS), D614G mutation and truncation of the cytoplasmic tail (delta21) of ancestral and Beta (B.1.351) Spike, the latter mutation to markedly improve IDLV membrane-tethering. BALB/c mice were injected once with IDLV delivering the different forms of Spike or the recombinant trimeric Spike protein with 2P substitutions and FCS mutations in association with a squalene-based adjuvant. Anti-receptor binding domain (RBD) binding Abs, nAbs and T cell responses were detected up to six months from a single immunization with escalating doses of vaccines in all mice, but with different levels and kinetics. Results indicated that IDLV delivering the Spike protein with all the combined modifications, outperformed the other candidates in terms of T cell immunity and level of both binding Abs and nAbs soon after the single immunization and persistence over time, showing the best capacity to neutralize all formerly circulating VoC Alpha, Beta, Gamma and Delta. Although present, the lowest response was detected against Omicron variants (BA.1, BA.2 and BA.4/5), suggesting that the magnitude of immune evasion may be related to the higher genetic distance of Omicron as indicated by increased number of amino acid substitutions in Spike acquired during virus evolution.
Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Animais , Humanos , Camundongos , Glicoproteína da Espícula de Coronavírus/genética , Integrases , Vacinas contra COVID-19 , SARS-CoV-2/genética , Anticorpos Neutralizantes , Modelos Animais de Doenças , Camundongos Endogâmicos BALB C , ImunidadeRESUMO
Integrase Defective Lentiviral Vectors (IDLVs) represent an attractive vaccine platform for delivering HIV-1 antigens, given their ability to induce specific and persistent immune responses in both mice and non-human primates (NHPs). Recent advances in HIV-1 immunogen design demonstrated that native-like HIV-1 Envelope (Env) trimers that mimic the structure of virion-associated Env induce neutralization breadth in rabbits and macaques. Here, we describe the development of an IDLV-based HIV-1 vaccine expressing either soluble ConSOSL.UFO.664 or membrane-tethered ConSOSL.UFO.750 native-like Env immunogens with enhanced bNAb epitopes exposure. We show that IDLV can be pseudotyped with properly folded membrane-tethered native-like UFO.750 trimers. After a single IDLV injection in BALB/c mice, IDLV-UFO.750 induced a faster humoral kinetic as well as higher levels of anti-Env IgG compared to IDLV-UFO.664. IDLV-UFO.750 vaccinated cynomolgus macaques developed unusually long-lasting anti-Env IgG antibodies, as underlined by their remarkable half-life both after priming and boost with IDLV. After boosting with recombinant ConM SOSIP.v7 protein, two animals developed neutralization activity against the autologous tier 1B ConS virus mediated by V1/V2 and V3 glycan sites responses. By combining the possibility to display stabilized trimeric Env on the vector particles with the ability to induce sustained humoral responses, IDLVs represent an appropriate strategy for delivering rationally designed antigens to progress towards an effective HIV-1 vaccine.
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The Eastern woodchuck (Marmota monax) is a useful experimental model for evaluating antiviral therapy against chronic HBV infection. In the present study, an immunogenic complex (IGC) composed of immune sera containing PreS/S heterologous antibodies (anti-HBs) and serum-derived WHV particles containing 10(7) WHV-DNA copies/50 µl was developed. The IGC was administered to WHV-negative woodchucks and natural chronic WHV carriers, with the final aim of evaluating the outcome of WHV infection in both groups. A control group of three animals, infected experimentally with viral particles only, was also evaluated. Following IGC administration, two WHV-negative woodchucks exhibited persistent infection, with WHV-DNA levels 3-6 logs lower than the WHV-DNA levels of the controls that developed persistent infection. WHeAg seroconversion to anti-WHe was observed in these two woodchucks and in two control woodchucks which developed self-limited infection. In two of the four chronic carriers, the WHV-DNA level decreased significantly (by 4-6 logs) following IGC administration, with no rebound in viral load during follow-up. WHeAg seroconversion to anti-WHe was observed also in these animals. Analyses of the sequences derived from envelope proteins confirmed that IGC did not induce the emergence of resistant viral variants. The results of this study indicate that the IGC could be useful for breaking the tolerance in hepadnaviral infection and for boosting the host's innate and adoptive immune response.
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Complexo Antígeno-Anticorpo/imunologia , Anticorpos Anti-Hepatite B/imunologia , Vírus da Hepatite B da Marmota/imunologia , Hepatite B/imunologia , Hepatite B/veterinária , Vírion/imunologia , Animais , Complexo Antígeno-Anticorpo/administração & dosagem , Sangue/virologia , DNA Viral/química , DNA Viral/genética , Modelos Animais de Doenças , Hepatite B/patologia , Hepatite B/virologia , Vírus da Hepatite B da Marmota/patogenicidade , Marmota , Dados de Sequência Molecular , Análise de Sequência de DNA , Resultado do Tratamento , Carga ViralRESUMO
Integrase-defective lentiviral vectors (IDLVs) represent an attractive platform for vaccine development as a result of the ability to induce persistent humoral- and cellular-mediated immune responses against the encoded transgene. Compared with the parental integrating vector, the main advantages for using IDLV are the reduced hazard of insertional mutagenesis and the decreased risk for vector mobilization by wild-type viruses. Here we report on the development and use in the mouse immunogenicity model of simian immunodeficiency virus (SIV)-based IDLV containing a long deletion in the U3 region and with the 3' polypurine tract (PPT) removed from the transfer vector for improving safety and/or efficacy. Results show that a safer extended deletion of U3 sequences did not modify integrase-mediated or -independent integration efficiency. Interestingly, 3' PPT deletion impaired integrase-mediated integration but did not reduce illegitimate, integrase-independent integration efficiency, contrary to what was previously reported in the HIV system. Importantly, although the extended deletion in the U3 did not affect expression or immunogenicity from IDLV, deletion of 3' PPT considerably reduced both expression and immunogenicity of IDLV.
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Integrase-defective lentiviral vectors (IDLVs) have been used as a safe and efficient delivery system in several immunization protocols in murine and non-human primate preclinical models as well as in recent clinical trials. In this work, we validated in preclinical murine models our vaccine platform based on IDLVs as delivery system for cancer immunotherapy. To evaluate the anti-tumor activity of our vaccine strategy we generated IDLV delivering ovalbumin (OVA) as a non-self-model antigen and TRP2 as a self-tumor associated antigen (TAA) of melanoma. Results demonstrated the ability of IDLVs to eradicate and/or controlling tumor growth after a single immunization in preventive and therapeutic approaches, using lymphoma and melanoma expressing OVA. Importantly, LV-TRP2 but not IDLV-TRP2 was able to break tolerance efficiently and prevent tumor growth of B16F10 melanoma cells. In order to improve the IDLV efficacy, the human homologue of murine TRP2 was used, showing the ability to break tolerance and control the tumor growth. These results validate the use of IDLV for cancer therapy.
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Vacinas Anticâncer/administração & dosagem , Vetores Genéticos/genética , Imunoterapia , Integrases/metabolismo , Lentivirus/genética , Melanoma/imunologia , Melanoma/terapia , Animais , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Vetores Genéticos/metabolismo , Humanos , Integrases/genética , Oxirredutases Intramoleculares/administração & dosagem , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/imunologia , Lentivirus/enzimologia , Lentivirus/metabolismo , Masculino , Melanoma/genética , Camundongos , Camundongos Endogâmicos C57BL , VacinaçãoRESUMO
Understanding how antibody responses to SARS-CoV-2 evolve during infection may provide important insight into therapeutic approaches and vaccination for COVID-19. Here we profile the antibody responses of 162 COVID-19 symptomatic patients in the COVID-BioB cohort followed longitudinally for up to eight months from symptom onset to find SARS-CoV-2 neutralization, as well as antibodies either recognizing SARS-CoV-2 spike antigens and nucleoprotein, or specific for S2 antigen of seasonal beta-coronaviruses and hemagglutinin of the H1N1 flu virus. The presence of neutralizing antibodies within the first weeks from symptoms onset correlates with time to a negative swab result (p = 0.002), while the lack of neutralizing capacity correlates with an increased risk of a fatal outcome (p = 0.008). Neutralizing antibody titers progressively drop after 5-8 weeks but are still detectable up to 8 months in the majority of recovered patients regardless of age or co-morbidities, with IgG to spike antigens providing the best correlate of neutralization. Antibody responses to seasonal coronaviruses are temporarily boosted, and parallel those to SARS-CoV-2 without dampening the specific response or worsening disease progression. Our results thus suggest compromised immune responses to the SARS-CoV-2 spike to be a major trait of COVID-19 patients with critical conditions, and thereby inform on the planning of COVID-19 patient care and therapy prioritization.
Assuntos
Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , COVID-19/mortalidade , SARS-CoV-2/imunologia , Idoso , Anticorpos Antivirais/imunologia , Formação de Anticorpos , Betacoronavirus/imunologia , COVID-19/virologia , Feminino , Humanos , Imunoglobulina G/imunologia , Cinética , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/imunologia , Taxa de SobrevidaRESUMO
Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in Escherichia coli, suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.
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Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Epitopos/imunologia , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/genética , Animais , Sítios de Ligação de Anticorpos/imunologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Feminino , Glicosilação , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Testes de Neutralização , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Tratamento Farmacológico da COVID-19RESUMO
Cellular immune responses play a fundamental role in controlling viral replication and AIDS progression in human immunodeficiency virus (HIV)-infected subjects and in simian immunodeficiency virus (SIV)-infected macaques. Integrase defective lentiviral vector (IDLV) represents a promising vaccine candidate, inducing functional and durable immune responses in mice and non-human primates. Here, we designed HIV- and SIV-based IDLVs to express the HIVACAT T cell immunogen (HTI), a mosaic antigen designed to cover vulnerable sites in HIV-1 Gag, Pol, Vif, and Nef. We observed that HTI expression during lentiviral vector production interfered profoundly with IDLV particles release because of sequestration of both HIV- and SIV-Gag proteins in the cytoplasm of the vector-producing cells. However, modifications in IDLV design and vector production procedures greatly improved recovery of both HIV- and SIV-based IDLV-HTI. Immunization experiments in BALB/c mice showed that both IDLVs elicited HTI-specific T cell responses. However, immunization with HIV-based IDLV elicited also a T cell response toward exogenous HIV proteins in IDLV particles, suggesting that SIV-based IDLV may be a preferable platform to assess the induction of transgene-specific immune responses against rationally designed HIV structural antigens. These data support the further evaluation of IDLV as an effective platform of T cell immunogens for the development of an effective HIV vaccine.
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Viral vectors represent an attractive technology for vaccine delivery. We exploited the integrase defective lentiviral vector (IDLV) as a platform for delivering relevant antigens within the context of the ADITEC collaborative research program. In particular, Influenza virus hemagglutinin (HA) and nucleoprotein (NP) were delivered by IDLVs while H1N1 A/California/7/2009 subunit vaccine (HAp) with or without adjuvant was used to compare the immune response in a murine model of immunization. In order to maximize the antibody response against HA, both IDLVs were also pseudotyped with HA (IDLV-HA/HA and IDLV-NP/HA, respectively). Groups of CB6F1 mice were immunized intramuscularly with a single dose of IDLV-NP/HA, IDLV-HA/HA, HAp alone, or with HAp together with the systemic adjuvant MF59. Six months after the vaccine prime all groups were boosted with HAp alone. Cellular and antibody responses to influenza antigens were measured at different time points after the immunizations. Mice immunized with HA-pseudotyped IDLVs showed similar levels of anti-H1N1 IgG over time, evaluated by ELISA, which were comparable to those induced by HAp + MF59 vaccination, but significantly higher than those induced by HAp alone. The boost with HAp alone induced an increase of antibodies in all groups, and the responses were maintained at higher levels up to 18 weeks post-boost. The antibody response was functional and persistent overtime, capable of neutralizing virus infectivity, as evaluated by hemagglutination inhibition and microneutralization assays. Moreover, since neuraminidase (NA)-expressing plasmid was included during IDLV preparation, immunization with IDLV-NP/HA and IDLV-HA/HA also induced functional anti-NA antibodies, evaluated by enzyme-linked lectin assay. IFNγ-ELISPOT showed evidence of HA-specific response in IDLV-HA/HA immunized animals and persistent NP-specific CD8+ T cell response in IDLV-NP/HA immunized mice. Taken together our results indicate that IDLV can be harnessed for producing a vaccine able to induce a comprehensive immune response, including functional antibodies directed toward HA and NA proteins present on the vector particles in addition to a functional T cell response directed to the protein transcribed from the vector.
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Vetores Genéticos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/administração & dosagem , Vacinas contra Influenza/administração & dosagem , Lentivirus/genética , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas do Core Viral/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , ELISPOT , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunoglobulina G/sangue , Integrases/genética , Interferon gama , Camundongos , Infecções por Orthomyxoviridae/imunologia , Vacinação/métodos , Proteínas do Core Viral/imunologiaRESUMO
Multiple Myeloma (MM) is the second most common hematological malignancy and is responsive to a limited number of drugs. Unfortunately, to date, despite the introduction of novel drugs, no relevant increase in survival rates has been obtained. Proton pump inhibitors (PPIs) have been shown to have significant antitumor action as single agents as well as in combination with chemotherapy. This study investigates the potential anti-tumor effectiveness of two PPIs, Lansoprazole and Omeprazole, against human MM cells. We found that Lansoprazole exerts straightforward efficacy against myeloma cells, even at suboptimal concentrations (50 µM), while Omeprazole has limited cytotoxic action. The Lansoprazole anti-MM effect was mostly mediated by a caspase-independent apoptotic-like cytotoxicity, with only a secondary anti-proliferative action. This study provides clear evidence supporting the use of Lansoprazole in the strive against MM with an efficacy proven much higher than current therapeutical approaches and without reported side effects. It is however conceivable that, consistent with the results obtained in other human tumors, Lansoprazole may well be combined with existing anti-myeloma therapies with the aim to improve the low level of efficacy of the current strategies.
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Caspases/metabolismo , Lansoprazol/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Omeprazol/farmacologia , Inibidores da Bomba de Prótons/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/patologia , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
EBV is a human herpesvirus associated with a number of malignancies. Both lymphoblastoid cell lines (LCLs), and EBV-infected nasopharyngeal carcinoma (NPC) cells have been demonstrated to release exosomes containing the EBV-encoded latent membrane protein 1 (LMP1), and mature micro-RNAs (EBV-miRNAs). Here we analyze the EBV protein and nucleic acid content of exosomes from different EBV-infected cells (LCL, 721 and Daudi) and we show for the first time that exosomes released from LCLs and 721 also contain EBV-encoded latent phase mRNAs. This confirms and strengthens exosomes pathogenetic potential, and might provide insights for development of novel diagnostic and therapeutic strategies.
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Transformação Celular Viral , Exossomos/genética , Herpesvirus Humano 4/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas Virais/genética , Linhagem Celular Tumoral , Antígenos Nucleares do Vírus Epstein-Barr/genética , Exossomos/metabolismo , Exossomos/virologia , Herpesvirus Humano 4/metabolismo , Humanos , Imunoprecipitação , MicroRNAs/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/metabolismoRESUMO
Studies focused on the understanding of the molecular mechanisms involved in recovery or progression to chronicity of HBV may take advantage of natural and experimental models that mimic its properties. This is also of relevance for associated diseases such as cirrhosis and hepatocellulocarcinoma. The eastern woodchuck (Marmota monax) infected by the hepadnavirus woodchuck Hepatitis B virus (WHV) has been applied as a predictive model to support development of new HBV vaccines, antivirals, immunotherapies and combination therapies. This report summarizes studies carried out by our and other groups, with the application of this model in natural and experimental infections. Using standardized viral inocula in neonate and adult animals and newly established assays, the presence of the specific patterns of markers of acute, chronic and resolved infections and their relationships in the different virus-host interactions have been shown. B and T cell responses and T(H)1 cytokine expression have been shown to play a crucial role in the outcome of infection. The availability of the WHV/Marmota monax model and specific standardized assays may allow evaluation of new formulations of multimodal therapeutic strategies based on antiviral chemotherapy and immunomodulation. These may also include specifically targeted immunocomplexes. Such therapies could constitute new frontiers for the treatment of HBV chronic disease.
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Present efforts of HBV vaccine research are aimed at defining targeted antigen compositions and adjuvancy systems for earlier and broader immune responses and optimization of immunotherapeutic approaches. We have demonstrated the applicability of the WHV/Marmota monax model for the evaluation of immunogenicity and protection of new formulations of HBV vaccines for human use. Protective activity was evaluated following the administrations of HBV CHO-PreS/S and adjuvanted S/Core vaccines. The administration of a complex constituted by HBV derived woodchuck PreS/S antibodies coupled with WHV particles was able to induce inhibition of viral replication. Future studies on treatment of HBV chronic infection should be addressed to the evaluation of therapies combined with antivirals, vaccines and immunomodulatory compounds.
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Vacinas contra Hepatite B/imunologia , Imunização , Animais , DNA Viral/sangue , Anticorpos Anti-Hepatite B/sangue , Lamivudina/farmacologia , Marmota , Vacinas Sintéticas/imunologia , Carga ViralRESUMO
To determine whether the addition of a pre-S/S human vaccine increases the antiviral activity of lamivudine, four woodchucks were treated with a daily dose of 100 mg/kg lamivudine and four 50 microg doses of CHO-derived pre-S/S human vaccine. WHV DNA titres decreased up to two logarithms in three woodchucks. At week 4, in three of the animals, the sequence analysis showed a predominant strain containing a nucleotide change from A to T at position 1696 of domain B of the WHV DNA polymerase. Vaccination did not further suppress WHV DNA, despite anti-HBs production in three animals. The woodchuck remains a useful model for characterising the biology and kinetics of the emergence of drug-resistant variants and could be used for pre-clinical studies of combinations of new antiviral drugs.
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Farmacorresistência Viral , Vacinas contra Hepatite B/genética , Vacinas contra Hepatite B/uso terapêutico , Vírus da Hepatite B da Marmota/genética , Vírus da Hepatite B da Marmota/imunologia , Hepatite B/imunologia , Hepatite B/virologia , Lamivudina/uso terapêutico , Marmota/virologia , Inibidores da Transcriptase Reversa/uso terapêutico , Animais , Células CHO , Cricetinae , Cricetulus , DNA Viral/química , Hepatite B/tratamento farmacológico , Inflamação/patologia , Inflamação/virologia , Cinética , Fígado/patologia , Fígado/virologia , Monócitos/imunologia , Monócitos/virologia , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , VacinaçãoRESUMO
We evaluated whether a non-adjuvanted vaccine derived from Chinese hamster ovary cells was capable of providing protection against woodchuck hepatitis virus (WHV). Three woodchucks were vaccinated with four 50-microg doses and challenged with a previously characterized virus isolate (WHV197). In all three animals, titre levels of antibodies against hepatitis B surface antigens (anti-HBs) exceeded 10 mIU/ml, peaking at 150 mIU/ml. Challenge resulted in productive acute infection in the two non-vaccinated woodchucks yet in none of the vaccinated woodchucks. In the vaccinated animals, there was evidence of abortive infection. The results demonstrate that a human vaccine is able to protect woodchucks from WHV infection.
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Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B da Marmota , Hepatite B/prevenção & controle , Animais , Células CHO , Cricetinae , Cricetulus , DNA Viral/análise , Modelos Animais de Doenças , Anticorpos Anti-Hepatite B/sangue , Vacinas contra Hepatite B/administração & dosagem , Vírus da Hepatite B da Marmota/genética , Vírus da Hepatite B da Marmota/imunologia , Vírus da Hepatite B da Marmota/isolamento & purificação , Fígado/virologia , Marmota , Reação em Cadeia da Polimerase , ViremiaRESUMO
The mechanism of action of the antiviral compound 3(2H)-isoflavene against Sabin type 2 poliovirus has been studied, and interference with virus uncoating was demonstrated. Isolation and sequencing of drug-resistant variants revealed single amino acid substitutions (I194M or D131V) in the VP1 capsid protein. While M194 is located in a hydrophobic pocket and should partially fill the space occupied by the isoflavene ring, V131 is exposed on the VP1 surface, forming a contact with VP4. The D131V mutation most likely induces local conformational changes in VP1 and/or VP4 that affect viral flexibility. Two dependent variants, N53S of VP1 and K58E of VP4, both located on the inner surface of the capsid, near the threefold axis of symmetry, were also selected. Both mutations affected viral stability, allowing the transition to 135S particles in the absence of drug, without the involvement of the viral receptor.