RESUMEN
The presence of replication-competent lentivirus (RCL) in lentiviral vector preparations is a major safety concern for clinical applications of such vectors. RCL are believed to emerge from rare recombinant vector genomes that are referred to as partial recombinants or Psi-Gag recombinants. To quantitatively determine the fraction of partial recombinants in lentiviral vector preparations and to analyze them at the DNA sequence level, we established a drug selection assay involving a lentiviral packaging construct containing a drug-resistance gene encoding blasticidin (BSD) resistance. Upon transduction of target cells, the BSD resistance gene confers BSD resistance to the transduced cells. The results obtained indicate that there were up to 156 BSD-resistant colonies in a total of 10(6) transducing vector particles. The predicted recombination events were verified by polymerase chain reaction using genomic DNA obtained from BSD-resistant cell clones and by DNA sequence analysis. In an attempt to reduce the emergence of partial recombinants, sequence overlaps between the packaging and the vector constructs were reduced by substituting the Rev response element (RRE) present in the vector construct using a heterologous RRE element derived from simian immunodeficiency virus (SIVmac239). The results obtained showed that a reduction of sequence overlaps resulted in an up to sevenfold reduction of the frequency of BSD-resistant colonies, indicating that the capacity to form partial recombinants was diminished.
Asunto(s)
Vectores Genéticos/análisis , Lentivirus/genética , Reacción en Cadena de la Polimerasa , Antibacterianos/farmacología , Antimicina A/análogos & derivados , Antimicina A/farmacología , Secuencia de Bases , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Vectores Genéticos/metabolismo , Genoma Viral , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Datos de Secuencia Molecular , Elementos de Respuesta/genética , Análisis de Secuencia de ADNRESUMEN
Previously, priming rhesus macaques with Adenovirus type 5 host range mutant-recombinants encoding Tat and Env and boosting with Tat and Env protein in MPL-SE controlled chronic viremia by 4 logs following homologous intravenous SHIV89.6P challenge. Here we evaluated Tat, Env, and Tat/Env regimens for immunogenicity and protective efficacy using clade C Env, alum adjuvant, and a heterologous intrarectal SHIV1157ipd3N4 challenge. Despite induction of strong cellular and humoral immunity, Tat/Env group T and B-cell memory responses were not significantly enhanced over Tat- or Env-only groups. Lack of viremia control post-challenge was attributed to lower avidity Env antibodies and no anamnestic ADCC response or SHIV1157ipd3N4 neutralizing antibody development post-challenge. Poor biologic activity of the Tat immunogen may have impaired Tat immunity. In the absence of sterilizing immunity, strong anamnestic responses to heterologous virus can help control viremia. Both antibody breadth and optimal adjuvanticity are needed to elicit high-quality antibody for protective efficacy.
Asunto(s)
Anticuerpos Antivirales/sangre , Memoria Inmunológica , Vacunas contra el SIDAS/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Viremia/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Animales , Anticuerpos Neutralizantes/sangre , Productos del Gen env/inmunología , Productos del Gen tat/inmunología , Humanos , Macaca mulatta , Vacunas contra el SIDAS/administración & dosificación , Virus de la Inmunodeficiencia de los Simios/inmunologíaRESUMEN
BACKGROUND: During human (HIV) and simian (SIV) immunodeficiency virus infection, loss of CD4+ T cells and progression to AIDS are associated with a decline in antibody titers to the viral Gag protein, while antibodies to the Env protein remain high, suggesting a T cell independent antibody response to Env. RESULTS: To explore differential regulation of Gag and Env antibody responses, immunocompetent BALB/c and T cell deficient nude mice were immunized with virus like particles (VLP) of simian immunodeficiency virus or adenoviral vectors expressing SIV Gag and Env. High levels of antibodies against Gag and Env could only be induced in immunocompetent mice, but not in the immunodeficient mice. Thus, neither cells expressing Env after adenoviral gene transfer nor VLPs induce a T cell independent primary anti-Env antibody response. However, secondary B cell responses to Env, but not to Gag, were observed in immunodeficient mice after transfer of primed B cells and boosting with VLPs or adenoviral vectors expressing Gag and Env. This T cell independent secondary antibody response to Env was reduced after stimulation with VLPs modified to contain monomeric membrane bound gp130 surface subunit of Env and undetectable after injection of soluble gp130. CONCLUSIONS: Membrane-bound trimeric Env seems to be responsible for the maintenance of high levels of anti-Env antibodies during progression to AIDS. This T cell independent secondary antibody response may prevent T cell-dependent affinity maturation and thus contribute to viral immune escape by favoring persistence of non-protective antibodies.
Asunto(s)
Formación de Anticuerpos , Linfocitos T CD4-Positivos/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Proteínas del Envoltorio Viral/inmunología , Síndrome de Inmunodeficiencia Adquirida/inmunología , Síndrome de Inmunodeficiencia Adquirida/virología , Adenoviridae , Animales , Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/virología , Membrana Celular/metabolismo , Progresión de la Enfermedad , Productos del Gen gag/inmunología , Vectores Genéticos , Evasión Inmune , Inmunización Secundaria , Huésped Inmunocomprometido , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Virus de la Inmunodeficiencia de los Simios/metabolismo , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Although priming with replicating adenovirus type 5 host range mutant (Ad5hr)-human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) recombinants, followed by HIV/SIV envelope boosting, has proven highly immunogenic, resulting in protection from SIV/simian-human immunodeficiency virus (SHIV) challenges, Ad5hr recombinant distribution, replication, and persistence have not been examined comprehensively in nonhuman primates. We utilized Ad5hr-green fluorescent protein and Ad5hr-SIV recombinants to track biodistribution and immunogenicity following mucosal priming of rhesus macaques by the intranasal/intratracheal, sublingual, vaginal, or rectal route. Ad recombinants administered by all routes initially targeted macrophages in bronchoalveolar lavage (BAL) fluid and rectal tissue, later extending to myeloid dendritic cells in BAL fluid with persistent expression in rectal mucosa 25 weeks after the last Ad immunization. Comparable SIV-specific immunity, including cellular responses, serum binding antibody, and mucosal secretory IgA, was elicited among all groups. The ability of the vector to replicate in multiple mucosal sites irrespective of delivery route, together with the targeting of macrophages and professional antigen-presenting cells, which provide potent immunogenicity at localized sites of virus entry, warrants continued use of replicating Ad vectors.
Asunto(s)
Adenovirus Humanos/genética , Células Dendríticas/inmunología , Vectores Genéticos , Macrófagos/inmunología , Vacunas contra el SIDAS/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Administración Intranasal , Administración Intravaginal , Administración Rectal , Administración Sublingual , Animales , Anticuerpos Antivirales/sangre , Femenino , Inmunoglobulina A Secretora/análisis , Leucocitos Mononucleares/inmunología , Macaca mulatta , Masculino , Vacunas contra el SIDAS/administración & dosificación , Vacunas contra el SIDAS/genética , Virus de la Inmunodeficiencia de los Simios/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
We have shown that sequential replicating adenovirus type 5 host range mutant human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) recombinant priming delivered first intranasally (i.n.) plus orally and then intratracheally (i.t.), followed by envelope protein boosting, elicits broad cellular immunity and functional, envelope-specific serum and mucosal antibodies that correlate with protection from high-dose SIV and simian/human immunodeficiency virus (SHIV) challenges in rhesus macaques. Here we extended these studies to compare the standard i.n./i.t. regimen with additional mucosal administration routes, including sublingual, rectal, and vaginal routes. Similar systemic cellular and humoral immunity was elicited by all immunization routes. Central and effector memory T cell responses were also elicited by the four immunization routes in bronchoalveolar lavage fluid and jejunal, rectal, and vaginal tissue samples. Cellular responses in vaginal tissue were more compartmentalized, being induced primarily by intravaginal administration. In contrast, all immunization routes elicited secretory IgA (sIgA) responses at multiple mucosal sites. Following a repeated low-dose intrarectal (i.r.) challenge with SIV(mac251) at a dose transmitting one or two variants, protection against acquisition was not achieved except in one macaque in the i.r. immunized group. All immunized macaques exhibited reduced peak viremia compared to that of controls, correlated inversely with prechallenge serum antienvelope avidity, antibody-dependent cellular cytotoxicity (ADCC) titers, and percent antibody-dependent cell-mediated viral inhibition. Both antibody avidity and ADCC titers were correlated with the number of exposures required for infection. Notably, we show for the first time a significant correlation of vaccine-induced sIgA titers in rectal secretions with delayed acquisition. Further investigation of the characteristics and properties of the sIgA should elucidate the mechanism leading to this protective effect.
Asunto(s)
Adenovirus Humanos/inmunología , Productos del Gen env/inmunología , Inmunoglobulina A Secretora/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Adenovirus Humanos/genética , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Citocinas/metabolismo , Femenino , Productos del Gen env/genética , Humanos , Inmunidad Mucosa , Memoria Inmunológica , Macaca mulatta , Masculino , Datos de Secuencia Molecular , Virus de la Inmunodeficiencia de los Simios/genética , Linfocitos T/inmunología , Vacunas Sintéticas/genética , Viremia/inmunologíaRESUMEN
An HIV Env immunogen capable of eliciting broad immunity is critical for a successful vaccine. We constructed and characterized adenovirus 5 host range mutant (Ad5hr) recombinants encoding HIV(SF162) gp160 (subtype B) and HIV(TV1) gp160 (subtype C). Immunization of mice with one or both induced cellular immunity to subtype B and C peptides by ELISpot, and antibody responses with high binding titers to HIV Env of subtypes A, B, C, and E. Notably, Ad5hr-HIV(TV1) gp160 induced better cellular immunity than Ad5hr-HIV(SF162) gp160, either alone or following co-administration. Thus, the TV1 Env recombinant alone may be sufficient for eliciting immune responses against both subtype B and C envelopes. Further studies of Ad5hr-HIV(TV1) gp160 in rhesus macaques will evaluate the suitability of this insert for a future phase I clinical trial using a replication-competent Ad4 vector.
Asunto(s)
Vacunas contra el SIDA/inmunología , Adenoviridae/inmunología , Proteínas gp160 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Vacunas contra el SIDA/biosíntesis , Adenoviridae/genética , Animales , Formación de Anticuerpos , Especificidad de Anticuerpos , Femenino , Anticuerpos Anti-VIH/sangre , VIH-1/inmunología , Inmunidad Celular , Ratones , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: We investigated the interplay between complement and antibodies upon priming with single-cycle replicating viral vectors (SCIV) encoding SIV antigens combined with Adeno5-SIV or SCIV pseudotyped with murine leukemia virus envelope boosting strategies. The vaccine was applied via spray-immunization to the tonsils of rhesus macaques and compared with systemic regimens. RESULTS: Independent of the application regimen or route, viral loads were significantly reduced after challenge with SIVmac239 (p < 0.03) compared to controls. Considerable amounts of neutralizing antibodies were induced in systemic immunized monkeys. Most of the sera harvested during peak viremia exhibited a trend with an inverse correlation between complement C3-deposition on viral particles and plasma viral load within the different vaccination groups. In contrast, the amount of the observed complement-mediated lysis did not correlate with the reduction of SIV titres. CONCLUSION: The heterologous prime-boost strategy with replication-deficient viral vectors administered exclusively via the tonsils did not induce any neutralizing antibodies before challenge. However, after challenge, comparable SIV-specific humoral immune responses were observed in all vaccinated animals. Immunization with single cycle immunodeficiency viruses mounts humoral immune responses comparable to live-attenuated immunodeficiency virus vaccines.
Asunto(s)
Anticuerpos Antivirales/inmunología , Proteínas del Sistema Complemento/inmunología , Vacunas contra el SIDAS/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/inmunología , Adenovirus Humanos/genética , Aerosoles , Animales , Anticuerpos Antivirales/sangre , Vectores Genéticos , Virus de la Leucemia Murina/genética , Macaca mulatta , Pruebas de Neutralización , Tonsila Palatina/virología , Virus de la Inmunodeficiencia de los Simios/genética , Carga ViralRESUMEN
BACKGROUND: HIV prevalence time trends vary in sub-Saharan African countries. In the present study, we studied time trends and regional differences in the prevalence of HIV infection among women attending antenatal care clinics (ANC) in 7 sites located in 2 provinces in Cameroon. METHODOLOGY: As part of ANC, 16,626 women consented to HIV testing from 2000 to 2006. Sociodemographic and risk factor information was collected during the initial 3 years of the study. This information was aggregated within sites and used as site-level covariate in multilevel logistic regression analysis. RESULTS: HIV prevalence decreased significantly in women younger than 20 years from 13% in 2000 to 5% in 2006. Age-specific prevalence varied among the sites, with a peak prevalence occurring more often at a higher age in 2004-2006 versus 2000-2003, suggesting a reduction of HIV incidence over time. There was a substantial heterogeneity across sites in HIV prevalence, which was lower in sites where women had earlier sexual debut and were less well educated. CONCLUSIONS: ANC surveillance indicates a decreasing trend in HIV prevalence in the studied sites in Cameroon. Cultural differences might have accounted for the heterogeneity of HIV infection observed across sites, which call for tailored interventions.
Asunto(s)
Infecciones por VIH/epidemiología , Infecciones por VIH/prevención & control , Complicaciones Infecciosas del Embarazo/prevención & control , Adolescente , Adulto , Camerún/epidemiología , Femenino , Humanos , Embarazo , Prevalencia , Factores de Tiempo , Adulto JovenRESUMEN
Exosomes have been proposed as candidates for therapeutic immunization. The present study demonstrates that incorporation of the G protein of vesicular stomatitis virus (VSV-G) into exosome-like vesicles (ELVs) enhances their uptake and induces the maturation of dendritic cells. Targeting of VSV-G and ovalbumin as a model antigen to the same ELVs increased the cross-presentation of ovalbumin via an endosomal acidification mechanism. Immunization of mice with VSV-G and ovalbumin containing ELVs led to an increased IgG2a antibody response, expansion of antigen-specific CD8 T cells, strong in vivo CTL responses, and protection from challenge with ovalbumin expressing tumor cells. Thus, incorporation of VSV-G and targeting of antigens to ELVs are attractive strategies to improve exosomal vaccines.
Asunto(s)
Células Dendríticas/inmunología , Glicoproteínas de Membrana/inmunología , Vesículas Secretoras/inmunología , Vacunas/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos/sangre , Presentación de Antígeno , Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica , Endosomas/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias/prevención & control , Ovalbúmina/inmunología , Transporte de ProteínasRESUMEN
BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) has shown promising results as a cytokine adjuvant for antiviral vaccines and in various models of tumor gene therapy. To explore whether the targeting of antigens to GM-CSF receptors on antigen-presenting cells enhances antigen-specific CD8 T-cell responses, fusion proteins of GM-CSF and ovalbumin (OVA) were expressed by DNA and adenoviral vector vaccines. In addition, bicistronic vectors allowing independent expression of the antigen and the cytokine were tested in parallel. RESULTS: In vitro, the GM-CSF ovalbumin fusion protein (GM-OVA) led to the better stimulation of OVA-specific CD8+ T cells by antigen-presenting cells than OVA and GM-CSF given as two separate proteins. However, prime-boost immunizations of mice with DNA and adenoviral vector vaccines encoding GM-OVA suppressed CD8+ T-cell responses to OVA. OVA-specific IgG2a antibody levels were also reduced, while the IgG1 antibody response was enhanced. Suppression of CD8+ T cell responses by GM-OVA vaccines was associated with the induction of neutralizing antibodies to GM-CSF. In contrast, the coexpression of GM-CSF and antigens in DNA prime adenoviral boost immunizations led to a striking expansion of polyfunctional OVA-specific CD8+ T cells without the induction of autoantibodies. CONCLUSION: The induction of autoantibodies suggests a general note of caution regarding the use of highly immunogenic viral vector vaccines encoding fusion proteins between antigens and host proteins. In contrast, the expansion of polyfunctional OVA-specific CD8+ T cells after immunizations with bicistronic vectors further support a potential application of GM-CSF as an adjuvant for heterologous prime-boost regimens with genetic vaccines. Since DNA prime adenoviral vector boost regimenes are presently considered as one of the most efficient ways to induce CD8+ T cell responses in mice, non-human primates and humans, further enhancement of this response by GM-CSF is a striking observation.
Asunto(s)
Adenoviridae/inmunología , Autoinmunidad/inmunología , Linfocitos T CD8-positivos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Vacunas de ADN/inmunología , Adyuvantes Inmunológicos , Animales , Autoanticuerpos/biosíntesis , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Femenino , Vectores Genéticos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The development of needle-free vaccines is one of the recently defined "grand challenges in global health" (H. Varmus, R. Klausner, R. Klausner, R. Zerhouni, T. Acharya, A. S. Daar, and P. A. Singer, Science 302:398-399, 2003). To explore whether a natural pathway to the inductive site of the mucosa-associated lymphatic tissue could be exploited for atraumatic immunization purposes, replication-deficient viral vector vaccines were sprayed directly onto the tonsils of rhesus macaques. Tonsillar immunization with viral vector vaccines encoding simian immunodeficiency virus (SIV) antigens induced cellular and humoral immune responses. Viral RNA levels after a stringent SIV challenge were reduced, providing a level of protection similar to that observed after systemic immunization with the same vaccines. Thus, atraumatic oral spray immunization with replication-deficient vectors can overcome the epithelial barrier, deliver the vaccine antigen to the mucosa-associated lymphatic tissue, and avoid induction of tolerance, providing a novel approach to circumvent acceptability problems of syringe and needle vaccines for children and in developing countries.
Asunto(s)
Administración Oral , Vacunas contra el SIDAS/administración & dosificación , Vacunas contra el SIDAS/inmunología , Animales , Macaca mulatta , Tonsila Palatina/inmunología , ARN Viral/sangre , Vacunas contra el SIDAS/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/inmunología , Carga ViralRESUMEN
BACKGROUND: Proteins of human and animal viruses are frequently expressed from RNA polymerase II dependent expression cassettes to study protein function and to develop gene-based vaccines. Initial attempts to express the G protein of vesicular stomatitis virus (VSV) and the F protein of respiratory syncytial virus (RSV) by eukaryotic promoters revealed restrictions at several steps of gene expression. RESULTS: Insertion of an intron flanked by exonic sequences 5'-terminal to the open reading frames (ORF) of VSV-G and RSV-F led to detectable cytoplasmic mRNA levels of both genes. While the exonic sequences were sufficient to stabilise the VSV-G mRNA, cytoplasmic mRNA levels of RSV-F were dependent on the presence of a functional intron. Cytoplasmic VSV-G mRNA levels led to readily detectable levels of VSV-G protein, whereas RSV-F protein expression remained undetectable. However, RSV-F expression was observed after mutating two of four consensus sites for polyadenylation present in the RSV-F ORF. Expression levels could be further enhanced by codon optimisation. CONCLUSION: Insufficient cytoplasmic mRNA levels and premature polyadenylation prevent expression of RSV-F by RNA polymerase II dependent expression plasmids. Since RSV replicates in the cytoplasm, the presence of premature polyadenylation sites and elements leading to nuclear instability should not interfere with RSV-F expression during virus replication. The molecular mechanisms responsible for the destabilisation of the RSV-F and VSV-G mRNAs and the different requirements for their rescue by insertion of an intron remain to be defined.
Asunto(s)
Expresión Génica , Técnicas Genéticas , Glicoproteínas de Membrana/biosíntesis , ARN Polimerasa II/metabolismo , Virus Sincitiales Respiratorios/genética , Virus de la Estomatitis Vesicular Indiana/genética , Proteínas del Envoltorio Viral/biosíntesis , Proteínas Virales de Fusión/biosíntesis , Línea Celular , Codón/genética , Citoplasma/química , Exones , Vectores Genéticos , Humanos , Intrones , Glicoproteínas de Membrana/genética , Plásmidos , Señales de Poliadenilación de ARN 3'/genética , Proteínas del Envoltorio Viral/genética , Proteínas Virales de Fusión/genéticaRESUMEN
Previously we demonstrated that the expression of fusogenic membrane proteins (FMG) of measles virus (MV-H/F) can synergistically enhance chemotherapy. In this study, we used median-effect analysis to evaluate whether the expression of respiratory syncytial virus (RSV-F), as well as vesicular stomatitis virus (VSV-G) can also synergistically enhance chemotherapy. Furthermore we elucidated by western blot analysis some molecular pathways that might be responsible for this effect. We showed in colorectal cancer cell lines that the expression of MV-H/F, but also of RSV-F, as well as VSV-G can synergistically enhance p53-independent clinically relevant chemotherapy (FOLFOX) over most of the cytotoxic dose range. In a subcutaneous HT-29 colorectal xenograft model, we demonstrated that the administration of replication-deficient adenovirus vectors encoding MV-H/F, RSV-F or VSV-G in combination with FOLFOX significantly enhanced treatment outcome when compared to the treatment with each compound individually. The anti-neoplastic efficacy of RSV-F was somewhat better than that of MV-H/F and both were statistically significantly more efficacious than VSV-G alone or in combination with chemotherapy. Treatment efficacy was further significantly improved when the replication-deficient FMG encoding vectors were trans-complemented for replication with a replication-restricted oncolytic adenovirus to improve tumor transduction efficiency. The combination of FMG expression, chemotherapy and trans-complementing oncolytic vectors resulted in a significantly better treatment efficacy than treatment with its components as single- or double-agent therapy. Our data indicates that FMG expression (i.e., RSV-F and MV-H/F) in combination with chemotherapy and viral oncolysis warrants further investigations.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Colorrectales/terapia , Terapia Genética , Virus Sincitiales Respiratorios/genética , Vesiculovirus/genética , Proteínas Virales de Fusión/genética , Animales , Anexina A5/metabolismo , Apoptosis , Caspasas/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Terapia Combinada , Femenino , Fluorouracilo/uso terapéutico , Humanos , Leucovorina/uso terapéutico , Fusión de Membrana , Ratones , Ratones Desnudos , Mitocondrias/enzimología , Compuestos Organoplatinos/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Infection with the SARS-associated coronavirus (SARS-CoV) induces an atypical pulmonary disease with a high lethality rate. Although the initial SARS epidemic was contained, sporadic outbreaks of the disease still occur, suggesting a continuous need for a vaccine against this virus. We therefore explored exosome-based vaccines containing the spike S proteins of SARS-CoV. S-containing exosomes were obtained by replacing the transmembrane and cytoplasmic domains of the S protein by those of VSV-G. The immunogenicity and efficacy of the S-containing exosomes were tested in mice and compared to an adenoviral vector vaccine expressing the S protein. Both, S-containing exosomes and the adenoviral vector vaccine induced neutralizing antibody titers. After priming with the SARS-S exosomal vaccine and boosting with the adenoviral vector the neutralizing antibody titers exceeded those observed in the convalescent serum of a SARS patient. Both approaches were effective in a SARS-S-expressing tumor challenge model and thus warrant further investigation.
Asunto(s)
Glicoproteínas de Membrana/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Vesículas Transportadoras/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/sangre , Línea Celular , Chlorocebus aethiops , Modelos Animales de Enfermedad , Humanos , Inmunización Secundaria , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Neoplasias/inmunología , Neoplasias/patología , Pruebas de Neutralización , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Glicoproteína de la Espiga del Coronavirus , Vacunas Sintéticas/inmunología , Células Vero , Proteínas del Envoltorio Viral/genéticaRESUMEN
Preferential integration into transcriptionally active regions of genomes has been observed for retroviral vectors based on gamma-retroviruses and lentiviruses. However, differences in the integration site preferences were detected, which might be explained by differences in viral components of the preintegration complexes. Viral determinants of integration site preferences have not been defined. Therefore, integration sites of simian immunodeficiency virus (SIV)-based vectors produced in the absence of accessory genes or lacking promoter and enhancer elements were compared. Similar integration patterns for the different SIV vectors indicate that vif, vpr, vpx, nef, env, and promoter or enhancer elements are not required for preferential integration of SIV into transcriptionally active regions of genomes.
Asunto(s)
Vectores Genéticos/genética , Virus de la Inmunodeficiencia de los Simios/genética , Integración Viral/genética , Células Cultivadas , Mapeo Cromosómico , Genoma Humano , HumanosRESUMEN
Vaccination with exogenous antigens such as recombinant viral proteins, immunodeficiency virus-derived whole inactivated virus particles, or virus-like particles (VLP) has generally failed to provide sufficient protection in animal models for AIDS. Pseudotyping VLPs with the vesicular stomatitis virus G protein (VSV-G), which is known to mediate entry into dendritic cells, might allow more efficient stimulation of immune responses. Therefore, we pseudotyped noninfectious immunodeficiency virus-like particles with VSV-G and carried out a preliminary screen of their immunogenicity and vaccination efficacy. Incorporation of VSV-G into HIV-1 VLPs led to hundred-fold higher antibody titers to HIV-1 Gag and enhancement of T cell responses in mice. Repeated vaccination of rhesus monkeys for 65 weeks with VSV-G pseudotyped simian immunodeficiency virus (SIV)-like particles (VLP[G]) provided initial evidence for efficient suppression of viral load after mucosal challenge with the SIVmac239 virus. Challenge of monkeys after a 28 week vaccination regimen with VLP[G] led to a reduction in peak viremia, but persistent suppression of viral load was not achieved. Due to limitations in the number of animals available for this study, improved efficacy of VSV-G pseudotyped VLPs in nonhuman primates could not be demonstrated. However, mouse experiments revealed that pseudotyping of VLPs with fusion-competent VSV-G clearly improves their immunogenicity. Additional strategies, particularly adjuvants, should be considered to provide greater protection against a challenge with pathogenic immunodeficiency virus.
Asunto(s)
Vacunas contra el SIDA/inmunología , Glicoproteínas de Membrana/inmunología , Virus de la Estomatitis Vesicular Indiana , Proteínas del Envoltorio Viral/inmunología , Vacunas contra el SIDA/administración & dosificación , Animales , Línea Celular , Esquema de Medicación , Anticuerpos Anti-VIH/sangre , Humanos , Macaca mulatta/inmunología , Glicoproteínas de Membrana/genética , Ratones , ARN Viral/sangre , Factores de Tiempo , Proteínas del Envoltorio Viral/genética , Carga Viral , Virión/genética , Virión/inmunologíaRESUMEN
BACKGROUND: The potential of lentiviral vectors for clinical gene therapy has not yet been evaluated. One of the reasons is the cytotoxicity of lentiviral packaging genes which makes the generation of stable producer cell lines difficult. Therefore, a novel packaging system for lentiviral vectors based on transient expression of packaging genes by recombinant adenoviruses was developed. METHODS: Adenoviral vectors expressing VSV-G, codon-optimized HIV-1 gag-pol, and codon-optimized SIV gag-pol under the control of a tetracycline-regulatable promoter (adenoviral lenti-pack vectors) were constructed and the production levels of this vector system were evaluated. RESULTS: The generated adenoviral lenti-pack vectors could be grown to high titers when transgene expression was suppressed and no evidence for instabilities was obtained. Cells stably transfected with a SIV-based vector construct were converted into lentiviral vector producer cells by infection with the adenoviral lenti-pack vectors. Lentiviral vector titers obtained were as high as vector titers obtained by transient cotransfection experiments. A protocol was developed that allowed preparation of lentiviral vector stocks with undetectable levels of contaminating adenoviral lenti-pack vectors. CONCLUSIONS: The adenoviral lenti-pack vectors described should provide a convenient alternative approach to inducible packaging cell lines for large-scale lentiviral vector production. Transient expression of cytotoxic lentiviral packaging genes by the adenoviral lenti-pack vectors circumvents loss of titers during prolonged culture of packaging cell lines. The design of the adenoviral lenti-pack vectors should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the lentiviral vector constructs that can be packaged.
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Adenoviridae/genética , VIH-1/genética , Virus de la Inmunodeficiencia de los Simios/genética , Animales , Línea Celular , Chlorocebus aethiops , Proteínas de Fusión gag-pol/genética , Vectores Genéticos , Humanos , Regiones Promotoras Genéticas , Tetraciclina/farmacología , Transducción GenéticaRESUMEN
Granulocyte/macrophage-colony stimulating factor (GM-CSF) is a valuable adjuvant to enhance induction of cellular immune responses in rodents. Less information is available regarding its use as an adjuvant in primates or humans. We explored recombinant human GM-CSF for potential vaccine studies in rhesus macaques and focused on its effect on peripheral monocytes as progenitors of dendritic cells and its potential immunogenicity. Application of human GM-CSF to nine animals led to an average 32-fold increase in monocyte numbers. This was not observed upon re-treatment, which coincided with GM-CSF-specific neutralising antibodies. These also neutralised the activity of rhesus macaque GM-CSF. The data underscore the need to use species-specific GM-CSF for immunomodulation in primates.
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Anticuerpos Bloqueadores/biosíntesis , Anticuerpos Bloqueadores/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Vacunas/inmunología , Animales , Western Blotting , Separación Celular , Clonación Molecular , Citocinas/farmacología , Femenino , Humanos , Macaca mulatta , Masculino , Monocitos/inmunología , Pruebas de NeutralizaciónRESUMEN
The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia with a fatal outcome in approximately 10% of patients. SARS-CoV is not closely related to other coronaviruses but shares a similar genome organization. Entry of coronaviruses into target cells is mediated by the viral S protein. We functionally analyzed SARS-CoV S using pseudotyped lentiviral particles (pseudotypes). The SARS-CoV S protein was found to be expressed at the cell surface upon transient transfection. Coexpression of SARS-CoV S with human immunodeficiency virus-based reporter constructs yielded viruses that were infectious for a range of cell lines. Most notably, viral pseudotypes harboring SARS-CoV S infected hepatoma cell lines but not T- and B-cell lines. Infection of the hepatoma cell line Huh-7 was also observed with replication-competent SARS-CoV, indicating that hepatocytes might be targeted by SARS-CoV in vivo. Inhibition of vacuolar acidification impaired infection by SARS-CoV S-bearing pseudotypes, indicating that S-mediated entry requires low pH. Finally, infection by SARS-CoV S pseudotypes but not by vesicular stomatitis virus G pseudotypes was efficiently inhibited by a rabbit serum raised against SARS-CoV particles and by sera from SARS patients, demonstrating that SARS-CoV S is a target for neutralizing antibodies and that such antibodies are generated in SARS-CoV-infected patients. Our results show that viral pseudotyping can be employed for the analysis of SARS-CoV S function. Moreover, we provide evidence that SARS-CoV infection might not be limited to lung tissue and can be inhibited by the humoral immune response in infected patients.
Asunto(s)
Hígado/virología , Glicoproteínas de Membrana/metabolismo , Síndrome Respiratorio Agudo Grave/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Proteínas del Envoltorio Viral/metabolismo , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Carcinoma Hepatocelular , Línea Celular , Línea Celular Tumoral , Humanos , Riñón/citología , Riñón/virología , Lentivirus/genética , Hígado/citología , Neoplasias Hepáticas , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Pruebas de Neutralización , Conejos , Síndrome Respiratorio Agudo Grave/inmunología , Glicoproteína de la Espiga del Coronavirus , Transfección , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , ViriónRESUMEN
To reduce the risks associated with live-attenuated immunodeficiency virus vaccines, single-cycle immunodeficiency viruses (SCIVs) were developed by primer complementation and production of the vaccine in the absence of vif in a vif-independent cell line. After a single intravenous injection of SCIVs into rhesus monkeys, peak viral RNA levels of 10(3) to 10(4) copies/ml plasma were observed, indicating efficient expression of SCIV in the vaccinee. After booster immunizations with SCIVs, SIV-specific humoral and cellular immune responses were observed. Although the vaccine doses used in this pilot study could not protect vaccinees from subsequent intravenous challenge with pathogenic SIVmac239, our results demonstrate that the novel SCIV approach allows us to uncouple in vivo expression levels from the viral replicative capacity facilitating the analysis of the relationship between viral expression levels or viral genes and immune responses induced by SIV.