RESUMEN
UNLABELLED: In previous work, a prototypic recombinant vesicular stomatitis virus Indiana serotype (rVSIV) vector expressing simian immunodeficiency virus (SIV) gag and human immunodeficiency virus type 1 (HIV-1) env antigens protected nonhuman primates (NHPs) from disease following challenge with an HIV-1/SIV recombinant (SHIV). However, when tested in a stringent NHP neurovirulence (NV) model, this vector was not adequately attenuated for clinical evaluation. For the work described here, the prototypic rVSIV vector was attenuated by combining specific G protein truncations with either N gene translocations or mutations (M33A and M51A) that ablate expression of subgenic M polypeptides, by incorporation of temperature-sensitive mutations in the N and L genes, and by deletion of the VSIV G gene to generate a replicon that is dependent on trans expression of G protein for in vitro propagation. When evaluated in a series of NHP NV studies, these attenuated rVSIV variants caused no clinical disease and demonstrated a very significant reduction in neuropathology compared to wild-type VSIV and the prototypic rVSIV vaccine vector. In spite of greatly increased in vivo attenuation, some of the rVSIV vectors elicited cell-mediated immune responses that were similar in magnitude to those induced by the much more virulent prototypic vector. These data demonstrate novel approaches to the rational attenuation of VSIV NV while retaining vector immunogenicity and have led to identification of an rVSIV N4CT1gag1 vaccine vector that has now successfully completed phase I clinical evaluation. IMPORTANCE: The work described in this article demonstrates a rational approach to the attenuation of vesicular stomatitis virus neurovirulence. The major attenuation strategy described here will be most likely applicable to other members of the Rhabdoviridae and possibly other families of nonsegmented negative-strand RNA viruses. These studies have also enabled the identification of an attenuated, replication-competent rVSIV vector that has successfully undergone its first clinical evaluation in humans. Therefore, these studies represent a major milestone in the development of attenuated rVSIV, and likely other vesiculoviruses, as a new vaccine platform(s) for use in humans.
Asunto(s)
Vacunas contra el SIDA/inmunología , Sistema Nervioso Central/virología , Vectores Genéticos/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Macaca fascicularis , Virus de la Estomatitis Vesicular Indiana/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/genética , Animales , Anticuerpos Antivirales/inmunología , Sistema Nervioso Central/inmunología , Modelos Animales de Enfermedad , Vectores Genéticos/genética , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , VIH-1/genética , Humanos , Macaca fascicularis/genética , Macaca fascicularis/inmunología , Macaca fascicularis/virología , Masculino , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Virus de la Estomatitis Vesicular Indiana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/administración & dosificación , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent "blips" in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication.
Asunto(s)
Inmunización Secundaria/métodos , Interleucina-12/administración & dosificación , Vacunas contra el SIDAS/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/inmunología , Vacunación/métodos , Vacunas de ADN/inmunología , Adenoviridae/genética , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/genética , Animales , Vectores Genéticos , Interleucina-12/genética , Ganglios Linfáticos/virología , Macaca mulatta , ARN Viral/aislamiento & purificación , Vacunas contra el SIDAS/administración & dosificación , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Vacunas de ADN/administración & dosificación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Carga Viral , Viremia/prevención & controlRESUMEN
Assessment of in vivo viral replication of live attenuated recombinant vesicular stomatitis virus (rVSV) vaccine vector candidates encoding HIV gag requires comprehensive preclinical safety studies, and development of sensitive assays to monitor the outcome of vaccination of animals is important. In this study, two 2-step quantitative real-time RT-PCR assays were developed; a singleplex assay to detect VSV genomic RNA from ferrets inoculated intra-cranially (IC) or intra-nasally (IN) with either a wild-type (wt) virus or an attenuated rVSV vector engineered to express HIV gag protein, and a duplex assay to simultaneously detect VSV-N and HIV-gag mRNAs from cynomolgus macaques inoculated intra-thalamically (IT) with the same viruses. Using synthetic oligonucleotides as standards, the lower limit of detection of VSV-N and HIV-gag was 50 copies. Results showed high levels of wt VSV(IN) genomic RNA and mRNA in ferret and macaque tissues, respectively, and significantly lower levels of VSV genomic RNA and VSV-N and HIV-gag mRNAs in tissues from animals inoculated with the attenuated rVSV vector. These assays correlated with both the course of infection for these animals, and the infectious viral load measured by a standard plaque assay, and could be used to determine the safety profile of rVSV vaccine vectors.
Asunto(s)
Vacunas contra el SIDA , Productos del Gen gag/aislamiento & purificación , VIH/genética , ARN Viral/aislamiento & purificación , Virus de la Estomatitis Vesicular Indiana/genética , Vacunas contra el SIDA/genética , Animales , Terapia Antirretroviral Altamente Activa , Hurones , Productos del Gen gag/genética , Vectores Genéticos , Macaca , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Virus de la Estomatitis Vesicular Indiana/aislamiento & purificación , Carga Viral , Replicación ViralRESUMEN
This report describes a transfection-independent system for packaging alphavirus replicon vectors using modified vaccinia virus Ankara (MVA) vectors to express all of the RNA components necessary for the production of Venezuelan equine encephalitis (VEE) virus replicon particles (VRP). Infection of mammalian cells with these recombinant MVA vectors resulted in robust expression of VEE structural genes, replication of the alphavirus vector and high titers of VRP. In addition, VRP packaging was achieved in a cell type (fetal rhesus lung) that has been approved for the manufacturing of vaccines destined for human use.
Asunto(s)
ADN Viral/administración & dosificación , ADN Viral/genética , Sistemas de Liberación de Medicamentos/métodos , Virus de la Encefalitis Equina Venezolana/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Replicón/genética , Transfección/métodos , Virus Vaccinia/genética , Virión/genética , Alphavirus/genética , Regulación Viral de la Expresión GénicaRESUMEN
Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert.
Asunto(s)
Virus del Moquillo Canino/fisiología , Portadores de Fármacos , Expresión Génica , Productos del Gen gag/biosíntesis , Vectores Genéticos , Inestabilidad Genómica , Replicación Viral , Abdomen/virología , Animales , Encéfalo/virología , Virus del Moquillo Canino/genética , Hurones , Productos del Gen gag/genética , Tejido Linfoide/virología , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Vacunas contra el SIDAS/administración & dosificación , Vacunas contra el SIDAS/genética , Virus de la Inmunodeficiencia de los Simios/genética , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genéticaRESUMEN
Alphavirus replicon particles are being exploited for a variety of purposes both in vitro as gene expression vectors, and in vivo as vaccines or gene therapy vectors. There is a need for a simple and universal method of titration of replicon particles that is independent of expression of the foreign protein. We devised a method that uses modified vaccinia virus Ankara (MVA) as an indicator virus, to deliver a Venezuelan equine encephalitis virus (VEE) defective helper RNA encoding green fluorescent protein (GFP). Co-infection of cells with the MVA-based indicator and Venezuelan equine encephalitis virus replicon particles (VRP) results in expression of the GFP gene. VRP titer is readily determined by counting fluorescent cells.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/fisiología , Virus Defectuosos/genética , Virus de la Encefalitis Equina Venezolana/genética , Proteínas Luminiscentes/genética , Replicón/genética , Animales , Chlorocebus aethiops , Proteínas Fluorescentes Verdes , Virus Vaccinia/genética , Células VeroRESUMEN
The structure of the HIV-1 envelope membrane-proximal external region (MPER) is influenced by its association with the lipid bilayer on the surface of virus particles and infected cells. To develop a replicating vaccine vector displaying MPER sequences in association with membrane, Env epitopes recognized by the broadly neutralizing antibodies 2F5, 4E10, or both were grafted into the membrane-proximal stem region of the vesicular stomatitis virus (VSV) glycoprotein (G). VSV encoding functional G-MPER chimeras based on G from the Indiana or New Jersey serotype propagated efficiently, although grafting of both epitopes (G-2F5-4E10) modestly reduced replication and resulted in the acquisition of one to two adaptive mutations in the grafted MPER sequence. Monoclonal antibodies 2F5 and 4E10 efficiently neutralized VSV G-MPER vectors and bound to virus particles in solution, indicating that the epitopes were accessible in the preattachment form of the G-MPER chimeras. Overall, our results showed that the HIV Env MPER could functionally substitute for the VSV G-stem region implying that both perform similar functions even though they are from unrelated viruses. Furthermore, we found that the MPER sequence grafts induced low but detectable MPER-specific antibody responses in rabbits vaccinated with live VSV, although additional vector and immunogen modifications or use of a heterologous prime-boost vaccination regimen will be required to increase the magnitude of the immune response.
Asunto(s)
Anticuerpos Anti-VIH/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Vesiculovirus/fisiología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Femenino , Glicoproteínas de Membrana/inmunología , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Vesiculovirus/genética , Vesiculovirus/crecimiento & desarrollo , Vesiculovirus/inmunología , Proteínas del Envoltorio Viral/inmunología , Replicación Viral , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
We are investigating canine distemper virus (CDV) as a vaccine vector for the delivery of HIV envelope (Env) that closely resembles the native trimeric spike. We selected CDV because it will promote vaccine delivery to lymphoid tissues, and because human exposure is infrequent, reducing potential effects of pre-existing immunity. Using SIV Env as a model, we tested a number of vector and gene insert designs. Vectors containing a gene inserted between the CDV H and L genes, which encoded Env lacking most of its cytoplasmic tail, propagated efficiently in Vero cells, expressed the immunogen on the cell surface, and incorporated the SIV glycoprotein into progeny virus particles. When ferrets were vaccinated intranasally, there were no signs of distress, vector replication was observed in the gut-associated lymphoid tissues, and the animals produced anti-SIV Env antibodies. These data show that live CDV-SIV Env vectors can safely induce anti-Env immune responses following intranasal vaccination.
Asunto(s)
Virus del Moquillo Canino/genética , Portadores de Fármacos , Vacunas contra el SIDAS/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Vacunación/métodos , Proteínas del Envoltorio Viral/inmunología , Administración Intranasal , Animales , Anticuerpos Antivirales/sangre , Hurones , Tracto Gastrointestinal/virología , Tejido Linfoide/virología , Vacunas contra el SIDAS/administración & dosificación , Vacunas contra el SIDAS/genética , Virus de la Inmunodeficiencia de los Simios/genética , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Proteínas del Envoltorio Viral/genéticaRESUMEN
Broadly neutralizing antibodies (bNAbs), which develop over time in some HIV-1-infected individuals, define critical epitopes for HIV vaccine design. Using a systematic approach, we have examined neutralization breadth in the sera of about 1800 HIV-1-infected individuals, primarily infected with non-clade B viruses, and have selected donors for monoclonal antibody (mAb) generation. We then used a high-throughput neutralization screen of antibody-containing culture supernatants from about 30,000 activated memory B cells from a clade A-infected African donor to isolate two potent mAbs that target a broadly neutralizing epitope. This epitope is preferentially expressed on trimeric Envelope protein and spans conserved regions of variable loops of the gp120 subunit. The results provide a framework for the design of new vaccine candidates for the elicitation of bNAb responses.
Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , África del Sur del Sahara , Subgrupos de Linfocitos B/inmunología , Epítopos/inmunología , Anticuerpos Anti-VIH/biosíntesis , Proteína gp120 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/inmunología , Humanos , Memoria Inmunológica , Activación de Linfocitos , Pruebas de Neutralización , Fragmentos de Péptidos/inmunología , Multimerización de Proteína , Proteínas Recombinantes/inmunologíaRESUMEN
Widespread use of a live-attenuated influenza vaccine (LAIV) in the United States (licensed as FluMist) raises the possibility that vaccine viruses will contribute gene segments to the type A influenza virus gene pool. Progeny viruses possessing new genotypes might arise from genetic reassortment between circulating wild-type (wt) and vaccine strains, but it will be difficult to predict whether they will be viable or exhibit novel properties. To begin addressing these uncertainties, reverse-genetics was used to generate 34 reassortant viruses derived from wt influenza virus A/Sydney/5/97 and the corresponding live vaccine strain. The reassortants contained different combinations of vaccine and wt PB2, PB1, PA, NP, M, and NS gene segments whereas all strains encoded wt HA and NA glycoproteins. The phenotypes of the reassortant strains were compared to wt and vaccine viruses by evaluating temperature-sensitive (ts) plaque formation and replication attenuation (att) in ferrets following intranasal inoculation. The results demonstrated that the vaccine virus PB1, PB2, and NP gene segments were dominant when introduced into the wt A/Sydney/5/97 genetic background, producing recombinant viruses that expressed the ts and att phenotypes. A dominant attenuated phenotype also was evident when reassortant strains contained the vaccine M or PA gene segments, even though these polypeptides are not temperature-sensitive. Although the vaccine M and NS gene segments typically are not associated with temperature sensitivity, a number of reassortants containing these vaccine gene segments did exhibit a more restricted ts phenotype. Overall, no reassortant strains were more virulent than wt, and in fact, 33 of the 34 recombinant viruses replicated less efficiently in infected ferrets. These results suggest that genetic reassortment between wt and vaccine strains is unlikely to produce viruses having novel properties that differ substantially from either progenitor, and that the likely outcome of reassortment will be attenuated viruses.
Asunto(s)
Genes Virales , Virus de la Influenza A/genética , Vacunas contra la Influenza/biosíntesis , Virus Reordenados/genética , Recombinación Genética , Vacunas Atenuadas/genética , Proteínas Virales/genética , Animales , Hurones , Ingeniería Genética , Genotipo , Virus de la Influenza A/inmunología , Virus de la Influenza A/fisiología , Fenotipo , ARN Polimerasa Dependiente del ARN/metabolismo , Virus Reordenados/fisiología , Temperatura , Células Tumorales Cultivadas , Vacunas Atenuadas/química , Vacunas Atenuadas/inmunología , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Ensayo de Placa Viral , Proteínas Virales/química , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
The genetic and phenotypic stability of viruses isolated from young children following intranasal administration of the trivalent live-attenuated influenza virus vaccine (LAIV, marketed in the United States as FluMist) was evaluated by determination of genomic sequence and assessment of the cold-adapted (ca), temperature-sensitive (ts) and attenuated (att) phenotypes. The complete genomic sequence was determined for 56 independent isolates obtained from children following vaccination (21 type A/H1N1, 12 A/H3N2, 1 A/H3N1 and 22 type B viruses), 20% of which had no nucleotide misincorporations compared with administered vaccine. The remaining isolates had from one to seven changes per genome. None of the observed misincorporations resulted in predicted amino acid codon substitutions at sites previously shown to contribute to the ca, ts or att phenotypes, and all vaccine-derived isolates retained ca and ts phenotypes consistent with the observation that none of the vaccine recipients displayed distinctive symptoms. The results indicate that LAIV strains undergo very limited genetic change following replication in vaccine recipients and that those changes did not affect vaccine attenuation.
Asunto(s)
Adaptación Fisiológica , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Animales , Guarderías Infantiles , Preescolar , Frío , Femenino , Hurones , Humanos , Inmunización , Lactante , Recién Nacido , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Virus de la Influenza B/genética , Virus de la Influenza B/crecimiento & desarrollo , Vacunas contra la Influenza/efectos adversos , Vacunas contra la Influenza/inmunología , Masculino , Nasofaringe/microbiologíaRESUMEN
The International AIDS Vaccine Initiative has established a consortium to elucidate mechanisms of protection conferred by live attenuated simian immunodeficiency virus vaccines in monkeys. Here, the strategies defining key components of the protective immune response elicited by these vaccines are discussed.
Asunto(s)
Vacunas contra el SIDA , Síndrome de Inmunodeficiencia Adquirida/prevención & control , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Haplorrinos , Humanos , Vacunas AtenuadasRESUMEN
FluMist is a live-attenuated, trivalent influenza vaccine (LAIV) recently approved for intranasal administration. To demonstrate genetic stability during manufacture of the vaccine viruses in LAIV and a similar vaccine in development (CAIV-T), full genome consensus sequences were determined at multiple manufacturing stages for four influenza type A and five type B strains. The critical cold-adapted (ca), temperature-sensitive (ts) and attenuated (att) mutations were preserved in the virus manufacturing intermediates. Moreover, sequence identity was observed for all vaccine intermediates of the same strain. Minor sequence differences were noted in the shared gene segments of the vaccine viruses and their common progenitor master donor virus (MDV) and several of the hemagglutinin (HA) and neuraminidase (NA) genes contained nucleotide differences when compared to the wild-type parent. Nonetheless, all vaccine viruses retained the ca, ts, and att phenotypes. Thus, genetic and phenotypic stability of the vaccine viruses is maintained during the manufacture of LAIV/CAIV-T vaccines.
Asunto(s)
Adaptación Fisiológica/fisiología , Virus de la Influenza A/genética , Vacunas contra la Influenza/genética , Cultivo de Virus , Administración Intranasal , Aerosoles , Frío , Virus de la Influenza A/química , Virus de la Influenza A/fisiología , Vacunas contra la Influenza/química , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genéticaRESUMEN
A herpes simplex virus type 2 (HSV-2) UL24 beta-glucuronidase (UL24-betagluc) insertion mutant was derived from HSV-2 strain 186 via standard marker transfer techniques. Cell monolayers infected with UL24-betagluc yielded cytopathic effect with syncytium formation. UL24-betagluc replicated to wild-type viral titers in three different cell lines. UL24-betagluc was not virulent after intravaginal inoculation of BALB/c mice in that all inoculated animals survived doses up to 400 times the 50% lethal dose (LD50) of the parental virus. Furthermore, few UL24-betagluc-inoculated mice developed any vaginal lesions. Intravaginal inoculation of guinea pigs with UL24-betagluc at a dose equivalent to the LD50 of parental virus (approximately 5 x 10(3) PFU) was not lethal (10/10 animals survived). Although genital lesions developed in some UL24-betagluc-inoculated guinea pigs, both the overall number of lesions and the severity of disease were far less than that observed for animals infected with parental strain 186.
Asunto(s)
Modelos Animales de Enfermedad , Herpes Simple/etiología , Herpesvirus Humano 2/genética , Proteínas Virales/genética , Animales , Secuencia de Bases , Femenino , Genoma Viral , Cobayas , Herpesvirus Humano 2/patogenicidad , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Timidina Quinasa/fisiología , Virulencia , Replicación ViralRESUMEN
We have designed a peptide-based immunotherapeutic vaccine for treatment of androgen-responsive prostate cancer. The vaccine targets the luteinizing hormone-releasing hormone (LHRH) decapeptide that results in an androgen-deprivation immunotherapy. The design elements of the peptide immunogens are the LHRH peptide or B cell epitope synthetically linked to different promiscuous helper T cell (Th) sequences, the UBITh epitopes, derived from four natural pathogens for effective immunogenicity in outbred populations, and in some cases, also linked to an adjuvanting peptide from Yersinia invasin (Inv) protein. The UBITh LHRH immunogens are adsorbed on Alhydrogel or formulated as several different oil-based emulsions and tested in rodents, dogs, and a non-human primate, baboons. The immunogens generate an anti-LHRH antibody response specific to the LHRH decapeptide element in contrast to LHRH conjugate-carrier protein vaccines where only a small portion of the antibody response is directed to the target epitope and epitopic suppression is noted. Individual UBITh peptide domains, but not the LHRH and Inv peptide domains, are stimulatory in lymphocyte cultures. The UBITh LHRH immunogens in a clinically applicable formulation, controlled the growth of Dunning R3327-H androgen-responsive prostate tumor cells in rats. The results demonstrate universal responsiveness and long duration of androgen deprivation from three diverse species, and thus vaccine efficacy.
Asunto(s)
Andrógenos/metabolismo , Hormona Liberadora de Gonadotropina/inmunología , Neoplasias de la Próstata/terapia , Vacunas/inmunología , Animales , Perros , Diseño de Fármacos , Ensayo de Inmunoadsorción Enzimática , Hormona Liberadora de Gonadotropina/sangre , Humanos , Inmunoterapia , Masculino , Trasplante de Neoplasias , Papio , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/prevención & control , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , Testosterona/sangre , Vacunas/síntesis química , Vacunas de Subunidad/síntesis química , Vacunas de Subunidad/inmunologíaRESUMEN
In this study, we evaluated the potential of a genetically modified cholera toxin, CT-E29H as an adjuvant for recombinant Norwalk virus like particle (NV-VLP) vaccine. This detoxified mutant, containing E to H substitution at amino acid 29 of the CT-A1 subunit, was administered with a recombinant Norwalk virus like particle vaccine to Balb/c mice by mucosal routes to monitor the induction of mucosal, humoral and cellular responses. We observed that a low dose of NV-VLP (5 microg) with the adjuvant delivered by the intranasal route (IN) was more effective than the highest dose (200 microg) delivered by oral route at inducing both cellular and NV-VLP specific IgG and IgA responses. Higher counts of antigen specific IgA secreting cells were observed in the Peyer's Patches (PP) following delivery of the vaccine with CT-E29H as compared to delivery of vaccine by mucosal routes without CT-E29H. Furthermore, there was an increase in antigen specific cells producing IL-4 from animals that received the vaccine with the adjuvant. Delivery of the vaccine by the oral route results in antigen specific CD4(+) and CD8(+) T cells in PP and spleen. Addition of CT-E29H results in an increase of antigen specific CD4(+) cell population in PP and both CD4(+) and CD8(+) populations in the spleen. These cellular and cytokine responses suggest that combining the vaccine with CT-E29H results in a stronger Th2 type response. Collectively, these results indicate that immune responses to NV-VLP vaccine are qualitatively and quantitatively improved when the vaccine is delivered along with CT-E29H, and thus merits its further consideration as a mucosal adjuvant.