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.
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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
The ultimate vaccine against infections caused by Nipah virus should be capable of providing protection at the respiratory tractâthe most probable port of entry for this pathogen. Intranasally delivered vaccines, which target nasal-associated lymphoid tissue and induce both systemic and mucosal immunity, are attractive candidates for enabling effective vaccination against this lethal disease. Herein, the water-soluble polyphosphazene delivery vehicle assembles into nanoscale supramolecular constructs with the soluble extracellular portion of the Hendra virus attachment glycoproteinâa promising subunit vaccine antigen against both Nipah and Hendra viruses. These supramolecular constructs signal through Toll-like receptor 7/8 and promote binding interactions with mucinâan important feature of effective mucosal adjuvants. High mass contrast of phosphorus-nitrogen backbone of the polymer enables a successful visualization of nanoconstructs in their vitrified state by cryogenic electron microscopy. Here, we characterize the self-assembly of polyphosphazene macromolecule with biologically relevant ligands by asymmetric flow field flow fractionation, dynamic light scattering, fluorescence spectrophotometry, and turbidimetric titration methods. Furthermore, a polyphosphazene-enabled intranasal Nipah vaccine candidate demonstrates the ability to induce immune responses in hamsters and shows superiority in inducing total IgG and neutralizing antibodies when benchmarked against the respective clinical stage alum adjuvanted vaccine. The results highlight the potential of polyphosphazene-enabled nanoassemblies in the development of intranasal vaccines.
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Administración Intranasal , Virus Nipah , Compuestos Organofosforados , Polímeros , Vacunas de Subunidad , Vacunas Virales , Compuestos Organofosforados/química , Compuestos Organofosforados/administración & dosificación , Polímeros/química , Virus Nipah/inmunología , Animales , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/química , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/química , Vacunas de Subunidad/administración & dosificación , Tamaño de la Partícula , Ensayo de Materiales , Materiales Biocompatibles/química , Nanopartículas/química , InmunizaciónRESUMEN
Two classes of compounds, thiocarbamates 1 and triazoles 2, have been identified as HIV RT RNase H inhibitors using a novel FRET-based HTS assay. The potent analogs in each series exhibited selectivity and were active in cell-based assays. In addition, saturable, 1:1 stoichiometric binding to target was established and time of addition studies were consistent with inhibition of RT-mediated HIV replication.
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Fármacos Anti-VIH/química , Inhibidores Enzimáticos/química , Ribonucleasa H del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Tiocarbamatos/química , Triazoles/química , Secuencia de Aminoácidos , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Línea Celular , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Transferencia Resonante de Energía de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Humanos , Datos de Secuencia Molecular , Ribonucleasa H del Virus de la Inmunodeficiencia Humana/metabolismo , Relación Estructura-Actividad , Tiocarbamatos/síntesis química , Tiocarbamatos/farmacología , Triazoles/síntesis química , Triazoles/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
Recent occurrences of filoviruses and the arenavirus Lassa virus (LASV) in overlapping endemic areas of Africa highlight the need for a prophylactic vaccine that would confer protection against all of these viruses that cause lethal hemorrhagic fever (HF). We developed a quadrivalent formulation of VesiculoVax that contains recombinant vesicular stomatitis virus (rVSV) vectors expressing filovirus glycoproteins and that also contains a rVSV vector expressing the glycoprotein of a lineage IV strain of LASV. Cynomolgus macaques were vaccinated twice with the quadrivalent formulation, followed by challenge 28 days after the boost vaccination with each of the 3 corresponding filoviruses (Ebola, Sudan, Marburg) or a heterologous contemporary lineage II strain of LASV. Serum IgG and neutralizing antibody responses specific for all 4 glycoproteins were detected in all vaccinated animals. A modest and balanced cell-mediated immune response specific for the glycoproteins was also detected in most of the vaccinated macaques. Regardless of the level of total glycoprotein-specific immune response detected after vaccination, all immunized animals were protected from disease and death following lethal challenges. These findings indicate that vaccination with attenuated rVSV vectors each expressing a single HF virus glycoprotein may provide protection against those filoviruses and LASV most commonly responsible for outbreaks of severe HF in Africa.
Asunto(s)
Anticuerpos Antivirales/inmunología , Vectores Genéticos , Inmunoglobulina G/inmunología , Fiebre de Lassa/prevención & control , Virus Lassa/inmunología , Vesiculovirus , Vacunas Virales/inmunología , Animales , Humanos , Fiebre de Lassa/genética , Fiebre de Lassa/inmunología , Virus Lassa/genética , Macaca fascicularis , Vacunas Virales/genéticaRESUMEN
BACKGROUND: The safety and immunogenicity of a highly attenuated recombinant vesicular stomatitis virus (rVSV) expressing HIV-1 gag (rVSVN4CT1-HIV-1gag1) was shown in previous phase 1 clinical studies. An rVSV vector expressing Ebola virus glycoprotein (EBOV-GP) in place of HIV-1 gag (rVSVN4CT1-EBOVGP1) showed single-dose protection from lethal challenge with low passage Ebola virus in non-human primates. We aimed to evaluate the safety and immunogenicity of the rVSVN4CT1-EBOVGP1 vaccine in healthy adults. METHODS: We did a randomised double-blind, placebo-controlled, phase 1 dose-escalation study at a single clinical site (Optimal Research) in Melbourne, FL, USA. Eligible participants were healthy men and non-pregnant women aged 18-60 years, with a body-mass index (BMI) of less than 40 kg/m2, no history of filovirus infection, VSV infection, or receipt of rVSV in previous studies, and who had not visited regions where Ebola virus outbreaks have occurred. Three cohorts were enrolled to assess a low (2·5â×â104 plaque forming units [PFU]), intermediate (2â×â105 PFU), or high dose (1·8â×â106 PFU) of the vaccine. Participants within each cohort were randomly allocated (10:3) to receive vaccine or placebo by intramuscular injection in a homologous prime and boost regimen, with 4 weeks between doses. All syringes were masked with syringe sleeves; participants and study site staff were not blinded to dose level but were blinded to active vaccine and placebo. The primary outcomes were safety and tolerability; immunogenicity, assessed as GP-specific humoral immune response (at 2 weeks after each dose) and cellular immune response (at 1 and 2 weeks after each dose), was a secondary outcome. All randomised participants were included in primary and safety analyses. This trial is registered with ClinicalTrials.gov, NCT02718469. FINDINGS: Between Dec 22, 2015, and Sept 15, 2016, 39 individuals (18 [46%] men and 21 [54%] women, mean age 51 years [SD 10]) were enrolled, with ten participants receiving the vaccine and three participants receiving placebo in each of three cohorts. One participant in the intermediate dose cohort was withdrawn from the study because of a diagnosis of invasive ductal breast carcinoma 24 days after the first vaccination, which was considered unrelated to the vaccine. No severe adverse events were observed. Solicited local adverse events occurred in ten (26%) of 39 participants after the first dose and nine (24%) of 38 participants after the second dose; the events lasted 3 days or less, were predominantly injection site tenderness (17 events) and injection site pain (ten events), and were either mild (19 events) or moderate (ten events) in intensity. Systemic adverse events occurred in 13 (33%) of 39 participants after the first dose and eight (21%) of 38 participants after the second dose; the events were mild (45 events) or moderate (11 events) in severity, and the most common events were malaise or fatigue (13 events) and headache (12 events). Arthritis and maculopapular, vesicular, or purpuric rash distal to the vaccination site(s) were not reported. A GP-specific IgG response was detected in all vaccine recipients after two doses (and IgG response frequency was 100% after a single high dose), and an Ebola virus neutralising response was detected in 100% of participants in the high-dose cohort. INTERPRETATION: The rVSVN4CT1-EBOVGP1 vaccine was well tolerated at all dose levels tested and was immunogenic despite a high degree of attenuation. The combined safety and immunogenicity profile of the rVSVN4CT1-EBOVGP1 vaccine vector support phase 1-2 clinical evaluation. FUNDING: US Department of Defense Joint Program Executive Office for Chemical, Biological, Radiological and Nuclear Defense: Joint Project Manager for Chemical, Biological, Radiological and Nuclear Medical.
Asunto(s)
Vacunas contra el Virus del Ébola/inmunología , Ebolavirus/inmunología , Glicoproteínas/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Inmunogenicidad Vacunal , Seguridad , Método Doble Ciego , Vacunas contra el Virus del Ébola/administración & dosificación , Femenino , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Vacunación , Vacunas Atenuadas/inmunologíaRESUMEN
Immunotherapy for HPVPOS malignancies is attractive because well-defined, viral, non-self tumor antigens exist as targets. Several approaches to vaccinate therapeutically against HPV E6 and E7 antigens have been adopted, including viral platforms such as VSV. A major advantage of VSV expressing these antigens is that VSV also acts as an oncolytic virus, leading to direct tumor cell killing and induction of effective anti-E6 and anti-E7 T cell responses. We have also shown that addition of immune adjuvant genes, such as IFNß, further enhances safety and/or efficacy of VSV-based oncolytic immunovirotherapies. However, multiple designs of the viral vector are possible-with respect to levels of immunogen expression and method of virus attenuation-and optimal designs have not previously been tested head-to-head. Here, we tested three different VSV engineered to express a non-oncogenic HPV16 E7/6 fusion protein for their immunotherapeutic and oncolytic properties. We assessed their profiles of efficacy and toxicity against HPVPOS and HPVNEG murine tumor models and determined the optimal route of administration. Our data show that VSV is an excellent platform for the oncolytic immunovirotherapy of tumors expressing HPV target antigens, combining a balance of efficacy and safety suitable for evaluation in a first-in-human clinical trial.
RESUMEN
OBJECTIVE: To develop a SIV-rhesus macaque (Rh) model of AIDS that more closely approximates HIV pathogenesis in humans. DESIGN: The pathogenesis of SIV was compared in two different types of Rh, the Chinese (Ch) and Indian (Ind) subspecies. METHODS: Ch Rh and Ind Rh origin were identified genetically and infected with the SIV(mac)239 molecular clone. Plasma viral loads, depletion of intestinal lymphocytes with memory phenotype, humoral immune responses and CD4/CD8 cell ratios were compared during acute and steady-state periods of infection. RESULTS: Plasma viral loads from 7 days after infection through 240 days were significantly lower in Rh of Ch origin compared with Ind Rh. Viral loads in Ch Rh were closer to viral loads observed in untreated humans infected with HIV-1. Depletion of intestinal effector cells was less evident in SIV-infected Ch Rh compared with Ind Rh. An index of intestinal pathogenesis was devised that closely paralleled viral load and severity of infection. There were no rapid progressors to AIDS among 10 Ch Rh. In contrast, three of four Ind Rh progressed rapidly to AIDS. CONCLUSIONS: Compared with Ind Rh, SIV(mac) pathogenesis in Ch Rh was closer to HIV-1 infections in untreated adult humans. The differences were statistically significant. The Ch Rh subspecies is a suitable AIDS model and may have advantages over the rapid and highly pathogenic Ind Rh model. Moreover, Ind Rh supplies are limited and use of Ch Rh provides a new resource.
Asunto(s)
Modelos Animales de Enfermedad , Macaca mulatta/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Síndrome de Inmunodeficiencia Adquirida/inmunología , Síndrome de Inmunodeficiencia Adquirida/fisiopatología , Síndrome de Inmunodeficiencia Adquirida/virología , Animales , Anticuerpos Antivirales/análisis , Relación CD4-CD8 , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , China , Progresión de la Enfermedad , India , Macaca mulatta/clasificación , Macaca mulatta/genética , Macaca mulatta/inmunología , ARN Viral/sangre , Síndrome de Inmunodeficiencia Adquirida del Simio/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/fisiopatología , Virus de la Inmunodeficiencia de los Simios/genética , Carga ViralRESUMEN
Despite repeated high-risk exposure to infectious HIV-1, some individuals remain HIV-1 seronegative and apparently uninfected. The use of nonhuman primate model systems to study SIVmac transmission may help to elucidate the factors responsible for protection in exposed, seronegative (ESN) humans. In an earlier vaccination study, three control rhesus macaques that were exposed to three sequential intravaginal challenges with pathogenic SIVmac251 failed to show evidence of infection after 5 years of observation. 51Cr release assay results suggested that these animals had low-level cytotoxic T lymphocyte responses to SIVmac proteins. We hypothesized that these responses might be an important component of protection from mucosal challenge. We performed an additional intravaginal challenge of all three macaques and monitored SIV-specific T cell responses in peripheral blood, using the sensitive enzyme-linked immunospot (ELISpot) assay. After the fourth challenge, one animal became infected; this animal did not mount a strong SIV-specific T cell response. Two other macaques remained uninfected as determined by peripheral blood mononuclear cell (PBMC) coculture, polymerase chain reaction (PCR), and branched DNA (bDNA) analysis of peripheral blood and lymphoid tissues, but demonstrated boosting of SIV-specific T cell responses after challenge. These results support a protective role for SIVmac-specific T cells in repeatedly exposed, persistently seronegative rhesus macaques.
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Seronegatividad para VIH/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Linfocitos T Citotóxicos/inmunología , Vagina/virología , Animales , ADN Viral/análisis , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Macaca mulatta , Reacción en Cadena de la Polimerasa , Virus de la Inmunodeficiencia de los Simios/inmunologíaRESUMEN
Vaccination and preexposure prophylaxis (PrEP) with antiretrovirals have shown only partial protection from HIV-1 infection in human trials. Oral Truvada (emtricitabine/tenofovir disoproxil fumarate) is FDA approved as PrEP but partial adherence reduces efficacy. If combined as biomedical preventions (CBP), an HIV vaccine could protect when PrEP adherence is low and PrEP could prevent vaccine breakthroughs. The efficacy of combining oral PrEP with an HIV vaccine has not been evaluated in humans. We determined the efficacy of combining a DNA/virus-like particle (VLP) vaccine with partially effective intermittent PrEP in Indian rhesus macaques (RM). Eight RM received intramuscular inoculations of five DNA plasmids encoding four HIV-1 Clade B primary isolate Envs and SIVmac239 Gag (at weeks 0 and 4), followed by intramuscular and intranasal inoculations of homologous Gag VLPs and four Env VLPs (at weeks 12, 16, and 53). At week 61, we initiated weekly rectal exposures with heterologous SHIV162p3 (10 TCID50) along with oral Truvada (TDF, 22 mg/kg; FTC 20 mg/kg) dosing 2 h before and 22 h after each exposure. This PrEP regimen previously demonstrated 50% efficacy. Five controls (no vaccine, no PrEP) received weekly SHIV162p3. All controls were infected after a median of four exposures; the mean peak plasma viral load (VL) was 3.9×10(7) vRNA copies/ml. CBP protected seven of eight (87.5%) RM. The one infected CBP RM had a reduced peak VL of 8.8×10(5) copies/ml. SHIV exposures during PrEP amplified Gag and Env antibody titers in protected RM. These results suggest that combining oral PrEP with HIV vaccines could enhance protection against HIV-1 infection.
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Fármacos Anti-VIH/administración & dosificación , Desoxicitidina/análogos & derivados , Compuestos Organofosforados/administración & dosificación , Profilaxis Pre-Exposición/métodos , Vacunas contra el SIDAS/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Vacunación/métodos , Vacunas de Partículas Similares a Virus/inmunología , Administración Intranasal , Administración Oral , Animales , Anticuerpos Antivirales/sangre , Quimioprevención/métodos , Terapia Combinada/métodos , Desoxicitidina/administración & dosificación , Combinación de Medicamentos , Combinación Emtricitabina y Fumarato de Tenofovir Disoproxil , Femenino , VIH-1/genética , VIH-1/inmunología , Inyecciones Intramusculares , Macaca mulatta , Masculino , Vacunas contra el SIDAS/administración & dosificación , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología , Resultado del Tratamiento , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Carga ViralRESUMEN
BACKGROUND: Pre-exposure prophylaxis (PrEP) is a novel intervention strategy for the prevention HIV transmission. Because several clinical trials are at various stages of completion, it is important to understand the impact of PrEP treatment on the development of the immune response to HIV, particularly in individuals who exhibit breakthrough infections despite PrEP. METHODS: A model of HIV infection, using rhesus macaques and the simian/human immunodeficiency virus (SHIV), was used to evaluate the effects of PrEP on the evolution of the humoral immune response. Time to seroconversion, neutralizing and binding antibody levels, and antibody avidity were measured in 12 rhesus macaques infected during daily or intermittent PrEP with FTC (emtricitabine) or Truvada (FTC/tenofovir combination) and compared with 11 untreated, simian HIV-infected controls. RESULTS: Macaques that became infected while receiving PrEP exhibited significantly lower peak virus loads during acute infection as compared with untreated animals. Although the timing of seroconversion and SHIV binding and neutralizing antibody levels were not impacted by treatment, lower maturation rates of antibody avidity for anti-p27, gp120, gp160, and gp41 were observed. CONCLUSIONS: This study suggests that reduced virus loads associated with PrEP treatment have little impact on timing of seroconversion and neutralizing/binding antibody levels; however, maturation of antibody avidity was suppressed.
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Fármacos Anti-VIH/uso terapéutico , Anticuerpos Antivirales/fisiología , Afinidad de Anticuerpos , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Adenina/administración & dosificación , Adenina/análogos & derivados , Adenina/uso terapéutico , Administración Oral , Animales , Fármacos Anti-VIH/administración & dosificación , Anticuerpos Antivirales/sangre , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Combinación de Medicamentos , Emtricitabina , VIH , Anticuerpos Anti-VIH/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana/inmunología , Macaca mulatta , Organofosfonatos/administración & dosificación , Organofosfonatos/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Tenofovir , Carga ViralRESUMEN
Plasmid DNA (pDNA) vaccines are effective at eliciting immune responses in a wide variety of animal model systems, however, pDNA vaccines have generally been incapable of inducing robust immune responses in clinical trials. Therefore, to identify means to improve pDNA vaccine performance, we compared various post-transcriptional and post-translational genetic modifications for their ability to improve antigen-specific CMI responses. Mice vaccinated using a sub-optimal 100 mcg dose of a pDNA encoding an unmodified primary isolate HIV-1(6101) env gp160 failed to demonstrate measurable env-specific CMI responses. In contrast, significant env-specific CMI responses were seen in mice immunized with pDNA expression vectors encoding env genes modified by RNA optimization or codon optimization. Further modification of the RNA optimized env gp160 gene by the addition of (i) a simian retrovirus type 1 constitutive RNA transport element; (ii) a murine intracisternal A-particle derived RNA transport element; (iii) a tissue plasminogen activator protein signal leader sequences; (iv) a beta-catenin derived ubiquitination target sequence; or (v) a monocyte chemotactic protein-3 derived signal sequence failed to further improve the induction of env-specific CMI responses. Therefore, modification of the env gp160 gene by RNA or codon optimization alone is necessary for high-level rev-independent expression and results in robust env-specific CMI responses in immunized mice. Importantly, further modification(s) of the env gene to alter cellular localization or increase proteolytic processing failed to result in increased env-specific immune responses. These results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection.
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Vacunas contra el SIDA/inmunología , Codón/genética , Proteínas gp160 de Envoltorio del VIH/genética , Infecciones por VIH/prevención & control , ARN/genética , Linfocitos T/inmunología , Vacunas de ADN/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/genética , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Anticuerpos Anti-VIH/sangre , Proteínas gp160 de Envoltorio del VIH/inmunología , Proteínas gp160 de Envoltorio del VIH/metabolismo , Infecciones por VIH/inmunología , VIH-1/genética , VIH-1/inmunología , VIH-1/metabolismo , Inmunidad Celular , Interferón gamma/biosíntesis , Ratones , Ratones Endogámicos BALB C , Plásmidos/genética , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genéticaRESUMEN
The effectiveness of plasmid DNA (pDNA) vaccines can be improved by the co-delivery of plasmid-encoded molecular adjuvants. We evaluated pDNAs encoding GM-CSF, Flt-3L, IL-12 alone, or in combination, for their relative ability to serve as adjuvants to augment humoral and cell-mediated immune responses elicited by prototype pDNA vaccines. In Balb/c mice we found that co-administration of plasmid-based murine GM-CSF (pmGM-CSF), murine Flt-3L (pmFlt-3L) or murine IL-12 (pmIL-12) could markedly enhance the cell-mediated immune response elicited by an HIV-1 env pDNA vaccine. Plasmid mGM-CSF also augmented the immune response elicited by DNA vaccines expressing HIV-1 Gag and Nef-Tat-Vif. In addition, the use of pmGM-CSF as a vaccine adjuvant appeared to markedly increase antigen-specific proliferative responses and improved the quality of the resulting T-cell response by increasing the percentage of polyfunctional memory CD8(+) T cells. Co-delivery of pmFlt-3L with pmGM-CSF did not result in a further increase in adjuvant activity. However, the co-administration of pmGM-CSF with pmIL-12 did significantly enhance env-specific proliferative responses and vaccine efficacy in the murine vaccinia virus challenge model relative to mice immunized with the env pDNA vaccine adjuvanted with either pmGM-CSF or pmIL-12 alone. These data support the testing of pmGM-CSF and pmIL-12, used alone or in combination, as plasmid DNA vaccine adjuvants in future macaque challenge studies.
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Vacunas contra el SIDA/inmunología , Adyuvantes Inmunológicos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interleucina-12/farmacología , Proteínas de la Membrana/farmacología , Vacunas de ADN/inmunología , Adyuvantes Inmunológicos/genética , Animales , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , VIH-1/genética , VIH-1/inmunología , Interleucina-12/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Plásmidos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Since human immunodeficiency virus (HIV)-specific cell-mediated immune (CMI) responses are critical in the early control and resolution of HIV infection and correlate with postchallenge outcomes in rhesus macaque challenge experiments, we sought to identify a plasmid DNA (pDNA) vaccine design capable of eliciting robust and balanced CMI responses to multiple HIV type 1 (HIV-1)-derived antigens for further development. Previously, a number of two-, three-, and four-vector pDNA vaccine designs were identified as capable of eliciting HIV-1 antigen-specific CMI responses in mice (M. A. Egan et al., Vaccine 24:4510-4523, 2006). We then sought to further characterize the relative immunogenicities of these two-, three-, and four-vector pDNA vaccine designs in nonhuman primates and to determine the extent to which in vivo electroporation (EP) could improve the resulting immune responses. The results indicated that a two-vector pDNA vaccine design elicited the most robust and balanced CMI response. In addition, vaccination in combination with in vivo EP led to a more rapid onset and enhanced vaccine-specific immune responses. In macaques immunized in combination with in vivo EP, we observed a 10- to 40-fold increase in HIV-specific enzyme-linked immunospot assay responses compared to those for macaques receiving a 5-fold higher dose of vaccine without in vivo EP. This increase in CMI responses translates to an apparent 50- to 200-fold increase in pDNA vaccine potency. Importantly, in vivo EP enhanced the immune response against the less immunogenic antigens, resulting in a more balanced immune response. In addition, in vivo EP resulted in an approximate 2.5-log(10) increase in antibody responses. The results further indicated that in vivo EP was associated with a significant reduction in pDNA persistence and did not result in an increase in pDNA associated with high-molecular-weight DNA relative to macaques receiving the pDNA without EP. Collectively, these results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection.
Asunto(s)
Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/inmunología , Anticuerpos Anti-VIH/sangre , VIH-1/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Vacunas contra el SIDA/genética , Animales , Electroporación , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , VIH-1/genética , Humanos , Interferón gamma/biosíntesis , Leucocitos Mononucleares/inmunología , Macaca mulatta , Masculino , Modelos Animales , Pruebas de Neutralización , Plásmidos , Vacunas de ADN/genéticaRESUMEN
Respiratory syncytial virus (RSV) is a major cause of acute respiratory tract disease in humans. Towards development of a prophylactic vaccine, we genetically engineered Venezuelan equine encephalitis virus (VEEV) replicons encoding the fusion (Fa) or attachment (Ga or Gb) proteins of the A or B subgroups of RSV. Intramuscular immunization with a formulation composed of equal amounts of each replicon particle (3vRSV replicon vaccine) generated serum neutralizing antibodies against A and B strains of RSV in BALB/c mice and rhesus macaques. When contrasted with purified natural protein or formalin-inactivated RSV formulated with alum, the 3vRSV replicon vaccine induced balanced Th1/Th2 T cell responses in mice. This was evident in the increased number of RSV-specific IFN-gamma(+) splenocytes following F or G peptide stimulation, diminished quantity of eosinophils and type 2 T cell cytokines in the lungs after challenge, and increased in vivo lysis of RSV peptide-loaded target cells. The immune responses in mice were also protective against intranasal challenge with RSV. Thus, the replicon-based platform represents a promising new strategy for vaccines against RSV.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/inmunología , Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/inmunología , Vacunas Sintéticas/inmunología , Proteínas Estructurales Virales/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/sangre , Citocinas/biosíntesis , Virus de la Encefalitis Equina Venezolana/genética , Femenino , Inyecciones Intramusculares , Pulmón/inmunología , Pulmón/virología , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Infecciones por Virus Sincitial Respiratorio/inmunología , Vacunas contra Virus Sincitial Respiratorio/genética , Virus Sincitiales Respiratorios/genética , Bazo/inmunología , Linfocitos T/inmunología , Vacunas Sintéticas/genética , Proteínas Estructurales Virales/genética , Vacunas Virales/genéticaRESUMEN
Given the importance of the HIV-specific cell-mediated immune response in the early control and resolution of HIV infection and the observed correlation between pre-challenge vaccine elicited CTL responses and post challenge outcome in SHIV/rhesus macaque experiments, we sought to identify several candidate plasmid DNA (pDNA) vaccine designs capable of eliciting robust and balanced cell-mediated immune responses to multiple HIV-1 derived antigens in mice for further vaccine development. To rationally construct candidate vaccines for immunogenicity testing, we determined the relative immunogenicity of the individual HIV-derived vaccine antigens (env, gag, pol, nef, tat and vif) and the relative strength of various transcriptional control elements (HCMV, SCMV, HSV Lap1) in Balb/c mice. Next, a number of 1-, 2-, 3- and 4-vector pDNA vaccine designs were tested for their ability to elicit HIV-1 antigen-specific CMI responses. For these studies, Balb/c mice were immunized with a fixed total pDNA vaccine dose of 100 mcg in combination with 25 mcg plasmid-based murine IL-12 and tested for the induction of HIV-1 antigen-specific CMI responses by IFN-gamma ELISpot analysis. The results of this study indicate that all pDNA vaccine designs were capable of eliciting CMI responses to multiple HIV-1 antigens. As a result of this iterative comparative analysis, we have identified a number of pDNA vaccine candidates capable of eliciting potent, balanced CMI responses to multiple HIV-1 derived antigens. These results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection.
Asunto(s)
Antígenos VIH/inmunología , Inmunidad Celular , Plásmidos , Vacunas de ADN/inmunología , Animales , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , VIH-1/genética , VIH-1/inmunología , Ratones , Regiones Promotoras Genéticas , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genéticaRESUMEN
Information on the establishment of immunodeficiency virus infection through transmission of infected cells is sparse. Dendritic cells (DCs) and T cells may be central to the onset and subsequent spread of infection following mucosal exposure. To directly investigate the consequences of virus being introduced by DCs or T cells, we reinjected ex vivo simian immunodeficiency virus (SIV)-loaded autologous immature DCs and T cells subcutaneously (s.c.) into healthy macaques. s.c. injection of cell-bound virus was used to mirror what may happen if virus-loaded cells pass through an epithelium or perhaps DCs and T cells that immediately entrap cell-free virus, having just crossed an epithelial barrier. Virus load in the plasma was monitored along with combined in situ hybridization and immunohistochemistry to identify the cells replicating virus in the lymphoid tissues. Both DCs and T cells transmitted infection after being pulsed with either wild-type or nef-defective (delta nef) SIVmac239. As seen in animals infected intravenously, replication of delta nef was attenuated compared to that of wild-type virus when introduced in either cell-bound form. Upon examination of the draining lymph nodes (LNs) during the first days of infection, virus-producing CD4(+) T cells predominated in control animals that received s.c. cell-free virus. In dramatic contrast, both SIV-positive macrophages and T cells were detected in the LNs of monkeys infected with cell-associated SIV. Therefore, although both cell-free and cell-associated viruses are infectious, the initial cells amplifying the virus differ. This may have important implications for the subsequent dissemination of infection and/or induction of antiretroviral immunity.