RESUMO
The ability of vaccines to induce immunity both in mucosal and systemic compartments may be required for prevention of HIV infection and AIDS. We compared DNA-MVA vaccination regimens adjuvanted by IL-12 DNA, administered intramuscularly and nasally or only nasally. Most of the vaccinated Rhesus macaques developed mucosal and systemic humoral and cell-mediated SHIV-specific immune responses. Stimulation of mucosal anti-Env IgA responses was limited. After rectal challenge with SHIV 89.6P, all vaccinated and naive animals became infected. However, most of the vaccinated animals showed significant control of viremia and protection from CD4(+) T cell loss and AIDS progression compared to the control animals. The levels of CD4(+) and CD8(+) T cell virus-specific responses measured on the day of challenge correlated with the level of viremia control observed later during the chronic infection. Postchallenge viremia levels inversely correlated with the preservation of SHIV-specific CD4(+)/IL-2(+) and CD8(+)/TNF-alpha(+) T cells but not with CD4(+)/IFN-gamma(+) T cells measured over time after challenge. We also found that during the early chronic infection SHIV vaccination permitted a more significant preservation of both naive and memory CD4(+) T cells compared to controls. In addition, we observed a more significant and prolonged preservation of memory CD4(+) T cells after SHIV vaccination and challenge than that observed after SIV vaccination and challenge. As the antiviral immunity stimulated by vaccination is present in the memory CD4(+) T cell subpopulations, its more limited targeting by SHIV compared to SIV may explain the better control of X4 tropic SHIV than R5 tropic SIVs by vaccination.
Assuntos
Vacinas contra a AIDS/imunologia , Linfócitos T CD4-Positivos/imunologia , Memória Imunológica , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vacinas de DNA/imunologia , Vacinas contra a AIDS/administração & dosagem , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/farmacologia , Administração Intranasal , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/sangue , Contagem de Linfócito CD4 , Linfócitos T CD8-Positivos/imunologia , Imunidade nas Mucosas , Imunoglobulina A/análise , Injeções Intramusculares , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-2/análise , Macaca mulatta , Masculino , Vacinas contra a SAIDS/administração & dosagem , Subpopulações de Linfócitos T/química , Subpopulações de Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/análise , Vacinas de DNA/administração & dosagem , Vaccinia virus/genética , Carga Viral , Viremia/imunologia , Viremia/prevenção & controleRESUMO
PURPOSE: The poor immunogenicity of tumor antigens and the antigenic heterogeneity of tumors call for vaccine strategies to enhance T-cell responses to multiple antigens. Two antigens expressed noncoordinately on most human carcinomas are carcinoembryonic antigen (CEA) and MUC-1. We report here the construction and characterization of two viral vector vaccines to address these issues. EXPERIMENTAL DESIGN: The two viral vectors analyzed are the replication-competent recombinant vaccinia virus (rV-) and the avipox vector, fowlpox (rF-), which is replication incompetent in mammalian cells. Each vector encodes the transgenes for three human costimulatory molecules (B7-1, ICAM-1, and LFA-3, designated TRICOM) and the CEA and MUC-1 transgenes (which also contain agonist epitopes). The vectors are designated rV-CEA/MUC/TRICOM and rF-CEA/MUC/TRICOM. RESULTS: Each of the vectors is shown to be capable of faithfully expressing all five transgenes in human dendritic cells (DC). DCs infected with either vector are shown to activate both CEA- and MUC-1-specific T-cell lines to the same level as DCs infected with CEA-TRICOM or MUC-1-TRICOM vectors. Thus, no evidence of antigenic competition between CEA and MUC-1 was observed. Human DCs infected with rV-CEA/MUC/TRICOM or rF-CEA/MUC/TRICOM are also shown to be capable of generating both MUC-1- and CEA-specific T-cell lines; these T-cell lines are in turn shown to be capable of lysing targets pulsed with MUC-1 or CEA peptides as well as human tumor cells endogenously expressing MUC-1 and/or CEA. CONCLUSION: These studies provide the rationale for the clinical evaluation of these multigene vectors in patients with a range of carcinomas expressing MUC-1 and/or CEA.
Assuntos
Vacinas Anticâncer/genética , Vetores Genéticos/genética , Transgenes/genética , Animais , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Antígenos CD58/genética , Antígenos CD58/imunologia , Vacinas Anticâncer/imunologia , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Citotoxicidade Imunológica/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Citometria de Fluxo , Vírus da Varíola das Aves Domésticas/genética , Humanos , Immunoblotting , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Interferon gama/biossíntese , Mucina-1/genética , Mucina-1/imunologia , Recombinação Genética , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transfecção , Transgenes/imunologia , Vaccinia virus/genéticaRESUMO
CD4+ T cells play an important role in the induction and maintenance of an effective antiviral and antitumor immune response. However, standardized monitoring of antigen-specific CD4+ T cells has not been established at the single-cell level. We now present a sensitive, specific, and simple methodology in which purified memory CD4+ T cells are expanded from PBMC in a single cycle of antigen-driven stimulation and quantitatively assayed by interferon-gamma ELISPOT. Issues of nonspecific background in assays were resolved with the use of innovative target cells, autologous PHA-expanded CD4+ T cells (T-APC). Remarkably, T-APC could not only present peptide epitopes from model antigens NY-ESO-1 and influenza nucleoprotein, but could also process full-length antigen endogenously expressed from recombinant fowlpox vector. This approach makes it possible to monitor CD4+ T cells in large series of patients, regardless of HLA haplotype, against the full peptide repertoire of a given antigen.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Imunoensaio/métodos , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Humanos , Separação Imunomagnética , Neoplasias Pulmonares , Melanoma/imunologiaRESUMO
We explored the use of a simian-human immunodeficiency virus (SHIV) DNA vaccine as an effective mucosal priming agent to stimulate a protective immune response for AIDS prevention. Rhesus macaques were vaccinated rectally with a DNA construct producing replication-defective SHIV particles, and boosted with either the same DNA construct or recombinant modified vaccinia virus Ankara (MVA) expressing SIV Gag, SIV Pol, and HIV Env (MVA-SHIV). Virus-specific mucosal and systemic humoral and cell-mediated immune responses could be stimulated by this approach but were present inconsistently among the vaccinated animals. Rectal vaccination with either SHIV DNA alone or SHIV DNA followed by MVA-SHIV induced SIV Gag/Pol- or HIV gp120-specific IgA in rectal secretions of four of seven animals. However, the gp120-specific rectal IgA antibody responses were not durable and had become undetectable in all but one animal shortly before rectal challenge with pathogenic SHIV 89.6P. Only the macaques primed with SHIV DNA and boosted with MVA-SHIV demonstrated SHIV-specific IgG in plasma. In addition, these animals developed more consistent antiviral cell-mediated responses and had better preservation of CD4 T cells following challenge with SHIV 89.6P. Our study demonstrates the utility of a rectal DNA/MVA vaccination protocol for the induction of diverse responses in different immunological compartments. In addition, the immunity achieved with this mucosal vaccination regimen is sufficient to delay progression to AIDS.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vacinas contra a AIDS/genética , Administração Retal , Animais , Proteínas de Fusão gag-pol/genética , Proteínas de Fusão gag-pol/imunologia , Imunidade nas Mucosas , Macaca mulatta , Vacinas contra a SAIDS/administração & dosagem , Vacinas contra a SAIDS/genética , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/isolamento & purificação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vaccinia virus/genética , Vaccinia virus/imunologia , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologiaRESUMO
We evaluated replication-defective poxvirus vectors (modified vaccinia Ankara [MVA] and fowlpox [FPV]) in a homologous and heterologous vector prime-boost vaccination regimen containing matching HIV inserts (MVA-HIV and FPV-HIV) given at months 0, 1, 3, 5 and 7 in 150 healthy HIV-negative vaccinia-naïve participants. FPV-HIV alone was poorly immunogenic, while the high dose (10(9)pfu/2 ml) of MVA-HIV alone elicited maximal responses after two injections: CD4+ and CD8+ T-cell responses in 26/55 (47.3%) and 5/60 (8.3%) of participants, respectively, and IFN-γ ELISpot responses in 28/62 (45.2%). The infrequent CD8+ T-cell responses following MVA-HIV priming were boosted only by the heterologous (FPV-HIV) construct in 14/27 (51.9%) of participants post 4th vaccination. Alternatively, HIV envelope-specific binding antibodies were demonstrated in approximately two-thirds of recipients of the homologous boosting regimen, but in less than 20% of subjects after the heterologous vector boost. Thus, a heterologous poxvirus vector prime-boost regimen can induce HIV-specific CD8+ T-cell and CD4+ T-cell responses, which may be an important feature of an optimal regimen for preventive HIV vaccination.
Assuntos
Vacinas contra a AIDS/imunologia , Portadores de Fármacos , Vírus da Varíola das Aves Domésticas/genética , Vetores Genéticos , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Vaccinia virus/genética , Vacinas contra a AIDS/genética , Adolescente , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Anticorpos Anti-HIV/sangue , HIV-1/genética , Humanos , Imunização Secundária/métodos , Masculino , Fatores de Tempo , Vacinação/métodos , Adulto JovemRESUMO
As the diversity of potential immunogens increases within certain classes of vectors, the possibility has arisen of employing heterologous prime/boost immunizations using diverse members of the same family of vectors. The present study was initiated to explore the use of divergent pox vectors in a prime/boost regimen to elicit high-frequency cellular immune responses to human immunodeficiency virus type 1 envelope and simian immunodeficiency virus gag in rhesus monkeys. We demonstrated that monkeys vaccinated with a recombinant modified vaccinia virus Ankara (rMVA) prime/recombinant fowlpox virus (rFPV) boost regimen and monkeys vaccinated with a recombinant vaccinia virus prime/rFPV boost regimen developed comparable cellular immune responses that were greater in magnitude than those elicited by a homologous prime/boost with rMVA. Nevertheless, comparable magnitude recall cellular immune responses were observed in monkeys vaccinated with heterologous and homologous recombinant poxvirus following challenge with the CXCR4-tropic SHIV-89.6P. Consistent with this finding, comparable levels of containment of viral replication and CD4(+) T-lymphocyte preservation were seen in these groups of recombinant poxvirus-vaccinated monkeys. This study supports further exploration of combining recombinant vectors of the same family in prime/boost immunization strategies to optimize vaccine-elicited cellular immune responses.
Assuntos
Vetores Genéticos/imunologia , Imunização Secundária/métodos , Poxviridae/imunologia , Vacinação/métodos , Vaccinia virus/imunologia , Animais , Anticorpos Antivirais/sangue , Contagem de Linfócito CD4 , Vetores Genéticos/genética , Imunidade Celular , Macaca mulatta , Poxviridae/genética , RNA Viral/sangue , Vaccinia virus/genética , Replicação ViralRESUMO
Poxvirus vaccine vectors, although capable of eliciting potent immune responses, pose serious health risks in immunosuppressed individuals. We therefore constructed five novel recombinant vaccinia virus vectors which contained overlapping deletions of coding regions for the B5R, B8R, B12R, B13R, B14R, B16R, B18R, and B19R immunomodulatory gene products and assessed them for both immunogenicity and pathogenicity. All five of these novel vectors elicited both cellular and humoral immunity to the inserted HIV-BH10 env comparable to that induced by the parental Wyeth strain vaccinia virus. However, deletion of these immunomodulatory genes did not increase the immunogenicity of these vectors compared with the parental vaccinia virus. Furthermore, four of these vectors were slightly less virulent and one was slightly more virulent than the Wyeth strain virus in neonatal mice. Attenuated poxviruses have potential use as safer alternatives to current replication-competent vaccinia virus. Improved vaccinia virus vectors can be generated by deleting additional genes to achieve a more significant viral attenuation.
Assuntos
Vacinas contra a AIDS/imunologia , Vetores Genéticos/genética , Infecções por HIV/imunologia , Vaccinia virus/genética , Vaccinia virus/patogenicidade , Animais , Feminino , Deleção de Genes , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/genética , Imunização , Imunização Secundária , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Sintéticas/imunologia , VirulênciaRESUMO
A successful HIV vaccine may need to stimulate antiviral immunity in mucosal and systemic immune compartments, because HIV transmission occurs predominantly at mucosal sites. We report here the results of a combined DNA-modified vaccinia virus Ankara (MVA) vaccine approach that stimulated simian/human immunodeficiency virus (SHIV)-specific immune responses by vaccination at the nasal mucosa. Fifteen male rhesus macaques, divided into three groups, received three nasal vaccinations on day 1, wk 9, and wk 25 with a SHIV DNA plasmid producing noninfectious viral particles (group 1), or SHIV DNA plus IL-2/Ig DNA (group 2), or SHIV DNA plus IL-12 DNA (group 3). On wk 33, all macaques were boosted with rMVA expressing SIV Gag-Pol and HIV Env 89.6P, administered nasally. Humoral responses were evaluated by measuring SHIV-specific IgG and neutralizing Abs in plasma, and SHIV-specific IgA in rectal secretions. Cellular responses were monitored by evaluating blood-derived virus-specific IFN-gamma-secreting cells and TNF-alpha-expressing CD8+ T cells, and blood- and rectally derived p11C tetramer-positive T cells. Many of the vaccinated animals developed both mucosal and systemic humoral and cell-mediated anti-SHIV immune responses, although the responses were not homogenous among animals in the different groups. After rectal challenge of vaccinated and naive animals with SHIV89.6P, all animals became infected. However a subset, including all group 2 animals, were protected from CD4+ T cell loss and AIDS development. Taken together, these data indicate that nasal vaccination with SHIV-DNA plus IL-2/Ig DNA and rMVA can provide significant protection from disease progression.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Infecções por HIV/prevenção & controle , Interleucina-2/administração & dosagem , Mucosa Nasal/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vacinas de DNA/administração & dosagem , Vaccinia virus/imunologia , Viremia/prevenção & controle , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/genética , Administração Intranasal , Animais , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos/métodos , Anticorpos Anti-HIV/biossíntese , Infecções por HIV/imunologia , Humanos , Imunidade Celular , Imunidade nas Mucosas , Imunoglobulina A/biossíntese , Interleucina-2/genética , Interleucina-2/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/virologia , Macaca mulatta , Masculino , Mucosa Nasal/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/genética , Vacinas de DNA/imunologia , Vaccinia virus/genética , Viremia/imunologiaRESUMO
Induction of mucosal anti-human immunodeficiency virus type 1 (HIV-1) T-cell responses in males and females will be important for the development of a successful HIV-1 vaccine. An HIV-1 envelope peptide, DNA plasmid, and recombinant modified vaccinia virus Ankara (rMVA) expressing the H-2D(d)-restricted cytotoxic T lymphocyte P18 epitope were used as immunogens to test for their ability to prime and boost anti-HIV-1 T-cell responses at mucosal and systemic sites in BALB/c mice. We found of all prime-boost combinations tested, an HIV-1 Env peptide subunit mucosal prime followed by systemic (intradermal) boosting with rMVA yielded the maximal induction of gamma interferon (IFN-gamma) spot-forming cells in the female genital tract and colon. However, this mucosal prime-systemic rMVA boost regimen was minimally immunogenic for the induction of genital, colon, or lung anti-HIV-1 T-cell responses in male mice. We determined that a mucosal Env subunit immunization could optimally prime an rMVA boost in female but not male mice, as determined by the magnitude of antigen-specific IFN-gamma responses in the reproductive tracts, colon, and lung. Defective mucosal priming in male mice could not be overcome by multiple mucosal immunizations. However, rMVA priming followed by an rMVA boost was the optimal prime-boost strategy for male mice as determined by the magnitude of antigen-specific IFN-gamma responses in the reproductive tract and lung. Thus, prime-boost immunization strategies able to induce mucosal antigen-specific IFN-gamma responses were identified for male and female mice. Understanding the cellular and molecular basis of gender-determined immune responses will be important for optimizing induction of anti-HIV-1 mucosal immune responses in both males and females.
Assuntos
Vacinas contra a AIDS/imunologia , Linfócitos T CD8-Positivos/imunologia , Colo/imunologia , Genitália/imunologia , HIV-1/imunologia , Vacinas Sintéticas/imunologia , Animais , Feminino , Imunização , Interferon gama/biossíntese , Interleucina-2/biossíntese , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Caracteres Sexuais , Vaccinia virus/imunologiaRESUMO
A therapeutic vaccine for individuals infected with HIV-1 and treated with antiretroviral therapy (ART) should be able to replenish virus-specific CD4+ T-cells and broaden the virus-specific CD8+ T-cell response in order to maintain CD8+ T-cell function and minimize viral immune escape after ART cessation. Because a combination of DNA and recombinant poxvirus vaccine modalities induces high levels of virus-specific CD4+ T-cell response and broadens the cytolytic activity in naive macaques, we investigated whether the same results could be obtained in SIVmac251-infected macaques. The macaques studied here were long-term nonprogressors that naturally contained viremia but were nevertheless treated with a combination of antiviral drugs to assess more carefully the effect of vaccination in the context of ART. The combination of a DNA expressing the gag and pol genes (DNA-SIV-gp) of SIVmac239 followed by a recombinant fowlpox expressing the same SIVmac genes (FP-SIV-gp) was significantly more immunogenic than two immunizations of FP-SIV-gp in SIVmac251-infected macaques treated with ART. The DNA/FP combination significantly expanded and broadened Gag-specific T-cell responses measured by tetramer staining, ELISPOT, and intracellular cytokine staining and measurement of ex vivo cytolytic function. Importantly, the combination of these vaccine modalities also induced a sizeable expansion in most macaques of Gag-specific CD8-(CD4+) T-cells able to produce TNF-alpha. Hopefully, this modality of vaccine combination may be useful in the clinical management of HIV-1-infected individuals.
Assuntos
Epitopos Imunodominantes/imunologia , Vírus da Influenza A/genética , Macaca mulatta/imunologia , Macaca mulatta/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Progressão da Doença , Vírus da Influenza A/fisiologia , VacinaçãoRESUMO
Nearly all human immunodeficiency virus (HIV) infections are acquired mucosally, and the gut-associated lymphoid tissues are important sites for early virus replication. Thus, vaccine strategies designed to prime virus-specific cytotoxic T lymphocyte (CTL) responses that home to mucosal compartments may be particularly effective at preventing or containing HIV infection. The Salmonella type III secretion system has been shown to be an effective approach for stimulating mucosal CTL responses in mice. We therefore tested DeltaphoP-phoQ attenuated strains of Salmonella enterica serovar Typhimurium and S. enterica serovar Typhi expressing fragments of the simian immunodeficiency virus (SIV) Gag protein fused to the type III-secreted SopE protein for the ability to prime virus-specific CTL responses in rhesus macaques. Mamu-A*01(+) macaques were inoculated with three oral doses of recombinant Salmonella, followed by a peripheral boost with modified vaccinia virus Ankara expressing SIV Gag (MVA Gag). Transient low-level CTL responses to the Mamu-A*01 Gag(181-189) epitope were detected following each dose of Salmonella. After boosting with MVA Gag, strong Gag-specific CTL responses were consistently detected, and tetramer staining revealed the expansion of Gag(181-189)-specific CD8(+) T-cell responses in peripheral blood. A significant percentage of the Gag(181-189)-specific T-cell population in each animal also expressed the intestinal homing receptor alpha4beta7. Additionally, Gag(181-189)-specific CD8(+) T cells were detected in lymphocytes isolated from the colon. Yet, despite these responses, Salmonella-primed/MVA-boosted animals did not exhibit improved control of virus replication following a rectal challenge with SIVmac239. Nevertheless, this study demonstrates the potential of mucosal priming by the Salmonella type III secretion system to direct SIV-specific cellular immune responses to the gastrointestinal mucosa in a primate model.