RESUMO
Ab-dependent cellular cytotoxicity (ADCC) Abs stimulate NK cell effector functions and play a role in protecting from and controlling viral infections. We characterized ADCC Abs in a cross-sectional cohort of 80 HIV-infected subjects not on antiretroviral therapy. We analyzed ADCC response by killing fluorescently labeled target cells, as well as expression of IFN-gamma and the degranulation marker CD107a from activated NK cells as measured by a novel intracellular cytokine assay. HIV-specific ADCC directed toward Envelope proteins were present in the majority of 80 untreated HIV-infected individuals measured by killing function. Similarly, most subjects had HIV-specific Abs that mediated degranulation or cytokine expression by NK cells. Interestingly, there was a poor correlation between ADCC-mediated killing of fluorescently labeled whole Envelope protein-pulsed cell lines and Ab-mediated expression of IFN-gamma by NK cells. However, in contrast to healthy donor NK cells, autologous patient NK cells more effectively degranulated granzyme B in response to ADCC activation. Activation of NK cells in response to stimulation by HIV-specific Abs occurs at least as rapidly as activation of Gag-specific CTLs. Our studies highlight the complexity of ab-mediated NK cell activation in HIV infection, and suggest new avenues toward studying the utility of ADCC in controlling HIV infection.
Assuntos
Especificidade de Anticorpos , Degranulação Celular/imunologia , Anticorpos Anti-HIV/fisiologia , HIV-1/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/imunologia , Adulto , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linhagem Celular Transformada , Estudos de Coortes , Estudos Transversais , Citocinas/análise , Citocinas/biossíntese , Fluoresceínas , Corantes Fluorescentes , Anticorpos Anti-HIV/sangue , Antígenos HIV/sangue , Antígenos HIV/imunologia , Infecções por HIV/sangue , Infecções por HIV/imunologia , Humanos , Líquido Intracelular/química , Líquido Intracelular/imunologia , Células Matadoras Naturais/virologia , Succinimidas , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Effective immunotherapies for HIV are needed. Drug therapies are life-long with significant toxicities. Dendritic-cell based immunotherapy approaches are promising but impractical for widespread use. A simple immunotherapy, reinfusing fresh autologous blood cells exposed to overlapping SIV peptides for 1 hour ex vivo, was assessed for the control of SIV(mac251) replication in 36 pigtail macaques. An initial set of four immunizations was administered under antiretroviral cover and a booster set of three immunizations administered 6 months later. Vaccinated animals were randomized to receive Gag peptides alone or peptides spanning all nine SIV proteins. High-level, SIV-specific CD4 and CD8 T-cell immunity was induced following immunization, both during antiretroviral cover and without. Virus levels were durably approximately 10-fold lower for 1 year in immunized animals compared to controls, and a significant delay in AIDS-related mortality resulted. Broader immunity resulted following immunizations with peptides spanning all nine SIV proteins, but the responses to Gag were weaker in comparison to animals only immunized with Gag. No difference in viral outcome occurred in animals immunized with all SIV proteins compared to animals immunized against Gag alone. Peptide-pulsed blood cells are an immunogenic and effective immunotherapy in SIV-infected macaques. Our results suggest Gag alone is an effective antigen for T-cell immunotherapy. Fresh blood cells pulsed with overlapping Gag peptides is proceeding into trials in HIV-infected humans.
Assuntos
Produtos do Gene gag/administração & dosagem , Imunoterapia/métodos , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Viremia/terapia , Animais , Transfusão de Componentes Sanguíneos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Feminino , Produtos do Gene gag/imunologia , Longevidade , Macaca nemestrina , Masculino , Síndrome de Imunodeficiência Adquirida dos Símios/imunologiaRESUMO
Oxford University and Nairobi University are jointly developing a HIVA.DNA/modified vaccinia Ankara (MVA) prime-boost vaccine for the potential prevention of infection with HIV subtype A. The vaccination strategy consists of priming with a DNA vaccine made from HIV-1 clade A gag p24/p17 consensus sequence (pTHr.HIVA) then boosting with a MVA virus expressing HIVA (MVA.HIVA). Phase II clinical trials of the vaccine are underway in Kenya and the UK.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Infecções por HIV/prevenção & controle , Vacinas de DNA/administração & dosagem , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Animais , Ensaios Clínicos Fase II como Assunto , Infecções por HIV/imunologia , Humanos , Quênia , Reino Unido , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vaccinia virus/genética , Vaccinia virus/imunologiaRESUMO
The impaired functional activity of cytotoxic T lymphocytes and natural killer cells during HIV-1 infection has recently been attributed to decreased intracellular levels of perforin and granzyme B. In sera from individuals chronically infected with HIV-1 we report increased levels of extracellular perforin compared with uninfected individuals. Increased perforin was also observed during experimental SIV/SHIV infection. The combination of reduced intracellular perforin levels and an increased serum level indicates that HIV infection induces aberrant perforin secretion.
Assuntos
Infecções por HIV/sangue , HIV-1 , Glicoproteínas de Membrana/sangue , Síndrome de Imunodeficiência Adquirida dos Símios/sangue , Vírus da Imunodeficiência Símia , Doença Aguda , Animais , Antirretrovirais/uso terapêutico , Doença Crônica , Infecções por HIV/tratamento farmacológico , Humanos , Macaca fascicularis , Perforina , Proteínas Citotóxicas Formadoras de Poros , Carga ViralRESUMO
The correlate of protection in human immunodeficiency virus (HIV) infection is not known, but preclinical and clinical studies support the involvement of both antibodies and cellular immunity. In addition, the existence of multiple HIV clades makes HIV vaccine design especially challenging. We have constructed a vaccine platform with an HIV-1 subtype B DNA immunogen expressing full length consensus sequences from HIV-1 rev, nef, tat, and gag with additional cellular epitope clusters from the env and pol regions. Furthermore, this platform has been extended to three additional plasmids expressing the same immunogens but originating from subtypes A or C consensus or FGH ancestral sequences. Immunogenicity in BALB/c mice, by gene gun or intramuscular delivery, revealed strong IFN-gamma production in response to in vitro re-stimulation with a H-2d restricted gag peptide (AMQMLKETI) or even stronger toward an env epitope (RGPGRAFVTI). Weak humoral immunity was detected. Gene gun immunization with a cocktail of all four plasmids induced pre-challenge cellular immunity in C57Bl6/A2.01 mice and subsequently a robust frequency of protection (11/12 animals) after experimental challenge with subtype A or B HIV-1/Murine Leukemia Virus (HIV-1/MuLV). The cross-clade protection observed in this challenge experiment demonstrates that these multigene/multiepitope HIV DNA immunogens are likely to be potent immunogens also against the HIV-infection of human beings.
Assuntos
Vacinas contra a AIDS/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Vacinas de DNA/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Animais , Sequência Consenso , Modelos Animais de Doenças , Epitopos/genética , Epitopos/imunologia , Feminino , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Produtos do Gene nef/genética , Produtos do Gene nef/imunologia , Produtos do Gene pol/genética , Produtos do Gene pol/imunologia , Produtos do Gene rev/genética , Produtos do Gene rev/imunologia , Produtos do Gene tat/genética , Produtos do Gene tat/imunologia , Anticorpos Anti-HIV/sangue , Proteína gp160 do Envelope de HIV/genética , Proteína gp160 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Produtos do Gene pol do Vírus da Imunodeficiência Humana , Produtos do Gene rev do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
We have cloned and sequenced the chimpanzee (Pan troglodytes) cyclin T1 cDNA and performed functional HIV-1 Tat trans-activation studies. A unique codon deletion leading to a deleted asparagine residue in the N-terminal region of the first cyclin domain was discovered. This mutation does not significantly change the trans-activation of HIV-1, suggesting that Tat-Cyclin T1 mediated transcription is not a major barrier to HIV replication in the chimpanzee.
Assuntos
Ciclinas/genética , Deleção de Genes , HIV-1/genética , Pan troglodytes/virologia , Ativação Transcricional , Animais , Sequência de Bases , Feminino , Produtos do Gene tat , Masculino , Dados de Sequência Molecular , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Mutations in reverse transcriptase (RT) confer high levels of HIV resistance to drugs. However, while conferring drug resistance, they can lower viral replication capacity (fitness). The molecular mechanisms behind remain largely unknown. The aim of the study was to characterize the effect of drug-resistance mutations on HIV RT expression. Genes encoding AZT-resistant RTs with single or combined mutations D67N, K70R, T215F, and K219Q, and RTs derived from drug-resistant HIV-1 strains were designed and expressed in a variety of eukaryotic cells. Expression in transiently transfected cells was assessed by Western blotting and immunofluorescent staining with RT-specific antibodies. To compare the levels of expression, mutated RT genes were microinjected into the nucleus of the oocytes of Xenopus laevis. Expression of RT was quantified by sandwich ELISA. Relative stability of RTs was assessed by pulse-chase experiments. Xenopus oocytes microinjected with the genes expressed 2-50 pg of RT mutants per cell. The level of RT expression decreased with accumulation of drug-resistance mutations. Pulse-chase experiments demonstrated that poor expression of DR-RTs was due to proteolytic instability. Instability could be attributed to additional cleavage sites predicted to appear in the vicinity of resistance mutations. Accumulation of drug-resistance mutations appears to affect the level of eukaryotic expression of HIV-1 RT by inducing proteolytic instability. Low RT levels might be one of the determinants of impaired replication fitness of drug-resistant HIV-1 strains.
Assuntos
Transcriptase Reversa do HIV/genética , HIV-1/enzimologia , HIV-1/genética , Animais , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Farmacorresistência Viral/genética , Estabilidade Enzimática , Feminino , Expressão Gênica , Genes Virais , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , Humanos , Técnicas In Vitro , Mutação , Oócitos/enzimologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Transfecção , Xenopus laevis , Zidovudina/farmacologiaRESUMO
Natural killer (NK) cells have been suggested to play a protective role in HIV disease progression. One potent effector mechanism of NK cells is antibody-dependent cellular cytotoxicity (ADCC) mediated by antiviral antibodies binding to the FcγRIIIa receptor (CD16) on NK cells. We investigated NK cell-mediated ADCC function and the presence of ADCC antibodies in plasma from 20 HIV-1-infected patients and 10 healthy donors. The HIV-positive patients were divided into two groups: six who controlled viremia for at least 8 y without treatment (controllers), and 14 who were persistently viremic and not currently on treatment. Plasma from both patient groups induced NK cell IFN-γ expression and degranulation in response to HIV-1 envelope (Env) gp140-protein-coated cells. Patient antibodies mediating ADCC were largely directed towards the Env V3 loop, as identified by a gp140 protein lacking the V3 loop. Interestingly, in two controllers ADCC-mediating antibodies were more broadly directed to other parts of Env. A high viral load in patients correlated with decreased ADCC-mediated cytolysis of gp140-protein-coated target cells. NK cells from both infected patients and healthy donors degranulated efficiently in the presence of antibody-coated HIV-1-infected Jurkat cells. In conclusion, the character of ADCC-mediating antibodies differed in some controllers compared to viremic patients. NK cell ADCC activity is not compromised in HIV-infected patients.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Células Matadoras Naturais/imunologia , Viremia/imunologia , Adulto , Anticorpos Monoclonais/imunologia , Feminino , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/fisiologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/imunologia , Carga Viral , Viremia/virologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
We show here that it is possible to combine two different genetic immunogens, one designed to induce HIV-1 specific humoral immune responses (pKCMVgp160B) and one designed to induce cellular anti-HIV-1 immune responses (Auxo-GTU-MultiHIV), and still retain the major properties of both vaccine constructs. The two different constructs were delivered using two different methods; the gene-gun and the Biojector, which both are needle-free devices. In BALB/c mice we were able to induce high levels of HIV-1-specific T cell responses as well as high levels of anti-gp160 antibodies by co-administrating the vaccine constructs. The cellular immune responses, but not antibody responses, were moderately compromised from the combination. This study shows that it is a feasible strategy to combine different vaccines and modes of delivery, but that interference as to magnitude may occur to certain gene products.
Assuntos
Biolística , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Injeções a Jato , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Animais , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Anticorpos Anti-HIV/sangue , Proteína gp160 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Imunização , Injeções Intradérmicas , Injeções a Jato/instrumentação , Injeções a Jato/métodos , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/imunologia , Vacinas de DNA/genéticaRESUMO
Release of granzymes and perforin from the cytolytic granules of SIV-specific CD8 T cells is a critically important effector mechanism facilitating the elimination of SIV-infected cells. We sequenced granzyme A, B, and K and perforin in pigtail macaques and defined polymorphisms between humans, rhesus macaques, and pigtail macaques. The pigtail macaque sequences were similar to the corresponding rhesus sequences at the mRNA and protein level and (0.4-1.1% sequence differences) but substantially different from human sequences (3.8-8.1% sequence differences). We used this sequence information to develop multiplex PCR assays to detect these genes. We also successfully studied the release of perforin and granzyme B from deregulating SIV-specific CD8 T cells by flow cytometry. These sequences and tools enable further study of the cytolytic control of SIV in pigtail macaques.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Granzimas/metabolismo , Perforina/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Granzimas/genética , Humanos , Macaca mulatta , Macaca nemestrina , Dados de Sequência Molecular , Perforina/genética , RNA MensageiroRESUMO
Simple antibodies or vector-induced T cell immunity are unable to provide broad immunity to HIV. Although broadly reactive neutralising antibodies are a goal of vaccination, this remains elusive. There is growing evidence that HIV-specific antibodies that mediate their activity via the Fc-receptor, such as antibody dependent cellular cytotoxicity (ADCC), have an important role in controlling HIV infection. Newer assays are being developed that enable HIV-specific ADCC responses to be finely mapped. In turn, this should allow a more definitive analysis of the effectiveness of HIV-specific ADCC antibodies. However, progressive dysfunction of effector cells that mediate ADCC responses, such as NK cells, combined with immune escape variants that emerge from effective ADCC responses, likely undermine the utility of ADCC responses during chronic HIV infection. Nonetheless the utility of ADCC responses in preventing HIV infection requires urgent consideration.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Infecções por HIV/imunologia , HIV/imunologia , Animais , Humanos , Macaca mulattaRESUMO
HIV-1 only infects humans and chimpanzees. SIV or SHIV are, therefore, used as models for HIV in rhesus, cynomologus and pigtail macaques. Since conducting experiments in primate models does not fully mimic infection or vaccination against HIV-1 and is expensive, there is a great need for small-animal models in which it is possible to study HIV-1 infection, immunity and vaccine efficacy. This review summarizes the available murine models for studying HIV-1 infection with an emphasis on our experience of the HIV-1-infected-cell challenge as a model for evaluating candidate HIV-1 vaccines. In the cell-based challenge model, several important factors that, hopefully, can be related to vaccine efficacy in humans were discovered: the efficiency of combining plasmid DNA representing several of the viral genes originating from multiple clades of HIV-1, the importance of adjuvants activating innate and induced immunity and the enhanced HIV eradication by drug-conjugated antibody.
Assuntos
Vacinas contra a AIDS , Infecções por HIV/prevenção & controle , Modelos Animais , Vacinas contra a AIDS/imunologia , Animais , Linhagem Celular , Infecções por HIV/imunologia , HIV-1/genética , Humanos , Camundongos , Camundongos SCID , TransgenesRESUMO
BACKGROUND: Live attenuated SIV vaccines are highly efficacious, but how they mediate protection is poorly understood. A feature of the effectiveness of live attenuated vaccines is their ability to control high dose challenge viruses early, without a large peak of acute viraemia. We hypothesized that long-lived antigen exposure from live attenuated SIV may result in CD8+ cytotoxic T lymphocytes persistently capable of rapidly cytolytic potential. METHODS: We employed a kinetic degranulation assay to study multiple tetramer+ SIV-specific CTL specificities before and after the SIV(mac251) challenge of pigtail macaques inoculated with a live attenuated SIV. RESULTS: Live attenuated SIV-vaccinated animals rapidly controlled a subsequent challenge, with minimal viraemia after exposure. For over 9 months after the initial vaccination with live attenuated SIV we could detect both Gag- and Tat-specific CTLs that maintained a long-term capacity to rapidly degranulate (CD107a expression) and release granzyme B within 30 minutes of antigen exposure. This rapid cytolytic phenotype was maintained throughout the early period after challenge, despite the absence of a marked enhancement in CTL frequencies. CONCLUSIONS: Our results suggest that highly functional CTLs may contribute to the remarkable efficacy of live attenuated SIV vaccines. Studying the killing kinetics of CTLs induced by other, safer, HIV vaccines could facilitate a better understanding of the requirements for an effective HIV vaccine.
Assuntos
Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Linfócitos T Citotóxicos/imunologia , Animais , Epitopos de Linfócito T/genética , Memória Imunológica/imunologia , Cinética , Macaca nemestrina , Vacinas contra a SAIDS/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Fatores de Tempo , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologiaRESUMO
Persistent gag-specific T cell immunity would be a useful component of an effective HIV vaccine. The Flavivirus Kunjin replicon was previously engineered to persistently express HIV gag and was shown to induce protective responses in mice. We evaluated Kunjin replicon virus-like-particles expressing SIVgag-pol in pigtail macaques. Kunjin-specific antibodies were induced, but no SIV-specific T cell immunity were detected. Following SIVmac251 challenge, there was no difference in SIV viremia or retention of CD4 T cells between Kunjin-SIVgag-pol vaccine immunized animals and controls. An amnestic SIV gag-specific CD8 T cell response associated with control of viremia was observed in 1 of 6 immunized animals. Refinements of this vector system and optimization of the immunization doses, routes, and schedules are required prior to clinical trials.
Assuntos
Replicon , Vacinas contra a SAIDS , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Vacinas Sintéticas/imunologia , Vírus do Nilo Ocidental , Vacinas contra a AIDS , Animais , Anticorpos Antivirais/sangue , Proteínas de Fusão gag-pol/genética , Proteínas de Fusão gag-pol/imunologia , Proteínas de Fusão gag-pol/metabolismo , Engenharia Genética , HIV-1/genética , HIV-1/imunologia , HIV-1/metabolismo , Ativação Linfocitária , Macaca nemestrina , Vacinas contra a SAIDS/administração & dosagem , Vacinas contra a SAIDS/genética , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/metabolismo , Vírus da Imunodeficiência Símia/patogenicidade , Linfócitos T/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/metabolismo , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/metabolismoRESUMO
Both the magnitude and function of vaccine-induced HIV-specific CD8+ CTLs are likely to be important in the outcome of infection. We hypothesized that rapid cytolysis by CTLs may facilitate control of viral challenge. Release kinetics of the cytolytic effector molecules granzyme B and perforin, as well as the expression of the degranulation marker CD107a and IFN-gamma were simultaneously studied in SIV Gag(164-172) KP9-specific CD8+ T cells from Mane-A*10+ pigtail macaques. Macaques were vaccinated with either prime-boost poxvirus vector vaccines or live-attenuated SIV vaccines. Prime-boost vaccination induced Gag-specific CTLs capable of only slow (after 3 h) production of IFN-gamma and with limited (<5%) degranulation and granzyme B release. Vaccination with live-attenuated SIV resulted in a rapid cytolytic profile of SIV-specific CTLs with rapid (<0.5 h) and robust (>50% of tetramer-positive CD8+ T cells) degranulation and granzyme B release. The cytolytic phenotype following live-attenuated SIV vaccinations were similar to that associated with the partial resolution of viremia following SIV(mac251) challenge of prime-boost-vaccinated macaques, albeit with less IFN-gamma expression. High proportions of KP9-specific T cells expressed the costimulatory molecule CD28 when they exhibited a rapid cytolytic phenotype. The delayed cytolytic phenotype exhibited by standard vector-based vaccine-induced CTLs may limit the ability of T cell-based HIV vaccines to rapidly control acute infection following a pathogenic lentiviral exposure.
Assuntos
Vacinas contra a AIDS/imunologia , Linfócitos T CD8-Positivos/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vacinação/métodos , Animais , Biomarcadores , Linfócitos T CD8-Positivos/metabolismo , Granzimas/metabolismo , Memória Imunológica/imunologia , Cinética , Macaca , Fenótipo , Infecções por Retroviridae/imunologia , Infecções por Retroviridae/virologia , Vacinas contra a SAIDS/imunologiaRESUMO
Many current-generation human immunodeficiency virus (HIV) vaccines induce specific T cells to control acute viremia, but their utility following infection with escape mutant virus is unclear. We studied reversion to wild type of an escape mutant simian-HIV in major histocompatibility complex-matched vaccinated pigtail macaques. High levels of vaccine-induced CD8+ T cells strongly correlated with maintenance of escape mutant virus during acute infection. Interestingly, in animals with lower CD8+ T-cell levels, transient reversion to wild-type virus resulted in better postacute control of viremia. Killing of wild-type virus facilitated by transient reversion outweighs the benefit of a larger CD8+ T-cell response that only maintains the less fit escape mutant virus. These findings have important implications for the further development of T-cell-based HIV vaccines where exposure to escape mutant viruses is common.
Assuntos
Vacinas contra a AIDS/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , HIV/imunologia , Mutação , Animais , HIV/genética , Infecções por HIV/virologia , Macaca nemestrina , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , ViremiaRESUMO
BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Nef, is an attractive vaccine target because it is involved in viral pathogenesis, is expressed early in the viral life cycle and harbors many T and B cell epitopes. Several clinical trials include gene-based vaccines encoding this protein. However, Nef has been shown to transform certain cell types in vitro. Based on these findings we performed a long-term toxicity and immunogenicity study of Nef, encoded either by Modified Vaccinia virus Ankara or by plasmid DNA. BALB/c mice were primed twice with either DNA or MVA encoding Nef and received a homologous or heterologous boost ten months later. In the meantime, the Nef-specific immune responses were monitored and at the time of sacrifice an extensive toxicological evaluation was performed, where presence of tumors and other pathological changes were assessed. RESULTS: The toxicological evaluation showed that immunization with MVAnef is safe and does not cause cellular transformation or other toxicity in somatic organs.Both DNAnef and MVAnef immunized animals developed potent Nef-specific cellular responses that declined to undetectable levels over time, and could readily be boosted after almost one year. This is of particular interest since it shows that plasmid DNA vaccine can also be used as a potent late booster of primed immune responses. We observed qualitative differences between the T cell responses induced by the two different vectors: DNA-encoded nef induced long-lasting CD8+ T cell memory responses, whereas MVA-encoded nef induced CD4+ T cell memory responses. In terms of the humoral immune responses, we show that two injections of MVAnef induce significant anti-Nef titers, while repeated injections of DNAnef do not. A single boost with MVAnef could enhance the antibody response following DNAnef prime to the same level as that observed in animals immunized repeatedly with MVAnef. We also demonstrate the possibility to boost HIV-1 Nef-specific immune responses using the MVAnef construct despite the presence of potent anti-vector immunity. CONCLUSION: This study shows that the nef gene vectored by MVA does not induce malignancies or other adverse effects in mice. Further, we show that when the nef gene is delivered by plasmid or by a viral vector, it elicits potent and long-lasting immune responses and that these responses can be directed towards a CD4+ or a CD8+ T cell response depending on the choice of vector.
RESUMO
Pre-clinical HIV-1 vaccine protocols, using multiple vaccine modalities and a potent adjuvant were assessed for vaccine efficacy in an experimental HIV-1 challenge model. C57Bl/6 mice were immunized with DNA plasmids encoding HIV-1 gp140, Gag and Tat alone or in combination with the corresponding recombinant proteins formulated in the adjuvant MF59. HIV-1 DNA alone or a DNA prime protein boost schedule resulted in complete protection against challenge with HIV-1/MuLV-infected murine cells. Although HIV-1 protein immunization in combination with MF59 resulted in partial protection, the DNA priming seemed to be crucial for obtaining full protection against the challenge. It is likely that the partial protection seen after immunization with protein alone is, to a certain extent, due to effects of the adjuvant since some animals that received the adjuvant MF59 alone were protected from the challenge. For the most part, antigen-specific cell-mediated immune responses as detected in the spleen (in contrast to responses detected in peripheral blood) of immunized animals appeared to be associated with protection in this study.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Produtos do Gene gag/imunologia , Produtos do Gene tat/imunologia , Infecções por HIV/prevenção & controle , HIV-1/patogenicidade , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Adjuvantes Imunológicos , Animais , Modelos Animais de Doenças , Produtos do Gene gag/genética , Produtos do Gene tat/genética , Anticorpos Anti-HIV/sangue , Infecções por HIV/virologia , Humanos , Imunização , Esquemas de Imunização , Imunoglobulina G/sangue , Vírus da Leucemia Murina , Camundongos , Camundongos Endogâmicos C57BL/genética , Camundongos Transgênicos , Polissorbatos , Esqualeno , Linfócitos T/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
A DNA plasmid encoding human immunodeficiency virus type 1 (HIV-1) env, nef and tat genes was used in mice in a prime-boost immunization regimen with the corresponding recombinant proteins. The genetic immunogen was delivered with a gene gun and the proteins were injected intramuscularly together with the adjuvant AS02A. Immunizations were followed by experimental challenge with pseudotyped HIV-1 subtype A or B virus. In an initial experiment in which animals were challenged four weeks after the final immunization, all single modality and prime-boost vaccinations resulted in a significant level of protection as compared to control animals. There was a trend for DNA-alone immunization yielding the highest protection. In a subsequent study, a late challenge was performed 19 weeks after the final immunization. All groups having received the DNA vaccine, either alone or in combination with adjuvanted protein, exhibited strong protection against HIV replication. The subtype-specific protection against the experimental HIV challenge was significantly stronger than the cross-protection. Cellular and humoral immune responses were assessed during immunization and after challenge, but without clear correlation to protection against HIV replication. The data suggest that either DNA or protein antigens alone provide partial protection against an HIV-1/MuLV challenge and that DNA immunization is essential for achieving very high levels of efficacy in this murine HIV-1 challenge model. While prime-boost combinations were more immunogenic than DNA alone, they did not appear to provide any further enhancement over DNA vaccine mediated efficacy. The DNA immunogen might prime low levels of CD8+ T cells responsible for virus clearance or possibly a yet unidentified mechanism of protection.
Assuntos
Vacinas contra a AIDS/imunologia , Adjuvantes Imunológicos , Infecções por HIV/prevenção & controle , Lipídeo A/análogos & derivados , Infecções por Retroviridae/prevenção & controle , Saponinas/imunologia , Vacinas de DNA/imunologia , Animais , Biolística , Modelos Animais de Doenças , Combinação de Medicamentos , Anticorpos Anti-HIV/sangue , Antígenos HIV/genética , Antígenos HIV/imunologia , HIV-1/imunologia , Humanos , Imunização Secundária , Injeções Intramusculares , Vírus da Leucemia Murina , Lipídeo A/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Linfócitos T/imunologia , Infecções Tumorais por Vírus/prevenção & controle , Vacinação , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
The results presented here are from the preclinical evaluation in BALB/c mice of a DNA prime/modified vaccinia virus Ankara (MVA) boost multi-gene multi-subtype human immunodeficiency virus-1 (HIV-1) vaccine intended for use in humans. The plasmid DNA vaccine was delivered intradermally using a Biojector, and the MVA was delivered intramuscularly by needle. This combination of recombinant DNA and MVA proved to induce extraordinarily strong cellular responses, with more than 80% of the CD8(+) T cells specific for HIV-1 antigens. Furthermore, we show that the DNA priming increases the number of T-cell epitopes recognized after the MVA boost. In the prime/boost-immunized animals, a significant proportion of CD8(+) T cells were stained positive for both interferon-gamma (IFN-gamma) and interleukin-2 (IL-2), a feature that has been associated with control of HIV-1 infection in long-term non-progressors. The HIV-1-specific antibody levels were moderate after the plasmid DNA immunizations but increased dramatically after the MVA boost. Although the initial injection of MVA induced significant levels of vaccinia-neutralizing antibodies, the HIV-specific responses were still significantly boosted by the second MVA immunization. The results from this study demonstrate the potency of this combination of DNA plasmids and MVA construct to induce broad and high levels of immune responses against several HIV-1 proteins of different subtypes.