RESUMO
Most HIV transmissions among men who have sex with men (MSM), the group that accounted for 67% of new US infections in 2014, occur via exposure to the rectal mucosa. However, it is unclear how the act of condomless receptive anal intercourse (CRAI) may alter the mucosal immune environment in HIV-negative MSM. Here, we performed a comprehensive characterization of the rectal mucosal immune environment for the phenotype and production of pro-inflammatory cytokines by CD4 and CD8 T cells, global transcriptomic analyses, and the composition of microbiota in HIV-negative MSM. Our results show that compared with men who had never engaged in anal intercourse, the rectal mucosa of MSM engaging in CRAI has a distinct phenotype characterized by higher levels of Th17 cells, greater CD8+ T cell proliferation and production of pro-inflammatory cytokines, molecular signatures associated with mucosal injury and repair likely mediated by innate immune cells, and a microbiota enriched for the Prevotellaceae family. These data provide a high-resolution model of the immunological, molecular, and microbiological perturbations induced by CRAI, will have direct utility in understanding rectal HIV transmission among MSM, and will enhance the design of future biomedical prevention interventions, including candidate HIV vaccines.
Assuntos
Infecções por Bacteroidaceae/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , Microbiota/genética , Mucosa/imunologia , Prevotella/genética , Reto/patologia , Células Th17/imunologia , Adulto , Proliferação de Células , Preservativos/estatística & dados numéricos , Citocinas/metabolismo , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , Soronegatividade para HIV , Homossexualidade Masculina , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Comportamento Sexual , Transcriptoma , Adulto JovemRESUMO
Protective immunity at the gut-associated mucosal tissue is induced primarily by oral/rectal immunization owing to the need for targeting antigen to the gut-resident dendritic cells (DCs). In this study we show that an adenovirus type 5 (Ad5)-based human immunodeficiency virus type 1 vaccine can prime a durable antigen-specific CD8 T-cell response in the gut following intramuscular (IM) immunization in mice. The ability of Ad5 to prime gut-homing CD8 T cells in vivo was associated with Ad5-induced expression of retinal dehydrogenase (RALDH) enzymes in conventional DCs. The Ad5-mediated induction of RALDH did not require signaling through Toll-like receptors, DNA-dependent activator of interferon regulatory factors and several mitogen-activated protein kinases, or replication capacity of the virus, but was dependent on nuclear factor-κB and granulocyte-macrophage colony-stimulating factor. These results provide an innate mechanism through which Ad5-stimulated DCs prime gut-homing CD8 T cells and have implications for the development of novel mucosal adjuvants for subunit vaccines administered via the IM route.
Assuntos
Adenoviridae/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Trato Gastrointestinal/imunologia , Retinal Desidrogenase/metabolismo , Vacinas contra a AIDS/imunologia , Animais , Técnicas de Cocultura , Células Dendríticas/virologia , Feminino , Trato Gastrointestinal/metabolismo , Regulação da Expressão Gênica/imunologia , HIV-1/imunologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/metabolismo , Transdução de Sinais/imunologia , Baço/imunologia , Serina-Treonina Quinases TOR/metabolismo , Tretinoína/metabolismoRESUMO
A vaccine consisting of DNA priming followed by recombinant modified vaccinia Ankara (rMVA) boosting has achieved long-term control of a pathogenic challenge with a chimera of simian and human immunodeficiency viruses (SHIV-89.6P) in rhesus macaques. Based on these results, clade B HIV-1 DNA and rMVA immunogens have been developed for trials in humans. We conducted a first-time in humans phase I safety trial using the pGA2/JS2 (JS2) HIV-1 DNA priming vector expressing Gag, Pol, Env, Tat, Rev, and Vpu. Thirty HIV-uninfected adults were vaccinated with 0.3 or 3 mg of JS2 DNA, or a saline placebo, by intramuscular injection at months 0 and 2. Both doses of DNA were safe and well-tolerated with no differences between the control, 0.3 mg, or 3 mg groups (n = 6, 12, and 12, respectively) through 12 months of postvaccination follow- up. A chromium-release assay using fresh peripheral blood mononuclear cells (PBMCs) and a validated IFN-gamma ELISpot assay with frozen PBMCs failed to detect CD4(+) or CD8(+) HIV-1-specific T cell responses. HIV-specific neutralizing antibodies were also not detected. The vaccine is being further developed as a priming vector for a combined DNA plus rMVA prime/boost HIV vaccination regimen.
Assuntos
Vacinas contra a AIDS/efeitos adversos , HIV-1/imunologia , Plasmídeos/efeitos adversos , Vacinas de DNA/efeitos adversos , Vaccinia virus/imunologia , Vacinas contra a AIDS/imunologia , Adulto , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos/efeitos adversos , Vetores Genéticos/imunologia , Anticorpos Anti-HIV/metabolismo , Humanos , Interferon gama/metabolismo , Masculino , Plasmídeos/imunologia , Estatísticas não Paramétricas , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/imunologia , Vaccinia virus/genéticaRESUMO
Heterologous prime/boost regimens have the potential for raising high levels of immune responses. Here we report that DNA priming followed by a recombinant modified vaccinia Ankara (rMVA) booster controlled a highly pathogenic immunodeficiency virus challenge in a rhesus macaque model. Both the DNA and rMVA components of the vaccine expressed multiple immunodeficiency virus proteins. Two DNA inoculations at 0 and 8 weeks and a single rMVA booster at 24 weeks effectively controlled an intrarectal challenge administered 7 months after the booster. These findings provide hope that a relatively simple multiprotein DNA/MVA vaccine can help to control the acquired immune deficiency syndrome epidemic.
Assuntos
Vacinas contra a AIDS/imunologia , Síndrome da Imunodeficiência Adquirida/prevenção & controle , Vacinas de DNA/imunologia , Vacinas contra a AIDS/administração & dosagem , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/virologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Centro Germinativo/imunologia , Anticorpos Anti-HIV/sangue , Anticorpos Anti-HIV/imunologia , HIV-1/genética , HIV-1/imunologia , HIV-1/fisiologia , Imunidade nas Mucosas , Imunização Secundária , Memória Imunológica , Interferon gama/biossíntese , Linfonodos/imunologia , Macaca mulatta , Vacinas contra a SAIDS/administração & dosagem , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Linfócitos T/imunologia , Vacinas de DNA/administração & dosagem , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vaccinia virus/imunologia , Carga ViralRESUMO
Apoptotic bodies can be used to target delivery of DNA-expressed immunogens into professional antigen-presenting cells (APCs). Here we show that antigen-laden apoptotic bodies created by vectors co-expressing influenza virus hemagglutinin (HA) or nucleoprotein (NP) genes and mutant caspase genes markedly increased T-cell responses. Both CD8 and CD4 T-cell responses were affected. The adjuvant activity was restricted to partially inactivated caspases that allowed immunogen expression before the generation of apoptotic bodies. Active-site mutants of murine caspase 2 and an autocatalytic chimera of murine caspase 2 prodomain and human caspase 3 induced apoptosis that did not interfere with immunogen expression. The adjuvant activity also enhanced B-cell responses, but to a lesser extent than T-cell responses. The large increases in T-cell responses represent one of the strongest effects to date of a DNA adjuvant on cellular immunity.
Assuntos
Apoptose , Caspases/genética , Caspases/imunologia , DNA/metabolismo , Células 3T3 , Animais , Anexina A5/farmacologia , Células Apresentadoras de Antígenos , Sítios de Ligação , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Relação Dose-Resposta a Droga , Proteínas de Fluorescência Verde , Hemaglutininas/biossíntese , Humanos , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Nucleoproteínas/biossíntese , Orthomyxoviridae/metabolismo , Plasmídeos/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Mouse serum raised against killed antigen preparations of Mycobacterium tuberculosis failed to recognize most of the recombinant antigens of M. tuberculosis that were originally identified by reactivity to tuberculosis (TB) patient sera. Similar results were obtained with serum from guinea pigs immunized with live and killed mycobacteria. Antibodies raised against seven random TB patient serum-reactive antigens detected each of these antigens in the sonicate preparation. The nucleotide sequences of the genes for these seven antigens revealed that all represented hitherto unreported genes of M. tuberculosis. Our results suggest differential presentation to the host immune system of the same antigens derived from live and killed mycobacteria.
Assuntos
Antígenos de Bactérias/imunologia , Mycobacterium tuberculosis/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/imunologiaRESUMO
Obtaining pure mRNA preparations from prokaryotes has been difficult, if not impossible, for want of a poly(A) tail on these messages. We have used poly(A) polymerase from yeast to effect specific polyadenylation of Escherichia coli polysomal mRNA in the presence of magnesium and manganese. The polyadenylated total mRNA, which could be subsequently purified by binding to and elution from oligo(dT) beads, had a size range of 0.4-4.0 kb. We have used hybridization to a specific plasmid-encoded gene to further confirm that the polyadenylated species represented mRNA. Withdrawal of Mg2+ from the polyadenylation reaction resulted in addition of poly(A) to 16S rRNA despite the presence of Mn2+, indicating the vital role of Mg2+ in maintaining the native structure of polysomes. Complete dissociation of polysomes into ribosomal subunits resulted in quantitative polyadenylation of both 16S and 23S rRNA species. Chromosomal lacZ gene-derived messages were quantitatively recovered in the oligo(dT)-bound fraction, as demonstrated by RT-PCR analysis. Potential advantages that accrue from the availability of pure total mRNA from prokaryotes is discussed.
Assuntos
Escherichia coli/genética , Poli A/metabolismo , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Northern Blotting , Southern Blotting , Magnésio/farmacologia , Manganês/farmacologia , Reação em Cadeia da Polimerase , Polinucleotídeo Adenililtransferase/metabolismo , Polirribossomos/metabolismo , RNA Bacteriano/isolamento & purificação , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/metabolismo , DNA Polimerase Dirigida por RNARESUMO
DNA obtained from a human sputum isolate of Mycobacterium tuberculosis, NTI-64719, which showed extensive dissemination in the guinea pig model resulting in a high score for virulence was used to construct an expression library in the lambda ZAP vector. The size of DNA inserts in the library ranged from 1 to 3 kb, and recombinants represented 60% of the total plaques obtained. When probed with pooled serum from chronically infected tuberculosis patients, the library yielded 176 recombinants with a range of signal intensities. Among these, 93 recombinants were classified into 12 groups on the basis of DNA hybridization experiments. The polypeptides synthesized by the recombinants were predominantly LacZ fusion proteins. Serum obtained from patients who were clinically diagnosed to be in the early phase of M. tuberculosis infection was used to probe the 176 recombinants obtained. Interestingly, some recombinants that gave very strong signals in the original screen did not react with early-phase serum; conversely, other whose signals were extremely weak in the original screen gave very intense signals with serum from recently infected patients. This indicates the differential nature of either the expression of these antigens or the immune response elicited by them as a function of disease progression.