RESUMO
Chronic gut inflammatory diseases are associated with disruption of intestinal epithelial barriers and impaired mucosal immunity. HIV-1 (HIV) causes depletion of mucosal CD4+ T cells early in infection and disruption of gut epithelium, resulting in chronic inflammation and immunodeficiency. Although antiretroviral therapy (ART) is effective in suppressing viral replication, it is incapable of restoring the "leaky gut," which poses an impediment for HIV cure efforts. Strategies are needed for rapid repair of the epithelium to protect intestinal microenvironments and immunity in inflamed gut. Using an in vivo nonhuman primate intestinal loop model of HIV/AIDS, we identified the pathogenic mechanism underlying sustained disruption of gut epithelium and explored rapid repair of gut epithelium at the intersection of microbial metabolism. Molecular, immunological, and metabolomic analyses revealed marked loss of peroxisomal proliferator-activated receptor-α (PPARα) signaling, predominant impairment of mitochondrial function, and epithelial disruption both in vivo and in vitro. To elucidate pathways regulating intestinal epithelial integrity, we introduced probiotic Lactobacillus plantarum into Simian immunodeficiency virus (SIV)-inflamed intestinal lumen. Rapid recovery of the epithelium occurred within 5 h of L. plantarum administration, independent of mucosal CD4+ T cell recovery, and in the absence of ART. This intestinal barrier repair was driven by L. plantarum-induced PPARα activation and restoration of mitochondrial structure and fatty acid ß-oxidation. Our data highlight the critical role of PPARα at the intersection between microbial metabolism and epithelial repair in virally inflamed gut and as a potential mitochondrial target for restoring gut barriers in other infectious or gut inflammatory diseases.
Assuntos
Metabolismo Energético/fisiologia , Microbioma Gastrointestinal/fisiologia , Intestinos/imunologia , Intestinos/microbiologia , Mitocôndrias/metabolismo , PPAR alfa/metabolismo , Animais , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Epitélio/imunologia , Infecções por HIV , Humanos , Imunidade nas Mucosas , Interleucina-1beta/metabolismo , Intestinos/patologia , Lactobacillus plantarum/fisiologia , Macaca mulatta , Masculino , Metabolômica , Mitocôndrias/efeitos dos fármacos , Probióticos/administração & dosagem , Probióticos/uso terapêutico , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologiaRESUMO
Subclinical viral infections (SVI), including cytomegalovirus (CMV), are highly prevalent in humans, resulting in lifelong persistence. However, the impact of SVI on the interplay between the host immunity and gut microbiota in the context of environmental exposures is not well defined. We utilized the preclinical nonhuman primate (NHP) model consisting of SVI-free (specific-pathogen-free [SPF]) rhesus macaques and compared them to the animals with SVI (non-SPF) acquired through natural exposure and investigated the impact of SVI on immune cell distribution and function, as well as on gut microbiota. These changes were examined in animals housed in the outdoor environment compared to the controlled indoor environment. We report that SVI are associated with altered immune cell subsets and gut microbiota composition in animals housed in the outdoor environment. Non-SPF animals harbored a higher proportion of potential butyrate-producing Firmicutes and higher numbers of lymphocytes, effector T cells, and cytokine-producing T cells. Surprisingly, these differences diminished following their transfer to the controlled indoor environment, suggesting that non-SPFs had increased responsiveness to environmental exposures. An experimental infection of indoor SPF animals with CMV resulted in an increased abundance of butyrate-producing bacteria, validating that CMV enhanced colonization of butyrate-producing commensals. Finally, non-SPF animals displayed lower antibody responses to influenza vaccination compared to SPF animals. Our data show that subclinical CMV infection heightens host immunity and gut microbiota changes in response to environmental exposures. This may contribute to the heterogeneity in host immune response to vaccines and environmental stimuli at the population level.IMPORTANCE Humans harbor several latent viruses that modulate host immunity and commensal microbiota, thus introducing heterogeneity in their responses to pathogens, vaccines, and environmental exposures. Most of our understanding of the effect of CMV on the immune system is based on studies of children acquiring CMV or of immunocompromised humans with acute or reactivated CMV infection or in ageing individuals. The experimental mouse models are genetically inbred and are completely adapted to the indoor laboratory environment. In contrast, nonhuman primates are genetically outbred and are raised in the outdoor environment. Our study is the first to report the impact of long-term subclinical CMV infection on host immunity and gut microbiota, which is evident only in the outdoor environment but not in the indoor environment. The significance of this study is in highlighting the impact of SVI on enhancing host immune susceptibility to environmental exposures and immune heterogeneity.
Assuntos
Bactérias/classificação , Infecções por Citomegalovirus/veterinária , Citomegalovirus/patogenicidade , Doenças dos Macacos/imunologia , Doenças dos Macacos/microbiologia , Animais , Bactérias/isolamento & purificação , Citocinas/metabolismo , Infecções por Citomegalovirus/imunologia , Modelos Animais de Doenças , Microbioma Gastrointestinal , Abrigo para Animais , Linfócitos/metabolismo , Macaca mulatta , Filogenia , Organismos Livres de Patógenos Específicos , Linfócitos T/imunologiaRESUMO
HIV causes rapid CD4+ T cell depletion in the gut mucosa, resulting in immune deficiency and defects in the intestinal epithelial barrier. Breakdown in gut barrier integrity is linked to chronic inflammation and disease progression. However, the early effects of HIV on the gut epithelium, prior to the CD4+ T cell depletion, are not known. Further, the impact of early viral infection on mucosal responses to pathogenic and commensal microbes has not been investigated. We utilized the SIV model of AIDS to assess the earliest host-virus interactions and mechanisms of inflammation and dysfunction in the gut, prior to CD4+ T cell depletion. An intestinal loop model was used to interrogate the effects of SIV infection on gut mucosal immune sensing and response to pathogens and commensal bacteria in vivo. At 2.5 days post-SIV infection, low viral loads were detected in peripheral blood and gut mucosa without CD4+ T cell loss. However, immunohistological analysis revealed the disruption of the gut epithelium manifested by decreased expression and mislocalization of tight junction proteins. Correlating with epithelial disruption was a significant induction of IL-1ß expression by Paneth cells, which were in close proximity to SIV-infected cells in the intestinal crypts. The IL-1ß response preceded the induction of the antiviral interferon response. Despite the disruption of the gut epithelium, no aberrant responses to pathogenic or commensal bacteria were observed. In fact, inoculation of commensal Lactobacillus plantarum in intestinal loops led to rapid anti-inflammatory response and epithelial tight junction repair in SIV infected macaques. Thus, intestinal Paneth cells are the earliest responders to viral infection and induce gut inflammation through IL-1ß signaling. Reversal of the IL-1ß induced gut epithelial damage by Lactobacillus plantarum suggests synergistic host-commensal interactions during early viral infection and identify these mechanisms as potential targets for therapeutic intervention.
Assuntos
Interleucina-1beta/biossíntese , Celulas de Paneth/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Animais , Imunofluorescência , Interações Hospedeiro-Parasita/imunologia , Imuno-Histoquímica , Interleucina-1beta/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/ultraestrutura , Mucosa Intestinal/virologia , Macaca mulatta , Masculino , Microscopia Eletrônica de Transmissão , Análise de Sequência com Séries de Oligonucleotídeos , Celulas de Paneth/metabolismo , Celulas de Paneth/virologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Junções Íntimas/ultraestrutura , Carga ViralRESUMO
BACKGROUND: The impact of HIV infection on pattern recognition receptor (PRR) expression in gut-associated lymphoid tissue and its association with dysbiosis is not well understood. METHODS: PRR and cytokine gene expression were examined in mesenteric lymph nodes (mLN) of rhesus macaques during acute and chronic (untreated and early antiretroviral (ART) treated) infections. Gene expression was correlated with microbial abundance in the gut and immune activation. RESULTS: PRR expression rapidly increases during acute infection and is significantly decreased in chronic infection. Early ART maintains elevated PRR expression. Correlation analysis revealed three distinct groups of bacterial taxa that were associated with gene expression changes in infection. CONCLUSIONS: PRR and cytokine gene expression in the gut-draining mLN are rapidly modulated in response to viral infection and are correlated with gut dysbiosis. These data suggest that the dysregulation of PRR and related cytokine expression may contribute to chronic immune activation in SIV infection.
Assuntos
Antirretrovirais/farmacologia , Citocinas/genética , Microbioma Gastrointestinal , Regulação da Expressão Gênica , Receptores de Reconhecimento de Padrão/genética , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Doença Aguda , Animais , Doença Crônica , Citocinas/metabolismo , Linfonodos/imunologia , Linfonodos/virologia , Receptores de Reconhecimento de Padrão/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologiaRESUMO
The threat of a smallpox-based bioterrorist event or a human monkeypox outbreak has heightened the importance of new, safe vaccine approaches for these pathogens to complement older poxviral vaccine platforms. As poxviruses are large, complex viruses, they present technological challenges for simple recombinant vaccine development where a multicomponent mixtures of vaccine antigens are likely important in protection. We report that a synthetic, multivalent, highly concentrated, DNA vaccine delivered by a minimally invasive, novel skin electroporation microarray can drive polyvalent immunity in macaques, and offers protection from a highly pathogenic monkeypox challenge. Such a diverse, high-titer antibody response produced against 8 different DNA-encoded antigens delivered simultaneously in microvolumes has not been previously described. These studies represent a significant improvement in the efficiency of the DNA vaccine platform, resulting in immune responses that mimic live viral infections, and would likely have relevance for vaccine design against complex human and animal pathogens.
Assuntos
Mpox/prevenção & controle , Vacina Antivariólica/imunologia , Vacinas de DNA/imunologia , Animais , Anticorpos Antivirais/sangue , Eletroporação/métodos , Humanos , Macaca mulatta , Vacina Antivariólica/administração & dosagem , Análise de Sobrevida , Vacinas de DNA/administração & dosagemRESUMO
DNA vaccines have undergone important enhancements in their design, formulation, and delivery process. Past literature supports that DNA vaccines are not as immunogenic in nonhuman primates as live vector systems. The most potent recombinant vector system for induction of cellular immune responses in macaques and humans is adenovirus serotype 5 (Ad5), an important benchmark for new vaccine development. Here, we performed a head-to-head evaluation of the Merck Ad5 SIV vaccine and an optimized electroporation (EP) delivered SIV DNA vaccine in macaques. Animals receiving the Ad5 vaccine were immunized three times, whereas the DNA-vaccinated animals were immunized up to four times based on optimized protocols. We observed significant differences in the quantity of IFNgamma responses by enzyme-linked immunosorbent spot (ELISpot), greater proliferative capacity of CD8(+) T cells, and increased polyfunctionality of both CD4(+) and CD8(+) T cells in the DNA-vaccinated group. Importantly, Ad5 immunizations failed to boost following the first immunization, whereas DNA responses were continually boosted with all four immunizations demonstrating a major advantage of these improved DNA vaccines. These optimized DNA vaccines induce very different immune phenotypes than traditional Ad5 vaccines, suggesting that they could play an important role in vaccine research and development.
Assuntos
Adenoviridae/genética , Vetores Genéticos/genética , Vacinas contra a SAIDS/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Macaca mulatta , Plasmídeos/genética , Vacinas de DNA/imunologiaRESUMO
Although antiretroviral therapy suppresses HIV replication, it does not eliminate viral reservoirs or restore damaged lymphoid tissue, posing obstacles to HIV eradication. Using the SIV model of AIDS, we investigated the effect of mesenchymal stem/stromal cell (MSC) infusions on gut mucosal recovery, antiviral immunity, and viral suppression and determined associated molecular/metabolic signatures. MSC administration to SIV-infected macaques resulted in viral reduction and heightened virus-specific responses. Marked clearance of SIV-positive cells from gut mucosal effector sites was correlated with robust regeneration of germinal centers, restoration of follicular B cells and T follicular helper (Tfh) cells, and enhanced antigen presentation by viral trapping within the follicular DC network. Gut transcriptomic analyses showed increased antiviral response mediated by pathways of type I/II IFN signaling, viral restriction factors, innate immunity, and B cell proliferation and provided the molecular signature underlying enhanced host immunity. Metabolic analysis revealed strong correlations between B and Tfh cell activation, anti-SIV antibodies, and IL-7 expression with enriched retinol metabolism, which facilitates gut homing of antigen-activated lymphocytes. We identified potentially new MSC functions in modulating antiviral immunity for enhanced viral clearance predominantly through type I/II IFN signaling and B cell signature, providing a road map for multipronged HIV eradication strategies.
Assuntos
Centro Germinativo , Mucosa Intestinal/imunologia , Células-Tronco Mesenquimais , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Citocinas/metabolismo , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Imunidade Humoral/imunologia , Macaca mulatta , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologiaRESUMO
Chronic viral infection is characterized by the functional impairment of virus-specific T-cell responses. Recent evidence has suggested that the inhibitory receptor programmed death 1 (PD-1) is specifically upregulated on antigen-specific T cells during various chronic viral infections. Indeed, it has been reported that human immunodeficiency virus (HIV)-specific T cells express elevated levels of PD-1 and that this expression correlates with the viral load and inversely with CD4(+) T-cell counts. More importantly, antibody blockade of the PD-1/PD-L1 pathway was sufficient to both increase and stimulate virus-specific T-cell proliferation and cytokine production. However, the mechanisms that mediate HIV-induced PD-1 upregulation are not known. Here, we provide evidence that the HIV type 1 (HIV-1) accessory protein Nef can transcriptionally induce the expression of PD-1 during infection in vitro. Nef-induced PD-1 upregulation requires its proline-rich motif and the activation of the downstream kinase p38. Further, inhibition of Nef activity by p38 MAPK inhibitor effectively blocked PD-1 upregulation, suggesting that p38 MAPK activation is an important initiating event in Nef-mediated PD-1 expression in HIV-1-infected cells. These data demonstrate an important signaling event of Nef in HIV-1 pathogenesis.
Assuntos
Antígenos CD/genética , Proteínas Reguladoras de Apoptose/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Síndrome da Imunodeficiência Adquirida/metabolismo , Antígenos CD/biossíntese , Proteínas Reguladoras de Apoptose/biossíntese , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Receptor de Morte Celular Programada 1 , Regulação para CimaRESUMO
Immune responses to the factor IX (F.IX) transgene product are a concern in gene therapy for the X-linked bleeding disorder hemophilia B. The risk for such responses is determined by several factors, including the vector, target tissue, and others. Previously, we have demonstrated that hepatic gene transfer with adeno-associated viral (AAV) vectors can induce F.IX-specific immune tolerance. Muscle-derived F.IX expression, however, is limited by a local immune response. Here, skeletal muscle was investigated as a target for supplemental gene transfer. Given the low invasiveness of intramuscular injections, this route would be ideal for secondary gene transfer, thereby boosting levels of transgene expression. However, this is feasible only if immune tolerance established by compartmentalization of expression to the liver extends to other sites. Immune tolerance to human F.IX established by prior hepatic AAV-2 gene transfer was maintained after subsequent injection of AAV-1 or adenoviral vector into skeletal muscle, and tolerized mice failed to form antibodies or an interferon (IFN)-gamma(+) T cell response to human F.IX. A sustained increase in systemic transgene expression was obtained for AAV-1, whereas an increase after adenoviral gene transfer was transient. A CD8(+) T cell response specifically against adenovirus-transduced fibers was observed, suggesting that cytotoxic T cell responses against viral antigens were sufficient to eliminate expression in muscle. In summary, the data demonstrate that supplemental F.IX gene transfer to skeletal muscle does not break tolerance achieved by liver-derived expression. The approach is efficacious, if the vector for muscle gene transfer does not express immunogenic viral proteins.
Assuntos
Fator IX/genética , Terapia Genética , Vetores Genéticos/uso terapêutico , Hemofilia B/genética , Músculo Esquelético , Animais , Anticorpos/sangue , Dependovirus/genética , Ensaio de Imunoadsorção Enzimática , Fator IX/imunologia , Fator IX/uso terapêutico , Expressão Gênica , Vetores Genéticos/administração & dosagem , Hepatócitos/imunologia , Humanos , Tolerância Imunológica , Injeções Intramusculares , Interferon gama/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Músculo Esquelético/virologiaRESUMO
Antiviral innate host defenses against acute viral infections include suppression of host protein synthesis to restrict viral protein production. Less is known about mechanisms by which viral pathogens subvert host antiviral innate responses for establishing their replication and dissemination. We investigated early innate defense against human immunodeficiency virus (HIV) infection and viral evasion by utilizing human CD4+ T cell cultures in vitro and a simian immunodeficiency virus (SIV) model of AIDS in vivo Our data showed that early host innate defense against the viral infection involves GCN2-ATF4 signaling-mediated suppression of global protein synthesis, which is exploited by the virus for supporting its own replication during early viral infection and dissemination in the gut mucosa. Suppression of protein synthesis and induction of protein kinase GCN2-ATF4 signaling were detected in the gut during acute SIV infection. These changes diminished during chronic viral infection. HIV replication induced by serum deprivation in CD4+ T cells was linked to the induction of ATF4 that was recruited to the HIV long terminal repeat (LTR) to promote viral transcription. Experimental inhibition of GCN2-ATF4 signaling either by a specific inhibitor or by amino acid supplementation suppressed the induction of HIV expression. Enhancing ATF4 expression through selenium administration resulted in reactivation of latent HIV in vitro as well as ex vivo in the primary CD4+ T cells isolated from patients receiving suppressive antiretroviral therapy (ART). In summary, HIV/SIV exploits the early host antiviral response through GCN2-ATF4 signaling by utilizing ATF4 for activating the viral LTR transcription to establish initial viral replication and is a potential target for HIV prevention and therapy.IMPORTANCE Understanding how HIV overcomes host antiviral innate defense response in order to establish infection and dissemination is critical for developing prevention and treatment strategies. Most investigations focused on the viral pathogenic mechanisms leading to immune dysfunction following robust viral infection and dissemination. Less is known about mechanisms that enable HIV to establish its presence despite rapid onset of host antiviral innate response. Our novel findings provide insights into the viral strategy that hijacks the host innate response of the suppression of protein biosynthesis to restrict the virus production. The virus leverages transcription factor ATF4 expression during the GCN2-ATF4 signaling response and utilizes it to activate viral transcription through the LTR to support viral transcription and production in both HIV and SIV infections. This unique viral strategy is exploiting the innate response and is distinct from the mechanisms of immune dysfunction after the critical mass of viral loads is generated.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Linfócitos T CD4-Positivos/virologia , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Imunidade Inata , Proteínas Serina-Treonina Quinases/metabolismo , Replicação Viral , Fator 4 Ativador da Transcrição/genética , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Trato Gastrointestinal/virologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/patogenicidade , Humanos , Evasão da Resposta Imune , Macaca mulatta , Proteínas Serina-Treonina Quinases/genética , Selênio/farmacologia , Transdução de Sinais , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia , Carga Viral , Latência ViralRESUMO
Chronic HIV infection results in impairment of gut-associated lymphoid tissue leading to systemic immune activation. We previously showed that in early SIV-infected rhesus macaques intestinal dysfunction is initiated with the induction of the IL-1ß pathway in the small intestine and reversed by treatment with an exogenous Lactobacillus plantarum strain. Here, we provide evidence that the transcriptomes of L. plantarum and ileal microbiota are not altered shortly after SIV infection. L. plantarum adapts to the small intestine by expressing genes required for tolerating oxidative stress, modifying cell surface composition, and consumption of host glycans. The ileal microbiota of L. plantarum-containing healthy and SIV+ rhesus macaques also transcribed genes for host glycan metabolism as well as for cobalamin biosynthesis. Expression of these pathways by bacteria were proposed but not previously demonstrated in the mammalian small intestine.
Assuntos
Microbioma Gastrointestinal , Perfilação da Expressão Gênica , Íleo/patologia , Lactobacillus plantarum/genética , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Animais , Íleo/microbiologia , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/microbiologiaRESUMO
UNLABELLED: Nontyphoidal Salmonella enterica serovar Typhimurium is a frequent cause of bloodstream infections in children and HIV-infected adults in sub-Saharan Africa. Most isolates from African patients with bacteremia belong to a single sequence type, ST313, which is genetically distinct from gastroenteritis-associated ST19 strains, such as 14028s and SL1344. Some studies suggest that the rapid spread of ST313 across sub-Saharan Africa has been facilitated by anthroponotic (person-to-person) transmission, eliminating the need for Salmonella survival outside the host. While these studies have not ruled out zoonotic or other means of transmission, the anthroponotic hypothesis is supported by evidence of extensive genomic decay, a hallmark of host adaptation, in the sequenced ST313 strain D23580. We have identified and demonstrated 2 loss-of-function mutations in D23580, not present in the ST19 strain 14028s, that impair multicellular stress resistance associated with survival outside the host. These mutations result in inactivation of the KatE stationary-phase catalase that protects high-density bacterial communities from oxidative stress and the BcsG cellulose biosynthetic enzyme required for the RDAR (red, dry, and rough) colonial phenotype. However, we found that like 14028s, D23580 is able to elicit an acute inflammatory response and cause enteritis in mice and rhesus macaque monkeys. Collectively, these observations suggest that African S. Typhimurium ST313 strain D23580 is becoming adapted to an anthroponotic mode of transmission while retaining the ability to infect and cause enteritis in multiple host species. IMPORTANCE: The last 3 decades have witnessed an epidemic of invasive nontyphoidal Salmonella infections in sub-Saharan Africa. Genomic analysis and clinical observations suggest that the Salmonella strains responsible for these infections are evolving to become more typhoid-like with regard to patterns of transmission and virulence. This study shows that a prototypical African nontyphoidal Salmonella strain has lost traits required for environmental stress resistance, consistent with an adaptation to a human-to-human mode of transmission. However, in contrast to predictions, the strain remains capable of causing acute inflammation in the mammalian intestine. This suggests that the systemic clinical presentation of invasive nontyphoidal Salmonella infections in Africa reflects the immune status of infected hosts rather than intrinsic differences in the virulence of African Salmonella strains. Our study provides important new insights into the evolution of host adaptation in bacterial pathogens.
Assuntos
Adaptação Biológica , Infecções por Salmonella/microbiologia , Salmonella typhimurium/enzimologia , Salmonella typhimurium/fisiologia , Estresse Fisiológico , África Subsaariana/epidemiologia , Animais , Catalase/genética , Catalase/metabolismo , Modelos Animais de Doenças , Epidemias , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Humanos , Macaca mulatta , Camundongos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Infecções por Salmonella/epidemiologia , Salmonella typhimurium/genética , Salmonella typhimurium/isolamento & purificaçãoRESUMO
Chronic immune activation despite long-term therapy poses an obstacle to immune recovery in HIV infection. The role of antigen presenting cells (APCs) in chronic immune activation during HIV infection remains to be fully determined. APCs, the frontline of immune defense against pathogens, are capable of distinguishing between pathogens and non-pathogenic, commensal bacteria. We hypothesized that HIV infection induces dysfunction in APC immune recognition and response to some commensal bacteria and that this may promote chronic immune activation. Therefore we examined APC inflammatory cytokine responses to commensal lactobacilli. We found that APCs from HIV-infected patients produced an enhanced inflammatory response to Lactobacillus plantarum WCFS1 as compared to APCs from healthy, HIV-negative controls. Increased APC expression of TLR2 and CD36, signaling through p38-MAPK, and decreased expression of MAP kinase phosphatase-1 (MKP-1) in HIV infection was associated with this heightened immune response. Our findings suggest that chronic HIV infection enhances the responsiveness of APCs to commensal lactobacilli, a mechanism that may partly contribute to chronic immune activation.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Infecções por HIV/sangue , Infecções por HIV/imunologia , Lactobacillus/imunologia , Adulto , Idoso , Antígenos CD36/metabolismo , Doença Crônica , Estudos de Coortes , Células Dendríticas/metabolismo , Feminino , Infecções por HIV/enzimologia , Infecções por HIV/microbiologia , Humanos , Imunidade/imunologia , Inflamação/sangue , Inflamação/imunologia , Inflamação/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Fosforilação , Receptores Imunológicos/metabolismo , Receptor 2 Toll-Like/metabolismo , Adulto Jovem , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
One limitation in the development of an improved cellular response needed for an effective HIV-vaccine is the inability to induce robust effector T-cells capable of suppressing a heterologous challenge. To improve cellular immune responses, we examined the ability of an optimized DNA vaccine to boost the cellular immune responses induced by a highly immunogenic Ad5 prime. Five Chinese rhesus macaques received pVax encoding consensus (con) gag/pol/env intramuscularly (IM) with electroporation followed by the Merck Ad5 gag/pol/nef vaccine. A second group of five animals were vaccinated with Merck Ad5 gag/pol/nef followed by pVax gag/pol/env. One year following vaccination, Ad5-prime DNA-boosted monkeys and four unvaccinated controls received an intrarectal challenge with 1000 ID50 SIV(mac)251. The quality and magnitude of the T-cell response was analyzed by ELISpot and polyfunctional flow cytometry. We observed that an Ad5-prime DNA-boost resulted in significantly elevated SIV-specific T-cell responses even compared with animals receiving a DNA-prime Ad5-boost. Ad5 prime DNA boosted animals were capable of suppressing a pathogenic SIV(mac)251 challenge. Peak control correlated with the expansion of HLA-DR(+) CD8(+) T-cells two weeks post-infection. These data illustrate that high optimization of a DNA vaccine can drive of immune responses primed by a robust vector system. This previously unachievable feature of these newly optimized DNAs warrants future studies of this strategy that may circumvent issues of serology associated with viral vector prime-boost systems.
Assuntos
Imunização Secundária/métodos , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vacinas de DNA/imunologia , Animais , ELISPOT , Citometria de Fluxo , Macaca mulatta , Vacinas contra a SAIDS/administração & dosagem , Linfócitos T/imunologia , Vacinas de DNA/administração & dosagemRESUMO
PURPOSE OF REVIEW: We present current findings about two subsets of CD4+ T cells that play an important part in the initial host response to infection with the HIV type 1: those producing IL-17 (Th17 cells) and those with immunosuppressive function (CD25+FoxP3+ regulatory T cells or T-reg). The role of these cells in the control of viral infection and immune activation as well as in the prevention of immune deficiency in HIV-infected elite controllers will be examined. We will also discuss the use of the simian immunodeficiency virus (SIV)-infected macaque model of AIDS to study the interplay between these cells and lentiviral infection in vivo. RECENT FINDINGS: Study of Th17 cells in humans and nonhuman primates (NHPs) has shown that depletion of these cells is associated with the dissemination of microbial products from the infected gut, increased systemic immune activation, and disease progression. Most impressively, having a smaller Th17-cell compartment has been found to predict these outcomes. T-reg have been associated with the reduced antiviral T-cell responses but not with the suppression of generalized T cell activation. Both cell subsets influence innate immune responses and, in doing so, may shape the inflammatory milieu of the host at infection. SUMMARY: Interactions between Th17 cells, T-reg, and cells of the innate immune system influence the course of HIV and SIV infection from its earliest stages, even before the appearance of adaptive immunity. Such interactions may be pivotal for elite control over disease progression.
Assuntos
Infecções por HIV/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Linfócitos T CD4-Positivos , Sobreviventes de Longo Prazo ao HIV , Humanos , Imunidade Inata , MacacaRESUMO
Costimulatory molecules play a central role in the development of cellular immunity. Understanding how costimulatory pathways can be directed to positively influence the immune response may be critical for the generation of an effective HIV vaccine. Here, we evaluated the ability of intravenous administration of a blocking monoclonal antibody (mAb) directed against the negative costimulatory molecule CTLA-4, and an agonist mAb directed against the positive costimulatory molecule 4-1BB, either alone or in combination, to augment intramuscular SIV DNA immunizations. We then tested the ability these of these responses to impact a high-dose SIVmac251 challenge. Following immunization, the groups infused with the anti-4-1BB mAb exhibited enhanced IFN-γ responses compared to the DNA vaccine only group. Interestingly, although CTLA-4 blockade alone did not enhance IFN-γ responses it did increase the proliferative capacity of the CD4(+) and CD8(+) T cells. The combination of both mAbs enhanced the magnitude of the polyfunctional CD8(+) T cell response. Following challenge, the group that received both mAbs exhibited a significant, â¼2.0 log, decrease in plasma viral load compared to the naïve group the included complete suppression of viral load in some animals. Furthermore, the use of the CTLA-4 blocking antibody resulted in significantly higher viral loads during chronic infection compared to animals that received the 4-1BB mAb, likely due to the higher CD4(+) T cell proliferative responses which were driven by this adjuvant following immunization. These novel studies show that these adjuvants induce differential modulation of immune responses, which have dramatically different consequences for control of SIV replication, suggesting important implications for HIV vaccine development.
Assuntos
Macaca fascicularis/microbiologia , Macaca fascicularis/virologia , Vírus da Imunodeficiência Símia/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Vacinas de DNA/imunologia , Adjuvantes Imunológicos , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/metabolismo , Feminino , Citometria de Fluxo , Masculino , Vírus da Imunodeficiência Símia/patogenicidade , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidoresRESUMO
While HIV-1-specific cellular immunity is thought to be critical for the suppression of viral replication, the correlates of protection have not yet been determined. Rhesus macaques (RM) are an important animal model for the study and development of vaccines against HIV/AIDS. Our laboratory has helped to develop and study DNA-based vaccines in which recent technological advances, including genetic optimization and in vivo electroporation (EP), have helped to dramatically boost their immunogenicity. In this study, RMs were immunized with a DNA vaccine including individual plasmids encoding SIV gag, env, and pol alone, or in combination with a molecular adjuvant, plasmid DNA expressing the chemokine ligand 5 (RANTES), followed by EP. Along with standard immunological assays, flow-based activation analysis without ex vivo restimulation and high-throughput gene expression analysis was performed. Strong cellular immunity was induced by vaccination which was supported by all assays including PBMC microarray analysis that identified the up-regulation of 563 gene sequences including those involved in interferon signaling. Furthermore, 699 gene sequences were differentially regulated in these groups at peak viremia following SIVmac251 challenge. We observed that the RANTES-adjuvanted animals were significantly better at suppressing viral replication during chronic infection and exhibited a distinct pattern of gene expression which included immune cell-trafficking and cell cycle genes. Furthermore, a greater percentage of vaccine-induced central memory CD8+ T-cells capable of an activated phenotype were detected in these animals as measured by activation analysis. Thus, co-immunization with the RANTES molecular adjuvant followed by EP led to the generation of cellular immunity that was transcriptionally distinct and had a greater protective efficacy than its DNA alone counterpart. Furthermore, activation analysis and high-throughput gene expression data may provide better insight into mechanisms of viral control than may be observed using standard immunological assays.
Assuntos
Leucócitos Mononucleares/metabolismo , Vacinas contra a SAIDS/imunologia , Vacinas de DNA/imunologia , Animais , Citometria de Fluxo , Perfilação da Expressão Gênica , Interferon gama/metabolismo , Macaca mulatta , Análise de Sequência com Séries de OligonucleotídeosRESUMO
In an effort to improve DNA vaccine immune potency electroporation has emerged as a method of delivery of plasmids to target tissues. However, few studies have examined the use of this technology to deliver plasmid vaccines to the skin. Here we studied the effect of electroporation on DNA vaccine potency and gene delivery using skin as a target tissue in larger animal species. Using a pig model, we determined that high plasmid concentrations resulted in improved gene expression for plasmid GFP delivered by the intradermal/subcutaneous (ID/SQ) route. In a macaque model, we observed higher cellular and humoral responses to an HIV DNA vaccine, which included plasmid-encoded IL-12, with electroporation compared to ID/SQ injection alone. The induced responses were TH1 mediated. These results support that skin electroporation may have importance as an immunization approach in larger animal models.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Eletroporação/métodos , Anticorpos Anti-HIV/sangue , Infecções por HIV/prevenção & controle , Plasmídeos/administração & dosagem , Células Th1/imunologia , Vacinas de DNA/administração & dosagem , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Injeções Intradérmicas , Injeções Subcutâneas , Interferon gama/biossíntese , Macaca mulatta , Masculino , Plasmídeos/genética , Plasmídeos/imunologia , Suínos , Vacinas de DNA/genética , Vacinas de DNA/imunologiaRESUMO
Enhancing the expression of DNA vaccines requires that specific conditions of delivery are optimized. We describe experiments using adaptive constant-current electroporation (EP) in mice and pigs examining parameters such as target muscle, delay between plasmid delivery and onset of EP pulses and DNA vaccine formulation; our studies show that concentrated formulations result in better expression and immunogenicity. Furthermore, various conditions of EP that limit the amount of muscle damage were measured. The results of these studies will help to advance the success of DNA vaccines in animals into success in human clinical trials.
Assuntos
Fosfatase Alcalina , Anticorpos/sangue , Eletroporação , Proteínas de Fluorescência Verde/genética , Músculos/metabolismo , Pele/metabolismo , Vacinas de DNA , Fosfatase Alcalina/sangue , Fosfatase Alcalina/genética , Fosfatase Alcalina/imunologia , Animais , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/sangue , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/genética , Plasmídeos/fisiologia , Suínos , Vacinação , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vacinas de DNA/metabolismoRESUMO
BACKGROUND: Previously, we examined the effects of in vivo CTLA-4 blockade using a fully human monoclonal antibody as a part of a DNA vaccination regimen in cynomolgus macaques (Macaca fascicularis). We observed that while the antibody had little effect on the IFN-gamma ELISpot response, CTLA-4 blockade enhanced antigen-specific cellular proliferation in both CD4(+) and CD8(+)T-cell compartments. METHODS: We examine the specific effects of CTLA-4 blockade on memory T-cell compartments following the third immunization and 10 months following a fourth immunization, during the memory phase of the immune response. RESULTS: CLTA-4 blockade enhanced CD4(+) and CD8(+) central memory (CD28(hi), CD95(hi)) T-cell responses as well as a short-lived CD8(+) effector (CD28(lo), CD95(hi)) T-cell response. CONCLUSIONS: These data suggest differing effects of CTLA-4 blockade on CD4(+) and CD8(+) T cells with implications on the clinical use of anti-CTLA-4 antibodies for enhancement of vaccine strategies or treatment of human disease.