RESUMO
BACKGROUND: Long-acting subcutaneous lenacapavir (LEN), a first-in-class HIV capsid inhibitor approved by the US FDA for the treatment of multidrug-resistant HIV-1 with twice yearly dosing, is under investigation for HIV-1 pre-exposure prophylaxis (PrEP). We previously derived a simian-tropic HIV-1 clone (stHIV-A19) that encodes an HIV-1 capsid and replicates to high titres in pigtail macaques (PTM), resulting in a nonhuman primate model well-suited for evaluating LEN PrEP in vivo. METHODS: Lenacapavir potency against stHIV-A19 in PTM peripheral blood mononuclear cells in vitro was determined and subcutaneous LEN pharmacokinetics were evaluated in naïve PTMs in vivo. To evaluate the protective efficacy of LEN PrEP, naïve PTMs received either a single subcutaneous injection of LEN (25 mg/kg, N = 3) or vehicle (N = 4) 30 days before a high-dose intravenous challenge with stHIV-A19, or 7 daily subcutaneous injections of a 3-drug control PrEP regimen starting 3 days before stHIV-A19 challenge (N = 3). FINDINGS: In vitro, LEN showed potent antiviral activity against stHIV-A19, comparable to its potency against HIV-1. In vivo, subcutaneous LEN displayed sustained plasma drug exposures in PTMs. Following stHIV-A19 challenge, while all vehicle control animals became productively infected, all LEN and 3-drug control PrEP animals were protected from infection. INTERPRETATION: These findings highlight the utility of the stHIV-A19/PTM model and support the clinical development of long-acting LEN for PrEP in humans. FUNDING: Gilead Sciences as part of a Cooperative Research and Development Agreement between Gilead Sciences and Frederick National Lab; federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024/HHSN261201500003I; NIH grant R01AI078788.
Assuntos
Fármacos Anti-HIV , Soropositividade para HIV , HIV-1 , Estados Unidos , Animais , Humanos , Macaca , Leucócitos Mononucleares , Administração Intravenosa , Proteínas do CapsídeoRESUMO
We described a novel HIV autologous isolation method based in coculturing macrophages and CD4+T-cell-enriched fractions from peripheral blood collected from antiretroviral-treated (ART) HIV patients. This method allows the isolation of high viral titers of autologous viruses, over 1010HIV RNA copies/ml, and reduces the time required to produce necessary amounts for virus for use as antigens presented by monocyte-derived myeloid cells in HIV therapeutic vaccine approaches. By applying these high titer and autologous virus produced in the patient-derived cells, we intended to elicit a boost of the immunological system response in HIV therapeutic vaccines in clinical trials.
RESUMO
The effectiveness of virus-specific strategies, including administered HIV-specific mAbs, to target cells that persistently harbor latent, rebound-competent HIV genomes during combination antiretroviral therapy (cART) has been limited by inefficient induction of viral protein expression. To examine antibody-mediated viral reservoir targeting without a need for viral induction, we used an anti-CD4 mAb to deplete both infected and uninfected CD4+ T cells. Ten rhesus macaques infected with barcoded SIVmac239M received cART for 93 weeks starting 4 days after infection. During cART, 5 animals received 5 to 6 anti-CD4 antibody administrations and CD4+ T cell populations were then allowed 1 year on cART to recover. Despite profound CD4+ T cell depletion in blood and lymph nodes, time to viral rebound following cART cessation was not significantly delayed in anti-CD4-treated animals compared with controls. Viral reactivation rates, determined based on rebounding SIVmac239M clonotype proportions, also were not significantly different in CD4-depleted animals. Notably, antibody-mediated depletion was limited in rectal tissue and negligible in lymphoid follicles. These results suggest that, even if robust viral reactivation can be achieved, antibody-mediated viral reservoir depletion may be limited in key tissue sites.
Assuntos
Antirretrovirais/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/imunologia , Animais , Fármacos Anti-HIV/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Antígenos CD4/antagonistas & inibidores , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Depleção Linfocítica , Tecido Linfoide/imunologia , Tecido Linfoide/virologia , Macaca mulatta , Masculino , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/fisiologia , Carga Viral/efeitos dos fármacos , Carga Viral/imunologia , Ativação Viral/efeitos dos fármacos , Ativação Viral/imunologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/imunologiaRESUMO
There is currently a need for proxy measures of the HIV rebound competent reservoir (RCR) that can predict viral rebound after combined antiretroviral treatment (cART) interruption. In this study, macaques infected with a barcoded SIVmac239 virus received cART beginning between 4- and 27 days post-infection, leading to the establishment of different levels of viral dissemination and persistence. Later treatment initiation led to higher SIV DNA levels maintained during treatment, which was significantly associated with an increased frequency of SIV reactivation and production of progeny capable of causing rebound viremia following treatment interruption. However, a 100-fold increase in SIV DNA in PBMCs was associated with only a 2-fold increase in the frequency of reactivation. These data suggest that the RCR can be established soon after infection, and that a large fraction of persistent viral DNA that accumulates after this time makes relatively little contribution to viral rebound.
Assuntos
Antirretrovirais/uso terapêutico , DNA Viral/genética , Leucócitos Mononucleares/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Ativação Viral , Animais , DNA Viral/metabolismo , Leucócitos Mononucleares/patologia , Macaca mulatta , Masculino , Prognóstico , Recidiva , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/crescimento & desenvolvimento , Vírus da Imunodeficiência Símia/patogenicidade , Tempo para o Tratamento/estatística & dados numéricos , Resultado do Tratamento , Carga Viral , Replicação ViralRESUMO
Reduction/elimination of HIV-1 reservoirs that persist despite combination antiretroviral therapy (cART) will likely require induction of viral expression by residual infected cells and enhanced clearance of these cells. TLR7 agonists have potential to mediate these activities. We evaluated immunologic and virologic effects of repeated doses of the TLR7 agonist GS-9620 in SIV-infected rhesus macaques receiving cART, which was initiated at 13 days after infection and was continued for 75 weeks prior to GS-9620 administration. During cART, GS-9620 induced transient upregulation of IFN-stimulated genes in blood and tissues, increases in plasma cytokines, and changes in immune cell population activation and phenotypes but did not result in measurable increases in plasma viremia or viral RNA-to-viral DNA ratio in PBMCs or tissues nor decreases in viral DNA in PBMC or tissues. SIV-specific CD8+ T cell responses, negligible prior to GS-9620 treatment, were not measurably boosted by treatment; a second course of GS-9620 administration overlapping with later cART discontinuation was associated with increased CD8+ T cell responses during viral recrudescence. These results confirm and extend evidence for GS-9620-mediated enhancement of antiviral immune responses in SIV-infected macaques but suggest that GS-9620-mediated viral induction may depend critically on the timing of initiation and duration of cART and resulting characteristics of viral reservoirs.
Assuntos
Antirretrovirais , Pteridinas , Síndrome de Imunodeficiência Adquirida dos Símios , Receptor 7 Toll-Like/agonistas , Viremia , Animais , Antirretrovirais/administração & dosagem , Antirretrovirais/farmacologia , Antirretrovirais/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Citocinas/metabolismo , Quimioterapia Combinada , Macaca mulatta , Masculino , Pteridinas/administração & dosagem , Pteridinas/farmacologia , Pteridinas/uso terapêutico , RNA Viral/genética , RNA Viral/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Regulação para Cima/efeitos dos fármacos , Carga Viral/efeitos dos fármacos , Viremia/tratamento farmacológico , Viremia/imunologia , Viremia/virologiaRESUMO
AIDS virus infections are rarely controlled by cell-mediated immunity, in part due to viral immune evasion and immunodeficiency resulting from CD4+ T-cell infection. One likely aspect of this failure is that antiviral cellular immune responses are either absent or present at low levels during the initial establishment of infection. To test whether an extensive, timely, and effective response could reduce the establishment of infection from a high-dose inoculum, we adoptively transferred large numbers of T cells that were molecularly engineered with anti-simian immunodeficiency virus (anti-SIV) activity into rhesus macaques 3 days following an intrarectal SIV inoculation. To measure in vivo antiviral activity, we assessed the number of viruses transmitted using SIVmac239X, a molecularly tagged viral stock containing 10 genotypic variants, at a dose calculated to transmit 12 founder viruses. Single-genome sequencing of plasma virus revealed that the two animals receiving T cells expressing SIV-specific T-cell receptors (TCRs) had significantly fewer viral genotypes than the two control animals receiving non-SIV-specific T cells (means of 4.0 versus 7.5 transmitted viral genotypes; P = 0.044). Accounting for the likelihood of transmission of multiple viruses of a particular genotype, the calculated means of the total number of founder viruses transmitted were 4.5 and 14.5 in the experimental and control groups, respectively (P = 0.021). Thus, a large antiviral T-cell response timed with virus exposure can limit viral transmission. The presence of strong, preexisting T-cell responses, including those induced by vaccines, might help prevent the establishment of infection at the lower-exposure doses in humans that typically transmit only a single virus. IMPORTANCE: The establishment of AIDS virus infection in an individual is essentially a race between the spreading virus and host immune defenses. Cell-mediated immune responses induced by infection or vaccination are important contributors in limiting viral replication. However, in human immunodeficiency virus (HIV)/SIV infection, the virus usually wins the race, irreversibly crippling the immune system before an effective cellular immune response is developed and active. We found that providing an accelerated response by adoptively transferring large numbers of antiviral T cells shortly after a high-dose mucosal inoculation, while not preventing infection altogether, limited the number of individual viruses transmitted. Thus, the presence of strong, preexisting T-cell responses, including those induced by vaccines, might prevent infection in humans, where the virus exposure is considerably lower.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Macaca mulatta/imunologia , Macaca mulatta/virologia , Vírus da Imunodeficiência Símia/imunologia , Transferência Adotiva/métodos , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Humanos , Imunidade Celular/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vacinação/métodos , Replicação Viral/genéticaRESUMO
Replication-competent human immunodeficiency virus (HIV) persists in infected people despite suppressive combination antiretroviral therapy (cART), and it represents a major obstacle to HIV functional cure or eradication. We have developed a model of cART-mediated viral suppression in simian human immunodeficiency virus (SIV) mac239-infected Indian rhesus macaques and evaluated the impact of the histone deacetylase inhibitor (HDACi) romidepsin (RMD) on viremia in vivo. Eight macaques virologically suppressed to clinically relevant levels (<30 viral RNA copies/ml of plasma), using a three-class five-drug cART regimen, received multiple intravenous infusions of either RMD (n = 5) or saline (n = 3) starting 31 to 54 weeks after cART initiation. In vivo RMD treatment resulted in significant transient increases in acetylated histone levels in CD4(+) T cells. RMD-treated animals demonstrated plasma viral load measurements for each 2-week treatment cycle that were significantly higher than those in saline control-treated animals during periods of treatment, suggestive of RMD-induced viral reactivation. However, plasma virus rebound was indistinguishable between RMD-treated and control-treated animals for a subset of animals released from cART. These findings suggest that HDACi drugs, such as RMD, can reactivate residual virus in the presence of suppressive antiviral therapy and may be a valuable component of a comprehensive HIV functional cure/eradication strategy.
Assuntos
Depsipeptídeos/uso terapêutico , Inibidores de Histona Desacetilases/uso terapêutico , Macaca mulatta/virologia , RNA Viral/sangue , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Carga Viral/efeitos dos fármacos , Acetilação , Animais , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos/metabolismo , Depsipeptídeos/farmacocinética , Inibidores de Histona Desacetilases/farmacocinética , Histonas/metabolismo , Viremia/tratamento farmacológico , Ativação Viral/efeitos dos fármacosRESUMO
Mucosal damage to the gastrointestinal (GI) tract with resulting microbial translocation is hypothesized to significantly contribute to the heightened and persistent chronic inflammation and immune activation characteristic to HIV infection. Here we employ a non-human primate model of chemically induced colitis in SIV-uninfected rhesus macaques that we developed using dextran sulfate sodium (DSS), to directly test this hypothesis. DSS treatment results in GI barrier damage with associated microbial translocation, inflammation and immune activation. The progression and severity of colitis are longitudinally monitored by a magnetic resonance imaging approach. DSS treatment of SIV-infected African green monkeys, a natural host species for SIV that does not manifest GI tract damage or chronic immune activation during infection, results in colitis with elevated levels of plasma SIV RNA, sCD14, LPS, CRP and mucosal CD4+ T-cell loss. Together these results support the hypothesis that GI tract damage leading to local and systemic microbial translocation, and associated immune activation, are important determinants of AIDS pathogenesis.
Assuntos
Colite/induzido quimicamente , Colite/patologia , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Vírus da Imunodeficiência Símia , Animais , Sulfato de Dextrana/toxicidade , Feminino , Trato Gastrointestinal/patologia , Macaca mulatta , MasculinoRESUMO
Even with prolonged antiretroviral therapy (ART), many human immunodeficiency virus-infected individuals have <500 CD4(+) T cells/µL, and CD4(+) T cells in lymphoid tissues remain severely depleted, due in part to fibrosis of the paracortical T-cell zone (TZ) that impairs homeostatic mechanisms required for T-cell survival. We therefore used antifibrotic therapy in simian immunodeficiency virus-infected rhesus macaques to determine whether decreased TZ fibrosis would improve reconstitution of peripheral and lymphoid CD4(+) T cells. Treatment with the antifibrotic drug pirfenidone preserved TZ architecture and was associated with significantly larger populations of CD4(+) T cells in peripheral blood and lymphoid tissues. Combining pirfenidone with an ART regimen was associated with greater preservation of CD4(+) T cells than ART alone and was also associated with higher pirfenidone concentrations. These data support a potential role for antifibrotic drug treatment as adjunctive therapy with ART to improve immune reconstitution.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Terapia Antirretroviral de Alta Atividade/métodos , Linfócitos T CD4-Positivos/imunologia , Piridonas/uso terapêutico , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Contagem de Linfócito CD4 , Linfonodos/patologia , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Resultado do TratamentoRESUMO
Infection of macaques with chimeric viruses based on SIVMAC but expressing the HIV-1 envelope (Env) glycoproteins (SHIVs) remains the most powerful model for evaluating prevention and therapeutic strategies against AIDS. Unfortunately, only a few SHIVs are currently available. Furthermore, their generation has required extensive adaptation of the HIV-1 Env sequences in macaques so they may not accurately represent HIV-1 Env proteins circulating in humans, potentially limiting their translational utility. We developed a strategy for generating large numbers of SHIV constructs expressing Env proteins from newly transmitted HIV-1 strains. By inoculating macaques with cocktails of multiple SHIV variants, we selected SHIVs that can replicate and cause AIDS-like disease in immunologically intact rhesus macaques without requiring animal-to-animal passage. One of these SHIVs could be transmitted mucosally. We demonstrate the utility of the SHIVs generated by this method for evaluating neutralizing antibody administration as a protection against mucosal SHIV challenge.
Assuntos
Infecções por HIV/virologia , HIV-1/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Animais , Anticorpos Neutralizantes/imunologia , Modelos Animais de Doenças , Expressão Gênica , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/transmissão , HIV-1/metabolismo , Humanos , Macaca , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/transmissão , Vírus da Imunodeficiência Símia/metabolismo , Vírus da Imunodeficiência Símia/patogenicidade , Cultura de Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Nonhuman primate models are needed for evaluations of proposed strategies targeting residual virus that persists in HIV-1-infected individuals receiving suppressive combination antiretroviral therapy (cART). However, relevant nonhuman primate (NHP) models of cART-mediated suppression have proven challenging to develop. We used a novel three-class, six-drug cART regimen to achieve durable 4.0- to 5.5-log reductions in plasma viremia levels and declines in cell-associated viral RNA and DNA in blood and tissues of simian immunodeficiency virus SIVmac239-infected Indian-origin rhesus macaques, then evaluated the impact of treatment with the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA; Vorinostat) on the residual virus pool. Ex vivo SAHA treatment of CD4(+) T cells obtained from cART-suppressed animals increased histone acetylation and viral RNA levels in culture supernatants. cART-suppressed animals each received 84 total doses of oral SAHA. We observed SAHA dose-dependent increases in acetylated histones with evidence for sustained modulation as well as refractoriness following prolonged administration. In vivo virologic activity was demonstrated based on the ratio of viral RNA to viral DNA in peripheral blood mononuclear cells, a presumptive measure of viral transcription, which significantly increased in SAHA-treated animals. However, residual virus was readily detected at the end of treatment, suggesting that SAHA alone may be insufficient for viral eradication in the setting of suppressive cART. The effects observed were similar to emerging data for repeat-dose SAHA treatment of HIV-infected individuals on cART, demonstrating the feasibility, utility, and relevance of NHP models of cART-mediated suppression for in vivo assessments of AIDS virus functional cure/eradication approaches.
Assuntos
Antirretrovirais/uso terapêutico , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Modelos Animais de Doenças , Quimioterapia Combinada , Histonas/metabolismo , Macaca mulatta , RNA Viral/sangue , RNA Viral/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Carga Viral/efeitos dos fármacos , VorinostatRESUMO
UNLABELLED: HIV transmission efficiency is greatly increased when viruses are transmitted at virological synapses formed between infected and uninfected cells. We have previously shown that virological synapses formed between HIV-pulsed mature dendritic cells (DCs) and uninfected T cells contain interdigitated membrane surfaces, with T cell filopodia extending toward virions sequestered deep inside invaginations formed on the DC membrane. To explore membrane structural changes relevant to HIV transmission across other types of intercellular conjugates, we used a combination of light and focused ion beam scanning electron microscopy (FIB-SEM) to determine the three-dimensional (3D) architectures of contact regions between HIV-1-infected CD4(+) T cells and either uninfected human CD4(+) T cells or human fetal astrocytes. We present evidence that in each case, membrane extensions that originate from the uninfected cells, either as membrane sheets or filopodial bridges, are present and may be involved in HIV transmission from infected to uninfected cells. We show that individual virions are distributed along the length of astrocyte filopodia, suggesting that virus transfer to the astrocytes is mediated, at least in part, by processes originating from the astrocyte itself. Mechanisms that selectively disrupt the polarization and formation of such membrane extensions could thus represent a possible target for reducing viral spread. IMPORTANCE: Our findings lead to new insights into unique aspects of HIV transmission in the brain and at T cell-T cell synapses, which are thought to be a predominant mode of rapid HIV transmission early in the infection process.
Assuntos
Membrana Celular/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Sinapses/virologia , Astrócitos/ultraestrutura , Astrócitos/virologia , Linfócitos T CD4-Positivos/ultraestrutura , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Membrana Celular/ultraestrutura , Infecções por HIV/transmissão , Humanos , Imageamento Tridimensional , Microscopia Eletrônica de Transmissão , Sinapses/ultraestruturaRESUMO
Although the study of non-human primates has resulted in important advances for understanding HIV-specific immunity, a clear correlate of immune control over simian immunodeficiency virus (SIV) replication has not been found to date. In this study, CD8(+) T-cell cytotoxic capacity was examined to determine whether this function is a correlate of immune control in the rhesus macaque (RM) SIV infection model as has been suggested in chronic HIV infection. SIVmac251-infected human reverse transcriptase (hTERT)-transduced CD4(+) T-cell clone targets were co-incubated with autologous macaque effector cells to measure infected CD4(+) T-cell elimination (ICE). Twenty-three SIV-infected rhesus macaques with widely varying plasma viral RNA levels were evaluated in a blinded fashion. Nineteen of 23 subjects (83%) were correctly classified as long-term nonprogressor/elite controller (LTNP/EC), slow progressor, progressor or SIV-negative rhesus macaques based on measurements of ICE (weighted Kappa 0.75). LTNP/EC had higher median ICE than progressors (67.3% [22.0-91.7%] vs. 23.7% [0.0-58.0%], pâ=â0.002). In addition, significant correlations between ICE and viral load (râ=â-0.57, pâ=â0.01), and between granzyme B delivery and ICE (râ=â0.89, p<0.001) were observed. Furthermore, the CD8(+) T cells of LTNP/EC exhibited higher per-cell cytotoxic capacity than those of progressors (pâ=â0.004). These findings support that greater lytic granule loading of virus-specific CD8(+) T cells and efficient delivery of active granzyme B to SIV-infected targets are associated with superior control of SIV infection in rhesus macaques, consistent with observations of HIV infection in humans. Therefore, such measurements appear to represent a correlate of control of viral replication in chronic SIV infection and their role as predictors of immunologic control in the vaccine setting should be evaluated.
Assuntos
Linfócitos T CD8-Positivos/patologia , Imunidade , Macaca mulatta/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Linfócitos T Citotóxicos/patologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Progressão da Doença , Interações Hospedeiro-Patógeno , Macaca mulatta/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Replicação ViralRESUMO
BACKGROUND: Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections induce robust, generalized inflammatory responses that begin during acute infection and lead to pathological systemic immune activation, fibrotic damage of lymphoid tissues, and CD4⺠T-cell loss, pathogenic processes that contribute to disease progression. METHODS: To better understand the contribution of tumor necrosis factor (TNF), a key regulator of acute inflammation, to lentiviral pathogenesis, rhesus macaques newly infected with SIVmac239 were treated for 12 weeks in a pilot study with adalimumab (Humira), a human anti-TNF monoclonal antibody. RESULTS: Adalimumab did not affect plasma SIV RNA levels or measures of T-cell immune activation (CD38 or Ki67) in peripheral blood or lymph node T cells. However, compared with untreated rhesus macaques, adalimumab-treated rhesus macaques showed attenuated expression of proinflammatory genes, decreased infiltration of polymorphonuclear cells into the T-cell zone of lymphoid tissues, and weaker antiinflammatory regulatory responses to SIV infection (ie, fewer presumed alternatively activated [ie, CD163âº] macrophages, interleukin 10-producing cells, and transforming growth factor ß-producing cells), along with reduced lymphoid tissue fibrosis and better preservation of CD4⺠T cells. CONCLUSIONS: While HIV/SIV replication drives pathogenesis, these data emphasize the contribution of the inflammatory response to lentiviral infection to overall pathogenesis, and they suggest that early modulation of the inflammatory response may help attenuate disease progression.
Assuntos
Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Inflamação/metabolismo , Linfonodos/patologia , Ativação Linfocitária/efeitos dos fármacos , Retrovirus dos Símios , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Fator de Necrose Tumoral alfa/metabolismo , Adalimumab , Animais , Contagem de Linfócito CD4 , Movimento Celular/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/genética , Linfonodos/imunologia , Macaca mulatta , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Masculino , RNA Viral/metabolismo , Distribuição Aleatória , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Carga Viral/efeitos dos fármacosRESUMO
Although xenotropic murine leukemia virus-related virus (XMRV) has been previously linked to prostate cancer and myalgic encephalomyelitis/chronic fatigue syndrome, recent data indicate that results interpreted as evidence of human XMRV infection reflect laboratory contamination rather than authentic in vivo infection. Nevertheless, XMRV is a retrovirus of undefined pathogenic potential that is able to replicate in human cells. Here we describe a comprehensive analysis of two male pigtailed macaques (Macaca nemestrina) experimentally infected with XMRV. Following intravenous inoculation with >10(10) RNA copy equivalents of XMRV, viral replication was limited and transient, peaking at ≤2,200 viral RNA (vRNA) copies/ml plasma and becoming undetectable by 4 weeks postinfection, though viral DNA (vDNA) in peripheral blood mononuclear cells remained detectable through 119 days of follow-up. Similarly, vRNA was not detectable in lymph nodes by in situ hybridization despite detectable vDNA. Sequencing of cell-associated vDNA revealed extensive G-to-A hypermutation, suggestive of APOBEC-mediated viral restriction. Consistent with limited viral replication, we found transient upregulation of type I interferon responses that returned to baseline by 2 weeks postinfection, no detectable cellular immune responses, and limited or no spread to prostate tissue. Antibody responses, including neutralizing antibodies, however, were detectable by 2 weeks postinfection and maintained throughout the study. Both animals were healthy for the duration of follow-up. These findings indicate that XMRV replication and spread were limited in pigtailed macaques, predominantly by APOBEC-mediated hypermutation. Given that human APOBEC proteins restrict XMRV infection in vitro, human XMRV infection, if it occurred, would be expected to be characterized by similarly limited viral replication and spread.
Assuntos
Modelos Animais de Doenças , Macaca nemestrina , Infecções por Retroviridae/virologia , Replicação Viral , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/fisiologia , Animais , Anticorpos Antivirais/imunologia , Humanos , Masculino , Filogenia , Infecções por Retroviridae/imunologia , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/classificação , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/genéticaRESUMO
Studies using transformed human cell lines suggest that most SIV strains use CCR5 as co-receptor. Our analysis of primary rhesus macaque CD4(+) T-cell clones revealed marked differences in susceptibility to SIV(mac)239 infection. We investigated whether different levels of CCR5 expression account for clonal differences in SIV(mac)239 susceptibility. Macaque CD4(+) T-cells showed significant CCR5 downregulation 1-2days following CD3 mAb stimulation, which gradually recovered at resting state, 7-10days after activation. Exposure of clones to SIV(mac)239 during their CCR5(low) or CCR5(high) expression states revealed differences in SIV susceptibility independent of surface CCR5 levels. Furthermore, a CCR5 antagonist similarly reduced SIV(mac)239 infection of clones during their CCR5(low) or CCR5(high) expression states. Our data suggest a model where i) very low levels of CCR5 are sufficient for efficient SIV infection, ii) CCR5 levels above this threshold do not enhance infection, and iii) low level infection can occur in the absence of CCR5.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Regulação para Baixo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores CCR5/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/patogenicidade , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antagonistas dos Receptores CCR5 , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , DNA Viral/análise , Feminino , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Produtos do Gene gag/metabolismo , Humanos , Macaca mulatta , Masculino , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores CCR5/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologiaRESUMO
The efficiency of HIV infection is greatly enhanced when the virus is delivered at conjugates between CD4+ T cells and virus-bearing antigen-presenting cells such as macrophages or dendritic cells via specialized structures known as virological synapses. Using ion abrasion SEM, electron tomography, and superresolution light microscopy, we have analyzed the spatial architecture of cell-cell contacts and distribution of HIV virions at virological synapses formed between mature dendritic cells and T cells. We demonstrate the striking envelopment of T cells by sheet-like membrane extensions derived from mature dendritic cells, resulting in a shielded region for formation of virological synapses. Within the synapse, filopodial extensions emanating from CD4+ T cells make contact with HIV virions sequestered deep within a 3D network of surface-accessible compartments in the dendritic cell. Viruses are detected at the membrane surfaces of both dendritic cells and T cells, but virions are not released passively at the synapse; instead, virus transfer requires the engagement of T-cell CD4 receptors. The relative seclusion of T cells from the extracellular milieu, the burial of the site of HIV transfer, and the receptor-dependent initiation of virion transfer by T cells highlight unique aspects of cell-cell HIV transmission.
Assuntos
Células Dendríticas/virologia , HIV/fisiologia , Linfócitos T/virologia , Vírion/fisiologia , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/ultraestrutura , Células Apresentadoras de Antígenos/virologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/ultraestrutura , Linfócitos T CD4-Positivos/virologia , Comunicação Celular , Células Dendríticas/metabolismo , Células Dendríticas/ultraestrutura , Interações Hospedeiro-Patógeno , Humanos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Linfócitos T/metabolismo , Linfócitos T/ultraestruturaRESUMO
Plasma viremia decreases coincident with the appearance of virus-specific CD8(+) T cells during acute HIV or SIV infection. This finding, along with demonstrations of viral mutational escape from CD8(+) T cell responses and transient increase in plasma viremia after depletion of CD8(+) T cells in SIV-infected monkeys strongly suggest a role for CD8(+) T cells in controlling HIV/SIV. However, direct quantitative or qualitative correlates between CD8(+) T cell activity and virus control have not been established. To directly assess the impact of large numbers of virus-specific CD8(+) T cells present at time of SIV infection, we transferred in vitro expanded autologous central and effector memory-derived Gag CM9-, Nef YY9-, and Vif WY8-specific CD8(+) T cell clones to acutely infected rhesus macaques. The cells persisted in PBMCs between 4 and 9 d, but were not detected in gut-associated lymphoid tissue or lymph nodes. Interestingly, a high frequency of the infused cells localized to the lungs, where they persisted at high frequency for >6 wk. Although persisting cells in the lungs were Ag reactive, there was no measurable effect on virus load. Sequencing of virus from the animal receiving Nef YY9-specific CD8(+) T cells demonstrated an escape mutation in this epitope <3 wk postinfection, consistent with immune selection pressure by the infused cells. These studies establish methods for adoptive transfer of autologous SIV-specific CD8(+) T cells for evaluating immune control during acute infection and demonstrate that infused cells retain function and persist for at least 2 mo in specific tissues.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Quimiotaxia de Leucócito/imunologia , Memória Imunológica , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Transferência Adotiva , Animais , Sequência de Bases , Células Clonais , DNA Viral/genética , Mapeamento de Epitopos , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Citometria de Fluxo , Evasão da Resposta Imune/genética , Ativação Linfocitária/imunologia , Macaca mulatta , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Vírus da Imunodeficiência Símia/genética , Viremia/imunologiaRESUMO
The lack of a primate model that utilizes HIV-1 as the challenge virus is an impediment to AIDS research; existing models generally employ simian viruses that are divergent from HIV-1, reducing their usefulness in preclinical investigations. Based on an understanding of species-specific variation in primate TRIM5 and APOBEC3 antiretroviral genes, we constructed simian-tropic (st)HIV-1 strains that differ from HIV-1 only in the vif gene. We demonstrate that such minimally modified stHIV-1 strains are capable of high levels of replication in vitro in pig-tailed macaque (Macaca nemestrina) lymphocytes. Importantly, infection of pig-tailed macaques with stHIV-1 results in acute viremia, approaching the levels observed in HIV-1-infected humans, and an ensuing persistent infection for several months. stHIV-1 replication was controlled thereafter, at least in part, by CD8+ T cells. We demonstrate the potential utility of this HIV-1-based animal model in a chemoprophylaxis experiment, by showing that a commonly used HIV-1 therapeutic regimen can provide apparently sterilizing protection from infection following a rigorous high-dose stHIV-1 challenge.
Assuntos
Modelos Animais de Doenças , Infecções por HIV/virologia , HIV-1/genética , Macaca , Animais , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Quimioprevenção , Engenharia Genética , Viremia , Replicação ViralRESUMO
The rapid onset of massive, systemic viral replication during primary HIV or simian immunodeficiency virus (SIV) infection and the immune evasion capabilities of these viruses pose fundamental problems for vaccines that depend upon initial viral replication to stimulate effector T cell expansion and differentiation. We hypothesized that vaccines designed to maintain differentiated effector memory T cell (TEM cell) responses at viral entry sites might improve efficacy by impairing viral replication at its earliest stage, and we have therefore developed SIV protein-encoding vectors based on rhesus cytomegalovirus (RhCMV), the prototypical inducer of life-long TEM cell responses. RhCMV vectors expressing SIV Gag, Rev-Tat-Nef and Env persistently infected rhesus macaques, regardless of preexisting RhCMV immunity, and primed and maintained robust, SIV-specific CD4+ and CD8+ TEM cell responses (characterized by coordinate tumor necrosis factor, interferon-gamma and macrophage inflammatory protein-1beta expression, cytotoxic degranulation and accumulation at extralymphoid sites) in the absence of neutralizing antibodies. Compared to control rhesus macaques, these vaccinated rhesus macaques showed increased resistance to acquisition of progressive SIVmac239 infection upon repeated limiting-dose intrarectal challenge, including four macaques who controlled rectal mucosal infection without progressive systemic dissemination. These data suggest a new paradigm for AIDS vaccine development--vaccines capable of generating and maintaining HIV-specific TEM cells might decrease the incidence of HIV acquisition after sexual exposure.