RESUMO
Autologous transplantation and engraftment of HIV-resistant cells in sufficient numbers should recapitulate the functional cure of the Berlin Patient, with applicability to a greater number of infected individuals and with a superior safety profile. A robust preclinical model of suppressed HIV infection is critical in order to test such gene therapy-based cure strategies, both alone and in combination with other cure strategies. Here, we present a nonhuman primate (NHP) model of latent infection using simian/human immunodeficiency virus (SHIV) and combination antiretroviral therapy (cART) in pigtail macaques. We demonstrate that transplantation of CCR5 gene-edited hematopoietic stem/progenitor cells (HSPCs) persist in infected and suppressed animals, and that protected cells expand through virus-dependent positive selection. CCR5 gene-edited cells are readily detectable in tissues, namely those closely associated with viral reservoirs such as lymph nodes and gastrointestinal tract. Following autologous transplantation, tissue-associated SHIV DNA and RNA levels in suppressed animals are significantly reduced (p ≤ 0.05), relative to suppressed, untransplanted control animals. In contrast, the size of the peripheral reservoir, measured by QVOA, is variably impacted by transplantation. Our studies demonstrate that CCR5 gene editing is equally feasible in infected and uninfected animals, that edited cells persist, traffic to, and engraft in tissue reservoirs, and that this approach significantly reduces secondary lymphoid tissue viral reservoir size. Our robust NHP model of HIV gene therapy and viral persistence can be immediately applied to the investigation of combinatorial approaches that incorporate anti-HIV gene therapy, immune modulators, therapeutic vaccination, and latency reversing agents.
Assuntos
Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Receptores CCR5/genética , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Vírus da Imunodeficiência Símia/fisiologia , Carga Viral/fisiologia , Animais , Antirretrovirais/uso terapêutico , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Macaca nemestrina , Masculino , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Transplante Autólogo , Latência Viral , Replicação ViralRESUMO
The process by which drug-resistant HIV-1 arises and spreads spatially within an infected individual is poorly understood. Studies have found variable results relating how HIV-1 in the blood differs from virus sampled in tissues, offering conflicting findings about whether HIV-1 throughout the body is homogeneously distributed. However, most of these studies sample only two compartments and few have data from multiple time points. To directly measure how drug resistance spreads within a host and to assess how spatial structure impacts its emergence, we examined serial sequences from four macaques infected with RT-SHIVmne027, a simian immunodeficiency virus encoding HIV-1 reverse transcriptase (RT), and treated with RT inhibitors. Both viral DNA and RNA (vDNA and vRNA) were isolated from the blood (including plasma and peripheral blood mononuclear cells), lymph nodes, gut, and vagina at a median of four time points and RT was characterized via single-genome sequencing. The resulting sequences reveal a dynamic system in which vRNA rapidly acquires drug resistance concomitantly across compartments through multiple independent mutations. Fast migration results in the same viral genotypes present across compartments, but not so fast as to equilibrate their frequencies immediately. The blood and lymph nodes were found to be compartmentalized rarely, while both the blood and lymph node were more frequently different from mucosal tissues. This study suggests that even oft-sampled blood does not fully capture the viral dynamics in other parts of the body, especially the gut where vRNA turnover was faster than the plasma and vDNA retained fewer wild-type viruses than other sampled compartments. Our findings of transient compartmentalization across multiple tissues may help explain the varied results of previous compartmentalization studies in HIV-1.
Assuntos
Farmacorresistência Viral , Infecções por HIV/virologia , Transcriptase Reversa do HIV/genética , HIV-1/enzimologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Animais , DNA Viral/sangue , Feminino , Trato Gastrointestinal/virologia , HIV-1/genética , Humanos , Leucócitos Mononucleares , Linfonodos/virologia , Macaca mulatta , Especificidade de Órgãos , RNA Viral/sangue , Inibidores da Transcriptase Reversa/uso terapêutico , Vírus da Imunodeficiência Símia/genética , Vagina/virologia , ViremiaRESUMO
Gut-homing α4ß7high CD4+ T lymphocytes have been shown to be preferentially targeted by human immunodeficiency virus type 1 (HIV-1) and are implicated in HIV-1 pathogenesis. Previous studies demonstrated that HIV-1 envelope protein gp120 binds and signals through α4ß7 and that this likely contributes to the infection of α4ß7high T cells and promotes cell-to-cell virus transmission. Structures within the second variable loop (V2) of gp120, including the tripeptide motif LDV/I, are thought to mediate gp120-α4ß7 binding. However, lack of α4ß7 binding has been reported in gp120 proteins containing LDV/I, and the precise determinants of gp120-α4ß7 binding are not fully defined. In this work, we report the novel finding that fibronectins mediate indirect gp120-α4ß7 interactions. We show that Chinese hamster ovary (CHO) cells used to express recombinant gp120 produced fibronectins and other extracellular matrix proteins that copurified with gp120. CHO cell fibronectins were able to mediate the binding of a diverse panel of gp120 proteins to α4ß7 in an in vitro cell binding assay. The V2 loop was not required for fibronectin-mediated binding of gp120 to α4ß7, nor did V2-specific antibodies block this interaction. Removal of fibronectin through anion-exchange chromatography abrogated V2-independent gp120-α4ß7 binding. Additionally, we showed a recombinant human fibronectin fragment mediated gp120-α4ß7 interactions similarly to CHO cell fibronectin. These findings provide an explanation for the apparently contradictory observations regarding the gp120-α4ß7 interaction and offer new insights into the potential role of fibronectin and other extracellular matrix proteins in HIV-1 biology.IMPORTANCE Immune tissues within the gut are severely damaged by HIV-1, and this plays an important role in the development of AIDS. Integrin α4ß7 plays a major role in the trafficking of lymphocytes, including CD4+ T cells, into gut lymphoid tissues. Previous reports indicate that some HIV-1 gp120 envelope proteins bind to and signal through α4ß7, which may help explain the preferential infection of gut CD4+ T cells. In this study, we demonstrate that extracellular matrix proteins can mediate interactions between gp120 and α4ß7 This suggests that the extracellular matrix may be an important mediator of HIV-1 interaction with α4ß7-expressing cells. These findings provide new insight into the nature of HIV-1-α4ß7 interactions and how these interactions may represent targets for therapeutic intervention.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , Integrinas/metabolismo , Animais , Linfócitos T CD4-Positivos/virologia , Células CHO , Cricetinae , Cricetulus , Fibronectinas/metabolismo , Infecções por HIV/virologia , Humanos , Ligação ProteicaRESUMO
The envelope (Env) glycoprotein of HIV is expressed on the surface of productively infected cells and can be used as a target for cytotoxic immunoconjugates (ICs), in which cell-killing moieties, including toxins, drugs, or radionuclides, are chemically or genetically linked to monoclonal antibodies (MAbs) or other targeting ligands. Such ICs could be used to eliminate persistent reservoirs of HIV infection. We have found that MAbs which bind to the external loop of gp41, e.g., MAb 7B2, make highly effective ICs, particularly when used in combination with soluble CD4. We evaluated the toxicity, immunogenicity, and efficacy of the ICs targeted with 7B2 in mice and in simian-human immunodeficiency virus-infected macaques. In the macaques, we tested immunotoxins (ITs), consisting of protein toxins bound to the targeting agent. ITs were well tolerated and initially efficacious but were ultimately limited by their immunogenicity. In an effort to decrease immunogenicity, we tested different toxic moieties, including recombinant toxins, cytotoxic drugs, and tubulin inhibitors. ICs containing deglycosylated ricin A chain prepared from ricin toxin extracted from castor beans were the most effective in killing HIV-infected cells. Having identified immunogenicity as a major concern, we show that conjugation of IT to polyethylene glycol limits immunogenicity. These studies demonstrate that cytotoxic ICs can target virus-infected cells in vivo but also highlight potential problems to be addressed. IMPORTANCE: It is not yet possible to cure HIV infection. Even after years of fully effective antiviral therapy, a persistent reservoir of virus-infected cells remains. Here we propose that a targeted conjugate consisting of an anti-HIV antibody bound to a toxic moiety could function to kill the HIV-infected cells that constitute this reservoir. We tested this approach in HIV-infected cells grown in the lab and in animal infections. Our studies demonstrated that these immunoconjugates are effective both in vitro and in test animals. In particular, ITs constructed with the deglycosylated A chain prepared from native ricin were the most effective in killing cells, but their utility was blunted because they provoked immune reactions that interfered with the therapeutic effects. We then demonstrated that coating of the ITs with polyethylene glycol minimized the immunogenicity, as has been demonstrated with other protein therapies.
Assuntos
Fármacos Anti-HIV/farmacologia , Desenho de Fármacos , Proteína gp160 do Envelope de HIV/antagonistas & inibidores , Imunoconjugados/farmacologia , Animais , Fármacos Anti-HIV/química , Anticorpos Monoclonais/imunologia , Células Cultivadas , Modelos Animais de Doenças , Proteína gp160 do Envelope de HIV/imunologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Humanos , Imunoconjugados/química , Imunotoxinas/farmacologia , Macaca nemestrina , Camundongos , Polietilenoglicóis/químicaRESUMO
UNLABELLED: HIV-1 establishes persistent infection in part due to its ability to evade host immune responses. Occlusion by glycans contributes to masking conserved sites that are targets for some broadly neutralizing antibodies (bNAbs). Previous work has shown that removal of a highly conserved potential N-linked glycan (PNLG) site at amino acid residue 197 (N7) on the surface antigen gp120 of HIV-1 increases neutralization sensitivity of the mutant virus to CD4 binding site (CD4bs)-directed antibodies compared to its wild-type (WT) counterpart. However, it is not clear if the role of the N7 glycan is conserved among diverse HIV-1 isolates and if other glycans in the conserved regions of HIV-1 Env display similar functions. In this work, we examined the role of PNLGs in the conserved region of HIV-1 Env, particularly the role of the N7 glycan in a panel of HIV-1 strains representing different clades, tissue origins, coreceptor usages, and neutralization sensitivities. We demonstrate that the absence of the N7 glycan increases the sensitivity of diverse HIV-1 isolates to CD4bs- and V3 loop-directed antibodies, indicating that the N7 glycan plays a conserved role masking these conserved epitopes. However, the effect of the N7 glycan on virus sensitivity to neutralizing antibodies directed against the V2 loop epitope is isolate dependent. These findings indicate that the N7 glycan plays an important and conserved role modulating the structure, stability, or accessibility of bNAb epitopes in the CD4bs and coreceptor binding region, thus representing a potential target for the design of immunogens and therapeutics. IMPORTANCE: N-linked glycans on the HIV-1 envelope protein have been postulated to contribute to viral escape from host immune responses. However, the role of specific glycans in the conserved regions of HIV-1 Env in modulating epitope recognition by broadly neutralizing antibodies has not been well defined. We show here that a single N-linked glycan plays a unique and conserved role among conserved glycans on HIV-1 gp120 in modulating the exposure or the stability of the receptor and coreceptor binding site without affecting the integrity of the Env in mediating viral infection or the ability of the mutant gp120 to bind to CD4. The observation that the antigenicity of the receptor and coreceptor binding sites can be modulated by a single glycan indicates that select glycan modification offers a potential strategy for the design of HIV-1 vaccine candidates.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Polissacarídeos/análise , Antígenos de Superfície/química , Antígenos de Superfície/imunologia , Sítios de Ligação , Epitopos/química , Epitopos/imunologia , Glicosilação , Antígenos HIV/química , Antígenos HIV/imunologia , Proteína gp120 do Envelope de HIV/química , HIV-1/química , HumanosRESUMO
UNLABELLED: The envelope glycoprotein (Env) is the major target for HIV-1 broadly neutralizing antibodies (bNAbs). One of the mechanisms that HIV has evolved to escape the host's immune response is to mask conserved epitopes on Env with dense glycosylation. Previous studies have shown that the removal of a particular conserved glycan at N197 increases the neutralization sensitivity of the virus to antibodies targeting the CD4 binding site (CD4bs), making it a site of significant interest from the perspective of vaccine design. At present, the structural consequences that result from the removal of the N197 glycan have not been characterized. Using native-like SOSIP trimers, we examine the effects on antigenicity and local structural dynamics resulting from the removal of this glycan. A large increase in the binding of CD4bs and V3-targeting antibodies is observed for the N197Q mutant in trimeric Env, while no changes are observed with monomeric gp120. While the overall structure and thermostability are not altered, a subtle increase in the flexibility of the variable loops at the trimeric interface of adjacent protomers is evident in the N197Q mutant by hydrogen-deuterium exchange mass spectrometry. Structural modeling of the glycan chains suggests that the spatial occupancy of the N197 glycan leads to steric clashes with CD4bs antibodies in the Env trimer but not monomeric gp120. Our results indicate that the removal of the N197 glycan enhances the exposure of relevant bNAb epitopes on Env with a minimal impact on the overall trimeric structure. These findings present a simple modification for enhancing trimeric Env immunogens in vaccines. IMPORTANCE: The HIV-1 Env glycoprotein presents a dense patchwork of host cell-derived N-linked glycans. This so-called glycan shield is considered to be a major protective mechanism against immune recognition. While the positions of many N-linked glycans are isolate specific, some are highly conserved and are believed to play key functional roles. In this study, we examine the conserved, CD4 binding site-proximal N197 glycan and demonstrate that its removal both facilitates neutralizing antibody access to the CD4 binding site and modestly impacts the structural dynamics at the trimer crown without drastically altering global Env trimer stability. This indicates that surgical glycosylation site modification may be an effective way of sculpting epitope presentation in Env-based vaccines.
Assuntos
Sítios de Ligação/imunologia , Antígenos CD4/imunologia , Polissacarídeos/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Epitopos/imunologia , Glicoproteínas/imunologia , Glicosilação , Células HEK293 , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1 , HumanosRESUMO
UNLABELLED: Soluble forms of trimeric HIV-1 envelope glycoprotein (Env) have long been sought as immunogens and as reagents for analysis of Env structure and function. Isolation of trimers that mimic native Env, derived from diverse viruses, however, represents a major challenge. Thus far, the most promising native-like (NL) structures have been obtained by engineering trimer-stabilizing mutations, termed SOSIP, into truncated Env sequences. However, the abundances of NL trimeric conformers vary among Envs, necessitating purification by monoclonal antibodies (MAbs) like PGT145, which target specific epitopes. To surmount this inherent limitation, we developed an approach that uses lectin affinity chromatography, ion-exchange chromatography, hydrophobic-interaction chromatography (HIC), and size exclusion chromatography (SEC) to isolate NL trimers from nonnative Env species. We validated this method with SOSIP trimers from HIV-1 clades A and B. Analyses by SEC, blue native PAGE, SDS-PAGE, and dynamic light scattering indicated that the resulting material was homogeneous (>95% pure), fully cleaved, and of the appropriate molecular weight and size for SOSIP trimers. Negative-stain electron microscopy further demonstrated that our preparations were composed of NL trimeric structures. By hydrogen/deuterium-exchange mass spectrometry, these HIC-pure trimers exhibited structural organization consistent with NL trimers and inconsistent with profiles seen in nonnative Envs. Screened for antigenicity, some Envs, like BS208.b1 and KNH1144 T162A, did not present the glycan/quaternary structure-dependent epitope for PGT145 binding, suggesting that these SOSIPs would be challenging to isolate by existing MAb affinity methods. By selecting based on biochemical rather than antigenic properties, our method offers an epitope-independent alternative to MAbs for isolation of NL Env trimers. IMPORTANCE: The production and purification of diverse soluble Env trimers that maintain native-like (NL) structure present technical challenges that must be overcome in order to advance vaccine development and provide reagents for HIV research. Low levels of NL trimer expression amid heterogeneous Env conformers, even with the addition of stabilizing mutations, have presented a major challenge. In addition, it has been difficult to separate the NL trimers from these heterogeneous mixtures. While MAbs with specificity for quaternary NL trimer epitopes have provided one approach to purifying the desirable species, such methods are dependent on the Env displaying the proper epitope. In addition, MAb affinity chromatography can be expensive, the necessary MAb may be in limited supply, and large-scale purification may not be feasible. Our method based on biochemical separation techniques offers an epitope-independent approach to purification of NL trimers with general application to diverse Envs.
Assuntos
Antígenos Virais/imunologia , Epitopos/química , HIV-1/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Vacinas contra a AIDS/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Cromatografia de Afinidade/métodos , Cromatografia por Troca Iônica/métodos , Epitopos/imunologia , Genes env/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Humanos , Multimerização Proteica/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
UNLABELLED: Poxvirus prime-protein boost used in the RV144 trial remains the only immunization strategy shown to elicit a modest level of protection against HIV-1 acquisition in humans. Although neutralizing antibodies (NAb) were generated, they were against sensitive viruses, not the more resistant "tier 2" isolates that dominate circulating strains. Instead, risk reduction correlated with antibodies recognizing epitopes in the V1/V2 region of HIV-1 envelope glycoprotein (Env). Here, we examined whether tier 2 virus NAb and V1/V2-specific non-NAb could be elicited by a poxvirus prime-gp120 boost strategy in a rabbit model. We studied two clade B Envs that differ in multiple parameters, including tissue origin, neutralization sensitivity, and presence of the N197 (N7) glycan that was previously shown to modulate the exposure of conserved epitopes on Env. We demonstrate that immunized rabbits generated cross-reactive neutralizing activities against >50% of the tier 2 global HIV-1 isolates tested. Some of these activities were directed against the CD4 binding site (CD4bs). These rabbits also generated antibodies that recognized protein scaffolds bearing V1/V2 sequences from diverse HIV-1 isolates and mediated antibody-dependent cellular cytotoxicity. However, there are subtle differences in the specificities and the response rates of V1/V2-specific antibodies between animals immunized with different Envs, with or without the N7 glycan. These findings demonstrate that antibody responses that have been correlated with protection against HIV-1 acquisition in humans can be elicited in a preclinical model by a poxvirus prime-gp120 boost strategy and that improvements may be achievable by optimizing the nature of the priming and boosting immunogens. IMPORTANCE: The only vaccine approach shown to elicit any protective efficacy against HIV-1 acquisition is based on a poxvirus prime-protein boost regimen (RV144 Thai trial). Reduction of risk was associated with nonneutralizing antibodies targeting the V1/V2 loops of the envelope protein gp120. However, the modest efficacy (31.2%) achieved in this trial highlights the need to examine approaches and factors that may improve vaccine-induced responses, including cross-reactive neutralizing activities. We show here that rabbits immunized with a novel recombinant vaccinia virus prime-gp120 protein boost regimen generated antibodies that recognize protein scaffolds bearing V1/V2 sequences from diverse HIV-1 isolates and mediated antibody-dependent cellular cytotoxicity. Importantly, immunized rabbits also showed neutralizing activities against heterologous tier 2 HIV-1 isolates. These findings may inform the design of prime-boost immunization approaches and help improve the protective efficacy of candidate HIV-1 vaccines.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Imunização/métodos , Animais , Portadores de Fármacos , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Coelhos , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vaccinia virus/genéticaRESUMO
Antibody-antigen interactions are governed by recognition of specific residues and structural complementarity between the antigen epitope and antibody paratope. While X-ray crystallography has provided detailed insights into static conformations of antibody-antigen complexes, factors such as conformational flexibility and dynamics, which are not readily apparent in the structures, can also have an impact on the binding event. Here we investigate the contribution of dynamics in the HIV-1 gp120 glycoprotein to antibody recognition of conserved conformational epitopes, including the CD4- and coreceptor-binding sites, and an inner domain site that is targeted by ADCC-active antibodies. Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) was used to measure local structural dynamics across a panel of variable loop truncation mutants of HIV-1 gp120, including full-length gp120, ΔV3, ΔV1/V2, and extended core, which includes ΔV1/V2 and V3 loop truncations. CD4-bound full-length gp120 was also examined as a reference state. HDX-MS revealed a clear trend toward an increased level of order of the conserved subunit core resulting from loop truncation. Combined with biolayer interferometry and enzyme-linked immunosorbent assay measurements of antibody-antigen binding, we demonstrate that an increased level of ordering of the subunit core was associated with better recognition by an array of antibodies targeting complex conformational epitopes. These results provide detailed insight into the influence of structural dynamics on antibody-antigen interactions and suggest the importance of characterizing the structural stability of vaccine candidates to improve antibody recognition of complex epitopes.
Assuntos
Complexo Antígeno-Anticorpo/química , Reações Antígeno-Anticorpo , Epitopos/química , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Sequência de Aminoácidos , Sítios de Ligação de Anticorpos , Antígenos CD4/química , Antígenos CD4/metabolismo , Cristalografia por Raios X , Epitopos/metabolismo , Células HEK293 , HIV-1/imunologia , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação ProteicaRESUMO
Recent studies have demonstrated that genetically modified hematopoietic stem cells (HSCs) can reduce HIV viremia. We have developed an HIV/AIDS-patient model in Simian/human immunodeficiency virus (SHIV)-infected pigtailed macaques that are stably suppressed on antiretroviral therapy (ART: raltegravir, emtricitabine and tenofovir). Following SHIV infection and ART, animals undergo autologous HSC transplantation (HSCT) with lentivirally transduced cluster of differentiation (CD)34(+) cells expressing the mC46 anti-HIV fusion protein. We show that SHIV(+), ART-treated animals had very low gene marking levels after HSCT. Pretransduction CD34(+) cells contained detectable levels of all three ART drugs, likely contributing to the low gene transfer efficiency. Following HSCT recovery and the cessation of ART, plasma viremia rebounded, indicating that myeloablative total body irradiation cannot completely eliminate viral reservoirs after autologous HSCT. The kinetics of recovery following autologous HSCT in SHIV(+), ART-treated macaques paralleled those observed following transplantation of control animals. However, T-cell subset analyses demonstrated a high percentage of C-C chemokine receptor 5 (CCR5)-expressing CD4(+) T-cells after HSCT. These data suggest that an extended ART interruption time may be required for more efficient lentiviral transduction. To avoid complications associated with ART interruption in the context of high percentages of CD4(+)CCR5(+)T-cells after HSCT, the use of vector systems not impaired by the presence of residual ART may also be beneficial.
Assuntos
Terapia Antirretroviral de Alta Atividade , Terapia Genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Transdução Genética , Animais , Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Imunofenotipagem , Contagem de Linfócitos , Macaca nemestrina , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/efeitos da radiação , Subpopulações de Linfócitos T/virologia , Transgenes , Condicionamento Pré-Transplante , Carga ViralRESUMO
Despite continued progress in the development of novel antiretroviral therapies, it has become increasingly evident that drug-based treatments will not lead to a functional or sterilizing cure for HIV(+) patients. In 2009, an HIV(+) patient was effectively cured of HIV following allogeneic transplantation of hematopoietic stem cells (HSCs) from a CCR5(-/-) donor. The utility of this approach, however, is severely limited because of the difficulty in finding matched donors. Hence, we studied the potential of HIV-resistant stem cells in the autologous setting in a nonhuman primate AIDS model and incorporated a fusion inhibitor (mC46) as the means for developing infection-resistant cells. Pigtail macaques underwent identical transplants and Simian-Human Immunodeficiency Virus (SHIV) challenge procedures with the only variation between control and mC46 macaques being the inclusion of a fusion-inhibitor expression cassette. Following SHIV challenge, mC46 macaques, but not control macaques, showed a positive selection of gene-modified CD4(+) T cells in peripheral blood, gastrointestinal tract, and lymph nodes, accounting for >90% of the total CD4(+) T-cell population. mC46 macaques also maintained high frequencies of SHIV-specific, gene-modified CD4(+) T cells, an increase in nonmodified CD4(+) T cells, enhanced cytotoxic T lymphocyte function, and antibody responses. These data suggest that HSC protection may be a potential alternative to conventional antiretroviral therapy in patients with HIV/AIDS.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proteínas Recombinantes de Fusão/genética , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Antígenos Virais/imunologia , Linfócitos B/imunologia , Contagem de Linfócito CD4 , Relação CD4-CD8 , Linfócitos T CD4-Positivos/virologia , Terapia Baseada em Transplante de Células e Tecidos , Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Macaca nemestrina , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Carga Viral , Viremia/imunologia , Viremia/virologiaRESUMO
The HIV-1 envelope glycoprotein (Env) mediates viral entry into host cells and is the sole target of neutralizing antibodies. Much of the sequence diversity in the HIV-1 genome is concentrated within Env, particularly within its gp120 surface subunit. While dramatic functional diversity exists among HIV-1 Env isolates-observable even in the context of monomeric gp120 proteins as differences in antigenicity and immunogenicity-we have little understanding of the structural features that distinguish Env isolates and lead to isolate-specific functional differences, as crystal structures of truncated gp120 "core" proteins from diverse isolates reveal a high level of structural conservation. Because gp120 proteins are used as prospective vaccine immunogens, it is critical to understand the structural factors that influence their reactivity with antibodies. Here, we studied four full-length, glycosylated gp120 monomers from diverse HIV-1 isolates by using small-angle X-ray scattering (SAXS) to probe the overall subunit morphology and hydrogen/deuterium-exchange with mass spectrometry (HDX-MS) to characterize the local structural order of each gp120. We observed that while the overall subunit architecture was similar among isolates by SAXS, dramatic isolate-specific differences in the conformational stability of gp120 were evident by HDX-MS. These differences persisted even with the CD4 receptor bound. Furthermore, surface plasmon resonance (SPR) and enzyme-linked immunosorbance assays (ELISAs) showed that disorder was associated with poorer recognition by antibodies targeting conserved conformational epitopes. These data provide additional insight into the structural determinants of gp120 antigenicity and suggest that conformational dynamics should be considered in the selection and design of optimized Env immunogens.
Assuntos
Antígenos Virais/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Vacinas contra a AIDS , Anticorpos Neutralizantes , Antígenos Virais/metabolismo , Antígenos CD4/metabolismo , Medição da Troca de Deutério , Desenho de Fármacos , Glicosilação , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Conformação Proteica , Espalhamento a Baixo Ângulo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ressonância de Plasmônio de SuperfícieRESUMO
BACKGROUND: Pathogenic infection with human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) is characterized by a loss of CD4+ T cells and chronic lymphocyte activation even during suppressive antiretroviral therapy (ART). METHODS: Using NanoString, expression of >100 molecules associated with inflammation or immune activation was evaluated in mesenteric lymph nodes and ileum of macaques infected with a pathogenic SIV/HIV virus, RT-SHIV, during viremia or during suppressive ART and compared to uninfected controls. RESULTS: Of the 105 RNA species quantified in the tissues, expression of 33 genes was altered significantly in one or both tissues during viremia but returned to normal levels during ART. However, 29 additional genes were altered in expression levels in the tissues of both viremic and ART-suppressed macaques. CONCLUSIONS: Identification of the mechanisms of chronic inflammation in specific cells and in different tissues may help determine whether early ART initiation or novel therapies can prevent it.
Assuntos
Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Síndrome da Imunodeficiência Adquirida/imunologia , Regulação da Expressão Gênica , Íleo/imunologia , Linfonodos/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome da Imunodeficiência Adquirida/virologia , Animais , Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , HIV-1/imunologia , Ativação Linfocitária , Macaca nemestrina , Mesentério , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologia , Viremia/imunologiaRESUMO
Research involving nonhuman primates (NHPs) has played a vital role in many of the medical and scientific advances of the past century. NHPs are used because of their similarity to humans in physiology, neuroanatomy, reproduction, development, cognition, and social complexity-yet it is these very similarities that make the use of NHPs in biomedical research a considered decision. As primate researchers, we feel an obligation and responsibility to present the facts concerning why primates are used in various areas of biomedical research. Recent decisions in the United States, including the phasing out of chimpanzees in research by the National Institutes of Health and the pending closure of the New England Primate Research Center, illustrate to us the critical importance of conveying why continued research with primates is needed. Here, we review key areas in biomedicine where primate models have been, and continue to be, essential for advancing fundamental knowledge in biomedical and biological research.
Assuntos
Modelos Animais de Doenças , Primatas , Experimentação Animal/ética , Animais , Humanos , Estados UnidosRESUMO
The gp120 subunit of the HIV Env glycoprotein is responsible for receptor interactions leading to viral entry and is a primary target for neutralizing antibodies. Most structural studies have focused on the heavily truncated, deglycosylated gp120 core, leaving fundamental aspects of the glycoprotein that are responsible for immune evasion and receptor-induced activation unresolved. Here we investigate full-length, glycosylated HIV gp120 in unliganded and CD4-bound forms by using small-angle X-ray scattering to visualize global structural reorganization and hydrogen/deuterium exchange to track changes in local conformational dynamics. The studies revealed unliganded full-length gp120 to be considerably more dynamic, particularly at the CD4 binding site, than suggested by previous studies of the subunit core alone. The large V1/V2 loops, previously unmapped, are positioned to mask the coreceptor binding site in an orientation that recapitulates that observed in the Env trimer. CD4 binding shifts V1/V2 to unmask the coreceptor binding site and triggers profound dynamic changes in gp120 spanning from the binding site to the gp41-interactive face of gp120. These findings provide further insights on the structural basis of Env antigenicity and immunogenicity and of allosteric effects upon receptor binding.
Assuntos
Antígenos CD4/química , Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Espalhamento a Baixo ÂnguloRESUMO
Human immunodeficiency virus type 1 (HIV-1) infection is characterized by persistent viral replication in the context of CD4(+) T cell depletion and elevated immune activation associated with disease progression. In contrast, simian immunodeficiency virus (SIV) infection of African-origin sooty mangabeys (SM) generally does not result in simian AIDS despite high viral loads and therefore affords a unique model in which to study the immunologic contributions to a nonpathogenic lentiviral disease outcome. A key feature of these natural SIV infections is the maintenance of low levels of immune activation during chronic infection. Our goal was to delineate the contribution of monocytes to maintaining low levels of immune activation in SIV-infected SM. Utilizing an ex vivo whole-blood assay, proinflammatory cytokine production was quantified in monocytes in response to multiple Toll-like receptor (TLR) ligands and a specific, significant reduction in the tumor necrosis factor alpha (TNF-α) response to lipopolysaccharide (LPS) was observed in SIV-infected SM. In contrast, monocytes from hosts of pathogenic infections (HIV-infected humans and SIV-infected Asian macaques) maintained a robust TNF-α response. In SIV-infected SM, monocyte TNF-α responses to low levels of LPS could be augmented by the presence of plasma from uninfected control animals. The impact of LPS-induced TNF-α production on immune activation was demonstrated in vitro, as TNF-α blocking antibodies inhibited downstream CD8(+) T cell activation in a dose-dependent manner. These data demonstrate an association between nonpathogenic SIV infection of SM and a reduced monocyte TNF-α response to LPS, and they identify a role for monocytes in contributing to the suppressed chronic immune activation observed in these natural hosts.
Assuntos
Cercocebus atys/imunologia , Ativação Linfocitária , Monócitos/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/patogenicidade , Fator de Necrose Tumoral alfa/biossíntese , Animais , Linfócitos T CD8-Positivos/imunologia , Cercocebus atys/virologia , HIV-1/imunologia , Humanos , Interleucina-10/genética , Lipopolissacarídeos/imunologia , Monócitos/metabolismo , Fagocitose , Fito-Hemaglutininas/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Vírus da Imunodeficiência Símia/metabolismo , Ácidos Teicoicos/imunologia , Receptor 4 Toll-Like/genética , Fator de Necrose Tumoral alfa/genética , Carga ViralRESUMO
Vaccinia virus (VV) has been used to express a variety of heterologous proteins, including HIV envelope (Env) glycoproteins. The Env protein is synthesized as a precursor molecule, gp160, which is cleaved into the surface antigen gp120 and the transmembrane protein gp41. Even though production of gp160 by the VV expression system has been described, its use for gp120 production is not well documented. Here we report a new procedure for the purification of gp120 from serum-containing culture supernatant of VV-infected cells. The gp120 protein was enriched to a purity better than 60% on a snowdrop (Galanthus nivalis) lectin affinity column in the presence of 0.25% zwitterionic detergent Empigen BB. After additional DEAE anion exchange and Superdex size exclusion chromatography steps, the gp120 monomer was purified free of contamination as determined by SDS-PAGE. The retention of structural integrity was confirmed by determining the affinity constant of purified gp120s to soluble CD4 and a monoclonal antibody IgG1b12, using surface plasmon resonance analysis. The purification procedure is robust and reproducible, and may find general use for glycoprotein purifications from sources where the presence of serum is desirable.
Assuntos
Proteína gp120 do Envelope de HIV/isolamento & purificação , Vaccinia virus/metabolismo , Cromatografia em Gel , Detergentes/química , Eletroforese em Gel de Poliacrilamida , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp160 do Envelope de HIV/metabolismo , Compostos Orgânicos/química , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Nonhuman primates (NHPs) are an important model organism for studies of HIV pathogenesis and preclinical evaluation of anti-HIV therapies. The successful translation of NHP-derived data to clinically relevant anti-HIV studies will require better understanding of the viral strains and NHP species used and their responses to existing antiretroviral therapies (ART). METHODS: Five pigtailed macaques (Macaca nemestrina) were productively infected with the SIV/HIV chimeric virus SHIV-1157 ipd3N4 following intravenous challenge. After 8 or 27 weeks, ART (PMPA, FTC, raltegravir) was initiated. Viral load, T-cell counts, and production of SHIV-specific antibodies were monitored throughout the course of infection and ART. RESULTS: ART led to a rapid and sustained decrease in plasma viral load. Suppression of plasma viremia by ART was independent of the timing of initiation during chronic infection. CONCLUSIONS: We present a new NHP model of HIV infection on antiretroviral therapy, which should prove applicable to multiple clinically relevant anti-HIV approaches.
Assuntos
Antirretrovirais/administração & dosagem , Modelos Animais de Doenças , Infecções por Lentivirus/tratamento farmacológico , Lentivirus de Primatas/efeitos dos fármacos , Macaca nemestrina , Animais , Doença Crônica/tratamento farmacológico , Quimioterapia Combinada , Carga Viral , Viremia/tratamento farmacológico , Replicação Viral/efeitos dos fármacosRESUMO
The retroviral restriction factor TRIMCyp, derived from the TRIM5 gene, blocks replication at a postentry step. TRIMCyp has so far been found in four species of Asian macaques, Macaca fascicularis, M. mulatta, M. nemestrina, and M. leonina. M. fascicularis is commonly used as a model for AIDS research, but TRIMCyp has not been analyzed in detail in this species. We analyzed the prevalence of TRIMCyp in samples from Indonesia, Indochina, the Philippines, and Mauritius. We found that TRIMCyp is present at a higher frequency in Indonesian than in Indochinese M. fascicularis macaques and is also present in samples from the Philippines. TRIMCyp is absent in Mauritian M. fascicularis macaques. We then analyzed the restriction specificity of TRIMCyp derived from three animals of Indonesian origin. One allele, like the prototypic TRIMCyp alleles described for M. mulatta and M. nemestrina, restricts human immunodeficiency virus type 2 (HIV-2) and feline immunodeficiency virus (FIV) but not HIV-1. The others restrict HIV-1 and FIV but not HIV-2. Mutagenesis studies confirmed that polymorphisms at amino acid residues 369 and 446 in TRIMCyp (or residues 66 and 143 in the cyclophilin A [CypA] domain) confer restriction specificity. Additionally, we identified a polymorphism in the coiled-coil domain that appears to affect TRIMCyp expression or stability. Taken together, these data show that M. fascicularis has the most diverse array of TRIM5 restriction factors described for any primate species to date. These findings are relevant to our understanding of the evolution of retroviral restriction factors and the use of M. fascicularis models in AIDS research.
Assuntos
Antivirais/metabolismo , Lentivirus de Primatas/imunologia , Macaca fascicularis/genética , Macaca fascicularis/imunologia , Proteínas/genética , Proteínas/metabolismo , Alelos , Animais , China , Indonésia , Maurício , Dados de Sequência Molecular , Filipinas , Polimorfismo Genético , Prevalência , Análise de Sequência de DNARESUMO
The impact of antiretroviral therapy (ART) on the genetics of simian immunodeficiency virus (SIV) or human immunodeficiency virus (HIV) populations has been incompletely characterized. We analyzed SIV genetic variation before, during, and after ART in a macaque model. Six pigtail macaques were infected with an SIV/HIV chimeric virus, RT-SHIV(mne), in which SIV reverse transcriptase (RT) was replaced by HIV-1 RT. Three animals received a short course of efavirenz (EFV) monotherapy before combination ART was started. All macaques received 20 weeks of tenofovir, emtricitabine, and EFV. Plasma virus populations were analyzed by single-genome sequencing. Population diversity was measured by average pairwise difference, and changes in viral genetics were assessed by phylogenetic and panmixia analyses. After 20 weeks of ART, viral diversity was not different from pretherapy viral diversity despite more than 10,000-fold declines in viremia, indicating that, within this range, there is no relationship between diversity and plasma viremia. In two animals with consistent SIV RNA suppression to <15 copies/ml during ART, there was no evidence of viral evolution. In contrast, in the four macaques with viremias >15 copies/ml during therapy, there was divergence between pre- and during-ART virus populations. Drug resistance mutations emerged in two of these four animals, resulting in virologic failure in the animal with the highest level of pretherapy viremia. Taken together, these findings indicate that viral diversity does not decrease with suppressive ART, that ongoing replication occurs with viremias >15 copies/ml, and that in this macaque model of ART drug resistance likely emerges as a result of incomplete suppression and preexisting drug resistance mutations.