RESUMO
Using coevolution network interference based on comparison of two phylogenetically distantly related isolates, one from the main group M and the other from the minor group O of HIV-1, we identify, in the C-terminal domain (CTD) of integrase, a new functional motif constituted by four noncontiguous amino acids (N222K240N254K273). Mutating the lysines abolishes integration through decreased 3' processing and inefficient nuclear import of reverse-transcribed genomes. Solution of the crystal structures of wild-type (wt) and mutated CTDs shows that the motif generates a positive surface potential that is important for integration. The number of charges in the motif appears more crucial than their position within the motif. Indeed, the positions of the K's could be permutated or additional K's could be inserted in the motif, generally without affecting integration per se Despite this potential genetic flexibility, the NKNK arrangement is strictly conserved in natural sequences, indicative of an effective purifying selection exerted at steps other than integration. Accordingly, reverse transcription was reduced even in the mutants that retained wt integration levels, indicating that specifically the wt sequence is optimal for carrying out the multiple functions that integrase exerts. We propose that the existence of several amino acid arrangements within the motif, with comparable efficiencies of integration per se, might have constituted an asset for the acquisition of additional functions during viral evolution.IMPORTANCE Intensive studies of HIV-1 have revealed its extraordinary ability to adapt to environmental and immunological challenges, an ability that is also at the basis of antiviral treatment escape. Here, by deconvoluting the different roles of the viral integrase in the various steps of the infectious cycle, we report how the existence of alternative equally efficient structural arrangements for carrying out one function opens up the possibility of adapting to the optimization of further functionalities exerted by the same protein. Such a property provides an asset to increase the efficiency of the infectious process. On the other hand, though, the identification of this new motif provides a potential target for interfering simultaneously with multiple functions of the protein.
Assuntos
Integrase de HIV/química , HIV-1/química , Motivos de Aminoácidos , Linhagem Celular Tumoral , Células HEK293 , Integrase de HIV/genética , HIV-1/genética , Humanos , Domínios ProteicosRESUMO
Fcɣ receptors (FcɣRs) are key immune regulatory receptors that connect antibody-mediated immune responses to cellular effector functions. They are involved in the control of various immune functions including responses to infections. Genetic polymorphisms of FcɣRs coding genes (FCGR) have been associated with the regulation of HIV infection and progression. In this study, we analyzed the potential impact of five candidate FcɣR SNPs on viral control by genotyping 251 HIV controllers and 250 progressors. The rs10800309 AA genotype of the FcɣRIIa coding gene FCGR2A was found to be significantly associated with HIV control and this association was independent of HLA-B57 and HLA-B27 (OR, 2.84; 95% CI, 1.20-6.89; Pcor = 0.033). We further confirmed the functional role of this polymorphism by showing an association of this same AA genotype with an increased in vitro FcɣRII expression on myeloid cells including dendritic cells (P = 0.0032). Together, these results suggest that the AA genotype of rs10800309 confers an improved immune response through FcɣRII upregulation and that this polymorphism may serve as an additional predictive marker of HIV control.
Assuntos
Infecções por HIV/genética , Infecções por HIV/imunologia , Antígenos HLA-B/imunologia , Antígeno HLA-B27/imunologia , Receptores de IgG/genética , Receptores de IgG/metabolismo , Linfócitos T CD8-Positivos/imunologia , Estudos de Coortes , Progressão da Doença , Feminino , Estudos de Associação Genética , HIV-1/imunologia , HIV-1/fisiologia , Interações entre Hospedeiro e Microrganismos , Humanos , Masculino , Pessoa de Meia-Idade , Células Mieloides , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , Carga Viral , Replicação ViralRESUMO
Plasmacytoid dendritic cells (pDC) poorly replicate human immunodeficiency virus type 1 (HIV-1) but efficiently transfer HIV-1 to adjacent CD4 T lymphocytes. We found that coculture with T lymphocytes downregulates SAMHD1 expression, enhances HIV-1 replication, and increases pDC maturation and alpha interferon (IFN-α) secretion. HIV-1 transfer to T lymphocytes is inhibited by broadly neutralizing antibody VRC01 with efficiency similar to that of cell-free infection of T lymphocytes. Interestingly, prevention of HIV-1 transmission by VRC01 retains IFN-α secretion. These results emphasize the multiple functions of VRC01 in protection against HIV-1 acquisition.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Linfócitos T CD4-Positivos/virologia , Células Dendríticas/virologia , Anticorpos Anti-HIV/farmacologia , Infecções por HIV/virologia , HIV-1/fisiologia , Anticorpos Amplamente Neutralizantes , Células Cultivadas , Infecções por HIV/prevenção & controle , HIV-1/efeitos dos fármacos , HumanosRESUMO
UNLABELLED: Human immunodeficiency virus type 1 (HIV-1) replication in dendritic cells (DCs) is restricted by SAMHD1. This factor is counteracted by the viral protein Vpx; Vpx is found in HIV-2 and simian immunodeficiency virus (SIV) from sooty mangabeys (SIVsm) or from macaques (SIVmac) but is absent from HIV-1. We previously observed that HIV-1 replication in immature DCs is stimulated by cocultivation with primary T and B lymphocytes, suggesting that HIV-1 restriction in DCs may be overcome under coculture conditions. Here, we aimed to decipher the mechanism of SAMHD1-mediated restriction in DC-lymphocyte coculture. We found that coculture with lymphocytes downregulated SAMHD1 expression and was associated with increased HIV-1 replication in DCs. Moreover, in infected DC-T lymphocyte cocultures, DCs acquired maturation status and secreted type 1 interferon (alpha interferon [IFN-α]). The blockade of DC-lymphocyte cross talk by anti-ICAM-1 antibody markedly inhibited the stimulation of HIV-1 replication and prevented the downregulation of SAMHD1 expression in cocultured DCs. These results demonstrate that, in contrast to purified DCs, cross talk with lymphocytes downregulates SAMHD1 expression in DCs, triggering HIV-1 replication and an antiviral immune response. Therefore, HIV-1 replication and immune sensing by DCs should be investigated in more physiologically relevant models of DC/lymphocyte coculture. IMPORTANCE: SAMHD1 restricts HIV-1 replication in dendritic cells (DCs). Here, we demonstrate that, in a coculture model of DCs and lymphocytes mimicking early mucosal HIV-1 infection, stimulation of HIV-1 replication in DCs is associated with downregulation of SAMHD1 expression and activation of innate immune sensing by DCs. We propose that DC-lymphocyte cross talk occurring in vivo modulates host restriction factor SAMHD1, promoting HIV-1 replication in cellular reservoirs and stimulating immune sensing.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/virologia , HIV-1/fisiologia , Linfócitos/imunologia , Proteínas Monoméricas de Ligação ao GTP/biossíntese , Replicação Viral , Técnicas de Cocultura , Regulação para Baixo , Humanos , Proteína 1 com Domínio SAM e Domínio HD , Cultura de VírusRESUMO
Dendritic cells (DCs) support only low levels of HIV-1 replication, but have been shown to transfer infectious viral particles highly efficiently to neighboring permissive CD4 T lymphocytes. This mode of cell-to-cell HIV-1 spread may be a predominant mode of infection and dissemination. In the present study, we analyzed the kinetics of fusion, replication, and the ability of HIV-1-specific Abs to inhibit HIV-1 transfer from immature DCs to autologous CD4 T lymphocytes. We found that neutralizing mAbs prevented HIV-1 transfer to CD4 T lymphocytes in trans and in cis, whereas nonneutralizing Abs did not. Neutralizing Abs also significantly decreased HIV-1 replication in DCs, even when added 2 hours after HIV-1 infection. Interestingly, a similar inhibition of HIV-1 replication in DCs was detected with some nonneutralizing Abs and was correlated with DC maturation. We suggest that the binding of HIV-1-specific Abs to FcγRs leads to HIV-1 inhibition in DCs by triggering DC maturation. This efficient inhibition of HIV-1 transfer by Abs highlights the importance of inducing HIV-specific Abs by vaccination directly at the mucosal portal of HIV-1 entry to prevent early dissemination after sexual transmission.
Assuntos
Anticorpos Neutralizantes/imunologia , Linfócitos T CD4-Positivos/virologia , Células Dendríticas/virologia , Infecções por HIV/transmissão , Infecções por HIV/virologia , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Citometria de Fluxo , HIV-1/imunologia , HumanosRESUMO
OBJECTIVES: Broadly neutralizing antibodies have been proposed as key actors for HIV vaccine development. However, they display features of highly matured antibodies, hampering their induction by vaccination. As protective broadly neutralizing antibodies should be induced rapidly after vaccination and should neutralize the early-transmitted founder (T/F) viruses, we searched whether such antibodies may be induced following HIV infection. DESIGN: Sera were collected during acute infection (Day 0) and at viral set point (Month 6/12) and the neutralizing activity against T/F strains was investigated. Neutralizing activity in sera collected from chronic progressor was analyzed in parallel. METHODS: We compared neutralizing activity against T/F strains with neutralizing activity against non-T/F strains using the conventional TZM-bL neutralizing assay. RESULTS: We found neutralizing antibodies (nAbs) preferentially directed against T/F viruses in sera collected shortly after infection. This humoral response evolved by shifting to nAbs directed against non-T/F strains. CONCLUSION: Although features associated with nAbs directed against T/F viruses need further investigations, these early-induced nAbs may display lesser maturation characteristics; therefore, this might increase their interest for future vaccine designs.
Assuntos
Infecções por HIV , Humanos , Infecções por HIV/prevenção & controle , Anticorpos Amplamente NeutralizantesRESUMO
OBJECTIVE: Spontaneous control of HIV replication without treatment in HIV-1 controllers (HICs) was associated with the development of an efficient T-cell response. In addition, increasing data suggest that the humoral response participates in viral clearance. DESIGN: In-depth characterization of Ab response in HICs may help to define new parameters associated with this control. METHODS: We assessed the levels of total and HIV-specific IgA and IgG subtypes induction and their functional potencies - that is, neutralization, phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), according to the individual's major histocompatibility complex class I (HLA)-B∗57 status, and compared it with nontreated chronic progressors. RESULTS: We found that despite an undetectable viral load, HICs displayed HIV-specific IgG levels similar to those of chronic progressors. Interestingly, our compelling multifunctional analysis demonstrates that the functional Ab profile, by itself, allowed to discriminate HLA-B∗57+ HICs from HLA-B∗57- HICs and chronic progressors. CONCLUSION: These results show that HICs display a particular HIV-specific antibody (Ab) profile that may participate in HIV control and emphasize the relevance of multifunctional Ab response analysis in future Ab-driven vaccine studies.
Assuntos
Infecções por HIV , HIV-1 , Anticorpos Anti-HIV , Paciente HIV Positivo não Progressor , Antígenos HLA-B , Humanos , Imunoglobulina G , Carga ViralRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the outbreak led to the coronavirus disease 2019 (COVID-19) pandemic. Receptor binding domain (RBD) of spike (S) protein of SARS-CoV-2 is considered as a major target for immunotherapy and vaccine design. Here, we generated and characterized a panel of anti-RBD monoclonal antibodies (MAbs) isolated from eukaryotic recombinant RBD-immunized mice by hybridoma technology. Epitope mapping was performed using a panel of 20-mer overlapping peptides spanning the entire sequence of the RBD protein from wild-type (WT) Wuhan strain by enzyme-linked immunosorbent assay (ELISA). Several hybridomas showed reactivity toward restricted RBD peptide pools by Pepscan analysis, with more focus on peptides encompassing aa 76-110 and 136-155. However, our MAbs with potent neutralizing activity which block SARS-CoV-2 spike pseudovirus as well as the WT virus entry into angiotensin-converting enzyme-2 (ACE2) expressing HEK293T cells showed no reactivity against these peptides. These findings, largely supported by the Western blotting results suggest that the neutralizing MAbs recognize mainly conformational epitopes. Moreover, our neutralizing MAbs recognized the variants of concern (VOC) currently in circulation, including alpha, beta, gamma, and delta by ELISA, and neutralized alpha and omicron variants at different levels by conventional virus neutralization test (CVNT). While the neutralization of MAbs to the alpha variant showed no substantial difference as compared with the WT virus, their neutralizing activity was lower on omicron variant, suggesting the refractory effect of mutations in emerging variants against this group of neutralizing MAbs. Also, the binding reactivity of our MAbs to delta variant showed a modest decline by ELISA, implying that our MAbs are insensitive to the substitutions in the RBD of delta variant. Our data provide important information for understanding the immunogenicity of RBD, and the potential application of the novel neutralizing MAbs for passive immunotherapy of SARS-CoV-2 infection.
RESUMO
SARS-CoV-2 spike (S) protein mediates virus attachment to the cells and fusion between viral and cell membranes. Membrane fusion is driven by mutual interaction between the highly conserved heptad-repeat regions 1 and 2 (HR1 and HR2) of the S2 subunit of the spike. For this reason, these S2 regions are interesting therapeutic targets for COVID-19. Although HR1 and HR2 have been described as transiently exposed during the fusion process, no significant antibody responses against these S2 regions have been reported. Here we designed chimeric proteins that imitate highly stable HR1 helical trimers and strongly bind to HR2. The proteins have broad inhibitory activity against WT B.1 and BA.1 viruses. Sera from COVID-19 convalescent donors showed significant levels of reactive antibodies (IgG and IgA) against the HR1 mimetic proteins, whereas these antibody responses were absent in sera from uninfected donors. Moreover, both inhibitory activity and antigenicity of the proteins correlate positively with their structural stability but not with the number of amino acid changes in their HR1 sequences, indicating a conformational and conserved nature of the involved epitopes. Our results reveal previously undetected spike epitopes that may guide the design of new robust COVID-19 vaccines and therapies.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Glicoproteína da Espícula de Coronavírus/química , Proteínas do Envelope Viral/química , Epitopos , Vacinas contra COVID-19 , Glicoproteínas de Membrana/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
The drivers of critical coronavirus disease 2019 (COVID-19) remain unknown. Given major confounding factors such as age and comorbidities, true mediators of this condition have remained elusive. We used a multi-omics analysis combined with artificial intelligence in a young patient cohort where major comorbidities were excluded at the onset. The cohort included 47 "critical" (in the intensive care unit under mechanical ventilation) and 25 "non-critical" (in a non-critical care ward) patients with COVID-19 and 22 healthy individuals. The analyses included whole-genome sequencing, whole-blood RNA sequencing, plasma and blood mononuclear cell proteomics, cytokine profiling, and high-throughput immunophenotyping. An ensemble of machine learning, deep learning, quantum annealing, and structural causal modeling were used. Patients with critical COVID-19 were characterized by exacerbated inflammation, perturbed lymphoid and myeloid compartments, increased coagulation, and viral cell biology. Among differentially expressed genes, we observed up-regulation of the metalloprotease ADAM9. This gene signature was validated in a second independent cohort of 81 critical and 73 recovered patients with COVID-19 and was further confirmed at the transcriptional and protein level and by proteolytic activity. Ex vivo ADAM9 inhibition decreased severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake and replication in human lung epithelial cells. In conclusion, within a young, otherwise healthy, cohort of individuals with COVID-19, we provide the landscape of biological perturbations in vivo where a unique gene signature differentiated critical from non-critical patients. We further identified ADAM9 as a driver of disease severity and a candidate therapeutic target.
Assuntos
COVID-19 , Proteínas ADAM , Inteligência Artificial , Humanos , Unidades de Terapia Intensiva , Proteínas de Membrana , Respiração Artificial , SARS-CoV-2RESUMO
Sexual transmission is the major route of HIV-1 infection worldwide. Dendritic cells (DCs) from the mucosal layers are considered to be the initial targets of HIV-1 and probably play a crucial role in HIV-1 transmission. We investigated the role of cell-to-cell contact between HIV-1-exposed immature DCs and various lymphocyte subsets in the stimulation of HIV-1 replication. We found that HIV-1 replication and production in DCs were substantially enhanced by the coculture of DCs with primary CD4 T or nonpermissive B lymphocytes but not with primary activated CD8 T lymphocytes or human transformed CD4 T lymphocytes. Most of the new virions released by cocultures of HIV-1-exposed immature DCs and primary B lymphocytes expressed the DC-specific marker CD1a and were infectious for both immature DCs and peripheral blood mononuclear cells (PBMCs). Cocultured DCs thus produced large numbers of infectious viral particles under these experimental conditions. The soluble factors present in the supernatants of the cocultures were not sufficient to enhance HIV-1 replication in DCs, for which cell-to-cell contact was required. The neutralizing monoclonal antibody IgG1b12 and polyclonal anti-HIV-1 sera efficiently blocked HIV-1 transfer to CD4 T lymphocytes but did not prevent the increase in viral replication in DCs. Neutralizing antibodies thus proved to be more efficient at blocking HIV-1 transfer than previously thought. Our findings show that HIV-1 exploits DC-lymphocyte cross talk to upregulate replication within the DC reservoir. We provide evidence for a novel mechanism that may facilitate HIV-1 replication and transmission. This mechanism may favor HIV-1 pathogenesis, immune evasion, and persistence.
Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/virologia , HIV-1/fisiologia , Liberação de Vírus , Replicação Viral , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , HIV-1/crescimento & desenvolvimento , HumanosRESUMO
One of the therapeutic strategies in HIV neutralization is blocking membrane fusion. In this process, tight interaction between the N-terminal and C-terminal heptad-repeat (NHR and CHR) regions of gp41 is essential to promote membranes apposition and merging. We have previously developed single-chain proteins (named covNHR) that accurately mimic the complete gp41 NHR region in its trimeric conformation. They tightly bind CHR-derived peptides and show a potent and broad HIV inhibitory activity in vitro. However, the extremely high binding affinity (sub-picomolar) is not in consonance with their inhibitory activity (nanomolar), likely due to partial or temporal accessibility of their target in the virus. Here, we have designed and characterized two single-chain covNHR miniproteins each encompassing one of the two halves of the NHR region and containing two of the four sub-pockets of the NHR crevice. The two miniproteins fold as trimeric helical bundles as expected but while the C-terminal covNHR (covNHR-C) miniprotein is highly stable, the N-terminal counterpart (covNHR-N) shows only marginal stability that could be improved by engineering an internal disulfide bond. Both miniproteins bind their respective complementary CHR peptides with moderate (micromolar) affinity. Moreover, the covNHR-N miniproteins can access their target in the context of trimeric native envelope proteins and show significant inhibitory activity for several HIV pseudoviruses. In contrast, covNHR-C cannot bind its target sequence and neither inhibits HIV, indicating a higher vulnerability of C-terminal part of CHR. These results may guide the development of novel HIV inhibitors targeting the gp41 CHR region.
Assuntos
Fármacos Anti-HIV/farmacologia , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/efeitos dos fármacos , HIV-1/efeitos dos fármacos , HIV-1/metabolismo , Sequência de Aminoácidos , Fusão de Membrana/efeitos dos fármacos , Modelos Moleculares , Peptídeos , Conformação Proteica , Engenharia de Proteínas , Multimerização Proteica , Proteínas do Envelope Viral/químicaRESUMO
Poly(L-lysine citramide) is a degradable bioresorbable polyanion whose polyamide chains are composed of citric acid and L-lysine building blocks. Its chemical and physicochemical properties were extensively investigated in the past for its interest as polymeric drug carrier. In this work, 4(S)-amino-3-(S)-hydroxy-5-phenylpentanoyl-isoleucyl-phenylalanine methyl ester, a pseudopeptide active against the HIV protease in vitro, was linked to poly(L-lysine citramide) in attempts to promote solubility and cell penetration. Conjugates were characterized by FTIR, NMR, SEC, DLS, amino-acid analyses, and toxicity in mice. They degraded slowly at pH 7.4 and more rapidly at pH 4.5, two pH values selected to mimic extra-cellular fluids and intralysosome medium, respectively. According to capillary zone electrophoresis, degradation did not release the peptide. The phenylalanyl-isoleucyl-phenylalanine methyl ester peptide, inactive against the protease in vitro, was used as negative control. The anti-HIV activities of the carrier, of the conjugates and of model molecules, including a fluorescence-labeled pseudopeptide conjugate, were evaluated comparatively in vitro using two cell lines, namely, CEM-SS and MT-4 cells, infected with HIV-1 LAI and IIIB isolates, respectively. Unexpectedly, all the conjugates showed in vitro antiviral activity independent of peptide release and of inhibition of the HIV protease. According to FACS analysis, the antiviral activity was related to the presence of peptide moieties along the polymer chains and depended on the order by which cells, viruses, and conjugates were presented to each other. Although it was not possible to determine whether the antiviral activity resulted from interactions between conjugates and cells or conjugates and virus or both, the conjugates appeared able to inhibit the binding of the virus to cells in vitro when introduced before cell infection. None of the conjugates exhibited acute toxicity in mice.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Polilisina/análogos & derivados , Animais , Fármacos Anti-HIV/química , Peso Corporal/efeitos dos fármacos , Linhagem Celular , Cromatografia em Camada Fina , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , HIV-1/metabolismo , Humanos , Concentração Inibidora 50 , Linfócitos/citologia , Linfócitos/metabolismo , Lisina/química , Espectroscopia de Ressonância Magnética , Camundongos , Fragmentos de Peptídeos/química , Polilisina/química , Polilisina/farmacologia , Polímeros , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Development of effective inhibitors of the fusion between HIV-1 and the host cell membrane mediated by gp41 continues to be a grand challenge due to an incomplete understanding of the molecular and mechanistic details of the fusion process. We previously developed single-chain, chimeric proteins (named covNHR) that accurately mimic the N-heptad repeat (NHR) region of gp41 in a highly stable coiled-coil conformation. These molecules bind strongly to peptides derived from the gp41 C-heptad repeat (CHR) and are potent and broad HIV-1 inhibitors. Here, we investigated two covNHR variants differing in two mutations, V10E and Q123R (equivalent to V38E and Q40R in gp41 sequence) that reproduce the effect of HIV-1 mutations associated with resistance to fusion inhibitors, such as T20 (enfuvirtide). A detailed calorimetric analysis of the binding between the covNHR proteins and CHR peptides (C34 and T20) reveals drastic changes in affinity due to the mutations as a result of local changes in interactions at the site of T20 resistance. The crystallographic structure of the covNHR:C34 complex shows a virtually identical CHR-NHR binding interface to that of the post-fusion structure of gp41 and underlines an important role of buried interfacial water molecules in binding affinity and in development of resistance against CHR peptides. Despite the great difference in affinity, both covNHR variants demonstrate strong inhibitory activity for a wide variety of HIV-1 strains. These properties support the high potential of these covNHR proteins as new potent HIV-1 inhibitors. Our results may guide future inhibition approaches.
Assuntos
Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Termodinâmica , Internalização do Vírus/efeitos dos fármacos , Sequência de Aminoácidos , Fármacos Anti-HIV/farmacologia , Cristalografia por Raios X , Enfuvirtida/antagonistas & inibidores , Proteína gp41 do Envelope de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Modelos Moleculares , Mutação , Peptídeos , Conformação Proteica , Análise de Sequência de ProteínaRESUMO
OBJECTIVE: In the semen, both free virus and infected cells are able to establish HIV infection during sexual intercourse. An efficient vaccine should therefore inhibit both infectious states. The aim of this study was to analyze the capacity of broadly neutralizing antibodies (bNAbs) to inhibit HIV transmission by the infected cells. DESIGN/METHODS: We developed an in-vitro model aiming to mimic mucosal HIV transmission via infected cells. PHA-activated CD4+ T cells stained with PKH26 from donor A were infected and co-cultured with CD4+ T cells and dendritic cells from donor B in the presence of bNAbs. RESULTS: We showed that dendritic cells were the preferential HIV target cells at early time points in this co-culture model. In the context of this co-culture model where infection and transmission occurred simultaneously, bNAbs efficiently inhibited HIV replication as well as HIV transmission from infected cells to allogenic dendritic cells and CD4+ T cells. CONCLUSION: Overall, our results indicate that dendritic cells, in addition to CD4+ T cells, are key cells that are efficiently infected by HIV and bNAbs are potent inhibitors of infection of both target cells. Future HIV prophylactic vaccine design should develop immune strategies able to prevent the infection of dendritic cells, in addition to the inhibition of CD4+ T-cell infection.
Assuntos
Anticorpos Neutralizantes/imunologia , Linfócitos T CD4-Positivos/virologia , Células Dendríticas/virologia , Transmissão de Doença Infecciosa/prevenção & controle , Anticorpos Anti-HIV/imunologia , Infecções por HIV/prevenção & controle , Doadores de Sangue , Células Cultivadas , Técnicas de Cocultura , Infecções por HIV/transmissão , Humanos , Modelos BiológicosRESUMO
The development of an effective vaccine against HIV-1 has proven to be challenging. Broadly neutralizing antibodies (bNAbs), whilst exhibiting neutralization breadth and potency, are elicited only in a small subset of infected individuals and have yet to be induced by vaccination. Case-control studies of RV144 identified an inverse correlation of HIV-1 infection risk with antibodies (Abs) to the V1V2 region of gp120 with high antibody-dependent cellular cytotoxicity (ADCC) activity. The neutralizing activity of Abs was not found to contribute to this protective outcome. Using primary effector and target cells and primary virus isolates, we studied the ADCC profile of different monoclonal Abs targeting the V1V2 loop of gp120 that had low or no neutralizing activity. We compared their ADCC activity to some bNAbs targeting different regions of gp120. We found that mAbs targeting the V1V2 domain induce up to 60% NK cell mediated lysis of HIV-1 infected PBMCs in a physiologically relevant ADCC model, highlighting the interest in inducing such Abs in future HIV vaccine trials. Our data also suggest that in addition to neutralization, lysis of infected cells by Abs can effectively participate in HIV protection, as suggested by the RV144 immune correlate analysis.
Assuntos
Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Reações Cruzadas/imunologia , Epitopos/imunologia , Proteína gp120 do Envelope de HIV/uso terapêutico , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , HIV-1/patogenicidade , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/virologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Domínios Proteicos/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologiaRESUMO
Mucosal tissues are the predominant sites for genital HIV-1 transmission. We investigated the mechanisms by which broadly neutralizing antibodies (bNAbs) inhibit HIV-1 replication in a coculture model including primary mucosal dendritic cells (DCs), such as Langerhans cells, interstitial dendritic cells, and CD4(+) T lymphocytes. We show that bNAbs efficiently prevent HIV-1 infection by inhibiting HIV-1 transmission to CD4(+) T lymphocytes. This inhibition of cell-to-cell transmission was observed with equal potency as the inhibition of cell-free infection of primary CD4(+) T lymphocytes. In addition, a decrease in HIV-1 replication in DCs and the induction of DC maturation were detected. This additional inhibition was Fc mediated as it was blocked by the use of specific anti-FcγR monoclonal Abs. The DC maturation by bNAbs during HIV transmission may contribute to mucosal protection. Therefore, multiple antibody-mediated inhibitory functions should be combined for the improvement of future preventive/therapeutic strategies to cure HIV.
Assuntos
Anticorpos Neutralizantes/administração & dosagem , Transmissão de Doença Infecciosa/prevenção & controle , Anticorpos Anti-HIV/administração & dosagem , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Imunização Passiva/métodos , Anticorpos Neutralizantes/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/virologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/virologia , Humanos , Modelos Biológicos , Replicação Viral/efeitos dos fármacosRESUMO
The synthesis of a series of d4T analogues bearing an acyclic chains between the sugar and the base moities, is described. New compounds were obtained readily using microwave irradiation and selective deprotection of sugar part. The compounds were characterized by 1H NMR and IR spectroscopy. Antiviral (HIV-1) properties of these compounds were examined.
Assuntos
Inibidores da Transcriptase Reversa/química , Estavudina/análogos & derivados , Linhagem Celular , HIV-1/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/farmacologia , Espectrofotometria Infravermelho , Estavudina/síntese química , Estavudina/farmacologiaRESUMO
Plasmacytoid dendritic cells (pDC) expressing FcγRIIa are antigen-presenting cells able to link innate and adaptive immunity and producing various cytokines and chemokines. Although highly restricted, they are able to replicate HIV-1. We determined the activity of anti-HIV-1 neutralizing antibodies (NAb) and non-neutralizing inhibitory antibodies (NNIAb) on the infection of primary pDC by HIV-1 primary isolates and analyzed cytokines and chemokines production. Neutralization assay was performed with primary pDC in the presence of serial antibodies (Ab) concentrations. In parallel, we measured the release of cytokines and chemokines by ELISA and CBA Flex assay. We found that NAb, but not NNIAb, inhibit HIV-1 replication in pDC. This inhibitory activity was lower than that detected for myeloid dendritic cells (mDC) infection and independent of FcγRIIa expressed on pDC. Despite the complete protection, IFN-α production was detected in the supernatant of pDC treated with NAb VRC01, 4E10, PGT121, 10-1074, 10E8, or polyclonal IgG44 but not with NAb b12. Production of MIP-1α, MIP-1ß, IL-6, and TNF-α by pDC was also maintained in the presence of 4E10, b12 and VRC01. These findings suggest that pDC can be protected from HIV-1 infection by both NAb and IFN-α release triggered by the innate immune response during infection.
Assuntos
Anticorpos Neutralizantes/imunologia , Células Dendríticas/virologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Receptores de IgG/biossíntese , Células Cultivadas , Quimiocina CCL3/biossíntese , Quimiocina CCL4/biossíntese , Células Dendríticas/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Humanos , Imunidade Inata , Interferon-alfa/biossíntese , Interleucina-6/biossíntese , Testes de Neutralização , Receptores de IgG/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Replicação Viral/imunologiaRESUMO
OBJECTIVE: The chronology of HIV infection in mucosal tissue after sexual transmission is unknown. Several potential HIV target cells are present at these sites, including dendritic cells, macrophages, and CD4(+) T lymphocytes. Dendritic cells and macrophages are antigen-presenting cells (APCs) and are thus involved in cross-talk with T cells. This close contact may favor efficient HIV-1 transfer to T lymphocytes, resulting in rapid HIV-1 dissemination. DESIGN: We investigated the role of APCs in HIV transfer to T cells by incubating Langerhans cells and interstitial dendritic cells (IDCs) or monocyte-derived macrophages (MDMs) with HIV for 2âh before addition of uninfected autologous CD4(+) T lymphocytes. METHODS: HIV infection was recorded after different time points. Following staining, the measurement of intracellular p24 in the different cell populations was analyzed by flow cytometry. RESULTS: We showed that Langerhans cells/IDCs and macrophages efficiently transferred HIV to CD4(+) T cells. Interestingly, a rapid HIV transfer in trans predominated in MDMs, whereas cis transfer mainly occurred in Langerhans cells/IDC cocultures. Neutralizing antibody 2G12, added to HIV-loaded APCs, efficiently blocked both the trans and the cis infection of T cells. CONCLUSION: These findings highlight the major contributions of various mucosal cells in HIV dissemination and suggest that HIV hijacks the different properties of APCs to favor its dissemination through the body. They emphasize the role of macrophages in the rapid transmission of HIV to T lymphocytes at mucosal sites, dendritic cells being prone to migration to lymphoid organ for subsequent dissemination by cis transfer.