ABSTRACT
BACKGROUND: HIV-1 protease is subjected to dual selection pressure exerted by protease inhibitors (PIs) and cytotoxic T lymphocytes (CTL). Recently, we identified KMIGGIGGF (KF9) as a HLA-B*1501-restricted CTL epitope, including several major PI resistance mutations (M46I/L, I47A/V, G48V, I50V). To assess potential interactions between KF9-specific CTL and emergence of these important resistance mutations, we studied CTL recognition of the mutations and analyzed protease sequences in an HLA-Ityped patient cohort. METHODS: CTL recognition of KF9 and resistance mutations in KF9 were studied in 38 HLA-B*1501-positive HIV-1infected patients using variant KF9 peptides in interferon-g enzyme-linked immunospot assays. Protease sequences were analyzed in 302 HLA-Ityped HIV-1infected patients. RESULTS: G48V abolished KF9 recognition by CTL in all patients. Furthermore, M46I, I47A, and I50V could impair or abolish CTL recognition in many patients. In contrast, M46L and I47V showed good CTL recognition in nearly all patients. HIV-1 protease sequence analysis showed no statistical correlation between the occurrence of resistance mutations in KF9 and HLA-B*1501. Viral load in patients failing therapy with KF9 mutations was significantly lower in HLA-B*1501-positive patients in comparison with HLA-B*1501-negative patients. CONCLUSIONS: PI mutations, G48V, M46I, and I47A, can abrogate CTL recognition, indicating potential interactions between development of drug resistance and CTL response. However, we could not find evidence that development of these PI mutations is influenced by KF9-specific CTL.
Subject(s)
Drug Resistance, Viral , HIV Protease Inhibitors/pharmacology , HIV Protease/immunology , HIV-1/drug effects , HIV-1/immunology , Mutation, Missense , T-Lymphocytes, Cytotoxic/immunology , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , HIV Infections/virology , HIV Protease/genetics , HIV-1/genetics , HIV-1/isolation & purification , HLA-B Antigens/immunology , HumansABSTRACT
INTRODUCTION: HIV type-1 (HIV-1) protease (PR) and cleavage site (CS) mutations accumulate in protease-inhibitor-resistant isolates. HIV-1 CS mutation 431V is the most frequent treatment-associated CS mutation; however, little is known about its origin in treatment-naive HIV-1 isolates. Recently, it has been shown that the CS mutation 431V is located within the human leukocyte antigen (HLA)-B*13-restricted cytotoxic T-lymphocyte (CTL) epitope RQANFLGKI (RI9). Therefore, we investigated whether the presence of CS mutation 431V might additionally be related to immune escape. METHODS: CTL recognition of RI9 and of RI9 variants carrying the 431V or the 436R mutation was analysed by ELISPOT in nine HLA-B*13-positive HIV-1-infected patients. Treatment-naive HIV-1-infected patients with primary drug-resistant HIV-1 isolates (n=58) or carrying 431V (n=4) were genotyped for HLA class I alleles. RESULTS: ELISPOT analysis showed different patterns of CTL recognition of RI9. CS mutation 431V could abrogate recognition by RI9-specific CTL in a subgroup of patients. Nevertheless, in our study, the occurrence of 431V in treatment-naive HIV-1 without primary drug resistance could not be explained by HLA-B*13-mediated immune selection. In patients with primary drug-resistant HIV-1 isolates, the frequency of HLA-B*13 was not increased and HLA-B*13 did not correlate with CS mutations 436R or 431V. CONCLUSIONS: HIV-1 CS mutation 431V can abrogate CTL recognition, indicating interactions between development of drug resistance and the CTL response. However, we could not find evidence that the presence of 431V in treatment-naive HIV-1 isolates with and without primary drug resistance is related to immune selection by HLA-B*13 or other HLA class I alleles.
Subject(s)
HIV Infections/immunology , HIV-1/genetics , Mutation , T-Lymphocytes, Cytotoxic/immunology , Case-Control Studies , Cohort Studies , Drug Resistance, Multiple, Viral , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , HIV-1/immunology , HIV-1/isolation & purification , HLA-B Antigens/immunology , Humans , Protease Inhibitors/therapeutic use , T-Lymphocytes, Cytotoxic/virology , gag Gene Products, Human Immunodeficiency Virus/immunologyABSTRACT
BACKGROUND: HIV-1-specific cytotoxic T lymphocytes, which recognize conserved epitopes of the virus, are correlated with prolonged survival of infected individuals. Unfortunately, most HIV-1-infected patients are unable to generate such an immune response. Antigen-specific cytotoxic T lymphocytes can be generated by T-cell receptor transfer. This is commonly done by retroviral transduction, which is complicated and poses the threat of stable genetic alteration of autologous cells. METHODS: We reprogrammed primary CD8+ T cells by electroporation of RNA, which encoded an HIV-1-pol- and an HIV-1-gag-specific T-cell receptor recognizing the human leukocyte antigen-A2 restricted epitopes ILKEPVHGV and SLYNTVATL, respectively. RESULTS: These reprogrammed cells specifically produced the proinflammatory cytokines interleukin-2, tumor necrosis factor-alpha, and interferon-gamma after stimulation with target cells that presented the corresponding peptides, and were able to lyse these targets efficiently and specifically. The lytic avidities of the HIV-1-pol- and HIV-1-gag-TCR-RNA-electroporated CD8+ T cells were within the same range than those of the parental cytotoxic T lymphocytes. Most importantly, HIV-1-gag-reprogrammed T cells recognized target cells that presented endogenously processed antigen, which resulted in cytokine production and lysis. CONCLUSION: It is shown here for the first time that functional transfer of virus-specific T-cell receptors by RNA electroporation is feasible, and represents an innovative, safe, and easy method to generate virus-specific T cells, avoiding the risks of retroviral transduction.
Subject(s)
HIV Infections/immunology , HIV Infections/therapy , HIV-1/immunology , RNA/pharmacology , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes, Cytotoxic/immunology , CD8-Positive T-Lymphocytes/immunology , Cloning, Molecular , Cytokines/analysis , Cytotoxicity Tests, Immunologic , Electroporation , Epitopes/immunology , Humans , Immunotherapy , Transcription, GeneticABSTRACT
OBJECTIVES: To study the role of cytotoxic T-lymphocyte (CTL) escape for disease progression in HIV-1 infection, we analyzed the CTL response to the dominant human leukocyte antigen (HLA)-B8-restricted CTL epitope FLKEKGGL (FL8) in HIV-1 Nef. METHODS: HIV-1 nef genes derived from 56 patients were analyzed by polymerase chain reaction (PCR)-based sequencing. T-cell responses against FL8 and mutated FL8 variants were detected by gamma-interferon (gamma-IFN) enzyme linked immunospot (ELISPOT) assay. RESULTS: The longitudinal analysis of an HIV-1-infected patient with good control of HIV-1 viremia for several years demonstrated an association of rising viremia with the emergence of CTL escape mutations within the HLA-B8-restricted Nef-specific CTL epitopes FLKEKGGL and WPAIRERM. Analysis of nef genes in 56 HIV-1-infected patients demonstrated a significant correlation between the occurrence of mutations in the FL8 epitope and the presence of HLA-B8. The mutations within the FL8 epitope could decrease CTL recognition; however, there was strong variation regarding the recognition of viral variants between individual donors. The presence of FL8 mutations was associated with lower CD4 cell counts and higher viral loads. CONCLUSIONS: Our data demonstrate a strong CTL selection pressure on the immunodominant HLA-B8-restricted CTL epitope FL8 in HIV-1 Nef. The association of FL8 mutations with lower CD4 cell counts indicates an important role of CTL escape mutations for disease progression.
Subject(s)
Acquired Immunodeficiency Syndrome/immunology , Epitopes, T-Lymphocyte , HLA-B8 Antigen/physiology , Immunodominant Epitopes , T-Lymphocytes, Cytotoxic/immunology , nef Gene Products, Human Immunodeficiency Virus/immunology , Acquired Immunodeficiency Syndrome/virology , Base Sequence , CD4 Lymphocyte Count , Female , HLA-B8 Antigen/analysis , Humans , Longitudinal Studies , Middle Aged , Molecular Sequence Data , Mutation , Viral Load , nef Gene Products, Human Immunodeficiency Virus/chemistryABSTRACT
To determine the influence of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells on the development of drug resistance mutations in the HIV-1 protease, we analyzed protease sequences from viruses from a human leukocyte antigen class I (HLA class I)-typed cohort of 94 HIV-1-positive individuals. In univariate statistical analyses (Fisher's exact test), minor and major drug resistance mutations as well as drug-associated polymorphisms showed associations with HLA class I alleles. All correlations with P values of 0.05 or less were considered to be relevant without corrections for multiple tests. A subset of these observed correlations was experimentally validated by enzyme-linked immunospot assays, allowing the definition of 10 new epitopes recognized by CD8+ T cells from patients with the appropriate HLA class I type. Several drug resistance-associated mutations in the protease acted as escape mutations; however, cells from many patients were still able to generate CD8+ T cells targeting the escape mutants. This result presumably indicates the usage of different T-cell receptors by CD8+ T cells targeting these epitopes in these patients. Our results support a fundamental role for HLA class I-restricted immune responses in shaping the sequence of the HIV-1 protease in vivo. This role may have important clinical implications both for the understanding of drug resistance pathways and for the design of therapeutic vaccines targeting drug-resistant HIV-1.
Subject(s)
Drug Resistance, Viral , HIV Protease Inhibitors/therapeutic use , HIV Protease/immunology , Histocompatibility Antigens Class I/immunology , Selection, Genetic , Alleles , Amino Acid Sequence , Amino Acid Substitution , CD4 Lymphocyte Count , CD8-Positive T-Lymphocytes/immunology , Cohort Studies , Epitopes, T-Lymphocyte , HIV Infections/drug therapy , HIV Infections/immunology , HIV Infections/virology , HIV Protease/chemistry , HIV Protease/genetics , HIV Protease/metabolism , HIV Protease Inhibitors/pharmacology , HIV-1/drug effects , HIV-1/enzymology , HIV-1/immunology , Histocompatibility Testing , Humans , Leukocytes, Mononuclear/immunology , Molecular Sequence Data , Viral LoadABSTRACT
The safety and immunogenicity of an HIV-1 nef-expressing modified vaccinia virus Ankara (MVA) was investigated in 14 HIV-1-positive patients (CD4 >400/microl) on highly active antiretroviral therapy (HAART). Patients were vaccinated at weeks 0, 4 and 16, followed by interruption of HAART at week 18. MVA-nef was well-tolerated except for local reactions, with only mild systemic side effects reported in a few patients. Vaccination with MVA-nef was associated with recognition of new HIV-1 T-cell epitopes (cytotoxic T-lymphocyte epitopes in 9/14 patients, CD4 epitope/recombinant Nef protein in 2/14) and an increase in CD4+ and CD8+ T cells. All patients had been vaccinated against smallpox and a strong T-cell and antibody response to MVA was induced in all patients. After interruption of HAART, viral load rebounded in all patients, but after a median time of 36 (4-76) weeks in 9/14 patients, viraemia remained below the pre-HAART viral load and CD4 counts stayed above the pre-HAART levels. While six patients have remained off therapy for a median time of 64 (57-76) weeks, HAART was resumed in 8/14 patients after a median treatment interruption time of 15 (4-38) weeks. This study has demonstrated that MVA-nef is safe and immunogenic in HIV-1-infected subjects and has provided encouraging data on the potential of therapeutic vaccinations.
Subject(s)
AIDS Vaccines/therapeutic use , Genetic Vectors , HIV Seropositivity/therapy , HIV-1/genetics , Immunotherapy , Vaccinia virus/genetics , AIDS Vaccines/administration & dosage , Adult , Amino Acid Sequence , Anti-Retroviral Agents/therapeutic use , Antibodies, Viral/blood , Antibody Specificity , Antiretroviral Therapy, Highly Active , CD4 Antigens/immunology , Drug Administration Schedule , Epitopes/immunology , Gene Products, nef/genetics , HIV Seropositivity/drug therapy , HIV Seropositivity/immunology , HIV Seropositivity/virology , HIV-1/isolation & purification , Humans , Lymphocyte Count , Middle Aged , Molecular Sequence Data , Sequence Alignment , T-Lymphocytes, Cytotoxic/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology , Vaccines, Synthetic/therapeutic use , Vaccinia virus/immunology , Viral Load , Withholding Treatment , nef Gene Products, Human Immunodeficiency VirusABSTRACT
We report on the first HLA B13-restricted minimal cytotoxic T lymphocyte (CTL) epitope RQDILDLWI (RI9, amino acids 106-114 in HIV-1 Nef). In most patients the frequency of RI9-specific CTL exceeded the number of CTL against other epitopes, indicating that RI9 is a dominant epitope in HLA B13-positive patients. Targeting this conserved Nef epitope may be an important factor for the published association of HLA B13 with a favourable course of HIV-1 infection.