Ellen G. Harrer

Specific recognition of lamivudine-resistant HIV-1 by cytotoxic T lymphocytes.

ObjectiveThe reverse transcriptase (RT) M184V mutation within the HLA-A2-restricted HIV-1 cytotoxic T lymphocyte (CTL) epitope VL9 (VIYQYMDDL; RT 179–187) not only induces drug escape against lamivudine but also abolished recognition by a CTL clone derived from a long-term non-progressor. To test whether the variant VL9 epitope containing the M184V mutation represents a new CTL epitope, we studied recognition of this epitope in a cohort of HLA-A2-positive HIV-1-infected patients. MethodsPeripheral blood mononuclear cells isolated from 28 HIV-1-infected patients were stimulated with the peptide VIYQYVDDL, containing the M184V mutation. Outgrowing cell lines were tested for specific recognition in a standard chromium-release assay. ResultsIn one subject, a CTL line could be isolated recognizing the peptide VIYQYVDDL in conjunction with HLA-A2. The CTL clone also recognized the M184I mutation, but it failed to recognize the wild-type epitope VIYQYMDDL. ConclusionCTL can specifically recognize lamivudine-resistant HIV-1 variants. Therefore, the cellular immune response could have an important influence on the control of drug-resistant virus. Furthermore, this demonstrates that the immune system can generate new CTL specificities even in patients with advanced disease, as the M184V HIV variants emerges only after drug treatment. Specific immunotherapy against this epitope might be helpful in delaying or preventing lamivudine resistance.

Dual Selection Pressure by Drugs and HLA Class I-Restricted Immune Responses on Human Immunodeficiency Virus Type 1 Protease

ABSTRACT 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 (Fishers 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.

A conserved HLA B13-restricted cytotoxic T lymphocyte epitope in Nef is a dominant epitope in HLA B13-positive HIV-1-infected patients.

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.

HIV-associated hypertrophic herpes simplex genitalis with concomitant early invasive squamous cell carcinoma mimicking advanced genital cancer: case report and literature review.

Hypertrophic herpes simplex genitalis (HHSG) is an uncommon anogenital manifestation of herpes simplex virus (HSV) infection in immunocompromised patients. To date, 24 cases of HHSG have been reported; 23 of them were affected human immune deficiency virus (HIV) type 1-positive patients. We describe the case of a 44-year-old African HIV-1-positive woman who presented with painful ulcerated nodular lesions of the vulva and perianal area measuring up to 7 cm in diameter. Macroscopically, the lesions were highly suspicious of widely invasive cancer. The histologic workup of the resection specimen revealed patchy high-grade vulvar intraepithelial neoplasia Grade 3 (VIN 3) and 2 microscopic foci of superficially invasive squamous cell carcinoma. The nodular lesions were caused by massive tumefactive plasma cell-rich inflammatory infiltrates extending into the subcutis. Multinucleated herpes simplex virus 1 and herpes simplex virus 2-positive epithelial cells with glassy intranuclear inclusions were detected at the borders of the ulcerations, consistent with HHSG. Despite repeated surgery and medical treatment, the patient had 3 recurrences of HHSG within 18 months. The presence of intraepithelial neoplasia in HHSG lesions is relatively rare and has been described in 6 of 18 resected HHSG lesions in the literature so far. With regard to invasive malignancy, the present case is the first report of a superficially invasive squamous cell carcinoma associated with HHSG. Awareness of this condition is necessary to avoid misinterpretation of HHSG as widely invasive squamous cell carcinoma with the hazard of surgical and oncological overtreatment.

Human T cells expressing two additional receptors (TETARs) specific for HIV-1 recognize both epitopes.

Adoptive TCR transfer against rapidly mutating targets, such as HIV-1 or cancer, must counteract corresponding immune escape. Hence, we generated T cells expressing two additional receptors (TETARs) specific for HIV-1 by TCR mRNA electroporation. An HLA-A2-restricted gag-specific TCR and an HLA-B13-restricted nef-specific TCR were chosen. When both TCRs were transfected simultaneously, strong competitive effects occurred that were overcome by replacing the human constant domains of one TCR with murine counterparts and adapting the amounts of TCR-RNA used for transfection. The resulting TETAR responded to both epitopes with cytokine secretion and cytotoxic function. Cell sorting revealed that one individual cell indeed recognized both epitopes. The T cells diminished their reactivity to each epitope after stimulation but sequentially killed targets that presented the gag epitope and then targets that presented the nef epitope, or vice versa. Taken together, TETARs represent a sophisticated tool to study TCR functionality and might be a useful strategy in immunotherapy.

Influence of Major HIV-1 Protease Inhibitor Resistance Mutations on CTL Recognition

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-I-typed patient cohort. Methods:CTL recognition of KF9 and resistance mutations in KF9 were studied in 38 HLA-B*1501-positive HIV-1-infected patients using variant KF9 peptides in interferon-γ enzyme-linked immunospot assays. Protease sequences were analyzed in 302 HLA-I-typed HIV-1-infected 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.

Role of cytotoxic T-lymphocyte-mediated immune selection in a dominant human leukocyte antigen-B8-restricted cytotoxic T-lymphocyte epitope in Nef.

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 γ-interferon (γ-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.

Cross-Reactivity Between Influenza Matrix- and HIV-1 P17-Specific CTL-A Large Cohort Study.

Background:It has been reported that HIV-1–specific cytotoxic T cells (CTL) recognizing the HLA-A2–restricted p17 epitope SLYNTVATL (SL9) can cross-react with the HLA-A2–restricted influenza matrix epitope GILGFVFTL (GL9). So far, the prevalence of GL9-cross-reacting HIV-1–specific CTL in larger cohorts of HIV-1–infected patients is unknown, and there are no data yet on whether SL9/GL9-cross-reactive CTL may influence the course of HIV-1 infection. Methods:We analyzed the presence of SL9/GL9-cross-reacting CTL in a cohort of 175 HLA-A2–positive HIV-1–infected patients. Peripheral blood mononuclear cells were stimulated in vitro with SL9 and GL9 peptides, and outgrowing cell lines regarding cross-reactivity and recognition of viral variants in &ggr;-interferon enzyme-linked immunospot assays were analyzed. Results:SL9- and GL9-specific CTL could be generated in 52.6% and 53.7% of 175 patients, respectively. Both SL9- and GL9-specific CTL were more frequently observed in patients on antiretroviral therapy (ART). Of the 92 SL9-specific CTL and the 94 GL9-specific CTL, 65.2% and 66%, respectively, showed at least partial SL9/GL9 cross-reactivity. SL9/GL9-cross-reactive CTL could be detected in 42.9% of the 175 patients. Recognition of SL9 was associated with lower viral loads and higher CD4+ cell counts in patients on ART. Patients with GL9/SL9 cross-reactivity displayed similar CD4+ cell counts than patients without GL9/SL9-cross-reactive cells. GL9/SL9-cross-reactive cells were associated with higher viral loads in patients on ART. Conclusions:Partially SL9/GL9-cross-reactive CTL are frequently observed in HIV-1–infected patients. So far, we could not detect a significant influence of the presence of SL9/GL9-cross-reacting CTL on the course of HIV-1 infection.

Analysis of immune selection as a potential cause for the presence of cleavage site mutation 431V in treatment-naive HIV type-1 isolates.

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.

Identification of HLA-C restricted, HIV-1-specific CTL epitopes by peptide induced upregulation of HLA-C expression.

HIV-1 negative regulatory factor (Nef) can inhibit CTL recognition by downregulation of HLA-A and HLA-B on the cell surface. In contrast, HLA-C is not affected by Nef and a growing number of studies demonstrate an important role of HLA-C for the control of HIV-1. So far, only a limited number of HLA-C restricted CTL epitopes are known. As the mapping of new CTL epitopes is time and labor intensive, we investigated a novel method for the identification of HLA-C restricted CTL epitopes. B-lymphoblastoid cell lines (B-LCLs) and T2-cells were incubated with HIV-1 specific peptides and subsequently stained for HLA-C surface expression using the HLA-C specific antibody DT9. Peptides that led to increased HLA-C surface expression were used for stimulation of PBMC from HIV-1-infected patients. Subsequently, outgrowing cells were tested for peptide recognition in IFN-γ ELISPOT assays and HLA restriction of the recognized peptides was analyzed in ELISPOT assays using HLA-matched B-LCL. We observed that known HLA-C binding peptides increase HLA-C surface expression on T2-cells and on HLA-C*0102 and HLA-C*0702 homozygous B-LCL. Moreover, screening of HIV-1 Nef with overlapping peptides for potential C*0702 restricted epitopes using this method revealed a total of 8 peptides which considerably increased cell surface expression of HLA-C. By epitope mapping and functional analysis of peptide-stimulated T-cell lines we were able to define the peptide YPLTFGWCY as a new C*0702-restricted CTL epitope. These results show that the analysis of peptide induced HLA-C upregulation on B-LCL and T2-cells enables the efficient identification of new HLA-C restricted CTL epitopes.