Marlinda E. M. Dings

Intraocular T cells of patients with herpes simplex virus (HSV)-induced acute retinal necrosis recognize HSV tegument proteins VP11/12 and VP13/14.

It has previously been shown that T cells specific for the triggering virus infiltrate the eye of patients with herpes simplex virus type 1 (HSV-1)-induced acute retinal necrosis (ARN). The T cells were mainly directed against 0.67-0.73 HSV-1 map region encoded antigens. The fine specificities of genetically different T cell clones (TCC), obtained from affected eyes of 3 patients with HSV-induced ARN and reactive toward this genomic region of HSV-1, were analyzed with recombinant HSV viruses and synthetic peptides. For 1 patient, the HSV-1 UL46 gene encoded tegument protein VP11/12 was identified as the target antigen. Two separate CD4(+) T cell epitopes were defined in VP11/12. TCC from the other 2 patients recognized the HSV-1 UL47 gene encoded tegument protein VP13/14. Two separate CD4(+) VP13/14 T cell epitopes were identified in these patients. Analysis of the data indicates that HSV-1 VP11/12 and VP13/14 are major target antigens for T cells obtained from vitreous fluid samples of the HSV-induced ARN patients studied.

T Cells Specific for the Triggering Virus Infiltrate the Eye in Patients with Herpes Simplex Virus-Mediated Acute Retinal Necrosis

Acute retinal necrosis (ARN) is a rare, potentially blinding retinal disease resulting from ocular infections with herpes simplex virus (HSV) or varicella-zoster virus (VZV). To determine the antigen specificity and functional characteristics of ocular infiltrating T cells in ARN, T cells were isolated and expanded nonspecifically from intraocular fluid (IOF) samples from 2 patients with HSV-1- and 3 with VZV-mediated ARN. HSV-specific T cell reactivity could be detected only in the IOF-derived T cell lines (TCLs) of the 2 patients with HSV-mediated ARN. These TCLs consisted of both HSV type-common and type-specific CD4+ and CD8+ T cell clones (TCCs) with differential T cell receptor usage. Irrespective of their phenotype, the TCCs were cytolytic and secreted interferon-gamma, tumor necrosis factor-alpha, interleukin-4, and interleukin-5. In both patients, the antigen specificity of a substantial number of HSV-1-specific TCCs could be mapped to approximately 0.67-0.73 HSV-1 map units. The data presented suggest the contribution of T cells, specific for the triggering virus, to the pathogenesis of ARN.

Fine-specificity of cytotoxic T lymphocytes which recognize conserved epitopes of the Gag protein of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) Gag-specific cytotoxic T lymphocyte (CTL) responses were studied in seven seropositive long-term asymptomatic individuals (CDC A1) with stable CD4 counts for more than 8 years. Using a set of partially overlapping peptides covering the whole Gag, five 15-20-mer peptides were found to contain CTL epitopes. Further characterization of these epitopes revealed a new HLA-A25-restricted CTL epitope in p24, p24(203-212) ETINEEAAEW. This region of Gag is highly conserved in clades B and D of HIV-1. Naturally occurring amino acid sequences, containing p24(203)D (consensus HIV-1 clades A, C, F, G and H) or p24(204)I (HIV-2ROD) were not recognized by CTL recognizing the index peptide. No virus variants with mutations in this sequence were found in peripheral blood mononuclear cells from the HIV-1-infected individual concerned during the 8 year observation period, indicating that the virus had not escaped from the observed CTL response.

Protection of rhesus macaques from SIV infection by immunization with different experimental SIV vaccines.

The immunogenicity and efficacy of an inactivated whole SIVmac (32H) preparation adjuvanted with muramyl dipeptide (SIV-MDP) and a gp120-enriched SIVmac (32H) ISCOM preparation (SIV-ISCOM), were compared by immunizing four rhesus macaques (Macaca mulatta) four times with SIV-MDP and four others in the same way with SIV-ISCOM. Two monkeys immunized with whole inactivated measles virus (MV) adjuvanted with MDP (MV-MDP) and two monkeys immunized with MV-ISCOM served as controls. In the SIV-ISCOM-immunized monkeys higher SIV-specific serum antibody titres were found than in the SIV-MDP-immunized monkeys. In contrast to the MV-immunized monkeys all SIV-MDP- and SIV-ISCOM-immunized monkeys were protected against intravenous challenge 2 weeks after the last immunization with 10 median monkey infectious doses (MID50) of a cell-free SIVmac (32H) challenge stock propagated in the human T-cell line C8166. After 43 weeks the protected monkeys were reboosted and 2 weeks later rechallenged with 10 MID50 of the same virus produced in peripheral blood mononuclear cells (PBMC) from a rhesus macaque. None of these animals proved to be protected against this challenge. In a parallel experiment in which the same numbers of monkeys were immunized in the same way, the animals were challenged intravenously with 10 MID50 of PBMC from an SIVmac (32H)-infected rhesus macaque. Two out of four SIV-MDP- and two out of four SIV-ISCOM-immunized monkeys proved to be protected from SIV infection.

CD8+ cytotoxic T lymphocytes of a cynomolgus macaque infected with simian immunodeficiency virus (SIV) mac32H-J5 recognize a nine amino acid epitope in SIV Gag p26.

A detailed analysis of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocyte (CTL) responses and the identification of the proteins and epitopes they target may improve the design of immunotherapeutic interventions and provide insights into AIDS pathogenesis. Here, we identified a new CTL epitope in the SIV Gag protein, recognized by CD8+ and MHC class I-restricted CTL clones from a long-term asymptomatic cynomolgus macaque (Macaca fascicularis) infected with SIVmac32H-J5. Using overlapping synthetic peptides, the optimal minimal epitope was characterized as a nine amino acid peptide representing amino acids 242-250 of p26 (SVDEQIQWM). CTL recognition was shown to be abolished by amino acid substitutions observed within homologous human immunodeficiency virus (HIV)-1 and HIV-2 sequences.

Chemical inactivation of recombinant vaccinia viruses and the effects on antigenicity and immunogenicity of recombinant simian immunodeficiency virus envelope glycoproteins

The efficiency of paraformaldehyde (PFA) and binary ethylenimine (BEI) in inactivating recombinant vaccinia virus (rVV), present in baby hamster kidney cells expressing simian immunodeficiency virus envelope glycoproteins (SIV-Env), was measured in a series of inactivation studies. Both compounds were shown to be effective in reducing rVV titres. The use of standard 3-day titration assays proved to be inadequate to measure PFA inactivation, since upon prolonged incubation, residual rVV infectivity was detected in cultures negative at 3 days. Different procedures using PFA or BEI were selected to assess their influence on the antigenicity and immunogenicity or rVV expressed SIV-Env. Antigenicity, as defined by the ability to react with a panel of monoclonal antibodies recognizing major antigenic sites, and immunogenicity, as defined by the ability to induce SIV envelope specific and virus neutralizing serum antibodies in rats, proved to be preserved after either inactivation procedure. These data show that both protocols using PFA or BEI can be used successfully as part of the procedures to remove residual rVV infectivity.

Simian immunodeficiency virus (SIV)-specific CD8+ cytotoxic T lymphocyte responses of naive and vaccinated cynomolgus macaques infected with SIVmac32H(J5): quantitative analysis by in vitro antigenic stimulation

Detailed analyses of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocyte (CTL) responses in vaccinated and infected macaques may help to clarify the role of CTL immunity in protection against lentiviruses. Here, the optimal conditions for the measurement of SIV Gag-specific CTL were investigated by bulk and limiting dilution assays of peripheral blood mononuclear cells (PBMC) from naive and vaccinated cynomolgus macaques (Macaca fascicularis) infected with SIVmac32H(J5). In vitro restimulation was generally required for CTL detection. Selective activation of CD8+ and MHC-restricted SIV Gag-specific CTL was induced by stimulation with autologous para-formaldehyde-fixed B-lymphoblastoid cell lines infected with a recombinant vaccinia virus expressing SIV Gag. Applied to limiting dilution assays, antigenic stimulation reproducibly demonstrated SIV Gag-specific CTL precursors (CTLp) in PBMC of all animals studied, including those lacking significant responses in standard bulk CTL assays.

Decline of Simian Immunodeficiency Virus (SIV)-Specific Cytotoxic T Lymphocytes in the Peripheral Blood of Long-Term Nonprogressing Macaques Infected with SIVmac32H-J5

The evolution of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocyte precursors (CTLps) and their relationship with virus replication were studied in SIV-infected macaques. After primary viremia, 3 of 8 macaques lost culturable virus and polymerase chain reaction-detectable provirus in peripheral blood. Although proviral DNA persisted in the spleen and lymph nodes, virus loads were below or barely above detection levels. Throughout the study, the 3 macaques remained asymptomatic, with stable CD4+ cell counts. These findings were associated with the detection of CTLps directed against both structural and regulatory SIV proteins. The response peaked during the first 7 months of infection but waned subsequently. CTLps increased after rechallenge of 1 macaque, suggesting that limited antigenic stimulation contributed to their disappearance from circulation. Transient viremia with increasing CTLp frequencies and antibody titers also suggested at least partial susceptibility to reinfection. These findings bear implications for vaccination strategies aimed at inducing protective CTLs against lentiviruses.