MHC-I-restricted cytotoxic responses are considered a critical component of protective immunity against viruses, including human immunodeficiency virus type 1 (HIV-1). CTLs directed against accessory and early regulatory HIV-1 proteins might be particularly effective; however, CTL epitopes in these proteins are rarely found. Novel artificial neural networks (ANNs) were used to quantitatively predict HLA-A2-binding CTL epitope peptides from publicly available full-length HIV-1 protein sequences. Epitopes were selected based on their novelty, predicted HLA-A2-binding affinity and conservation among HIV-1 strains. HLA-A2 binding was validated experimentally and binders were tested for their ability to induce CTL and IFN-c responses. About 69 % were immunogenic in HLA-A2 transgenic mice and 61 % were recognized by CD8 + T-cells from 17 HLA-A2 HIV-1-positive patients. Thus, 31 novel conserved CTL epitopes were identified in eight HIV-1 proteins, including the first HLA-A2 minimal epitopes ever reported in the accessory and regulatory proteins Vif, Vpu and Rev. Interestingly, intermediate-binding peptides of low or no immunogenicity (i.e. subdominant epitopes) were found to be antigenic and more conserved. Such epitope peptides were anchor-optimized to improve immunogenicity and further increase the number of potential vaccine epitopes. About 67 % of anchor-optimized vaccine epitopes induced immune responses against the corresponding non-immunogenic naturally occurring epitopes. This study demonstrates the potency of ANNs for identifying putative virus CTL epitopes, and the new HIV-1 CTL epitopes identified should have significant implications for HIV-1 vaccine development. As a novel vaccine approach, it is proposed to increase the coverage of HIV variants by including multiple anchor-optimized variants of the more conserved subdominant epitopes.

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Cytotoxic T-lymphocytes (CTL) are critical for immune control of infection with human immunodeficiency virus type-1 (HIV-1) and searches for relevant CTL epitopes for immune therapy are ongoing. Recently, we identified 28 HLA-A2-binding HIV-1 CTL epitopes (1). In this follow-up study we fully genome sequenced HIV-1 from 11 HLA-A2(+) patients to examine the sequence variation of these natural epitopes and compared them with the patient's CD8(+) T-cell recall response. Often the epitope was conserved but only a few patients showed a CD8(+) T-cell recall response. This infrequent targeting may be explained by immune subdominance. CD8(+) T-cell recall response to a natural epitope could be measured despite sequence differences in the patient's virus. T-cell cross-reaction between such variants could be demonstrated in HLA-A2 transgenic mice. Nine infrequently targeted but conserved or cross-reacting epitopes were identified in seven HIV-1 proteins. More immunogenic anchor amino acid optimized immunogens were designed that induced T-cell cross-reaction with these natural epitopes. It is concluded that most of the new CTL epitopes are conserved but subdominant during the infection. It is suggested that T-cell promiscuity may explain the observed CD8(+) T-cell reaction to epitope variants and it may be possible to use the selected immune optimized epitope peptides for therapeutic vaccination.

The establishment of effective regimens for a vaccine against human immunodeficiency virus type 1 (HIV-1) is urgently needed. In the present study we have produced HIV-1 gp120 from a vaccine-relevant primary R5 isolate in recombinant vaccinia (rVV)-infected Vero cells. We have investigated the effect of boosting with this protein in mixed modality immunisations of rhesus macaques following different immunisation. As reported earlier, animals were primed with codon-optimised HIV-1(BX08)env DNA delivered as plasmid or as replication-deficient recombinant human adenovirus type 5 (rAd5), which both induced specific antibody and cellular immune responses (1). Boosting with rAd5 temporarily had increased the anti-gp120 antibody titres approximately 1 log (rAd5+rAd5) or 3 log (DNA+rAd5) (1). However, secondary rAd5 boosting showed less effect due to the induced vector-specific immunity. To further boost the antibody response, the rgp120(BX08) was injected with Quadri A saponin adjuvant. The protein boosting resulted in a 1-2 log antibody increase and also boosting of the cell-mediated immune response. Neutralising antibodies to the heterologous HIV-1(MN) were detected; however, neutralising antibodies to the primary HIV-1(Bx08) isolate were seen only transiently after rAd5 but not the rgp120 immunisation. It is concluded that the rgp120(Bx08) reagent from rVV-infected Vero cells is functional and immunogenic in macaques, inducing both antibody and cellular immunity. The rgp120(Bx08) is a relevant model antigen that may be used to boost antibody and cellular immunity in mixed modality vaccine regimens against HIV-1 in higher animals.

In its natural host, porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to have a restricted tropism for cells of the monocyte/macrophage lineage. To date, cloned monkey kidney cell lines, such as MARC-145 and CL2621 cells which have been established from MA-104 cells, are the only non-porcine cells known to support PRRSV replication. In the present study, a binding assay was set up to follow by flow cytometry the attachment of PRRSV on the surface of porcine and non-porcine cells. PRRSV was found to be able to bind permissive cells like porcine alveolar macrophages and MARC-145. Further binding assays with porcine peripheral blood leukocytes showed that only monocytes can attach the virus. By their lack of binding factor, lymphocytes appeared to be refractory to PRRSV infection. Pre-incubation of MARC-145 cells with chymotrypsin and pronase E, but not neuraminidase, blocked their binding activity for PRRSV. The binding activity of the protease-treated cells was regenerated 8 hours after treatment, but cells remained unable to bind PRRSV if maintained in the presence of cycloheximide, thus confirming the proteinic nature of the specific binding factor(s). Experiments conduced with cells that have been previously characterized as non-permissive to PRRSV infection showed that many of them were able to bind the virus. Data obtained suggest that interaction of PRRSV with a specific binding factor on the surface of some cells is not sufficient to lead to a productive infection, and that a second putative receptor or other phenomena are probably required to pursue later events.