Activation of HIV-1-Specific CD8+ and CD4+ Autologous Memory T-Cells by Dendritic Cells and B-Cells Electroporated with mRNA Encoding Consensus or Autologous HIV-1 Proteins.

Abstract

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by dysfunction of HIV-1-specific T-lymphocytes. In order to suppress the virus and delay evolution to AIDS, antigen-loaded antigen-presenting cells (eg. dendritic cells (DC), B-lymphocytes) might be useful to boost and broaden HIV-1-specific T-cell responses. Monocyte-derived DC from untreated HIV-1-infected patients were electroporated with codon-optimized (“humanized”) mRNA coding for consensus HxB-2 (hHXB-2) Gag protein. These DC elicited a strong HIV-1 Gag-specific interferon (IFN)-γ response by an HLA-A2-restricted CD8+ T-cell line. Moreover, hHXB-2 gag mRNA-electroporated DC also triggered IFN-γ secretion by autologous peripheral blood mononuclear cells (PBMC), CD8+ T-cells and CD4+ T-cells from all patients tested. Similar observations were made with CD40-activated cultured autologous B-cells (from HIV-1-seropositive patients) electroporated with hHXB-2 gag mRNA. Gag mRNA-electroporated, but not mock-electroporated, DC or B-cells secreted Gag protein. Next, a novel strategy was developed, using autologous virus sequences. Proviral DNA was amplified by polymerase chain reaction (PCR) from PBMC and viral cDNA was amplified by reverse transcriptase PCR (RT-PCR) from plasma virus. Proviral and viral mRNA were then obtained by in vitro transcription of proviral DNA and plasma viral cDNA, respectively. Significant specific IFN-γ T-cell responses were induced in all patients tested by DC electroporated with patients’ autologous proviral and plasma viral mRNA, coding for Gag or Env. The stimulatory effect was seen on PBMC, CD8+ T-cells and CD4+ T-cells, demonstrating both major histocompatibility complex (MHC) class I and MHC class II antigen presentation. Moreover, a significant interleukin (IL)-2 T-cell response was induced by DC electroporated with hHxB-2 or proviral gag mRNA. Sequence analysis in 4 randomly chosen patients showed that they were infected by 4 different subtypes. In a heteroduplex mobility assay (HMA) up to 50% of the cloned amplified sequences exhibited a differential migration pattern; by sequencing a high degree of variation was demonstrated, particularly between clones derived from proviral DNA and plasma viral cDNA, with mutations in an immunodominant epitope (Gag) or mutations and deletions in non-immunodominant epitopes (Env). The stimulatory effect of autologous DC electroporated with autologous viral sequences opens a major perspective for the development of patient-specific immunotherapy for HIV-1 disease, that might be necessary to control the virus, in view of the major inter-patient and intra-patient sequence variability.