Daniele Saverino

Anti-HIV genetic treatment of antigen-specific human CD4 lymphocytes for adoptive immunotherapy of opportunistic infections in AIDS

HIV-1 infection results in the loss of CD4+ T helper lymphocytes which make up the immune repertoire. This leads to opportunistic infections that define AIDS. Here, we show that CD4 T cell lines from normal donors with specificity for different antigens can be rendered resistant to HIV-1 replication by retroviral transduction with an antisense vector directed to the HIV-1 tat gene. The genetic treatment did not affect the properties of antigen-specific CD4 lymphocytes such as proliferative response, lymphokine production and phenotypic markers. The HIV-1 challenge dose that resulted in productive infection was two to four logs higher for transduced cells as compared with control cells. Resistance was shown with the HXB2 strain, whose tat sequence was used to design the antisense gene, and with the SF2 strain, whose targeted tat sequence carries five nucleotide mismatches. Retroviral transduction was also performed on a Candida-specific T cell line from a seropositive individual. This line, derived from T cells infected in vivo, produced infectious virus when stimulated in vitro with antigen, but was no longer productive after transduction. In addition, a four log higher HIV-1 challenge dose was needed for a productive superinfection of this T cell line. The production of antigen-specific CD4 T cells resistant to HIV-1 replication to be used in adoptive immunotherapy of opportunistic infections may represent a new form of gene therapy of AIDS.

Human CD4+ T cells can discriminate the molecular and structural context of T epitopes of HIV gp120 and HIV p66

CD4+ T cell lines and clones specific for human immunodeficiency virus (HIV) antigens have been generated from peripheral lymphocytes of naive individuals by priming with the envelope protein gp120, the enzyme reverse transcriptase (p66), and their synthetic peptides. T cells were tested for proliferation to proteins, to peptides, and to HIV virions. Different patterns of reaction were identified. T cells primed in vitro with the whole antigen responded to the protein, but recognition of overlapping peptides occurred with a fraction of the lines or clones. The virus was recognized by some, but not all, of the gp120- and p66-specific T cells, with an efficiency 2 logs higher than the recombinant soluble proteins on a molar basis. One T cell line specific for gp120 responded to virions presented by B cells, but not by monocytes. In contrast, T cells induced with peptides did not always respond to the proteins. Generation of T cell lines from naive individuals may be an in vitro model for T cell immunization, and the response patterns may have implications for the design of vaccines aimed at inducing a T helper response. In fact our in vitro data suggest that (a) immunization with peptides does not always induce T cells recognizing the whole protein, (b) immunization with proteins does not always induce T cells recognizing the protein in the context of the HIV virus, and (c) recognition of gp120 in the context of HIV may be dictated by the type of presenting cells.

Human T leukaemia virus type 1 (HTLV-1) specific T-helper cell response: clonal fluctuations and repertoire heterogeneity.

The naive T‐helper (Th) repertoire specific for HTLV‐1 envelope (env) has been examined on antigen specific T‐cell lines and clones from non‐immune individuals. Clonal heterogeneity was determined by analysing the T‐cell receptor (TCR) Vβ gene usage and by sequencing the hypervariable regions of the TCR genes. Fluctuations in the Vβ gene usage were determined by comparing the TCR Vβ gene profiles of T‐cell lines at different times. We found that a diverse repertoire for HTLV‐1 env could be triggered in vitro. Diverse Vβ genes were used by the same line tested at different times, suggesting that clonal composition of an antigen‐specific T‐cell line is not constant in vitro. Clones in fact may be up‐ and down‐regulated and clonotypes undetectable at one time point can emerge upon subsequent restimulation. Therefore evaluation of the clonal composition of a T‐cell line gives a snapshot of the dominant clones at the time of analysis, and does not tell the whole picture of the antigen‐specific ensemble. Furthermore, by sequencing the TCR genes, we identified clones with identical Vβ gene usage which differed in hypervariable regions (CDR3), indicating their derivation from independent precursors and contributing to overall clonal heterogeneity. If these data can be extended to HTLV‐1‐infected patients studied in vivo, the Th cell repertoire specific for HTLV‐1 env may prove very heterogenous, with important implications for vaccine development.

Handling of retroviral antigens by human antigen-presenting cells

Antigen-specific T helper cells play an important role in retroviral infections. Indeed, they provide help for B-cell activation and antibody production and for clonal expansion of cytolytic lymphocytes. Therefore, we used retrovirus-specific human T helper clones in order to define modes of antigen presentation, antigen-presenting cells and the molecular context of Th epitopes that could be exploited in the design of immunogens aimed at optimizing the Th cell response. In particular, we describe several mechanisms of receptor-mediated antigen uptake that enhance the stimulation of human T-cell clones specific for HIV and HTLV-1 antigens; we report on the differential recognition of Th epitopes depending on the molecular-viral context; we show that dendritic cells are the most efficient presenting cells and are essential for the induction of in vitro primary Th cell responses; and finally, we propose that Th cells specific for internal, conserved antigens of HIV such as reverse transcriptase, may be candidates for intrastructural help resulting in induction of envelope specific antibodies.

Identification of Salmonella enterica Serovar Typhi DNA Fragments with Transcriptional Activity Under Different Growth Conditions

Salmonella enterica serovar Typhi is a human pathogenic microorganism with a very complex infective cycle, involving the transit of bacteria across different microenvironments; to optimize the performance of attenuated Salmonella strains suitable as live carriers of heterologous antigens, fine tuning of wild type bacteria gene expression is essential. Several DNA fragments were obtained from a Salmonella enterica serovar Typhi (vi+, fim+) blood isolate and 18 clones were selected according to the dimension of the insert (range <0.2-1.6 kb). These fragments showed a transcriptional ac- tivity in a promoterless vector cloned in Escherichia coli background, according to homogeneous parameters. The results obtained provide an insight about signals mediating gene activation in vivo, particularly in the microenvironments known to exist during the infectious process, even if the fragments are not promoters sequences. Finally, the functional charac- terization of several fragments showed that they possessed an efficient and homogeneous transcriptional activity, worth to be further investigated.