Streamlined design of a self-inactivating feline immunodeficiency virus vector for transducing ex vivo dendritic cells and T lymphocytes.

BACKGROUND: Safe and efficient vector systems for delivering antigens or immunomodulatory molecules to dendritic cells (DCs), T lymphocytes or both are considered effective means of eliciting adaptive immune responses and modulating their type, extent, and duration. As a possible tool toward this end, we have developed a self-inactivating vector derived from feline immunodeficiency virus (FIV) showing performance characteristics similar to human immunodeficiency virus-derived vectors but devoid of the safety concerns these vectors have raised. METHODS: The pseudotyped FIV particles were generated with a three-plasmid system consisting of: the packaging construct, providing Gag, Pol and the accessory proteins; the vector(s), basically containing FIV packaging signal (psi), Rev responsive element, R-U5 region at both ends, and the green fluorescent protein as reporter gene; and the Env plasmid, encoding the G protein of vesicular stomatitis virus (VSV-G) or the chimeric RD114 protein. Both packaging and vector constructs were derived from p34TF10, a replication competent molecular clone of FIV. The pseudotyped particles were produced by transient transfection in the Crandell feline fibroblast kidney (CrFK) or the human epithelial (293T) cell line. RESULTS: To broaden its species tropism, the final vector construct was achieved through a series of intermediate constructs bearing a longer psi, the FIV central polypurin tract sequence (cPPT), or the woodchuck hepatitis post-regulatory element (WPRE). These constructs were compared for efficiency and duration of transduction in CrFK or 293T cells and in the murine fibroblast cell line NIH-3T3. Whereas psi elongation and cPPT addition did not bring any obvious benefit, insertion of WPRE downstream GFP greatly improved vector performances. To maximize the efficiency of transduction for ex-vivo murine DCs and T-lymphocytes, this construct was tested with VSV-G or RD114 and using different transduction protocols. The results indicated that the FIV construct derived herein stably transduced both cell types, provided that appropriate vector makeup and transduction protocol were used. Further, transduced DCs underwent changes suggestive of an induced maturation. CONCLUSION: In contrast to previously described FIV vectors that were poorly efficient in delivering genetic material to DCs and T lymphocytes, the vector developed herein has potential for use in experimental immunization strategies.

Adoptive immunotherapy of feline immunodeficiency virus with autologous ex vivo-stimulated lymphoid cells modulates virus and T-cell subsets in blood.

The potential of immunotherapy with autologous virus-specific T cells to affect the course of feline immunodeficiency virus (FIV) infection was explored in a group of specific-pathogen-free cats infected with FIV a minimum of 10 months earlier. Popliteal lymph node cells were stimulated by cocultivation with UV-inactivated autologous fibroblasts infected with recombinant vaccinia viruses expressing either FIV gag or env gene products, followed by expansion in interleukin-2. One or two infusions of both Gag- and Env-stimulated cells resulted in a slow increase in FIV-specific gamma interferon-secreting T cells in the circulation of cats. In the same animals, viral set points fluctuated widely during the first 2 to 3 weeks after adoptive transfer and then returned to pretreatment levels. The preexisting viral quasispecies was also found to be modulated, whereas no novel viral variants were detected. Circulating CD4(+) counts underwent a dramatic decline early after treatment. CD4/CD8 ratios remained instead essentially unchanged and eventually improved in some animals. In contrast, a single infusion of Gag-stimulated cells alone produced no apparent modulations of infection.

Genetic and phylogenetic analyses of HIV-1 corroborate the transmission link hypothesis.

BACKGROUND: Phylogenetic and genetic analyses have proven a valuable tool to infer epidemiological links between human immunodeficiency virus type-1 (HIV-1) isolates. These methods were applied in the present report for studying the genetic relatedness of the viral strains involved in two episodes of suspected HIV-1 transmission. OBJECTIVES: Provide any evidence that may help establish or refute the transmission link. STUDY DESIGN: In the first case, a leukemic patient became HIV-1 positive following the transfusion of platelets from a donor who was subsequently found to have tested false HIV-seronegative and to be sexual partner to an infected woman. In the second, a wife claimed to have acquired the infection from her husband who had concealed his infected status. RESULTS AND CONCLUSIONS: The viral pairs detected in each of the suspected transmission cases exhibited common amino acid signatures and low genetic distances and segregated together in phylogenetic trees, thus showing a level of genetic relatedness similar to reference pairs known with certainty to be epidemiologically linked. These findings corroborated the existence of a direct transmission link in both the episodes with a high level of confidence.