Antibody-directed targeting of retroviral vectors via cell surface antigens.

Targeted stable transduction of specific cells is a highly desirable goal for gene therapy applications. We report an efficient and broadly applicable approach for targeting retroviral vectors to specific cells. We find that the envelope of the alphavirus Sindbis virus can pseudotype human immunodeficiency virus type 1- and murine leukemia virus-based retroviral vectors. When modified to contain the Fc-binding domain of protein A, this envelope gives a significant enhancement in specificity in combination with antibodies specific for HLA and CD4 relative to that without antibody. Unlike previous targeting strategies for retroviral transduction, the virus titers are relatively high and stable and can be further increased by ultracentrifugation. This study provides proof of principle for a targeting strategy that would be generally useful for many gene therapy applications.

An inducible human immunodeficiency virus type 1 (HIV-1) vector which effectively suppresses HIV-1 replication.

Recently, gene therapy vectors based upon the human immunodeficiency virus type 1 (HIV-1) genome have been developed. Here, we create an HIV-1 vector which is defective for all HIV-1 genes, but which maintains cis-acting elements required for efficient packaging, infection, and expression. In T cells transduced by this vector, vector expression is low but efficiently induced following HIV-1 infection. Remarkably, although the HIV-1 vector does not contain specific anti-HIV-1 therapeutic genes, the presence of the vector alone is sufficient to inhibit the spread of HIV-1 infection. The mechanism of inhibition is likely to be at the level of competition for limiting substrates required for either efficient packaging or reverse transcription, thereby selecting against propagation of wild-type HIV-1. These results provide proof of a concept for potential application of a novel HIV-1 vector in HIV-1 disease.

Human immunodeficiency virus type 1 (HIV-1) infection and transcytosis activity of a HIV-1 susceptible clone from HeLa cell.

In order to clarify the transmission process of human immunodeficiency virus type 1 (HIV-1) through the epithelial cell barrier, HeLa cells susceptible and non-susceptible to HIV-1 were cloned and designated as P6 HeLa and N7 HeLa cells, respectively. P6 HeLa cells could be infected with the LAI strain of HIV-1 and mediated HIV-1 transcytosis. In contrast, N7 HeLa cells exhibited neither HIV-1 infection nor transcytosis. CD4 and galactosylceramide as the receptors for HIV-1 were not detected on P6 HeLa cells, although an anti-CD4 monoclonal antibody (mAb) blocked HIV-1 infection. Since HIV-1-infected P6 HeLa cells exhibited no fusion and survived, we speculated that the P6 HeLa cells expressed molecules other than CD4 which facilitated HIV-1 infection. Two mAbs (A-14 ITK and C57 a9-9) which inhibited the HIV-1 infection of P6 HeLa cells were generated. Each mAb recognized distinct molecule(s) as shown by Western blotting. Transcytosis by the P6 HeLa cells was inhibited by C57 a9-9 but not by A-14 ITK or anti-CD4 mAb. Both infection and transcytosis may be responsible for HIV-1 transmission through epithelial cells in a complex manner. Although infection and transcytosis occurred via different mechanisms, the molecule(s) recognized by C57 a9-9 mAb may be associated with both processes.

Isolation of human T-cell leukemia virus type I from a transformed T-cell line derived spontaneously from lymphocytes of a seronegative Egyptian patient with mycosis fungoides.

Mycosis fungoides (MF) is a rare form of cutaneous T-cell lymphoma that may be associated with human T-cell leukemia virus type I (HTLV-I) infection. Using the polymerase chain reaction, the HTLV-I pX region was constantly detected in the genomic DNA extracted from peripheral blood mononuclear cells (PBMCs) of an HTLV-I antibody-seronegative Egyptian MF patient enrolled in a study to isolate HTLV-I from North Africa. A CD4+ and interleukin-2 (IL-2) receptor-positive T-cell line was established when the phytohemagglutinin-stimulated PBMCs of that patient were maintained in IL-2-containing culture medium. The cell line (EMF) was initially IL-2 dependent and then became IL-2 independent after gradual withdrawal of the IL-2. The cells reacted positively with monoclonal antibodies specific for the HTLV-I Env or HTLV-I Gag proteins. Using the Southern blot analysis, HTLV-I provirus could be detected in the genomic DNA extracted from the EMF cells. Limited nucleotide sequence of the env region showed more than 95% homology between the EMF provirus and other known HTLV-I isolates. Western blot analysis of the cell lysates showed the expression of the HTLV-I structural proteins. These data imply that a transforming HTLV-I provirus may be present, at least in certain cases of MF, regardless of the presence or absence of the specific antibodies.