A human mutant CD4 molecule resistant to HIV-1 binding restores helper T-lymphocyte functions in murine CD4-deficient mice

CD4 is a cell surface glycoprotein that acts as a co-receptor for the T cell antigen receptor by binding to a non-polymorphic portion of MHC molecules. CD4 also functions as a receptor for human immunodeficiency virus type-I (HIV-1) because the viral envelope glycoprotein gp120 binds to CD4 with a high affinity. We have previously demonstrated that introduction of mutations into CD4 abolished the binding of gp120 and prevented HIV-1 from entering cells and spreading. However, whether introduction of such mutations into CD4 causes decreased binding to MHC and loss of function is yet to be determined. We generated transgenic mouse lines by injecting a mutant human CD4 (muthCD4) gene under a murine CD4 enhancer/promoter to ensure tissue and stage specific expression. To exclude the influence of endogenous murine CD4, transgenic mice were crossed with murine CD4-targeted mice to produce muthCD4 transgenic mice lacking endogenous CD4 (muthCD4TG/KO mice). In these mice, T lymphocytes expressing muthCD4 expanded and matured in the thymus and were present in the spleen and lymph nodes. They also activated B cells to mount an antibody response to a T-dependent antigen. The results from this study suggest that a human variant of CD4 modified to be resistant to HIV-1 binding can rescue the signaling for T cell development in the thymus in vivo, having helper T cell functions. Thus, further characterization of muthCD4 molecules should open the way to new HIV treatment modalities.

Production of mouse anti-CD4 antibodies by DNA-based immunization.

The intramuscular injection of plasmid DNA encoding an antigenic protein has been developed recently as a tool for immunization. DNA-based immunization was shown to generate immune responses against the encoded antigen in diverse animal species. In this report, we present the use of DNA-based immunization for the production of antibodies to CD4, a human leukocyte surface molecule. Mice were injected intramuscularly with eukaryotic expression vector containing cDNA encoding CD4 protein, termed CD4-DNA, and were subsequently assayed for anti-CD4 antibody production by indirect immunofluorescence. Sera collected from 2 of 3 inoculated mice reacted with CD4 expressing transfected COS cells and Sup-T1 cells. Anti-CD4 antibody activity was abolished by adsorption with CD4 molecule expressing cells. CD4+ cell depleted lymphocytes were also used to confirm the specificity of the anti CD4 antibodies present in immune serum. CD4-DNA immune serum reacted with approximately 1/3 of freshly isolated lymphocytes but to very few cells in the CD4+ cells-depleted preparation. CD4-DNA immunized sera was used to enumerate CD4+ cells in the peripheral blood of 6 healthy donors and 2 AIDS patients. The number of CD4+ cells estimated by DNA immunized sera was very similar to estimates using standard anti-CD4 monoclonal antibody Leu3a. DNA-based immunization is therefore capable of raising antibodies to human leukocyte surface antigens. This technology may be useful for producing antibodies to other cell surface antigens in mice or other animals.