Characterization of the CD4-specific antibody response of a recombinant soluble CD4-immunized, HIV-1-infected human through the construction of a combinatorial Fab library.

摘要

It has previously been demonstrated that CD4-specific, HIV-1-neutralizing antibodies can be raised by immunizing nonhuman primates with recombinant soluble CD4 (rsCD4). Studies are described in this thesis which characterize the CD4-specific antibodies raised in humans by employing such an immunization strategy. A combinatorial Fab library was generated from bone marrow mRNA of a human rsCD4-immunized, HIV-1-infected human. This library was constructed using the pCOMB3 phagemid, which directs the expression of recombinant Fab-expressing M13 bacteriophage. Human rsCD4-specific Fab clones were selected by panning the library either against human rsCD4 or against performed complexes of human rsCD4 and recombinant HIV-1 gp120 (rgp120). While none of the rsCD4-specific Fabs selected from this library recognized human T lymphocytes, as assessed by flow cytometry, several bound to CD4-positive cells preincubated with either recombinant HIV-1 gp120 or infectious HIV-1 virions. The binding properties of one of these Fab clones (Fab 3-47) were characterized in detail. The possibility that this Fab was specific for the HIV-1 envelope protein was ruled out by showing that it did not react with recombinant HIV-1 gp120 or with HIV-1 envelope- expressing cells. The CD4 specificity of this clone was confirmed by demonstrating that Fab 3-47 bound to digitonin-permeablilized CD4-positive cells, and immunoprecipitated a 55 kDa cell surface protein corresponding to the predicted molecular weight of CD4 from a human CD4-positive T cell line. The binding specificity of Fab clone 3-47 suggests that HIV-1 induces a conformational change in cell surface-expressed CD4.; Preliminary studies suggested that Fab 3-47, by binding to this conformationally altered CD4 molecule, may inhibit postbinding events in HIV-1 infection. However, contaminants in these bacterially-expressed Fab preparations resulted in nonspecific cellular activation, making it difficult to assess the neutralizing activity of this Fab. Fab 3-47 is currently being synthesized as a full length immunoglobulin in a mammalian expression system in order to confirm its virus neutralizing activity. In addition to identifying novel targets for the design of antiviral therapeutics, this Fab, or Fabs with a similar binding specificity, may be valuable inhibitors of HIV infection in vivo.