Research InterestsDevelopment of Mucosal Vaccines Against HIV-1Consequent to a decade long collaboration with David Hone, Ph.D. of the Institute of Human Virology we have developed the first bacterial-vector vaccine against HIV-1 to go to Phase I clinical trials. This vaccine is based on an attenuated carrier strain of S. typhi developed originally in the Center for Vaccine Development that carries a stabilized plasmid encoding a truncated HIV-1 envelope gene that produces secreted gp120 fragment. This construct induces both humoral and cellular immunity in the systemic and mucosal compartments after a single oral inoculation in laboratory animals. It is currently under evaluation in humans via a collaborative project among the IHV, CVD, and AIDS Vaccine Evaluation Group at Johns Hopkins as a part of a prime-boost protocol in conjunction with a soluble, recombinant gp120 vaccine produced by VaxGen. We have developed second and third generation Salmonella based HIV-1 DNA vaccines that encode codon optmized HIV-1 genes on plasmids encoding eukaryotic promoters. We have shown these vaccines to elicit strong CD8+ T cell responses in experimental animals and a plan has been formulated to take these new vaccines to Phase I trials in 2000. This work has led to the award of a broad based patent to Drs. Lewis, Powell, and Hone that covers the delivery of any DNA vaccine by any bacterium. The mechansims whereby this form of transfection occurs is currently under investigation in our group. Additional studies on the development of mucosal vaccines against HIV-1 include the development of new envelope constructs that elicit broadly neutralizing antibodies and the fusion of these constructs with the biologically active moieties of cholera toxin or E. coli heat-labile enterotoxin. Additional collaborators on these projects include Dr. Nick Carbonetti, Dr. Anthony L. Devico, Dr. Tim Fouts, Dr. Roberta Kamin-Lewis, Dr. Marvin Reitz, and Dr. Carol Tacket. The Role of Glycosaminoglycan Binding in the Biological Activities of b-chemokines We are investigating the role glycosaminoglycan binding in the biological activities of b-chemokines. In a recent series of studies, we isolated a monoclonal antibody against the b-chemokine, RANTES, that blocks its anti-viral effect against HIV-1. Epitope mapping studies showed that this antibody recognizes an epitope that overlaps the glycosaminoglycan binding domain of RANTES, suggesting a role for this structure in the anti-viral effect of this b-chemokine. This suggestion has been borne out in subsequent experiments and extended by the finding that removal of cell surface GAGs abrogates the ability of RANTES to elicit intracellular signaling via canonical 7-TM domain receptors such as CCR5. This observation has been extended to a wide array of both a-chemokines and b-chemokines. Thus, GAG binding is a key determinant in the biological activities of chemokines and we are now probing the structural basis of specificity in GAG binding by site directed mutagenesis of the GAG-biding domain of RANTES and by determination of the sugar structures recognized by this b-chemokine. These studies should provide new information on the molecular basis of chemokine function and new reagents to track the expression of specific GAGs on the cell surface by flow cytometry. In addition, we have shown that soluble complexes of RANTES and GAGs exhibit enhanced anti-viral activity against HIV-1 while the ability to elicit intracellular Ca++ signals is abrogated. This finding suggests that soluble complexes of RANTES and GAGs could be used in vivo against HIV-1 without the complications of the other biological responses to RANTES that depend on signaling. Furthermore, this observation provides a new clue about the biological activity of RANTES that is secreted as a GAG complex by HIV-1 specific, CD8+ cytotoxic T cells. Our results suggest that such complexes will block infection of CD4+ T cells by HIV-1 without attracting uninfected cells into the proximity of the HIV-1 infected cell and the CTL. Collaborators on this project include Drs. Anthony L. DeVico and Robert Gallo, and Ms. Jennifer Burns. Identification of b-chemokine Secreting T Cell Subsets as Correlates of Protective Immunity Against HIV-1. There is emerging consensus that anti-viral b-chemokines produced by T cells are correlates of protective immunity against HIV-1. We are investigating whether the production of b-chemokines by HIV-1 specific T cell subsets is a correlate of protective immunity against HIV-1. We have developed both polyclonal and antigen-driven assays to identify T cell subsets that secrete b-chemokines in conjunction with other cytokines. These assays are based on 4 to 6 color flow cytometry and the combined staining for cell surface markers and intracellular chemokines or cytokines. These systems are being used in three clinical settings to identify T cell subsets that predict protective immunity against HIV-1. These include an acute seroconverter cohort, a cohort of exposed-uninfected individuals, and a longitudinal study of HIV-1 infected individuals immunized with therapeutic vaccines. As these studies are still underway, no conclusions can be drawn regarding subsets that predict protective immunity. Studies in normal individuals have shown that CD8+ T cells have a much greater capacity to synthesize anti-viral b-chemokines than do CD4+ T cells. Limited studies in infected individuals suggest further that b-chemokine synthesis is most pronounced in an expanded subpopulation of CD8+ T cells defined by CD45 isoforms and L-selection that is uncommon in normal individuals. Collaborators on this work include Drs. Roberta Kamin-Lewis, Anthony L. DeVico, Fiorenza Cocchi, William Blattner, Farley Cleghorn, and Robert C. Gallo. Publications
Burns, J.M., R.C. Gallo, A. DeVico, and G.K. Lewis. A new monoclonal antibody, mAb 4A12, indentifies a role for the glycosaminoglycan (GAG) binding domain of RANTES in the anti-viral effect against HIV-1 and intracellular Ca++ signaling. Journal of Experimental Medicine, in press. |
