Human cytomegalovirus (HCMV) infections are particularly severe in immunodeficient individuals. New therapies for treatment of these infections are warranted, particularly because of the recrudescence of HCMV discase following termination of therapy with presently approved drugs (ganciclovir [DHPG] and foscarnet). The new HCMV therapies developed in this project will utilize immunotoxins (ITs), which are hybrid molecules constructed by attaching a cytotoxin to an antibody (immunoglobulin, Ig) that specifically binds to HCMV-infected cells. This proposed research will develop an understanding of the effects of these ITs in vitro and in murine cytomegalovirus (MCMV)-infected severe combined immunodeficient (SCID) mice. This animal model, now established in our laboratory, responds to antiviral therapy in a manner similar to the response of HCMV infections in immunodeficient human patients. An hypothesis to be tested is that upon cessation of DHBG therapy, infected cells that have escaped eradication resume production of infectious virus and are, in part, responsible for CMV disease recrudescence. These cells, because they express CMV antigens, will be potential targets to attack by ITs which contain antibodies directed against those antigens. We propose to utilize IT/DBPG combination therapy. It is proposed that as a consequence of this targeted toxicity, the infected cells will be destroyed, resulting in a decrease in virus levels in tissues and an increase in time to CMV disease relapse. We hypothesize that variables in IT construction will modify these anti-CMV effects, and that the immunodeficient nature of the infected host will prevent significant humoral immunity from developing against the IT. We further hypothesize that CMV-neutralizing antibody will enhance the antiviral activity of the IT/DHPG therapy in the MCMV-infected SCID mouse. To test these hypotheses: a) Polyclonal anti-CMV Igs will be attached to the toxin gelonin and evaluated for specific cell-killing activity, nonspecific cytotoxicity and antiviral activity in vitro vs HCMV and MCMV. b) Variables in Ig (polyclonal or monoclonal, epitope specificity, isotype, species of origin, Ig fragments), type of toxin-to-Ig linkage, and type of toxin will be studied with representative ITs in vitro, and, with selected active materials, in vivo vs MCMV. c) Combinations of active ITs, DHPG, and neutralizing antibodies will be studied vs MCMV in SCID mice, with effects determined on infectious virus levels and potentially latent viral genome in tissues, the latter using polymerase chain reaction methodology. d) The humoral immune responses to the toxin and the immunoglobulin portions of the ITS will be characterized, and the ability of the possible resultant antibodies to block the IT activity will be measured.
NIH ExPorter
NIH ExPorter