Complement-mediated neutralization of HIV: Search for a new therapeutical approach

World wide, huge efforts have been undertaken to eliminate human immunodeficiency virus (HIV). However, all strategies developed so far have been only partially successful. Therefore, we try in this study to develop an alternative therapeutical approach by inducing complement-mediated cylotoxicity and virolysis to neutralise this retrovirus and destroy HIV-infected cells. The complement system represents one of the main branches of innate immunity and contributes to the humoral defence mechanisms of the host. Activation of the complement cascade results in deposition of C3-fragments on invading pathogens, a process called opsonisation. When an certain activation threshold is exceeded and enough complement is deposited on the surface, complement-dependent lysis of micro-organisms or infected cells will result. Therefore, nearly all pathogens which get in contact with human serum have developed strategies to escape from this immune defence. One of the pathogens which are resistant to complement is HIV. During recent years we could show that this virus has acquired membrane-anchored and humoral regulators of complement activation (RCA) and is therefore protected against complement--dependent virolysis (CDV). The aim of the project is to block these protecting molecules and to achieve lysis by a three step approach, Firstly, complement activation should be induced only on HIV and HIV-infected cells. To enhance complement activation further, secondly, attachment of humoral RCA should be selectively blocked on the viral surface and on infected cells and thirdly, rnembrane-anchored RCA should be neutralised on HIV and HIV-infected cells to guarantee that a certain activation threshold is overcome and complement-mediated lysis is induced. Hereby, complement activation should be directed in such a way that uninfected cells are not affected. Combination of selective complement activation and simultaneous blocking of inhibitors of complement activation (RCAs) on HIV and HIV-infected cells shall result in massive localised complement activation on and, thus lysis of HIV and HIV-infected cells. This might offer a new therapeutical approach to neutralise and clear HIV and HIV-infected cells by the host`s own immune system.

The complement system represents one of the main branches of innate immunity and contributes to the humoral defence mechanisms of the host. Although activating the complement system, the Human immunodeficiency virus (HIV) is protected against complement-mediated lysis (COML). The aim of the project was to block these protecting molecules and to achieve lysis, which might offer a new therapeutical approach to neutralise and clear HIV and HIV-infected cells by the host`s own immune system. To achieve this aim, we tried to: 1. to ensure specificity, 2. to create a "fH free" area on special targets selectively, 3. to neutralise membrane anchored RCA on special targets selectively, 4. to manipulate the law of mass action to combine specificity for HIV and HIV-infected cells with a "fH free" situation by a bifunctional reagent, 5. to manipulate the law of mass action to combine specificity for HIV and HIV-infected cells with neutralisation of RCAs by a multi-functional reagents. ad 1. All sera derived from HIV-infected individuals were able to activate complement. In the presence of the HIV-specific mAb 2G12, the activation was further enhanced. No C3-deposition (i.e. complement activation) was observed, when cell were tested, which were not infected with the virus. The findings were confirmed by in vivo experiments with HIV-infected individuals. Complement activation correlated with HIV neutralization in these patients (Stiegler et. al. AIDS, 2002). ad 2. An interaction site on HIV and HIV-infected cells was localized in SCR13 of fH (H. Stoiber et al. Immunobiology, 2001). This peptide induced COML similar as sera, which were depleted by fH, indicating that the SCR13 derived peptide creates a "fH free" area. ad 3 and 4, Fab fragments of CD59 and DAF were generated and used in the lysis assays. The completed antibody and the Fab fragments induced COML to similar amounts and was significantly enhanced, when the Fab fragments were chemically cross-linked to the mAb 2G12. ad 5, Further enhancement of COML by multi-functional constructs failed so far. To identify further antibodies, which are suitable for coupling to the complement constructs, we investigated sera of polytransfused patients ex vivo (Wilflingseder et. al. IAAI, 131:62-72). To test complement-based neutralization in vivo, we switched to the mouse model and included mouse mammary tumor virus (MMTV). Constructs, which are specific for mouse complement were generated and induced COML, similar to the HIV-specific compounds. Present, we generate constructs for tests in the animals. In total, the project resulted in 10 publications (see appendix), with 9 already published.