Synthesis of bacterial mimetics of HIV-1 gp120 glycan epitopes

Combating the worldwide pandemic caused by the human immunodeficiency virus (HIV-1) is still a formidable challenge despite the progress made in antiviral drug design. The development of broadly cross-protective and neutralizing antibodies is counteracted by various immune evasion strategies of the virus. Still, the tight glycan clusters at the viral cell envelope spikes being present in the oligomannosidic glycoprotein gp120 may be exploited as Achilles heel target for antibody recognition. A limited number of gp120 directed monoclonal antibodies has been elaborated with highly unique epitope specificities but due to the close similarity of these epitopes with mammalian glycoprotein glycans the binding of gp120-specific antibodies is usually characterized by a low immunogenicity and low affinity. Recently, a gp120-related oligomannosyl glycan has been found connected to the core unit of the Gram-negative lipopolysaccharide of the soil bacterium Rhizobium radiobacter. In preliminary experiments these glycan structures cross-reacted with HIV-specific antibodies with modest affinity. Structural data of a gp120 ligand and the bacterial glycan in the binding sites of two monoclonal anti HIV antibodies indicated that the bacterial scaffold might provide additional binding interactions with mannosyl residues. Moreover, since the bacterial sugars are inherently recognized as foreign by the immune system, an enhanced immune response against these antigens and reduced autoreactivity may be expected. Two major hypotheses may thus be put forward: Do novel, modified HIV-related bacterial glycans mimic the viral gp120 epitopes? Second, will these glycans be more efficiently recognized by anti-HIV monoclonal antibodies and could elicit neutralizing antibodies? The project will focus on the chemical synthesis of oligosaccharides up to the undecasaccharide level as modified and chain-extended mimics of gp120 glycan epitopes by combining the structurally conserved bacterial lipopolysaccharide component with two oligomannosyl chains. As controls, the mannosyl units will also be provided without the bacterial scaffold. The ligands will be prepared as spacer derivatives to be conjugated biotin and to protein carriers for testing of monoclonal anti-HIV antibodies. In addition large glycan clusters will be assembled, which should lead to a substantial increase in binding affinities due to the multivalent presentation of ligands. NMR spectroscopic studies will be performed to get insight into the detailed recognitions motifs of HIV-specific antibodies with the ligands. Immunochemical studies will be jointly performed with Dr. Kunert at the Department of Biotechnology (BOKU) and with Dr. Pantophlet at Simon Fraser University in Canada. Provided the binding studies are successful, future studies will include immunization with the neoglycoconjugates in order to elicit cross-reactive and neutralizing antibodies. The outcome of the project should thus substantially contribute to novel approaches in anti-HIV vaccine development and/or supplement combination therapies and will have significant impact on anti-AIDS immunogen design strategies.

Attempts to use the densely packed carbohydrate shield of the HIV envelope for the development of vaccines have met with very limited success over the last 30 years. The main reason is seen in the structural resemblance of the viral sugar coat with the mammalian surface carbohydrates thereby preventing immune activation. Within the framework of the FWF-project and in cooperation with international project collaborators this immune tolerance could be overcome by subtle variations of the basic carbohydrate structures thereby opening new perspectives in the field of vaccine development against HIV. The hypothesis on which the project was built could be confirmed by showing that the resemblance of a bacterial cell wall carbohydrate from a phytopathogenic soil bacterium with the viral cell surface sugars could be utilized to generate potent anti HIV antibodies. First, the bacterial cell wall fragment was synthesized, then elongated with synthetic viral mannose units and eventually coupled to a protein carrier (bovine serum albumin) in varying ligand densities and using different conjugation approaches. The immune reagents prepared in Vienna were then used by the Canadian project partner to immunize rats harboring a humanized immune system. It could then be shown that the resulting immune sera had neutralizing activity against five out of seven HIV strains. One of the target compounds showed an unexpectedly high binding affinity towards broadly neutralizing antibodies of the PGT antibody family. These antibodies develop in some AIDS patients in the course of a few years and show protective activities against many HIV strains. The molecular details of the binding motifs of the lead compound complexed to a broadly neutralizing PGT antibody were determined in cooperation with the Scripps Research Institute and thus confirmed the structural similarity of the viral and bacterial carbohydrate in the binding site. Thus, the novel synthetic immunoreagents allow for options to generate similar neutralizing and protective antibodies by active immunization in a relatively short time. For future applications, however, alternative protein carriers, that are registered for vaccine applications have to be used. In addition, the immunization scheme has to be optimized as well as maturation of the immunoglobulins (IgG). These research issues are presently being pursued in a follow-up project, funded by the National Institutes of Health (NIH, USA) in collaboration with Simon Fraser University (Canada).