Novel Immunogens based on the gp41 protein of Human Immunodeficiency Virus type 1 and Display Systems for their Affinity Maturation

Abstract

As the Human Immunodeficiency Virus type 1 (HIV-1) pandemic progresses, a prophylactic vaccine is highly desirable. Neutralizing antibodies are thought to be crucial to mediate sterilizing immunity. A few broadly neutralizing monoclonal antibodies (BNMAbs) against HIV-1 have been described which target highly conserved regions of the envelope protein. The membrane-proximal external region (MPER) of gp41 is particularly conserved and target for the potent BNMAbs 2F5 and 4E10. Past immunization efforts were not able to induce a strong neutralizing immune response against this target so far. Besides epitope exposure and stabilization, affinity maturation of immunogens to BNMAbs might be a way to enhance the quality of immune responses. However, as gp41 is a membrane-bound mammalian protein, only limited display strategies like retroviral display are suitable for this purpose, and a display system based on HIV-1 might be an ideal platform. The objective of this work was to design and evaluate novel immunogens based on the HIV-1 gp41 MPER with the potential to elicit cross-clade neutralizing antibodies. In a complementary approach, novel methods for the affinity maturation of gp41-based immunogens were to be established. The following strategies were pursued to build an effective immunogen: gp41 was truncated N-terminally in order to dispose of immunodominant, non-neutralizing sites and enhance the exposure of conserved regions. Heterologous zipper domains were introduced to stabilize a trimeric conformation. Resulting constructs were modelled in silico, and checked in vitro for the desired specifications. Cell surface exposure and selective binding to BNMAbs 2F5 and 4E10 could be shown by cytometric analyses. Incorporation into VLPs and preservation of antigenic structures were verified by electron microscopy studies. Crosslinking experiments revealed an oligomeric state of all constructs and its stabilization by zipper domains. The immunogens were tested in two rodent immunization studies, as stand-alone constructs in a homologous DNA prime and VLP boost regimen, or in combination with two existing gp41-derived soluble proteins. Although sufficient titers of anti-MPER antibodies were measured by IgG ELISA in some groups, neutralizing activity was mostly limited. Best results were obtained by a DNA prime with a truncated gp41 construct and a boost with a soluble protein engrafting the MPER and stabilized by trimerization domains. For affinity maturation of gp41 and its derivatives, a novel lentiviral display system based on HIV-1 was developed. Both packaging function and antigen library were integrated in a proviral genome. A mini-library, tailor-made for 4E10, was created and subjected to proof-of-concept studies. A second cell-based system using fluorescence-activated cell sorting (FACS) was adapted for use with gp41-derived antigens and further developed regarding the gating strategy. Both systems led to an enrichment of strong binders in only one round, with the cell-based system being more effective as it enables normalization by antigen expression level. Finally, a complex gp41 library was created to be screened for affinity-enhanced variants. Further refinements of the immunogens based on these results and successful affinity maturation might elicit functional immune responses towards the conserved MPER in future studies.