Targeting the humoral and cellular immune response

Internal image antibodies are a subclass of anti-idiotypic antibodies, which recognise the antigen-binding site of first generation antibodies comprising a replica of the epitope and are thus able to mimic the original antigen (antigenic site) functionally or even structurally. Based on this mechanism an anti-idiotypic antibody, termed Ab2/3H6 was developed against a promising human monoclonal antibody 2F5, which is able to neutralise a number of primary HIV-1 isolates. Since the development of 2F5-like antibodies has failed so far and the expression and passive administration of monoclonal antibodies (mAb) in general, is very expensive the demand for an active immunisation is still required. Ab2/3H6 is one of these potential vaccines candidates, which could be easily administered to HIV-1 infected patients as well as to healthy individuals. In the context of this FWF project the chimeric Ab2/3H6 will be completely humanized to (1) reduce the unspecific human anti mouse antibody (HAMA) response and to (2) improve the specific immune response directed against the antigen binding site of Ab2/3H6. Additionally we will focus on the cellular immune response since this type of immune reaction also plays a key role in the development of an active vaccine against HIV-1. This will be arranged out by a novel human Ab2/3H6/interleukin-15 fusion protein which will be further investigated in vitro and finally in vivo. We will also analyse the use of a specific tetanus toxin sequence that should enhance the specific immunogenicity of Ab2/3H6 by adding a tag to the antitdiotypic antibody. The in vivo studies will be performed in a rabbit animal model since this species is able to produce long complementarity determining regions sufficient for a 2F5-like immune response comparable to humans. Finally we will establish a co-crystallisation of 2F5 and Ab2/3H6 to disclose the secret of the remaining contact residues of mAb 2F5 which are essential for advanced vaccine design.

Despite substantial progress in therapy, HIV/AIDS is still a terminal illness. According to the World Health Organization (WHO) there was a worldwide decrease in people living with HIV (minus 13 %), becoming newly infected with HIV (minus 33 %) or died during the course of AIDS (minus 40 %) in the time period of 2006 to 2011. This effect can be counted to the increase (plus 26 %) of people getting access to antiretroviral treatment (ARVs). Today the worldwide coverage of people having access to ARVs is about 55 %. This equates HIV/AIDS to become a chronic disease. However the negative effects of the HIV/AIDS pandemic for social and economic areas are substantial. Thus, a vaccine that could reduce the number of new infections would count for health and economic benefits and could help to curtail the pandemic. However, even after decades of enormous efforts of the scientific community no HIV-1 vaccine has been developed so far. The task of this project was to evaluate the efficiency of an anti-idiotypic antibody (Ab2/3H6) that might be a candidate for such a HIV-1 vaccine. This Ab2/3H6 mimics the binding-site of the well-known and intensively researched anti-HIV-1 antibody 2F5. In theory the use of Ab2/3H6 as a vaccine should elicit 2F5-like antibodies which are able to neutralize the HIV-1 virus and thus prevent vaccinated people becoming infected with HIV-1. To address the question if Ab2/3H6 is capable of eliciting 2F5-like antibodies two major tasks were processed during the course of this project: 1) is it possible to engineer Ab2/3H6 that only the site that mimics the antigen is immunigenic; 2) does the use of immune-modulators as fusion partner to Ab2/3H6 enhances the synthesis of 2F5-like antibodies in a rabbit in-vivo model. In order to drive the immunogenicity to the site that mimics the antigen the mouse variable regions of Ab2/3H6 were subjected to three different humanization approaches, namely resurfacing, complementarity determining region (CDR)-grafting and superhumanization. Four different humanized Ab2/3H6 variants were developed and characterized for their binding affinity to 2F5 in comparison to the original Ab2/3H6. The resurfaced and the CDR-grafted variants showed similar or slightly reduced binding to 2F5 when compared to the original version, while the superhumanized type lost its binding ability. Immunogenicity analysis by bioinformatical prediction tools revealed that the humanization methods applied to Ab2/3H6 reduced potential immunogenic epitopes indicating that humanization methods were successfully applied to engineer improved Ab2/3H6. Further in a proof-of-concept study, Ab2/3H6 was produced as fusion protein with attached immune-modulators and used for immunization of rabbits to induce HIV-1 antibodies. Affinity purification of collected antisera indicated, that only a small portion of the total rabbit antibody fraction contained specific antibodies. The characterization of the induced 2F5-like antibodies showed specificity for the HIV-1 envelope protein gp140 and the linear epitope of 2F5 GGGELDKWASL. Interestingly only those rabbits immunized with Ab2/3H6 fused with the immune-modulators developed the HIV-1 specific antibodies. Despite specificity for the HIV-1 epitopes these antibodies were not able to exhibit any virus neutralization activities. These results suggest that Ab2/3H6 alone might not be suitable as a vaccine, but in combinational therapy it could be beneficial for induction of HIV-1 neutralizing antibodies.