Keywords: HIV; Vaccine; Neutralising Antibodies; transgenic mice
For improved protection from HIV infection during birth or through accidental exposure to HIV, antibodies that have a broad neutralising activity would be a valuable addition to antiretroviral treatment and to vaccines. So far, there are only a few antibodies with broad cross-clade neutralising properties available in the field of HIV applied research – b12, 2G12, 2F5 and 4E10. They recognise either epitopes presented only transiently during infection of cells, or epitopes not recognisable by unmodified immunoglobulins (2G12). Two experimental strategies are used in separate and in combination. One uses the lead given by extremely rare mutant antibody genes (i.e. 2G12) to generate mice with germ line modified immunoglobulin genes, which introduce extended mobility into the immunoglobulin protein backbone. Immunisation of these mice will allow recognition of novel expanded epitopes including glycane clusters and the development of monoclonal antibody. The other line of experiments employs HIV receptor-transgenic mice for immunisation to favour receptor mediated transitory stages of the viral envelope.
Previous experience and reagents from earlier EU-funded programmes (virosome incorporating HIV proteins) and new microbicide-vaccine combinations against HIV to present native oligomeric HIV envelope of clinically relevant HIV isolates will be used in addition to liposome-presented recombinant C-clade gp 140 oligomers and gp160 encoding highly effective DNA plasmid vaccines in CD4 transgenic mice to generate monoclonal antibodies with novel broadly neutralising properties. This proof of principle can directly give valuable reagents. It can also be further developed into humanised antibodies. If successful, a fully human immunoglobulin-based strategy will be chosen as the next step. The novel antibodies developed out of this programme will be valuable additions to the immunogens developed by the EUROVAC group in the context of the Fifth Framework Programme. The C-clade chosen is of major importance in the worldwide HIV epidemic.
Antibodies with potent, HIV cross-clade neutralising ability are limited so far to the antibodies b12 (CD4 binding site epitope), 2G12 (unique carbohydrate epitope), 2F5 and 4E10 (C-terminus of the external domain of gp41) as well as 447-D2 (the only potential V3 monoclonal antibody). The target of human immunodeficiency virus type 1 (HIV-1) neutralising antibodies (Abs) is suggested to be the trimer of gp120-gp41 heterodimers that covers the surface of HIV-1. In the case of gp41, it appears that antibody access to neutralising epitopes may be more restricted than access to those on gp120, since the relevant epitopes on gp41 probably become fully exposed only during HIV-1 envelope-mediated virus-cell membrane fusion (Golding, et al., 2002). The two anti-gp41 monoclonal Abs (Mabs) that are the most potent and broadly neutralising are the human immunoglobulin G (IgG) Mabs 2F5 and 4E10. 2F5 not only neutralises primary HIV-1 from several different subtypes but confers protection against challenge by immunodeficiency virus by passive transfer in animal models (Mascola, et al. 1999; Hofmann-Lehmann, et al. 2002). Thus, a justifiable interest has arisen in developing immunogens or immunisation technologies capable of eliciting 2F5-like Abs by immunisation. Unfortunately, despite many attempts, the neutralising activity of 2F5 has not been recapitulated by administering either gp41, gp160, or a variety of immunogens including the 2F5 core epitope in different contexts. The importance of residues flanking the recognition sequence in binding 2F5 has been revealed, which could explain the inability of some, but not all, of the immunogens tested to induce neutralising Abs. One interpretation of the inability to elicit 2F5-like Abs in animals is that it may be difficult to produce an Ab with a very long third complementarity-determining region of the heavy chain (CDR H3), as is found in 2F5. This 22-residue CDR H3 loop in 2F5 is much longer than the average length of CDR H3s in humans, rabbits, and mice, 13, 11 to 12, and 9 to 10 residues, respectively (Wu, et al. 1993). However, it is not clear whether a long H3 loop is actually a requirement for Abs to interact with the 2F5 epitope and neutralise HIV-1. On the other hand, the tip of the CDR H3 of 2F5 might be involved in a crucial interaction with an as yet unidentified determinant on the virion that might need to be incorporated in an immunogen designed to elicit 2F5-like Abs. Using membrane-anchored Env and antibodies in physiological conditions it has been recently shown that the C-terminal repeat region of gp41 undergoes conformational changes after gp120 binds the receptor. 2F5 appeared to bind both native and fusion-intermediate conformations of gp41 and neutralises virus at a relatively late step in virus entry (de Rosny et al., 2004).
The aim of this project is to develop transgenic mice with distinct genetic alterations useful for the production of antibodies with otherwise, at best, very rarely observed properties such as broad neutralisation activity against HIV, by generating an immunoglobulin backbone that provides greater flexibility of respective antibodies or expressing an approved HIV-1 fusion inhibitor on HIV target cells in order to force a specific conformation allowing the production of neutralising antibodies.
Furthermore it is foreseen to develop antigens based on synthetic HIV envelope genes useful for the assembly of infection intermediates displaying antigenic structures qualified for the generation of neutralising antibodies.
Besides their role for controlling HIV/AIDS, these technologies and transgenic animals should be also useful for the identification of new broadly neutralising antibodies and their subsequent production following humanisation.
Apart from the contribution to a better control of HIV infection in humans the suggested technology of learning from extremely rare natural mutations which modify immunoglobulins in a way to be able to recognise larger epitopes (‘suprastructures’) and translating this experiment of nature into biotechnological applications will open a new approach to recognise immunogens from many sources which were so far not recognisable due to steric constraints.
The transgenic mice expressing immunoglobulins with mobilised antigen recognition regions will be a very valuable tool for the generation of variant antibodies with, until now, unavailable recognition capacity for clustered epitopes which should lead to novel recognition patterns. This should strengthen the European platforms engaged in antibody research, this includes also the biotechnological programmes which develop antibody production strategies in plants (LSH 2002 1.25-2) and indirectly again the health sector as novel reactive substances may become available for so far not fully successful projects such as microbicide interventions (LSH 2002 2.3.0 – 2).
The careful characterisation of antibodies (binding affinities, complement fixation, neutralisation) and the capability to develop even GMP products is a work where ICSTM and IAM Vienna with POLYMUN Vienna form a strong team which can develop products right up to clinical trials. The products will be highly attractive for ECDTP and major EU-funded HIV vaccine clusters such as EUROVAC and enable synergy on active/passive immunisation schemes. The product will also have important applications in the European upcoming Eastern Europe and global fields. HIVAB will be a pilot project with potential impact on many other fields of medicine as novel diagnostic reagents and as preventive or therapeutic interventions. One example is already designated for future calls (neonatal vaccination strategies for poverty associated diseases).
Institute for Medical Microbiology and Hygiene (RIMMH/UREG)
University of Regensburg
Tel: +49 941 9446400
Fax: +49 941 9446402
|Official Address||Other Information|
|2 ||Ralf Wagner ||Institute for Medical Microbiology and Hygiene (RIMMH/UREG) |
University of Regensburg
|Tel: +49 941 9446452 |
Fax: +49 941 9446455
|3 ||Thomas Hehlgans ||Institute for Immunology (URII/UREG), |
University of Regensburg
|Tel: +49 941 944 6626 |
Fax: +49 941 944 6602
|4 ||Simon Jeffs ||The Imperial College of Science, Technology and Medicine (ICSTM) |
London SW7 2AZ
|Tel: +44 2075943918 |
Fax: +44 2075943906
|5 ||Dr. Renate Kunert ||Institute for Applied Microbiology (IAM/BOKU)) |
|Tel: +43 360066595 |
Fax: +43 13697615
|6 ||Frank Notka ||Geneart GmbH |
|Tel: +49 941 9427645 |
Fax: +49 941 9427611
|7 ||Hermann Katinger ||Polymun Scientific GmbH |
|Tel: +43 136006 6202 |
Fax: +43 13697615