5. Immunogen design and gene delivery

5.1 Delivery systems and adjuvant strategies

Proteins, VLPs, non-viral and viral delivery systems

Based on our basic studies of HIV assembly and morphogenesis (chapter 4), we were able to construct recombinant, HIV-1 Gag derived VLPs, which allow the presentation of either smaller peptides (40-60 amino acids) or oligomeric HIV-derived Env glycoproteins. Recombinant VLPs were shown to induce sustained Th1-type cellular immune responses as well as neutralizing Env-specific antibody responses both in small animal models as well as in non-human primates. Recent ex vivo analysis suggests that recombinant VLP purified from insect cells following infection with recombinant Baculovirus induce maturation of human monocyte-derived dendritic cells (upregulation of CD80, CD83, CD86, CD40, HLA-DR; secretion of IL6, IL12). Taken together, these results recommended recombinant VLPs as basic compound both for T cell- as well as B-cell vaccines.

Furthermore, exploiting our improved understanding on the HIV codon bias for late lentiviral gene expression, technologies have been developed enabeling the design and synthesis of HIV genes that allowed transgene expression in complete absence of the viral Rev/RRE system and additional cis-acting sequence elements. This strategy has been employed to establish synthetic GagPolNef and Env polygene constructs both of which are based on the 97CN54 virus isolate representing the clade C epidemic in China (chapter 2). These basic constructs have been selected to construct (i) a DNA vaccine (DNA-C; own work), a New-York Vaccinia strain (NYVAC-C; Aventis-Sanofi) and a Modified Vaccinia Ankara strain based vaccine (MVA-C, Prof. Esteban). These vaccines turned out to be safe and immunogenic in mouse models, nonhuman primates and two phase 1 clinical trials (chapter 7).

To broaden our spectrum of viral vectors for future prime boost regimens we have in collaboration with Dr. Osterrieder (Cornell University) established equine herpesviruses (EHV-1) as vector to deliver HIV immunogens via the mucosal route. EHV-1 efficiently infects human cells including DC at minimal MOIs, does hardly replicate in human cells, expresses transgenes at high levels and an extended period of time and is not neutralized by high titered anti-HSV1, -HSV2, -EBV, -Varicella and - CMV antisera. BALB/c mice developed upon intranasal immunization with Gag recombinant EHV high titers of Gag specific antibodies including sIgA as well as gag specific T cell reponses that were even more pronounced, when DNA-C was used for priming immunization.

As already outlined briefly in chapter 3, we are also testing various vaccine formulations (DNA-C, NYVAC-C, EHV-C, protein-adjuvant combinations etc.) ex vivo for their capacity (i) to induce maturation of  various murine and human DC preparations (direct infection or cross priming), (ii) to support MHC class I-restricted antigen presentation and (iii) to properly activate autologous T cells following coincubation with stimulated DC. The potency of the individual vaccine preparations to affect the above steps will be monitored using various technologies including tetramer staining, FACS analyses and - in selected cases - transcriptome and proteome analyses. These studies will be also extended and correlated with in vivo analyses in mouse models (chapter 6). Furthermore, the impact of selected adjuvants such as Mega-CD40L on the performance of the immunogens ex vivo and in vivo will be determined.

5.2 T cell vaccines

Our record of immunological, preclinical and clinical data obtained so far with NYVAC-C, DNA-C and MVA-C clearly underlines safety and immunogenicity of these vaccines.

However, although constructed in 1997 according to the best of our knowledge, the design of these GagPolNef antigens was at least in part driven by assumptions, which should - under the light of recent progress made in different fields - be systematically re-evaluated and improved. This view is supported by the finding that GagPolNef immunogens seem to be less efficient compared to Gag immunogens in priming HIV specific T cells in animals and that Env specific T cell responses dominate over GagPolNef responses in the first human trial. The systematic assessment and improvement of immunogen design takes recent advances into account regarding (i) posttranslational modifications and proteasomal degradation to enhance antigen processing and epitope presentation and (ii) the importance of intracellular trafficking and cross-priming to ensure both MHC class I- and II-restricted epitope presentation to properly induce virus-specific CD4 and CD8 positive T cells.

5.3 B cell vaccines

VLPs as delivery system for complex Env immunogens

The HIV Env antigen is the main target of neutralizing antibodies and a number of studies suggest that neutralizing antibodies should be an important component of an HIV-1 vaccine. First, it has been shown that passive transfer of broadly neutralizing monoclonal antibodies can protect from infection in animal models. Second, antibody treatment causes a delay of HIV-1 rebound after cessation of antiretroviral therapy. Despite these promising data, all attempts to induce neutralizing antibodies by means of active immunization failed so far. However, it is believed that the limited set of broadly cross neutralizing monoclonal antibodies might guide the development of novel immunogens capable of inducing such broadly neutralizing antibody responses.  
As evidenced above, we have previously shown that HIV-1 Gag derived VLPs support the presentation of oligomeric HIV-derived Env and heterologous glycoproteins. Recombinant VLPs were shown to induce Env-specific antibody responses that neutralize homologous virus strains. Furthermore, we have shown, that humoral immune responses raised against heterologous herpesviral envelope proteins protected mice from pathogenic EHV challenge and HIV-specific neutralizing antibody responses induced in rhesus macaques seemed to correlate with accelerated clearance following SHIV challence of animals that were vaccinated with recombinant VLPs presenting chimeric Env-glycoproteins. Taken together, recent insights (i) into envelope structures and the nature of neutralizing epitopes, (ii) a  better understanding of the molecular mechanisms underlying the immunological potency of recombinant VLPs together and (iii) state of the art lentiviral vector technology strongly suggests the exploitation of  lentiviral libraries to screen and optimize Env-derived, potentially neutralizing epitopes.



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