Summary:

The aim of this project is to select peptide epitopes that mimic neutralisation-sensitive domains of HIV-1 envelope and may function as candidate HIV-1 vaccines. To date, efforts to develop a truly prophylactic HIV-1 vaccine have been hindered by difficulties in identifying immunogens that elicit broadly neutralising antibodies. This lack of significant cross-protection raises further concerns on the capacity of classical Env-based vaccines to afford substantial protection against field isolates. Indeed, current unmodified gp120 or gp140 envelope-based vaccines in human subjects have shown little or no protection from heterologous HIV-1 isolates such as would be encountered in the field. This indicates that vaccine trials with currently available immunogens will afford low percentages of protection with a large number of vaccine breakthroughs and will raise ethical and financial issues concerning the treatment of any volunteers who become infected. It is of the highest priority that a focused effort is undertaken to develop novel Env antigens capable of inducing broad and potent neutralising antibodies to a wide variety of strains. Consistently, this consortium is developing a new generation of Env antigens based on complex but conserved epitopes to induce broad neutralising antibodies. This objective will be achieved by two complementary strategies: (i) screening of random peptide libraries with novel MAbs that neutralise primary HIV-1 isolates assigned to distinct clades; (ii) designing 30-40 amino acid peptides that mimic discontinuous regions of gp120 and gp41 that are sensitive to neutralisation by antibodies and are conserved among HIV strains of distinct clades. These include the CD4-binding domain, the bridging sheet and the pre-fusogenic harpin loop of gp41. During this two-year proposal the novel vaccine candidates will be validated in mice; however, the Consortium is endowed with the facilities and resources to proceed to monkey models of HIV-1 infection.

Background:

While the need for an effective HIV-1 vaccine is urgent, no truly prophylactic candidates are presently available. The current HIV-vaccine candidates tested to date pre-clinically or clinically have all failed to protect from primary infection and have afforded limited protection from disease progression if volunteers are exposed to heterologous isolates (1-3). In fact, efforts to develop a protective HIV-1 vaccine have been hindered by difficulties in identifying epitopes capable of inducing a broad neutralising antibody response together with an efficient T-cell mediated immune response at both the periphery and mucosal sites. The high mutation rate occurring in HIV envelope proteins and the complex structure of gp120 as an oligomer associated with gp41 result in a high degree of antigenic polymorphism (4, 5). To overcome these obstacles, this research proposal is focused on developing pools of innovative immunogens that mimic conserved regions of the viral envelope and are shared by a substantial percentage of primary viral isolates assigned to distinct clades.

The significance of the proposed work relies on the fact that the project directly addresses the critical issues that hold the key to the development of an effective HIV-1 vaccine.

1. The lack of a significant degree of protection afforded by current envelope-based immunogens

A number of studies in animal models have shown a protective role of antibodies (Abs) against acute infection with HIV-1 and simian HIV (SHIV) strains. Adoptive immunotherapy with anti-HIV envelope monoclonal antibodies (MAbs) and HIV-specific immunoglobulins (Igs) has resulted in protection from HIV-1 and SHIV challenge in non-human primates (6). Sterilising immunity was obtained by passive transfer of monoclonal antibodies and serum immunoglobulins (Igs) from HIV-1 infected subjects or monkeys chronically infected with HIV-1 isolates (7-8). These results show that protective antibodies are elicited in the course of natural HIV/SHIV infection and indicate that an effective vaccine should elicit an antibody response to HIV-1 envelope proteins. In this regard, although monkeys immunised with the envelope of a given HIV-1 isolate may be protected against a subsequent challenge with a viral strain carrying an homologous envelope, little or no protection is observed in monkeys challenged with heterologous viruses with a different gp160 (3). Accordingly, envelope-based vaccine trials in human subjects have shown little or no protection from HIV-1 infection (9).

2. The lack of effective delivery of HIV-1 immunogens capable of inducing substantial levels of HIV-specific antibody-mediated immunity.

The recombinant prime, peptide boost vaccination strategy has the potential to replace the classical but ineffective immunisation strategies proposed for HIV. This strategy will be utilised to focus the humoral immune response on to pre-selected peptide epitopes for broad cross-neutralisation. As a carrier, we plan to use liposome, which can overcome the problem of hydrophobic peptides. The immunogenicity and safety of liposomes has been validated in a completed phase III trial for the treatment of several inflammatory diseases.

Aims:

The main objective of this proposal is to select peptide epitopes that mimic neutralisation sensitive regions of HIV-1 envelope and may function as candidate HIV-1 vaccines. This objective will be achieved by two complementary strategies:

1. Screening of Random Peptide Libraries with monoclonal Abs that neutralise primary HIV-1 isolates assigned to distinct clades.
2. Designing 30-40 amino acid peptides that mimic discontinuous regions of gp120 and gp41 that are sensitive to neutralisation by antibodies and are conserved among HIV strains of distinct clades. These include the CD4-binding domain, the bridging sheet and the pre-fusogenic harpin loop of gp41.

Expected results:

1 Development of novel vaccine candidates
1.1 Selection of neutralising antibodies out of a pool of HIV-specific monoclonal antibobies
1.2 Isolation and characterisation of novel HIV-1 clade C viral isolates from recently infected subjects to be used for the neutralisation assay
1.3 Screening of untested monoclonal antibodies for neutralising activity against primary HIV-1 isolates
1.4 Isolation of novel HIV-1-specific epitopes by screening random peptide libraries (RPL) with a pool of neutralising MAbs
1.5 Rational design of short peptide sequences that mimic discrete domains of HIV-1 gp160
1.6 Site-directed mutagenesis of peptide epitopes to optimise their binding affinity and immunological fitness
2 Optimisation of vaccine delivery
2.1 Peptide mimotopes delivered as immunogenic dendrimers of 20-60 kDa particles
2.2 Peptide mimotopes delivered as fusion products with protein carriers that stimulate both systemic and mucosal immunity
2.3 Optimisation of liposome technology to deliver HIV-1 peptides and proteins
3 Immunological evaluation of vaccine candidates in rodents
3.1 Immunisation of mice with selected vaccine candidates
3.2 Assessment of neutralising activity of sera from immunised mice.

Potential applications:

The project deals with:

1. Novel molecular approaches to develop peptides and short amino acid sequences (30-40 residues) that function as antigenic and immunogenic mimics of specific envelope domains;
2. Complex immune responses to HIV proteins mediated by antibodies.

To this end, the proposal includes the development of new methodologies that will set new standards of specificity and sensitivity. These include:
a) An extensive screening of random peptide libraries displayed on filamentous phages to rapidly isolate pools of candidate epitopes. Furthermore, the peptide epitopes will be subjected to an in vitro affinity maturation by using extensive structural analysis and site-directed mutagenesis to improve their immunological fitness.
b) Exploitation of the available knowledge on the structural conformations of the HIV-1 gp120 and gp41 envelope proteins to devise by rational design peptide sequences that mimic discrete domains of HIV-1 gp160. These include the CD4-binding domain, the bridging sheet and gp41 eptad repeats.
c) Antibody neutralisation assays based on the analysis of the Ab-antigen binding affinity and avidity and endowed with greater sensitivity.

When completed, this study will set new standards and tools to develop vaccines for highly mutable infectious agents. In this regard, the expected outcomes are not restricted to HIV and could help in devising novel antigens for malaria and tuberculosis vaccines.

References:

1. Shiver JW, et al., Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity. Nature. 2002 Jan 17;415(6869):331-5.
2. Barouch DH, Kunstman J, Kuroda MJ, Schmitz JE, Santra S, Peyerl FW, Krivulka GR, Beaudry K, Lifton MA, Gorgone DA, Montefiori DC, Lewis MG, Wolinsky SM, Letvin NL. Eventual AIDS vaccine failure in a rhesus monkey by viral escape from cytotoxic T lymphocytes. Nature. 2002 Jan 17;415(6869):335-9.
3. Cho, M.W. et al. Polyvalent envelope glycoprotein vaccine elicits a broader neutralizing antibody response but is unable to provide sterilizing protection against heterologous simian/human immunodeficiency virus infection in pigtailed macaques. J. Virol. 75, 2224-2234 (2001).
4. Kwong, P. D., R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski, W. A. Hendrickson. 1998. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393:648.
5. Wyatt, R., P. D. Kwong, E. Desjardins, R. W. Sweet, J. Robinson, W. A. Hendrickson, J. G. Sodroski. 1998. The antigenic structure of the HIV gp120 envelope glycoprotein. Nature 393:705.
6. Wei X, Decker JM, Wang S, Hui H, Kappes JC, Wu X, Salazar-Gonzalez JF, Salazar MG, Kilby JM, Saag MS, Komarova NL, Nowak MA, Hahn BH, Kwong PD, Shaw GM. Antibody neutralization and escape by HIV-1. Nature. 2003 422:307-12.
7. Emini, E.A. et al. Prevention of HIV-1 infection in chimpanzees by gp120 V3 domain-specific monoclonal antibody. Nature 355, 728-730 (1992).
8. Mascola, J.R. et al. Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nature Med. 6, 207-210 (2000).
9. Connor, R.I. et al. Immunological and virological analyses of persons infected by human immunodeficiency virus type 1 while participating in trials of recombinant gp120 subunit vaccines. J. Virol. 72, 1552-1576 (1998).

Coordinator:

Partners:

Nº	Principal Scientific Participants	Official Address	Other Information
2	Vincenzo Pavone	Department of Chemistry University of Naples ‘Federico II’ Complesso Universitario Monte S. Angelo via Cinthia, IT-80126 Naples Italy	Tel: +39 081 674399 Fax: +39 081 674090 E-mail: pavone@chemistry.unina.it
3	David Davis	Foundation Biomedical Primate Research Center Department of Virology 3306 Lange  Kleiweg 139 NL-2280 GH Rijswijk The Netherlands	Tel: +31 15 284 2661 Fax: +31 15 284 3986 E-mail: davis@bprc.nl
4	Gabriela Stiegler	Polymun Scientific Immunbiologische ­Forschung GmbH - HIV research Nussdorfer Laende 11 AT-1190 Vienna Austria	Tel: +43 1 36006 6408 Fax: +43 1 3697615 E-mail: G.Stiegler@iam.boku.ac.at
5	Boris Ferko	University of Applied Life Sciences and Natural Resources Institute of Applied Microbiology Muthgasse 18B AT-1190 Vienna Austria	Tel: +43 1 36006 6594 Fax: +43 1 3697615 E-mail: B.Ferko@iam.boku.ac.at