Induction of a cytotoxic T-cell response to HIV-1 proteins with short synthetic peptides and human compatible adjuvants
Introduction
Significant progress in the battle against human immunodeficiency virus (HIV) infection has been achieved with the introduction of highly active antiretroviral therapy (HAART), resulting in dramatic reductions of viral burden and prolonged survival. However, most HIV infected individuals live in areas with limited financial resources and may never benefit from these therapeutic agents. Even in industrialised countries therapy is restrained due to poor tolerance of the drugs, the emergence of resistant viruses and the fact that HIV-1 persists in lymphoid organs [1], [2], [3]. The best hope of preventing AIDS globally therefore remains the development of an effective vaccine. The induction of an antibody response and a broad cytotoxic T cell (CTL) response against different HIV epitopes may eradicate the virus from the host during primary infection. In addition, although it may not provide total protection, it may have a positive impact on the course of the disease by lowering the viral load after primary infection, which in turn can be correlated with a slow progression to AIDS [4].
Several vaccine approaches for HIV-1 have been evaluated with variable success in chimpanzees and macaques, including attenuated or inactivated viruses, live vector-based vaccines, recombinant proteins and DNA injections [5]. Some of these approaches have met with scepticism, because attenuated viruses may regain their pathogenicity due to the high mutation rate, and live vectors may cause life-threatening infections in immunosuppressed HIV patients. Finally, inconveniences associated with DNA vaccination may be the incorporation of DNA into the host genome.
In this study we used short synthetic HIV peptides, which have the advantages of being totally safe and of generating an immune response directed only to the relevant epitope(s) of the pathogen. In order to generate a strong immune response, peptides must be associated with an immunostimulant such as an adjuvant or an immunogenic carrier. Aluminum hydroxide is currently widely used in humans, but may induce inflammation and local granuloma formation [6] and may not be appropriate to induce cytotoxic responses [7]. Novel adjuvants, such as the saponin QS21 [8], [9], the lipid A derived OM 174 [10] or the recently approved Montanide® (ISA 720) [11], [12], which is based on a natural metabolizable oil and a highly refined emulsifier from the mannide monooleate family, are under investigation. A promising approach is the incorporation of antigens into biodegradable microspheres (MS). MS were developed as drug carriers in the early 80's [13] and, since then, have been adapted to carry antigenic material for vaccination [14], [15], [16], [17], [18]. They are composed of either poly(d,l-lactide-co-glycolide) (PLGA) or poly(d,l-lactide) (PLA), are biocompatible and are degraded by hydrolysis to soluble non-toxic natural products, lactic and glycolic acid. In addition, depending on their polymer composition, MS with delayed peptide release patterns can be produced, offering the possibility to convert a conventional multiple dose vaccine into a single dose vaccine.
The goal of this study was the development of an immunisation procedure to induce a broad CTL response in HLA-A2.1 transgenic mice using a mixture of synthetic HIV peptides. HLA-A2.1 restricted epitopes were identified through the HIV database (Los Alamos National Laboratory, NM). Peptides that showed stable binding to the HLA-A2.1 molecule in an in vitro assay were able to elicit an immune response in HLA-A2.1 transgenic mice using either IFA, Montanide® or MS as adjuvants, indicating the feasibility to induce a specific CTL response using this approach in humans.
Section snippets
Peptide synthesis
The following peptides were synthesised by the F-moc, t-Bu strategy for solid phase systems as described previously by Atherton et al. [19]: influenza matrix protein MA 58–66 (Flu); tetanus toxoid peptide tt 947–967 (P30); HIV peptides: RT 476–484 (LR22); p17 77–85 (LR23); gp160 318–327 (LR25); RT 346–354 (LR26); gp41 814–823, (LR27); RT 956–964 (LR28) (Table 1). Peptides were purified with a G25 size exclusion chromatography and further subjected to mass spectrometric analysis (purity >80%).
Microspheres
Peptide-HLA-A2.1 binding and stability
HLA-A2.1 restricted epitopes have been identified through the HIV database (Los Alamos National Laboratory, NM). HLA-A2.1 was chosen because it is the most common human MHC class I molecule, which is found at high frequencies in most populations. The peptides studied here are derived from gag, pol and env proteins and their sequences are relatively well conserved. The binding of these peptides to the HLA-A2.1 molecule and the stability of the HLA-A2.1-peptide complex was assessed by measuring
Discussion
In the present study we have demonstrated that a strong CTL response in mice can be induced with short synthetic HIV peptides in different formulations. We have chosen peptides that are derived from various proteins of HIV including the env, gag and pol proteins (Table 1). All peptides, except LR25, showed the typical binding motif for HLA-A2.1 including a leucine or a methionine at position 2 and valine, leucine or isoleucine at positions 9 or 10. On the other hand, LR25 has a glycine at
Acknowledgements
This project was funded by the Swiss National Science Foundation, Commission of AIDS.
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