Elsevier

Vaccine

Volume 19, Issue 30, 20 July 2001, Pages 4121-4129
Vaccine

Induction of a cytotoxic T-cell response to HIV-1 proteins with short synthetic peptides and human compatible adjuvants

https://doi.org/10.1016/S0264-410X(01)00179-7Get rights and content

Abstract

The goal of this study was the induction of a strong CTL response against multiple CTL epitopes present in HIV proteins using short synthetic peptides. Four HLA-A2.1 restricted peptides (RT 476–484, p17 77–85, gp41 814–823, RT 956–964) that showed stable binding to the HLA-A2.1 molecule in an in vitro binding assay were able to elicit a strong specific immune response in HLA-A2.1 transgenic mice when injected with IFA or Montanide®. The use of biodegradable microspheres (MS) as adjuvant was also successfully tested for all peptides. When the peptides were injected as a mixture the response was weaker as compared to individual injections of the peptides indicating the occurrence of immunodominance (ID). We are currently investigating whether ID can be overcome by a combined injection of peptide loaded MS with different release patterns. Taken together, it seems feasible to induce a specific CTL response in HLA-A2.1 transgenic mice against several HIV proteins using 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.

References (46)

  • G.B. Lipford et al.

    In vivo CTL induction with point-substituted ovalbumin peptides: immunogenicity correlates with peptide-induced MHC class I stability

    Vaccine

    (1995)
  • T.W. Chun et al.

    Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy

    Proc. Natl. Acad. Sci. USA

    (1997)
  • D. Finzi et al.

    Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy

    Science

    (1997)
  • J.K. Wong et al.

    Recovery of replication-competent HIV despite prolonged suppression of plasma viremia

    Science

    (1997)
  • J.W. Mellors et al.

    Prognosis in HIV-1 infection predicted by the quantity of virus in plasma

    Science

    (1996)
  • N.L. Letvin

    Progress in the development of an HIV-1 vaccine

    Science

    (1998)
  • I. Roitt et al.

    Immunology

    (1993)
  • M.J. Newman et al.

    Saponin adjuvant induction of ovalbumin-specific CD8+ cytotoxic T lymphocyte responses

    J. Immunol.

    (1992)
  • M. Singh et al.

    Advances in vaccine adjuvants

    Nat. Biotechnol.

    (1999)
  • N. Onier et al.

    Cure of colon cancer metastasis in rats with the new lipid A OM 174. Apoptosis of tumor cells and immunization of rats

    Clin. Exp. Metastasis

    (1999)
  • A.A. Scalzo et al.

    Induction of protective cytotoxic T cells to murine cytomegalovirus by using a nonapeptide and a human-compatible adjuvant (Montanide ISA 720)

    J. Virol.

    (1995)
  • D. Wise et al.
  • J.H. Eldridge et al.

    Biodegradable and biocompatible poly(DL-lactide-co-glycolide) microspheres as an adjuvant for staphylococcal enterotoxin B toxoid which enhances the level of toxin-neutralizing antibodies

    Infect. Immun.

    (1991)
  • Cited by (37)

    • Inflammasome-activating nanoparticles as modular systems for optimizing vaccine efficacy

      2009, Vaccine
      Citation Excerpt :

      Because of the historical emphasis on eliciting humoral immune responses, current adjuvants, represented predominately by aluminum adjuvants (alum), are optimized for effective induction of Th2-biased responses and high antibody serum titers, but are less effective at eliciting a strong T cell-mediated immune response. While soluble antigen is poorly presented on MHC Class I to stimulate cytotoxic T lymphocytes, it has been demonstrated that internalized antigen encapsulated in polymer particles can be effectively cross-presented on MHC Class I [10,11] yielding an effective CD8+ T cell response [12,13]. Aluminum adjuvants while having a limited capacity to adsorb many antigens [14,15] have been a mainstay in current vaccine formulations.

    • PEGylation as a tool for the biomedical engineering of surface modified microparticles

      2008, Journal of Pharmaceutical Sciences
      Citation Excerpt :

      Microparticles have also been associated with immunomodulatory properties. In fact, microparticulate antigen delivery systems, when phagocytosed by APC, have been shown to cause crosspresentation on both MHC I and II molecules7 and thus elicit cytotoxic T lymphocyte (CTL) immunity in addition to T helper cell and antibody responses.8-11 Upon entry into the organism, microparticulates are instantly recognised as foreign and are efficiently removed and digested via unspecific phagocytosis by the mononuclear phagocytic system (MPS), which (in addition to other specialised organs and tissues) also comprises the phagocytes of the innate immune system.12

    • Adjuvant activity of polymer microparticles and Montanide ISA 720 on immune responses to Plasmodium falciparum MSP2 long synthetic peptides in mice

      2007, Vaccine
      Citation Excerpt :

      The reason for this is not known and may be related to the strain of mice used since CB6F1 mice are good responders (manuscript in preparation). Although a previous work showed a greater adjuvant activity of Montanide as compared to PLGA MP using HIV short synthetic peptides, this difference could be a consequence of the low antigen load into the performed MP (around 0.05%), insufficient to trigger immune responses after being uptaken by the APCs [34]. The MSP2 peptide loads into the MP we used in the present study, 4.6% for 3D7 and 2.9% for FC27, would overcome the required threshold for the immune activation of the APC.

    View all citing articles on Scopus
    View full text