Research paper
A Gag peptide encompassing B- and T-cell epitopes of the caprine arthritis encephalitis virus functions as modular carrier peptide

https://doi.org/10.1016/j.jim.2008.11.015Get rights and content

Abstract

Short synthetic peptides are important tools in biomedical research permitting to generate hapten specific polyclonal sera for analytical purposes or functional studies. In this paper we provide proof of principle that a peptide located in a highly conserved portion of the Gag protein of the caprine arthritis encephalitis virus and carrying an immunodominant T helper cell epitope functions as an efficient carrier peptide, mediating a strong antibody response to a peptidic hapten encompassing a well-characterized B cell epitope of Env. The carrier and hapten peptides were collinearly synthesized permutating their molecular arrangement. While the antibody response to the hapten was similar for both constructs, the antibody response to a B cell epitope overlapping the T helper cell epitope of the Gag carrier peptide was considerably different. This permits a modular use of the carrier peptide to generate antibody directed exclusively to the hapten peptide or a strong humoral response to both carrier- and hapten-peptide. Finally, we have mapped the epitopes involved in this polarized antibody response and discussed the potential immunological implications.

Introduction

We have previously described a peptide encoded in the gag region of the caprine arthritis encephalitis virus (CAEV) that contains immunodominant B- and T-cell epitopes and proposed that this peptide may be used as a universal carrier for peptide haptens in goats. This immunogenic site is located in the major homology region (MHR) of the gag gene of retroviruses (Mammano et al., 1994, Wills and Craven, 1991), suggesting that its immunodominance may be shared by different retroviruses. This peptide contains a carboxyterminal amphipathic alpha helix encompassing a T cell epitope and a rather unstructured aminoterminal portion encompassing a cryptic B cell epitope (Fluri et al., 2006).

In goats immunized with this Gag-peptide, the T cell epitope is able to induce a robust proliferative response and supports a strong antibody response to the B cell epitope that, however, is not protective (Nenci et al., 2007).

Polyclonal hapten-specific antisera directed to short peptides are important tools in biomedical research and a valid complement to the production and the use of monoclonal antibodies. They can be used to visualize specific ligands in different specimens such as cells or histological slides using different techniques, for instance, immunofluorescence and immunohistochemistry (Yalowitz et al., 2004, Kubota et al., 2006, Schulz et al., 2006). Single epitope specific antibody can be applied in different assays to study receptor function and expression levels, and to define functional domains of proteins (Chen et al., 2007, Pastori et al., 2008, Hyser et al., 2008). The use of goats for the production of such reagents is very interesting owing to the size of the animal and its established use in biotechnology (Huang et al., 2007, Baldassarre et al., 2004). In fact, large amounts of immunoglobulin can be easily purified from goat-serum, -milk or -colostrum (Baruah et al., 2006). The principal interest in peptide-based antigens, however, remains the prospective use of synthetic peptides in vaccinology (Azizi and Diaz-Mitoma, 2007). Epitope specific antisera permit us to define the potential neutralizing activity of antibodies targeting the linear B cell epitopes of virus antigens, and synthetic peptides encompassing such neutralizing epitopes are potential candidates for the development of synthetic peptide vaccines (Meloen et al., 2001, Wang et al., 2001, Dietzschold et al., 1990). In addition to antibodies, synthetic peptides can also induce cytotoxic T cell immune responses and such peptides are already used in clinical trials of cancer immunotherapy (Melief and Van der Burg, 2008).

An important drawback of synthetic peptides is their poor immunogenicity. To induce an efficient B- and T-cell immune response synthetic peptides must be coupled to a source of T helper cell determinants which can be provided by large carrier molecules such as ovalbumin or keyhole limpet haemocyanin (KLH). The use of strongly immunogenic carrier molecules, however, may divert the immune response from the peptide haptens (Herzenberg et al., 1980, Schutze et al., 1989). This suppression can be prevented by conjugating the haptens to short peptides carrying appropriate T helper cell epitopes (Sad et al., 1992).

Short synthetic peptides containing T helper cell epitopes do not need carrier proteins to induce an efficient immune response and the colinear synthesis of such peptides with B- and cytotoxic T-cell epitopes carrying peptides is a valid alternative to the use of carrier proteins (Borras-Cuesta et al., 1987, Partidos et al., 1991). The use of such peptides was shown to induce antibody responses that are comparable or superior to those induced by synthetic peptides coupled to carrier molecules.

To provide proof of principle that the Gag peptide of CAEV described above can function as universal carrier peptide, we constructed different chimeric peptides containing the Gag peptide as a source of T helper epitopes collinearly synthesized to a short peptide encompassing the SU5 antigenic region of the envelope glycoprotein (Env) of CAEV, known to contain an immunodominant B cell epitope (Mordasini et al., 2006).

In this report, we present a detailed analysis of the humoral immune response of goats to chimeric SU5-Gag peptides, permutated to fuse the SU5 peptide at the amino- or carboxy-terminal end of the Gag carrier peptide. We show that the Gag peptide is an excellent carrier and that the permutation of the chimeric peptide has a profound influence on the quality of the immune response.

Section snippets

Synthetic peptides

For all the listed synthetic peptides the amino acid positions refer to the published sequence of the CAEV-CO strain (Saltarelli et al., 1990). Two synthetic peptide chimeras containing the immunodominant SU5 sequence 607-EMPENYAKTRIINRKK-622 of the CAEV Env (Mordasini et al., 2006) as well as the previously mapped T cell epitope of the Gag peptide 152-PYEDFAARLLEAIDAE-167 (Fluri et al., 2006) were synthesized permutating their arrangement. The first chimeric peptide reflected the natural B-,

Primary antibody response to SU5

As shown in Table 1a, Table 2a and Fig. 1a, each goat seroconverted to the SU5 peptide in ELISA, between the 3rd and the 6th week post-immunization independently of permutations of the molecular arrangement of the SU5 peptide in respect of the Gag carrier peptide. Individual animals showed differences in the kinetic and strength of their antibody response to SU5, for instance, goat #14 showed an unusually rapid response to SU5-peptide reaching an OD value of above 1.9 already at 21 dpi (Table 1a

Discussion

The use of short synthetic peptides to produce monospecific polyclonal sera or vaccines permits the induction of a tightly targeted antibody response to well defined antigenic structures. The poor immunogenicity of such peptidic haptens can be overcome by coupling them to a source of T helper cell epitopes indispensable for the induction of an efficient humoral and cellular immune response. Conjugation to complex multivalent antigens such as KLH or tetanus toxoid may mediate an efficient immune

Acknowledgments

We are indebted to Ruth Parham for valuable linguistic help and to Gabriela Obexer-Ruff for MHC typing. This work was supported by the European Community 6th framework programs, contract no. INCOCT-2005-003713.

References (33)

  • AgarwalA. et al.

    B cell responses to a peptide epitope. I. The cellular basis for restricted recognition

    J. Immunol.

    (1996)
  • AziziA. et al.

    Viral peptide immunogens: current challenges and opportunities

    J. Pept. Sci.

    (2007)
  • BaldassarreH. et al.

    State of the art in the production of transgenic goats

    Reprod. Fertil. Dev.

    (2004)
  • BaruahG.L. et al.

    Purification of monoclonal antibodies derived from transgenic goat milk by ultrafiltration

    Biotechnol. Bioeng.

    (2006)
  • Borras-CuestaF. et al.

    Engineering of immunogenic peptides by co-linear synthesis of determinants recognized by B and T cells

    Eur. J. Immunol.

    (1987)
  • DietzscholdB. et al.

    Structural and immunological characterization of a linear virus-neutralizing epitope of the rabies virus glycoprotein and its possible use in a synthetic vaccine

    J. Virol.

    (1990)
  • 1

    Present address: Novartis Vaccines & Diagnostics srl., via Fiorentina 1, 53100 Siena, Italy.

    View full text