Characterisation of the antibody response to a totally synthetic immunocontraceptive peptide vaccine based on LHRH

Abstract

In this study we describe our attempts to improve the immunogenicity of a synthetic epitope-based vaccine. The vaccine consists of an epitope (P25) that is recognised by CD4+ helper T cells and the target epitope luteinising hormone releasing hormone (LHRH). We show that replacement of the single cysteine residue within P25 with amino acids such as alanine, aminobutyric acid, serine or with carboxymethylated cysteine leads to diminished immunogenicity of the vaccine and only the oxidised dimeric form of the peptide retains the full immunogenicity of the vaccine. Secondly, by measuring the serum antibody response and the number of the antigen secreting cells in spleen and bone marrow we found that three doses of 20 nmol per mouse induced the more consistent and higher immune responses than those induced by three doses of either 2 nmol or 80 nmol per mouse. A greater variation in antibody titre was observed in mice that received the 2 nmol or 80 nmol dose regimes. Last, by administering the vaccine in its lipidated form in the presence or absence of additional adjuvant we found that either inoculation regime elicited similar antibody responses. Only at low doses of antigen was a synergistic effect observed when lipopeptide was co-administered with additional adjuvant.

Introduction

In the development of synthetic peptide-based vaccines a number of stages are involved. The initial stage is the identification of pathogen or ligand-specific epitopes which include B cell, cytotoxic T cell and helper T cell epitopes. Recent progress in technologies using peptide libraries has improved the speed and efficiency of identification. The next stage is to assemble epitopes into a construct which can be presented correctly and effectively to antigen-presenting cells such as dendritic cells. Advances in peptide synthesis have also extended the methods by which epitopes may be assembled such as the inclusion of multiple copies of the same or different peptide determinants [1], [2] as well as allowing incorporation of adjuvanting moieties such as lipids (for review see [3]). The discovery and use of promiscuous T helper epitopes has also advanced the development of peptide vaccines in which only a single or a limited number of T helper cell epitopes are needed to successfully vaccinate an outbred population [4], [5].

Once the appropriate epitopes have been identified and assembled into a construct there are, however, other problems which still remain to be overcome in order to make peptides feasible alternatives to other forms of vaccines. One possible problem is the reactivity of cysteine-containing peptide vaccines. Although peptide vaccines tend to be stable in freeze-dried form and do not require cold-chain distribution. Nevertheless all protein-based vaccines including those that are peptide-based contain amino acid residues such as cysteine are susceptible to oxidative modification. Such oxidative processes are more likely to occur in the case of subunit protein vaccines that are stored in solution than with peptide-based vaccines that can be stored freeze-dried. Cysteine, however, notoriously unstable in air readily oxidising to form cysteine dimers is present in the helper T cell epitope P25 that we use in the LHRH vaccine. The possibility that formation of peptide dimers may occur as a result of oxidation therefore prompted us to determine the effect on immunogenicity of deliberately modifying this cysteine residue in a number of ways including dimerisation.

The second problem is to select the appropriate inoculation regimes, in particular with regard to dose, which may influence the magnitude and duration of the immune response as reported [11], [12], [13].

The third problem is that peptides, like all other soluble protein vaccines, are weak immunogens in the absence of adjuvant. Although many adjuvants are available for administration with vaccines, many of these are toxic and unsuitable for use in animals and humans. Others are not yet registered for use or ineffective. Significant progress has been made in recent years to incorporate lipid moieties into peptide-based vaccines [6], [7], [8], [9], [10] and such self-adjuvanting peptides do promise a solution to the problem of poor immunogenicity.

In earlier publications from this laboratory [10], [14] we have reported that a simple peptide construct consisting of a co-linear CD4+ helper T cell epitope and LHRH was very efficient at inducing high antibody titres and preventing fertility of mice. The CD4+ T helper cell epitope has the sequence KLIPNASLIENCTKAEL and is derived from the F protein of the mobillivirus canine distemper virus. This T_H epitope (P25) has been shown to be reactive in a variety of animals including dogs [15]. LHRH is a peptide hormone secreted by the hypothalamus and initiates a cascade of endocrine events which lead to the control of reproduction in female and male of most mammals in which the sequence is conserved. In the present study we have used this peptide vaccine to investigate the influence of substitution of the single cysteine residue within the peptide sequence on the antibody response and the effect of dose on antibody titre and the number of antigen-secreting cells present in spleen and bone marrow. We also compared the immune responses obtained by administration of the peptide in its lipidated form with or without an additional adjuvant Iscomatrix^®.