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Although VP1 region 140 to 160 of foot-and-mouth disease virus (FMDV) is able to elicit neutralizing antibody in cattle, the protection against virus challenge that is conferred by peptide immunization is often poor. Here, we show that bovine T cells primed with peptides derived from this region generally show no reactivity to intact FMDV. In contrast, T-cell epitope VP4[20-34] is able to prime for a virus-specific response.

There is a quest for the development of a new generation of vaccines consisting of well-defined subunit antigens. For a number of practical reasons it is attractive to develop vaccines on the basis of synthetic peptides. However, their efficacy may be limited by genetic restrictions imposed on T-cell recognition via major histocompatibility complex (MHC) polymorphism, as shown by many studies using inbred animal species. To study the effect of MHC polymorphism in an outbred species, we selected four cattle homozygous for different A-DR-DQ haplotypes, and another four cattle which shared one haplotype in combination with a haplotype of one of the MHC homozygous animals. We analysed responses to synthetic peptides comprising defined T-cell epitopes of foot-and-mouth disease virus (FMDV) in this selected group of FMDV-vaccinated cattle. This analysis shows that even in outbred animals. MHC polymorphism influences the responses to synthetic peptides. Interestingly, one of the peptides, VP4[20-34], was recognized in association with at least four different MHC haplotypes. Fine specificity analysis of this peptide revealed subtle shifts in the core epitope recognized. All peptides that induced lymphocyte proliferation in vitro were found to induce a T-helper type-1 (Th1) type of response, irrespective of the MHC haplotype involved. Together, these data support the notion that individuals carrying distinct MHC types can be vaccinated successfully by vaccines that include only a limited number of peptides. In the design of a peptide vaccine against FMDV we suggest inclusion of the highly conserved VP4 sequence 20-34.

Cathepsin D and cathepsin B are endosomal/lysosomal proteases that are thought to play a role during in vivo antigen processing, releasing fragments for binding to major histocompatibility complex class II products and subsequent presentation to T cells. Here we treated purified foot-and-mouth disease virus (FMDV) strain A10Holland with both enzymes. Cathepsin D, but not cathepsin B, was shown to release fragments from reduced or non-reduced FMDV under mild conditions in vitro. Twenty-eight predominant cathepsin D-released fragments were purified by HPLC and identified by amino acid composition analysis and sequencing. The unseparated set of fragments produced (the digest) was able to stimulate T cells from eight vaccinated cattle. With respect to the response to intact virus the extent of the response to the digest differed between animals: four animals could be classified as good responders, three as intermediate responders and one as a low responder. Subsequently, we investigated the proliferative T cell response to a large set of synthetic peptides in detail for two animals, one belonging to the group of good responders, the other being the low responder. The peptides covered all 28 cathepsin D-released fragments analysed and also several sequences not recovered from the digest. In this way seven T cell sites could be identified, five of which coincided with cathepsin D-released fragments. The other two T cell sites were VP2[54-72], being a homologue of a T cell site identified for FMDV strain O1K and the N terminus of VP4. Whether the most dominantly recognized T cell site was recovered from the digest or not was shown to be related to the good or low response to the digest. These findings suggest a role for cathepsin D in the release of some but not all T cell-stimulatory fragments from FMDV.

T cells specific for the mycobacterial 65 kDa heat-shock protein (hsp65) play a pivotal role in the development of adjuvant arthritis (AA) in Lewis rats. Upon adoptive transfer, CD4+ T cells recognizing a particular hsp65 epitope trigger the onset of disease. Activation of hsp65-reactive T cells can be achieved by immunization with heat-killed mycobacteria in mineral oil--complete Freund's adjuvant (CFA)--or with purified recombinant hsp65. Arthritis, however, will only develop after immunization with CFA. In fact, preimmunization with hsp65 protects against any subsequent attempt to induce AA. In this study, we examined polyclonal lymph node cell responses in Lewis rats, immunized with either CFA or purified recombinant hsp65 in incomplete Freund's adjuvant, to a set of hsp65 fragments generated by a mild digestion with cathepsin D. Proliferative responses to several hsp65 fragments varied with the type of antigen used for immunization. A cathepsin D-released fragment, identified as residues 376-408, preferentially triggered proliferation of rat T cells after hsp65 immunization. Preimmunization of Lewis rats with this peptide delayed the onset and reduced the severity of AA. Preimmunization with another fragment which was preferentially recognized after CFA immunization, representing residues 40-60, did not have such a protective effect. Our findings suggest the presence of mycobacterial hsp65 determinants that selectively trigger AA-regulating T cells and illustrate that cathepsin D may be used as an experimental tool to generate such determinants.

This study reports an identification of the major processing products of an exogenous protein antigen, viz, sperm-whale myoglobin, as obtained after cell-free processing with partially purified macrophage endosomes. It is demonstrated that such a system yields fragments that are indistinguishable by high performance liquid chromatography analysis from those generated after uptake of myoglobin inside live macrophages. The concerted action of the endosomal proteases cathepsin D and cathepsin B can account for nearly all cleavages observed. Cathepsin D appears to be mainly responsible for the initial cleavage of myoglobin, while cathepsin B catalyzes the C-terminal trimming of initially released fragments. The fragments released by cathepsin D contain most, if not all, major epitopes for murine myoglobin-specific helper T cells. Interestingly, each known T cell epitope of myoglobin is located at the very N terminus of a different myoglobin fragment released upon processing. In order to explain this correspondence, noted also in several other protein antigens, a structural relationship is proposed between antigen processing by cathepsin D and antigen recognition by major histocompatibility complex (MHC) class II products. As is demonstrated here, this relationship may be used as a predictive tool for the identification of MHC-binding sequences as well as of T cell epitopes in their naturally occurring form.

Adjuvant arthritis (AA) can be induced in genetically susceptible rats by immunization with heat-killed mycobacteria suspended in mineral oil. From our analysis of arthritogenic T cell clone A2b, obtained from an arthritic Lewis rat and specific for the 180-188 epitope of mycobacterial 65-kDa heat-shock protein (hsp 65), the possible origin of AA was explained by the existence of a molecular mimicry of the 180-188 epitope with a cartilage-associated self antigen. We now have shown that Lewis rats respond to the 180-188 epitope after Mycobacterium tuberculosis immunization and that arthritis-resistant Fisher and (Lewis x Fisher)F1 rats, although major histocompatibility complex class II identical with Lewis, do not respond to this epitope. However, in rare cases of arthritis in Fisher rats, responses to the epitope were seen. We obtained no evidence for a defect at the level of antigen processing and presentation or for suppression in Fisher rats. Thus, non-responsiveness in Fisher rats was likely due to a difference at the level of the T cell repertoire. Previously, we have reported that pretreatment with hsp 65 in experimental arthritis, and not only in AA, caused resistance to arthritis induction. We now present evidence that immunization with hsp 65 or in vitro stimulation with hsp 65 may lead to inhibition of responses specific for epitope 180-188. Thus the hsp 65-induced resistance to arthritis is probably caused by the induction of regulatory control specifically targeted at the 180-188 epitope. Especially in rats that tend to focus their responses on the critical 180-188 sequence, such as Lewis, regulation seems to develop following immunization with hsp 65. Since recent evidence suggests that hsp 65 and also the 180-188 epitope have a role in human arthritic conditions, the present findings are expected to contribute to further experimentation directed at exploiting hsp 65 or its epitopes for the development of new therapeutical approaches in humans.

PhoE is a pore-forming protein, abundantly expressed in the Escherichia coli outer membrane. Previous investigations have shown the possibility of inserting antigenic determinants in cell surface-exposed regions of PhoE by recombinant DNA techniques without disturbing the biogenesis and the functioning of the protein. This method proved to be successful for foot-and-mouth disease virus B cell determinants. We have now shown for the first time that PhoE can also be used as a carrier molecule for T cell epitopes. A well-characterized T cell epitope (180-188) of the 65-kDa heat-shock protein (hsp 65) of Mycobacterium tuberculosis was expressed in PhoE and tested for recognition by specific T cell clones. Specific and efficient T cell proliferation was found after stimulation with this protein construct in vitro. Interestingly, paraformaldehyde fixation of antigen-presenting cells did not abrogate T cell recognition. Thus, in contrast to hsp 65 itself, recognition of epitope 180-188 in the context of PhoE appeared to be independent of antigen-processing events. At the level of polyclonal T cell responses the epitope in the context of PhoE is recognized more efficiently than 180-188 as synthetic peptide or in the context of the hsp 65 molecule itself. These findings indicate that PhoE may serve as attractive vaccine carrier not only for B, but also for T cell epitopes. Furthermore, the possibility for expression of PhoE constructs in attenuated Salmonella typhimurium strains offers the exciting prospect of new types of live oral vaccines expressing selected combinations of B and T cell epitopes.