The cellular immune recognition of proteins expressed by an African swine fever virus random genomic library.

The cellular immune recognition of peptides expressed by an African swine fever virus (ASFV) random genomic library has been studied. DNA from the Malawi (LIL20/1) ASFV isolate was randomly sheared by sonication, cloned into a plasmid vector downstream of a bacteriophage T7 promoter, and 72 recombinant plasmids were arbitrarily selected. These plasmids were transiently expressed following transfection into major histocompatibility complex (MHC) class I(+) class II(-) matched pig skin cells, which had been co-infected with vTF7-3, a recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase. Such cells served as antigen presenting cells and each recombinant plasmid was screened in a proliferation assay for recognition by CD8(+) lymphocytes from inbred pigs previously exposed to ASFV. This assay was demonstrated to measure CD8(+) T cell proliferation, as predicted by the phenotype of the antigen presenting cell. Of the 72 randomly selected clones, 14 were reproducibly recognised by immune pig lymphocytes and 10 corresponded to non-overlapping and distinct nucleic acid sequences. This high frequency of ASFV encoded antigenic epitopes supports the concept that cellular immunity to the virus may play an important role in resistance to ASF.

African swine fever virus: a B cell-mitogenic virus in vivo and in vitro.

The two major characteristics of pathogenesis in African swine fever virus (ASFV) infections of domestic pigs are massive B-cell apoptosis and haemorrhage. The effects of ASFV on porcine B cells have therefore been systematically examined in vivo, by using virus-infected pigs and SCID-Beige mice reconstituted with porcine bone marrow, and in vitro, by using porcine B-cell lines and B cells from normal and ASFV-infected pigs. Secretion of porcine Ig was stimulated by ASFV both in vivo and in bone marrow cultures in vitro, with the virulent Malawi isolate of ASFV being the most effective. Stimulation of Ig secretion in vitro depended on the presence of ASFV-infected macrophages and did not occur with supernatants from ASFV-infected macrophages. Although the virus alone did not stimulate proliferation of purified B cells in vitro, it was co-stimulatory with CD154 (CD40 ligand). The B cells recovered from ASFV-infected porcine lymphoid tissue were of activated surface marker phenotypes and, interestingly, expressed diminished levels of the B-cell co-stimulatory surface molecule CD21. In addition, they were highly sensitive to IL-4 and CD154. These results may be integrated into a model of pathogenesis in which those B cells activated indirectly as a result of virulent ASFV infection of macrophages are not rescued from apoptosis through interaction with CD154, due to the drastic depletion of T cells that occurs early in infection. The consequently diminished specific anti-ASFV antibody response would favour survival of the virus, with the non-specific hypergammaglobulinaemia being perhaps another example of pathogen-mediated immune deviation.

Demonstration of bovine CD8+ T-cell responses to foot-and-mouth disease virus.

The aim of this study was to investigate the importance of cellular immunity in foot-and-mouth disease in cattle, in particular to determine whether a CD8+ T-cell response could be detected, as these cells may play a role in both immunity and virus persistence. As attempts to characterize classical cytotoxic T cells had yielded non-reproducible results, largely due to high backgrounds in control cultures, a proliferation assay was developed that was demonstrated to detect antigen-specific, MHC class I-restricted bovine CD8+ cells responding to foot-and-mouth disease virus (FMDV). Proliferative CD8+ T-cell responses were detected consistently from 10 to 14 days following infection with FMDV and typically lasted 3-4 weeks. The role of CD8+ T cells in control of the disease, in particular their relevance for the establishment of persistence, may now be investigated.

Heterotypic recognition of recombinant FMDV proteins by bovine T-cells: the polymerase (P3Dpol) as an immunodominant T-cell immunogen.

In this study we have examined the recognition of VP0, VP1, VP2, VP3 and P3Dpol by PBMC and CD4+ T-cells from infected, vaccinated-challenged, and multiply-vaccinated (O1, A24, C1 or ASIA1) cattle using recombinant proteins of an O1 serotype virus. The structural protein VP1 was recognised in an homotypic context whereas VP2, VP3, VP4 and P3Dpol were also recognised by T-cells from animals exposed to heterotypic viruses. Only the non-structural protein P3Dpol was consistently recognised by T-cells from the majority of animals examined and heterotypic recognition correlated with the presence of serologically detectable P3Dpol in purified virus. Thus, P3Dpol is a major cross-reactive immunodeterminant of FMDV, eliciting heterotypic T-cell responses and, therefore, with possible potential for inclusion in a subunit vaccine.

Modulation of T cell and monocyte function in the spleen following infection of pigs with African swine fever virus.

Infection of pigs with many strains of African Swine Fever Virus (ASFV) has been shown to cause a loss or marked decrease in the ability of splenocytes to respond to mitogens. These observations have been extended by cell fractionation and reconstitution experiments to show that the mitogen stimulated proliferative capacity of both the CD4+ and CD8+ T cells is affected. Similarly, monocytes which are directly infectable by virus, are functionally defective as antigen presenting cells when added to mitogen stimulated normal T cells. Interestingly, the same T cells which respond poorly in mitogenic assays can be activated by stimulation through the CD3 receptor. In contrast to the defective mitogenic response of T cells, B cell function, as assessed by stimulation through the CD40 ligand in vitro remains intact. There is no evidence for apoptosis in either the T cells or the B cells recovered from the spleens of ASFV infected animals 1-5 days following infection. Although the number of leucocytes which can be recovered from the infected spleen decreases rapidly with progression of the disease, the proportion of the different cell phenotypes remains constant. Thus decreased activity of lymphocytes in lymphoid tissue from ASFV infected animals appears to be directly attributable to infection of the monocytes.

Recognition of a unique peptide epitope of the mycobacterial and human heat shock protein 65-60 antigen by T cells of patients with recurrent oral ulcers.

T cell epitopes of the 65-kD heat shock protein (hsp) were investigated in patients with recurrent oral ulcers (ROU). Peripheral blood mononuclear cells were stimulated with overlapping synthetic peptide (15ers), derived from the sequence of the 65-kD hsp of Mycobacterium tuberculosis. Specific lymphoproliferative responses were stimulated only with peptide 91-105 in ROU, compared with healthy or disease controls (P < 0.01). This was confirmed by studying 760 short term cell lines generated with the 65-kD hsp and then stimulated with the peptides. The frequency of short term cells lines responding to peptide 91-105 in ROU was significantly greater than in healthy (P < 0.0001) or disease controls (P < 0.01). A comparative investigation with the homologous human 60-kD hsp peptide 116-130 also showed significantly greater lymphoproliferative responses in ROU than in healthy (P < 0.01) or disease controls (P < 0.001). The potential involvement of the T cell epitope 91-105 in the pathogenesis of ROU is supported by finding a significant increase in the lymphoproliferative responses stimulated with peptide 91-105 during the stage of ulceration, compared with remission in 9/11 patients studied sequentially (P < 0.05). The results suggest that oral ulceration might be initiated by the microbial hsp peptide 91-105 stimulating the mucosal Langerhans cells, which may generate autoreactive T cell clones primed to the homologous peptide 116-130.

T cell epitope expression of mycobacterial and homologous human 65-kilodalton heat shock protein peptides in short term cell lines from patients with Behçet's disease.

T cell epitopes of the 65-kDa heat shock protein (HSP) were mapped in patients with Behçet's disease (BD), by stimulating T cells with the overlapping synthetic peptides derived from the sequences of the Mycobacterium tuberculosis 65-kDa HSP. Significant lymphoproliferative responses were stimulated with four HSP peptides in BD, as compared with the related disease (recurrent oral ulcers), unrelated disease, and healthy controls (p < 0.05 to 0.005). In order to assess the relative frequency of sensitized lymphocytes by these peptides, 7353 short term cell lines were generated from the lymphocytes of patients and controls. Peptides 111-125, 154-172, and 311-325 (p < 0.001) and peptide 219-233 (p < 0.02) yielded significantly greater frequency of STCL in BD than in healthy and disease controls. All but peptide 154-172 stimulated only the CD4+ subset of T cells, although there was no evidence that reactivity to the selected peptides is restricted by DR2 to DR7 Ag. HLA-B51 is significantly associated with BD, but there was no evidence that B51 was a restricting element, when B51+ patients were compared with B51- patients with BD, and with B51+ healthy control subjects. A comparative investigation was then carried out between the corresponding mycobacterial and human HSP peptides. Similar or higher lympho-proliferative responses were stimulated by the human peptides compared with the mycobacterial peptides. These results suggest that the four peptide determinants within the 65-kDa HSP might be involved in the pathogenesis of BD. Whereas the high microbial load and associated stress proteins found in oral ulceration of BD may initiate an immune response to these conserved epitopes, expression of autoreactive T cell clones might be stimulated by immunodominant T cell epitopes of endogenous HSP which may induce immunopathologic changes.

The comparative frequency of human T cells responding to a native antigen and a synthetic peptide derived from Streptococcus mutans.

The frequency of human peripheral blood T cells responding to a 21-residue synthetic peptide (SP 21) derived from the sequence of a 3.8-kDa streptococcal antigen was estimated by limiting dilution analysis and compared with the frequency of cells responding to the native, cross-reactive 185-kDa streptococcal antigen. Frequency estimates were made by measuring both [3H]thymidine incorporation and IL 2 production in the same cell cultures. The results provided frequency estimates for SP 21-reactive cells of between 1:42 147 and 1:306 110, with a mean of 1:160 617 by [3H]thymidine incorporation, and 1:139 893 to 1:241 315 (mean 1:165 315) using the IL 2 assay. With the native 185-kDa streptococcal antigen, frequency estimates were between 1:38 393 and 1:86 142 (mean 1:169 934) according to the proliferative response and 1:22 462 and 1:100 400 (mean 1:61 189) by the IL 2 assay.

An analysis of synthetic peptide restriction by HLA-DR alleles in T cells from human subjects, naturally sensitized by Streptococcus mutans.

T cells from most human subjects show significant in vitro proliferative responses to a 185-kDa surface Ag from Streptococcus mutans as well as to synthetic peptides derived from the sequence of a Mr 3800 streptococcal Ag. T cells from subjects expressing each of the alleles from DR1 to DR7 responded to synthetic peptides of 17 or 21 amino acid residues. Furthermore, inhibition studies with mAb to HLA class I and class II Ag showed that the DR Ag was a restriction molecule for the proliferative responses. Mouse L cells transfected with DR1, DR2, DR4, DR5, and DR7 were used to confirm the permissive nature of the responses. An analysis of the fine specificity of the responses showed that the minimum peptides capable of stimulating T cells from subjects with different DR types varied by one or two residues. For DR2 and DR3 the shortest peptide was residues 6-15, an additional serine (residue 5) was required for DR1 and DR7 and an aspartic acid (residue 4) for DR4, DR5, and DR6. Successful oral-mucosal bacterial colonisation in humans, by a largely commensal Streptococcus, might be associated with the permissive nature of the HLA-DR restriction of the response to a major streptococcal cell surface peptide. The peptide recognised in association with the HLA-DR molecule may induce an immune response that prevents central entry of the organism from the peripheral mucosal site.

The reactivity of naturally sensitized human CD4 cells and IgG antibodies to synthetic peptides derived from the amino terminal sequences of a 3800 MW Streptococcus mutans antigen.

Natural immunity to synthetic peptides (SP) derived from the sequences of a 3800 MW streptococcal antigen (SA) was found in human subjects. Significant serum IgG antibodies were detected both to the native SA and to peptides consisting of residues 3-13, 1-15 and 1-20. Inhibition studies confirmed cross-reactivity between the native SA and SP. A series of short peptides with deletions at the amino and carboxy termini were then tested to determine the sequence of B-cell epitopes. Residues 8-13 and 1-6 bound significant serum IgG antibodies, but residues 8-13 were more effective and consistent in inhibiting human antibodies than residues 1-6. These results suggest that residues 8-13 constitute a major B-cell epitope but that residues 1-6 may represent a minor B-cell epitope. The human CD4 subset of T cells was then examined by stimulating the cells with SA or SP and measuring the uptake of [3H]thymidine [( 3H]TdR). The cells were found to be sensitized in vivo to both the native SA and the SP and cross-reactivity between the SA and SP was shown by enrichment and depletion experiments on antigen-coated monocytes. As with the B-cell epitope, the series of short peptides was used to stimulate CD4 cells, in order to determine the T-cell epitope. Residues 6-15 were the shortest SP which stimulated significant [3H]TdR uptake and this peptide was designated as a T-cell epitope. The results suggest that natural oral immunization with Streptococcus mutans induces serum antibodies and T-cell sensitization to a peptide in which a T-cell epitope (residues 6-15) overlaps with a B-cell epitope (residues 8-13). Furthermore, a comparison between linear and cycled peptides suggests that unlike immunogenicity which is commonly enhanced by the more rigid cyclized peptides, antigenicity is favoured by linear peptides. This was evident not only for antibodies but also for T-cell proliferative responses.

Identification of T- and B-cell epitopes in synthetic peptides derived from a Streptococcus mutans protein and characterization of their antigenicity and immunogenicity.

Natural immunity to synthetic peptides (SP) derived from the sequences of a 3800 Mr Streptococcus mutans antigen was found in human subjects. Significant serum IgG antibodies were detected both to the native streptococcal antigen and to the SP17, containing essentially residues 1-15. A series of short peptides with deletions at the amino- and carboxy-termini were then tested to identify the B-cell epitopes. Residues 8-13 and 1-6 bound significant serum IgG antibodies but only the former consistently inhibited human antibodies, suggesting that residues 8-13 constitute a major B-cell epitope. The human CD4 subset of T-cells was then examined and this showed a significant uptake of [3H]-thymidine when stimulated with both the native streptococcal antigen and the SP17. The series of short peptides was then used to stimulate CD4 cells, in order to determine the T-cell epitope. The synthetic peptide with residues 6-15 was the shortest peptide that stimulated significant [3H]-thymidine uptake and this peptide was designated as a T-cell epitope. The immunogenicity and antigenicity of SP17 was also investigated in macaques. Immunization of monkeys with the free SP17 failed to elicit serum antibodies or T-cell responses. However, immunization with SP17 linked to tetanus toxoid as a carrier elicited serum antibodies and proliferative responses of lymphocytes, not only to the synthetic peptide but also to the native streptococcal antigen. As in the human studies a B-cell epitope was found in residues 8-13, whereas an overlapping T-cell epitope was located in residues 7-15.(ABSTRACT TRUNCATED AT 250 WORDS)

T cell interactions generated by synthetic peptides covalently linked to a carrier.

We have attempted to extend the synthetic peptide-carrier bridge concept of T cell-B cell interaction to T cell-T cell interaction. DNA synthesis of human CD4 cells that were sensitized in vivo to a native streptococcal antigen (SA) was stimulated in vitro with synthetic peptides (SP) derived from the sequence of native SA. The SP were linked to tetanus toxoid (TT) as a carrier which was recognized by primed T cells. The uptake of [3H]thymidine was significantly greater when stimulated with covalently linked SP-TT than that with non-covalently mixed SP and TT. The TT- and SP-sensitized CD4 cells were then enriched and depleted by panning on TT- or SP-treated monocyte layers. When TT-enriched CD4 cells were reconstituted with SP-enriched cells, [3H]thymidine uptake was significantly greater with the linked SP-TT than with the mixed SP and TT. However, reconstitution of the TT-enriched with SP-depleted CD4 cells or the converse failed to increase significantly DNA synthesis by cells stimulated with the linked SP-TT. The production of interleukin 2 (IL 2) and expression of IL 2 receptors were then assayed to examine any difference in stimulation between TT and SP. Both IL2 and IL2 receptors were diminished and delayed when T cells were stimulated with SP as compared with TT. The results suggest that epitope-linked clusters of monocytes, TT-sensitized CD4 and SP-sensitized CD4 cells enable IL2 released by the TT-sensitized CD4 cells to stimulate the SP-sensitized CD4 cells that produce inadequate amounts of IL2. Indeed, addition of recombinant IL2 to T cells stimulated with mixed SP and TT induces an increase in DNA synthesis which becomes similar to that resulting from stimulation with the linked SP-TT.