Identification of T-cell epitopes in nonstructural proteins of foot-and-mouth disease virus.

Porcine T-cell recognition of foot-and-mouth disease virus (FMDV) nonstructural proteins (NSP) was tested using in vitro lymphoproliferative responses. Lymphocytes were obtained from outbred pigs experimentally infected with FMDV. Of the different NSP, polypeptides 3A, 3B, and 3C gave the highest stimulations in the in vitro assays. The use of overlapping synthetic peptides allowed the identification of amino acid regions within these proteins that were efficiently recognized by the lymphocytes. The sequences of some of these antigenic peptides were highly conserved among different FMDV serotypes. They elicited major histocompatibility complex-restricted responses with lymphocytes from pigs infected with either a type C virus or reinfected with a heterologous FMDV. A tandem peptide containing the T-cell peptide 3A[21-35] and the B-cell antigenic site VP1[137-156] also efficiently stimulated lymphocytes from infected animals in vitro. Furthermore, this tandem peptide elicited significant levels of serotype-specific antiviral activity, a result consistent with the induction of anti-FMDV antibodies. Thus, inclusion in the peptide formulation of a T-cell epitope derived from the NSP 3A possessing the capacity to induce T helper activity can allow cooperative induction of anti-FMDV antibodies by B cells.

Recombinant viruses expressing the foot-and-mouth disease virus capsid precursor polypeptide (P1) induce cellular but not humoral antiviral immunity and partial protection in pigs.

The importance of the induction of virus neutralizing antibodies to provide protection against foot-and-mouth disease virus (FMDV) infection is well established. However, recent studies with recombinant adenovirus expressing the precursor polypeptide of the viral capsid (P1) indicate that cattle inoculated with this recombinant vector developed partial protection against FMDV infection, in the absence of a detectable specific humoral response. Other viral vectors have been widely used to induce protective immunity against many pathogens, and it has been reported that the use of different vectors for priming and boosting injections can provide a synergistic effect on this response. In this work, we determined the immunogenicity of two recombinant viruses (adenovirus and vaccinia) expressing P1-FMDV, administered either individually or sequentially, and the protection that they induced against FMDV challenge in pigs. A double immunization with the adeno-P1 virus was the most effective strategy at inducing protective immunity. In contrast to previous reports, the use of two different vectors for priming and boosting did not show a synergistic effect on the protection induced against FMD. Interestingly, immunized pigs developed FMDV-specific T cell responses but not detectable antibodies. Thus, the protection observed was likely to be mediated by a cellular immune response.

Evidence of partial protection against foot-and-mouth disease in cattle immunized with a recombinant adenovirus vector expressing the precursor polypeptide (P1) of foot-and-mouth disease virus capsid proteins.

A recombinant live vector vaccine was produced by insertion of cDNA encoding the structural proteins (P1) of foot-and-mouth disease virus (FMDV) into a replication-competent human adenovirus type 5 vaccine strain (Ad5 wt). Groups of cattle (n = 3) were immunized twice, by the subcutaneous and/or intranasal routes, with either the Ad5 wt vaccine or with the recombinant FMDV Ad5-P1 vaccine. All animals were challenged by intranasal instillation of FMDV 4 weeks after the second immunizations. In the absence of a detectable antibody response to FMDV, significant protection against viral challenge was seen in all of the animals immunized twice by the subcutaneous route with the recombinant vaccine. The observed partial protection against clinical disease was not associated with a reduction in titre of persistent FMDV infections in the oropharynx of challenged cattle.

Molecular cloning, expression and immunological analysis of the capsid precursor polypeptide (P1) from swine vesicular disease virus.

Swine vesicular disease virus (SVDV) is the aetiological agent of a highly contagious viral disease of pigs, whose symptoms are indistinguishable from those caused by foot-and-mouth disease virus (FMDV). The gene coding for the capsid protein precursor of SVDV (P1) from a recent spanish isolate (SPA/1/'93) was cloned and expressed in bacteria, and the antigenicity and immunogenicity of the recombinant product were evaluated. The recombinant P1 was recognised by antibodies against SVDV induced in pigs infected experimentally with different SVDV strains. Immunisation of swine with recombinant P1-induced SVDV-specific cellular and humoral immune responses. The implications of these results in SVD diagnostic as well as in vaccine development are discussed.

Analysis of the B and T cell response in guinea pigs induced with recombinant vaccinia expressing foot-and-mouth disease virus structural proteins.

Recombinant vaccinia viruses expressing foot-and-mouth disease virus (FMDV) P1 and VP1 genes have been used to study the immune response induced by these viral polypeptides in guinea pigs. Anti-FMDV antibodies, but not neutralizing activity, were detected in the sera from immunized animals. The results indicate that both CD4+ and CD8+ FMDV-specific T cells were induced by the vaccinia recombinants. Consistently with the activation of CD4+ T cells, lymphocytes from immunized animals specifically proliferated in vitro in response to whole virus. The induction of virus-specific CD8+ T cells was determined by CTL assay of immune splenocytes restimulated in vitro with FMDV infected cells. Altogether, the results obtained indicate that both B and T cell immune responses to FMDV are elicited upon immunization of guinea pigs with vaccinia recombinants expressing FMDV structural polypeptides.

Infection with foot-and-mouth disease virus results in a rapid reduction of MHC class I surface expression.

The modulation of MHC class I molecule expression on the surface of cells as a consequence of foot-and-mouth disease virus (FMDV) infection has been examined. On cells infected with FMDV, class I expression was reduced to approximately 70% of the initial value 3 h after the infection and to 53% after 6 h. On cells depleted of surface class I complexes by acid treatment, the appearance of newly assembled class I-peptide complexes on the cell surface of non-infected cells increased immediately upon neutralization and original class I levels were recovered in about 20 h. In contrast, the appearance of new peptide-bound class I molecules on the cell surface was inhibited as early as 30 min after FMDV infection. Since the shut-down of FMDV-mediated host protein synthesis occurs approximately 2-3 h post-infection, this result suggests that an earlier event, which prevents the surface expression of newly synthesized complexes, is induced following FMDV infection. Thus, FMDV-infected cells rapidly become unable to present viral peptides in association with MHC class I molecules to T lymphocytes. Such a mechanism would assist virus evasion of the cytotoxic immune response of the host.

The structural protein p54 is essential for African swine fever virus viability.

Protein p54, one of the most antigenic structural African swine fever virus (ASFV) proteins, has been localized by immuno-electron microscopy in the replication factories of infected cells, mainly associated with membranes and immature virus particles. Attempts to inactivate the p54 gene from ASFV by targeted insertion of beta-galactosidase selection marker was uniformly unsuccessful, suggesting that this gene is essential for virus viability. To demonstrate that, we inserted in the TK (thymidine kinase) locus of the virus a construction containing a second copy of the p54 gene and beta-glucuronidase selection marker under the control of p54 and p73 promoters, respectively. Virus mutant clones expressing a second copy of p54 and beta-glucuronidase were used to achieve deletion mutants of the original copy of the gene. Virus mutants expressing only the second inserted copy of p54 and the two selection markers mentioned above were successfully obtained. Therefore, we have demonstrated that the p54 gene product plays an essential role in virus growth, characterizing for the first time in ASFV an essential virus gene.

Inhibition of tumor growth by histoincompatible cells expressing interleukin-2.

Murine tumor cells engineered to express IL-2 have been shown to be rejected by the syngeneic host, which is then protected against a subsequent tumorigenic challenge. To assess whether IL-2 has to be produced by the tumor cells themselves, or whether its local delivery would be sufficient to promote such beneficial effects, the syngeneic tumor cells were co-inoculated with allogeneic or xenogeneic cells secreting IL-2, selected after gene transfection. In several murine systems, it was observed that this is an efficient approach for controlling the growth of the syngeneic tumor. However, animals which rejected the tumor were not protected against a subsequent challenge. Several lines of evidence indicate that NK cells play a major role in tumor rejection induced by the IL-2 expressing histoincompatible vector cells. Thus, while local delivery of IL-2 in the vicinity of a tumor might not be sufficient to promote a systemic long-term specific antitumor immune response, it can control the growth of the primary syngeneic tumor. These experiments demonstrate the feasibility of using genetically engineered histoincompatible cells (which are rejected by the host's immune system) as a transient delivery system in vivo.

[Inhibition of the tumoral growth induced by the injection of histo-incompatible cells producing interleukin-2]. / Inhibition de la croissance tumorale induite par l'injection de cellules histo-incompatibles produisant de l'interleukine-2.

In murine models, we show that the growth of a transplanted tumor can be controlled when allogeneic or xenogeneic cells expressing high levels of interleukin-2 are co-injected with the syngeneic tumor cells. Thus, genetically modified allogeneic or xenogeneic cells could have some therapeutic potential as vectors for transient cytokine gene expression.

The structure and function of MHC molecules. Possible implications for the control of tumor growth by MHC-restricted T cells.

We briefly survey recent basic progress on the structure and function of MHC molecules. We call attention on a number of remaining unanswered questions, and focus on a few points which we think might be of importance in immune surveillance against tumors mediated by MHC restricted T cells. In particular, we discuss the possible peptidic nature of certain tumor antigens. Following these lines, we mention a recent approach for the in vivo activation of specific CTL.

Interleukin 2-dependent activation of tumor-specific cytotoxic T lymphocytes in vivo.

We have quantitatively studied the effect of interleukin (IL) 2 on the cytotoxic T lymphocyte (CTL) response to tumor cells in vivo. Mastocytoma P815 was transfected with murine IL 2 cDNA (P815-IL 2) and injected into syngeneic mice. The anti-tumor response was analyzed and compared with the response induced by the non-transfected cells. P815 parental cells are highly tumorigenic, causing death within 20-30 days. In contrast, IL 2-transfected cells were totally rejected. Co-injection of IL 2-secreting and parental cells resulted in the inhibition of growth of both type of tumors. In addition, the response induced by IL 2-secreting cells protected the mice against a subsequent challenge with P815. Long-term memory persisted in treated mice 3 months after tumor rejection. Frequencies of CTL precursors and CTL specific for P815 increased as a result of IL 2 secretion by the target cells. Estimates of CTL frequency at days 8 and 12 after injection were 2 to 3 times higher in mice inoculated with P815-IL 2 cells, and this correlated with tumor rejection.

A novel approach to the induction of specific cytolytic T cells in vivo.

The induction of specific cytotoxic T lymphocytes (CTL) is one component in the immune response which can effectively protect the host against the progression of many viral infections. CTL are also known to play an important role in immune defence against tumour growth. CTL induction is dependent the presence of the specific antigen, appropriately presented, and interleukin-2 (IL2), provided by T helper lymphocytes. We studied the specific CTL response induced by tumour cells transfected with murine IL2. Our results show that tumour cells manipulated to secrete IL2 induce an improved specific anti-tumour response which results in tumour rejection in mice. To further investigate the effect of IL2 on the CTL response to different antigens, we introduced synthetic peptides into IL2-secreting tumour cells and determined the specific CTL induction in syngeneic mice immunized with these cells. We report here that such IL2-secreting cells can effectively prime peptide-specific CTL in vivo. Our data are relevant to immunotherapy and vaccine development and open up the possibility that autologous cells, manipulated to secrete IL2 and located with one or a cocktail of peptides, could be used to stimulate a specific CTL response.

Cloning and characterization of a gene coding for a protein (KAP) associated with the kinetoplast of epimastigotes and amastigotes of Trypanosoma cruzi.

We have cloned and characterized a gene of Trypanosoma cruzi which encodes a protein, KAP (kinetoplasts-associated protein), expressed in the kinetoplasts of epimastigotes and amastigotes, the replicative stages of the parasite, but not in kinetoplasts of trypomastigotes. The single-copy gene is transcribed into a 3900-nt polyadenylated mRNA. Its trans-splicing acceptor site is preceded by a run of 15 adenosine residues. An open reading frame of 1052 codons is followed by a 3' untranslated region containing short sequences characteristic of rapidly degradable RNAs. The potential translation product of the KAP gene contains a central region composed of four blocks of repeats of a 9-amino-acid motif. Rabbit antibodies raised against three synthetic peptides containing KAP sequence recognized a 175-kDa protein in epimastigotes and amastigotes which appears by indirect immunofluorescence to be associated with their kinetoplasts. The antibodies do not recognize the kinetoplast of trypomastigotes. The amino terminus of KAP contains features compatible with mitochondrial topogenic sequences.

Synthetic peptide vaccine confers protection against murine malaria.

A synthetic peptide, (DPPPPNPN)2D, representing a subunit of the repeat domain of the Plasmodium berghei circumsporozoite protein, was conjugated to tetanus toxoid using bisdiazobenzidine. Immunization of mice and rats with the conjugate induced high serum titers of antibodies to the parasite, and most of the animals were completely protected from malaria infection when challenged with sporozoites.

Isolation and characterization of the main allergen of Dermatophagoides farinae by monoclonal antibodies that recognize IgE related epitopes.

Mouse monoclonal antibodies (MAbs) with different specificities against Dermatophagoides farinae (D. farinae) extract have been obtained. Fifteen of these antibodies reacted with allergen molecules contained in D. farinae and D. pteronyssinus extracts, immunoprecipitating the main allergen of D. farinae (DF29) and homologous allergen of D. pteronyssinus (DP28). In addition, the monoclonal antibody MADF2 immunoprecipitated DF29 together with two other polypeptides (mol. wt 20,000 and 40,000) from D. farinae extracts. Five monoclonal antibodies (MADF2, MADF5, MADF9, MADF10 and MADF13) were selected to study their epitope specificity and the relationship of the epitope location on the allergen with the IgE binding site. By cross-inhibition studies two different epitopes and two partly overlapping determinants were found. In addition, two of these epitopes, those defined by MADF13 and MADF5, are close to, or overlapping, IgE binding site(s) on the allergen molecule. DF29 allergen from D. farinae extract was purified by affinity chromatography using MADF5 coupled to Sepharose. The purified allergen had capacity to bind mite specific human IgE and demonstrated an allergenic activity of up to 70% of total extract of D. farinae. These results indicate that DF29 molecule is the main allergen from D. farinae extracts.

Isolation of the major IgE-binding protein from Parietaria judaica pollen using monoclonal antibodies.

Allergen molecules from Parietaria judaica pollen, a widely distributed allergy inducer in Southern and Western Europe, have been studied using specific monoclonal antibodies (MAbs). MAbs against IgE-binding components were selected in a 4-step radioimmunoassay. Three different MAbs (AC/1.1, AC/7.1 and AC/15.1) were obtained which recognized epitope(s) located on a polypeptide of 10 Kd (Pj10). This polypeptide displayed the highest IgE-binding ability under either native or SDS-denatured conditions, as determined by immunoadsorption and immunodetection after SDS-PAGE, respectively. The Pj10-containing allergen, purified on an AC/1.1 MAb-Sepharose column, was able to inhibit most of the binding of specific IgE to the pollen extract coupled to paper discs in an inhibition radioallergosorbent test (RAST). The affinity-purified allergen exhibited the same immunoelectrophoretic behaviour as the native allergen.

Identification of a novel allergen molecule from Dermatophagoides by monoclonal antibodies.

Six different monoclonal antibodies (MAb) have been obtained which bind to components contained in extracts from Dermatophagoides. One out of the six MAb recognized molecules displaying IgE binding ability. This MAb (MADP-1) immunoprecipitated allergenic polypeptides of 42 kDa and 30 kDa from 125I-labeled extracts of D. pteronyssinus and D. farinae respectively. Purified allergen preparations from both extracts have been obtained by affinity chromatography using a column of purified MADP-1 coupled to Sepharose. The purified fractions partially compete the binding of specific IgE contained in sera from sensitized patients to the whole extracts, in a radioallergosorbent inhibition test.