Interleukin-2 reverses T cell unresponsiveness to Plasmodium falciparum-antigen in malaria immune subjects

Peripheral blood mononuclear cells (PBMC) from a large proportion of 34 healthy adult native residents in a malaria endemic area showed null or marginal proliferative response (low-responders) to schizont-enriched Plasmodium falciparum malaria antigen (M.Ag) but good response to pokeweed mitogen. In contrast, substantial proliferative response to M.Ag was observed in 8/8 adult temporary residents with a history of one to three acute malaria episodes. Purified CD4+ T cells preferentially responded to M.Ag, however in low-responders CD4+ T cell proliferation was poor. Moreover, no inhibition of CD4+ T cell proliferation was observed when graded numbers of CD8+ T cells were added in culture. The addition of recombinant interleukin 2 (rIL-2) to M.Ag restored the proliferative response of low-responders' PBMC. This response was M.Ag-specific when CD4+ T cells grown in M.Ag plus rIL-2, but not in rIL-2 alone, were tested in secondary cultures.     

Monoclonal anti-parasite and anti-RBC antibodies produced by stable EBV-transformed B cell lines from malaria patients

To produce human monoclonal antibodies associated with infectious disease, peripheral blood lymphocytes (PBL) from patients with Plasmodium falciparum malaria were transformed with EB-virus in vitro. To enrich for malaria-specific B cells, PBL were incubated for 3 days with unsoluble P. falciparum antigen before EBV-transformation. Furthermore, cyclosporin A was added during and after transformation to eliminate T cell suppression of B cell growth. Microcultures were screened for antibodies against blood stage antigens of P. falciparum or of noninfected erythrocytes by ELISA and indirect immunofluorescence. Cultures producing anti-P. falciparum and/or anti-erythrocyte antibodies were developed from the lymphocytes of eight patients, including some individuals with their first infection. Positive cultures were cloned and propagated for several weeks. Seven of 15 clones producing antibody at a stable rate have now been kept in cultures for more than 1 yr. Of six cultures analyzed in detail, all produced IgM antibodies of either K or lambda isotype. Although three clones were monoclonal after one cloning, three were oligoclonal. Of the former, two produced P. falciparum-specific antibodies directed to an antigen associated with the surface of merozoites. One of the oligoclonal cultures produced anti-erythrocyte antibodies, and it was probably reacting with spectrin.     

Promiscuous malaria peptide epitope stimulates CD45Ra T cells from peripheral blood of nonexposed donors.

PBL from individuals with no history of malaria exposure, as well as cord blood lymphocytes, were tested for proliferation to T cell epitopes from the malaria circumsporozoite proteins of Plasmodium falciparum and Plasmodium vivax. Cells from many individuals proliferated in response to these peptides, but for two peptides (P. vivax317-336 and P. falciparum CS331-350) the response rate ranged from 64 to 93%, with the specific stimulation indices reaching as high as 38. The phenotype of the cells responding to PfCS331-350 was predominantly CD4+,CD8-,CD45Ra+,CD45Ro-, which was the inverse of the phenotype of the cells responding to tetanus toxoid with respect to CD45 isoforms. T cell clones from different individuals specific for PfCS331-350 were restricted by at least four different HLA-DR molecules and there was no evidence that the peptide was a "superantigen." Overlapping peptides were used to demonstrate that clones had different fine specificities although the peptide specificities of the DR4-restricted and DR11-restricted clones were similar. Although the individuals tested here have had no history of malaria exposure, these data demonstrate that they have T cells specific for malaria sequences present in high frequency that proliferate as intensely as some memory responses. Although one clone from an individual with a history of BCG vaccination did react strongly with PPD, the phenotype of these cells suggests that they are not classical memory cells for a cross-reactive recall Ag. Such cells may affect the induction or expression of malaria immunity.     

Nonpolymorphic regions of p190, a protein of the Plasmodium falciparum erythrocytic stage, contain both T and B cell epitopes

Two conserved regions from the genetically polymorphic p190 molecule of the malaria parasite Plasmodium falciparum have previously been expressed in Escherichia coli as separate polypeptides (190.L and 190.M) or as a single fusion protein (190.N). In the present study we investigated whether human B and T lymphocytes recognize these conserved regions. The more amino-terminal region, 190.L (corresponding to residues 188-363 of the encoded protein sequence) reacted preferentially with sera from donors living in a malaria-endemic area. Also, EBV-transformed B cells, from a healthy donor living in a malaria-mesoendemic area, were fused with a human-mouse hybrid line (SPM4-0), yielding two hybridomas whose products recognized both 190.L and the fusion protein 190.N, but not the 190.M polypeptide. A large number of p190-specific T cell clones were obtained from PBMC of a noninfected donor, after in vitro stimulation with the recombinant fusion protein 190.N. The clones reacted with intact, parasite-derived p190, as well as either 190.L or 190.M. Four clones that recognized the more amino-terminal fragment also responded to infected E. According to these results the more amino-terminal conserved sequences of p190 have the requisites to be immunogenic in humans.     

Malaria Antigens Stimulate Neopterin Secretion by PBMC and U937 Cells

Lysates of Plasmodium falciparum parasitized human erythrocytes stimulate U937 cells to secrete neopterin during a 48 hr co-culture period. Neopterin secretion by U937 cells was enhanced by the addition of human interferon gamma (IFN-γ). Several P. falciparum antigens, ‘FC27’ (a synthetic ‘S’ antigen), Ag16 (a recombinant ‘S’ antigen) and Ag44/RHOP3 (a recombinant merozoite rhoptry protein), also activated U937 cells to neopterin secretion and produced a similar effect on peripheral blood mononuclear cells (PBMC) from 2 of 3 normal healthy donors cultured with the antigens for 7 days. Plasma from six Nigerian malaria patients contained high neopterin concentrations ranging from 5.06 to 14.17 ng/ml. This preliminary pilot study lends support for further investigation incorporating a larger number of malaria patients and further culture experiments with U937 cells and PBMC with the aim of defining the cause and source of the large quantities of plasma neopterin produced in this infection.     

Production of human monoclonal antibodies against various antigens of erythrocyte stages of Plasmodium falciparum

Peripheral blood lymphocytes from 20 individuals living in a malaria endemic area (Burkina Faso) were transformed with Epstein-Barr virus. No antigen specific selection, nor stimulation of B-cells were performed prior to transformation. 20 cell lines were established, 14 secreted polyclonal antibodies directed against erythrocytic stages of Plasmodium falciparum. 4 lines were cloned and the supernatant analysed and characterised against Plasmodium falciparum antigens.     

Use of human universally antigenic tetanus toxin T cell epitopes as carriers for human vaccination.

Synthetic constructs were assembled as multiple Ag peptide systems containing repetitive sequences of Plasmodium falciparum and Plasmodium berghei, the causative agents of human and murine malaria respectively, and two universal human tetanus toxin T cell epitopes 830-843 and 947-967. These constructs were tested for antibody production in mice and for their capacity to stimulate human PBL and tetanus toxin-specific T cell clones. A high antibody titer can be obtained in mice when multiple Ag peptide systems are injected in various adjuvants or in PBS alone. Furthermore, all constructs can activate PBL from every donor tested. However, a variable response was obtained when different clones specific for the two tetanus toxin universal epitopes were used. These constructs may represent possible candidates for a malaria vaccine.     

Plasmodium falciparum liver-stage antigen-1 peptide-specific interferon-gamma responses are not suppressed during uncomplicated malaria in African children.

Liver-stage antigen (LSA)-1 is a candidate vaccine molecule for Plasmodium falciparum malaria, but knowledge of the evolution of naturally acquired immune responses to LSA-1 in African children is lacking. We therefore assessed cellular immune responses to two defined T cell epitopes of LSA-1, during and after uncomplicated P. falciparum malaria in a group of Gabonese children. In terms of their prevalence, interferon (IFN)-gamma responses of peripheral blood mononuclear cells (PBMC) to an LSA-1 N-terminal peptide, T1, were significantly higher when measured during the acute phase compared with convalescence. IFN-gamma responses to the LSA-J (hinge region) peptide showed a similar profile, but at a lower prevalence. Depletion experiments confirmed that CD8+ T cells are a major source of peptide-driven IFN-gamma, but both lymphoproliferation and the production of IL-10 in response to either of the peptides was low in all children at all times. PBMC from 25% of the children failed to produce IFN-gamma in response to either peptide at any time-point. The results suggest that lymphocytes producing IFN-gamma in response to at least one T cell epitope of LSA-1 are most frequent in the peripheral circulation during the acute phase of P. falciparum malaria. Thus, in this case, the generalised suppression of cell-mediated responses which characterises acute malaria does not affect liver-stage antigen-specific IFN-gamma production. These findings imply that measurements of the frequency of parasite antigen-specific cellular immune responses in clinically healthy individuals may represent significant underestimations, which has important implications for the design of field-based vaccine antigen-related studies.     

Defective in vitro production of anti-Plasmodium falciparum antibodies in some malaria-immune subjects

The cell requirements for immunoglobulin (Ig) and Plasmodium falciparum-specific antibody production in the presence of schizont-enriched malaria antigen (M.Ag) were studied in vitro. Cell donors were healthy immune adult Africans and Europeans who had experienced single P. falciparum acute infection. In the presence of M.Ag a dose-dependent increase in polyclonal IgM and IgG levels was observed with T/B cell cultures from 4/4 European and from 4/14 African donors (high-Ig producers). In 10/14 Africans M.Ag failed to induce significant Ig production (low-Ig producers). No differences in Ig levels between high- and low-Ig producers were observed in the presence of pokeweed mitogen (PWM). The addition of CD8+T cells to CD4+T/B cell cultures significantly suppressed the Ig production in PWM- but not in M.Ag-activated cultures. High levels of P. falciparum-specific antibodies were found in M.Ag-activated cultures from high-Ig producer Africans but not in cultures from Europeans or from low-Ig producer Africans. The in vitro production reflected differences in plasma levels of specific antibodies.     

Human T cells recognize polymorphic and non-polymorphic regions of the Plasmodium falciparum circumsporozoite protein.

In order to characterize T cell epitopes in the Plasmodium falciparum circumsporozoite (CS) protein sequence, we isolated T cell clones, from non-immune donors, which reacted with synthetic peptides corresponding to two predicted CS protein T cell epitopes. Peptide CS.T3 (corresponding to a non-polymorphic region of the CS protein, residues 378-398) was recognized in association with either DR2 or DRw9 restriction elements. T cell clones recognizing CS.T3 also reacted with the sporozoite-derived CS protein. Peptide CS.T2 corresponds to a polymorphic region (residues 325-341) of the CS protein. Unlike the CS.T3-specific clones, the CS.T2-specific clones did not recognize the CS protein. Since the CS.T2 peptide includes residues which are polymorphic in different P. falciparum isolates, we investigated whether these residues were critical for recognition of the peptide. We show here that a single amino acid substitution at a position of the CS protein which shows genetic polymorphism affects recognition of the sequence by human T cells. The implications of these data for malaria vaccine development are discussed.     

Human T clones reactive to the sexual stages of Plasmodium falciparum malaria. High frequency of gamete-reactive T cells in peripheral blood from nonexposed donors

Malarial gametocytes, which are taken up by mosquitoes during a blood meal, develop in the gut of the mosquito into gametes. Gametes and gametocytes contain the target antigens of transmission-blocking immunity. Here, we show that the peripheral blood of nonexposed donors contains Plasmodium falciparum gamete-reactive T cells at frequencies ranging from 1/300 to 1/4000. Studies on long-term clones demonstrated that these cells often recognized antigens shared between gametes and asexual stage parasites or even between heterologous gametes, although it has been possible to derive a P. falciparum gamete-specific T clone. The T clones examined were T3+, T4+, T8-, and either HLA-DR- or HLA-DQ-restricted. They responded to gametes by both proliferation and the secretion of gamma-interferon. The gamete-specific clone and other asexual cross-reactive clones examined could be stimulated in vitro by a preparation of mature gametocytes within RBC, but not by RBC alone, suggesting that gametocytes are immunogenic or can become immunogenic for T cells in vivo. The significance of these observations to mosquito transmission of malaria and development and application of a gamete vaccine are discussed.     

Major histocompatibility complex and T cell interactions of a universal T cell epitope from Plasmodium falciparum circumsporozoite protein

A 20-residue sequence from the C-terminal region of the circumsporozoite protein of the malaria parasite Plasmodium falciparum is considered a universal helper T cell epitope because it is immunogenic in individuals of many major histocompatibility complex (MHC) haplotypes. Subunit vaccines containing T* and the major B cell epitope of the circumsporozoite protein induce high antibody titers to the malaria parasite and significant T cell responses in humans. In this study we have evaluated the specificity of the T* sequence with regard to its binding to the human class II MHC protein DR4 (HLA-DRB1*0401), its interactions with antigen receptors on T cells, and the effect of natural variants of this sequence on its immunogenicity. Computational approaches identified multiple potential DR4-binding epitopes within T*, and experimental binding studies confirmed the following two tight binding epitopes: one located toward the N terminus (the T*-1 epitope) and one at the C terminus (the T*-5 epitope). Immunization of a human DR4 volunteer with a peptide-based vaccine containing the T* sequence elicited CD4+ T cells that recognize each of these epitopes. Here we present an analysis of the immunodominant N-terminal epitope T*-1. T*-1 residues important for interaction with DR4 and with antigen receptors on T*-specific T cells were mapped. MHC tetramers carrying DR4/T*-1 MHC-peptide complexes stained and efficiently stimulated these cells in vitro. T*-1 overlaps a region of the protein that has been described as highly polymorphic; however, the particular T*-1 residues required for anchoring to DR4 were highly conserved in Plasmodium sequences described to date.     

Antibodies and reactive T cells against the malaria heat-shock protein Pf72/Hsp70-1 and derived peptides in individuals continuously exposed to Plasmodium falciparum.

Pf72/Hsp70-1, a heat-shock protein of m.w. 72 kDa from Plasmodium falciparum is one of the Ag of interest to be included in a polyvalent vaccine against malaria. It is one of the major immunogens present in a fraction of purified blood stage parasites that elicited protection against experimental infection of Saimiri monkeys with blood stages of P. falciparum. It is present at all blood stages and one of its B cell epitopes is also detected on the surface of the infected hepatocyte. Moreover, Pf72 appears to be well conserved among different isolates of P. falciparum. We have examined the immune response against Pf72/Hsp70-1 in individuals from different age groups living in a holoendemic area (West Africa). The immune response against the native Ag (purified from schizonts and called Pf/Hsp70) was analyzed both at the humoral level by ELISA and at the cellular level by assessing in vitro proliferation and IFN-gamma production of PBMC. Of the individuals studied 52% had a statistically significant level of anti-Pf/Hsp70 antibodies as compared with unexposed individuals. These positive individuals showed a heterogeneous distribution because significant levels of antibodies were found in 70% of the adults but in only 26% of the children. The presence of Pf/Hsp70-specific reactive T cells in the blood was detected in 32% of the individuals. The total anti-Pf/Hsp70 antibody level (IgG+IgM) appeared strongly age related and correlated positively with parasite exposure, whereas the T cell response failed to correlate either with the antibody level or with age. Moreover, PBMC of donors responded to the Pf/Hsp70 in a dissociated way, namely, by either T cell proliferation or IFN-gamma production. Ten synthetic peptides based on sequences found in the C-terminal part of Pf72/Hsp70-1 were further tested as potential T cell epitopes. The proliferative response of PBMC from individuals continuously exposed to the parasite showed that three peptides more frequently trigger significant T cell proliferation (in 21% to 27% of the individuals) and three others less frequently (10%). None of these peptides allowed detection of reactive T cells in PBMC of Europeans with no previous exposure to malaria. Some of the stimulating peptides are highly similar to human heat-shock Hsc and Hsp70 with large stretches of identical amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human CD4+ T cells induced by synthetic peptide malaria vaccine are comparable to cells elicited by attenuated Plasmodium falciparum sporozoites

Peptide vaccines containing minimal epitopes of protective Ags provide the advantages of low cost, safety, and stability while focusing host responses on relevant targets of protective immunity. However, the limited complexity of malaria peptide vaccines raises questions regarding their equivalence to immune responses elicited by the irradiated sporozoite vaccine, the "gold standard" for protective immunity. A panel of CD4+ T cell clones was derived from volunteers immunized with a peptide vaccine containing minimal T and B cell epitopes of the Plasmodium falciparum circumsporozoite protein to compare these with previously defined CD4+ T cell clones from volunteers immunized with irradiated P. falciparum sporozoites. As found following sporozoite immunization, the majority of clones from the peptide-immunized volunteers recognized the T* epitope, a predicted universal T cell epitope, in the context of multiple HLA DR and DQ molecules. Peptide-induced T cell clones were of the Th0 subset, secreting high levels of IFN-gamma as well as variable levels of Th2-type cytokines (IL-4, IL-6). The T* epitope overlaps a polymorphic region of the circumsporozoite protein and strain cross-reactivity of the peptide-induced clones correlated with recognition of core epitopes overlapping the conserved regions of the T* epitope. Importantly, as found following sporozoite immunization, long-lived CD4+ memory cells specific for the T* epitope were detectable 10 mo after peptide immunization. These studies demonstrate that malaria peptides containing minimal epitopes can elicit human CD4+ T cells with fine specificity and potential effector function comparable to those elicited by attenuated P. falciparum sporozoites.     

In vitro study of malaria parasite induced disruption of blood-brain barrier

The mechanism of blood-brain barrier breakdown in the complex pathogenesis of cerebral malaria is not well understood. In this study, primary cultures of porcine brain capillary endothelial cells (PBCEC) were used as in vitro model. Membrane-associated malaria antigens obtained from lysed Plasmodium falciparum schizont-infected erythrocytes stimulated human peripheral blood mononuclear cells (PBMC) to secrete tumor necrosis factor alpha. In co-cultivation with the brain endothelial cell model, the malaria-activated PBMC stimulated the expression of E-selectin and ICAM-1 on the PBCEC. Using electric cell-substrate impedance sensing, we detected a significant decrease of endothelial barrier function within 4h of incubation with the malaria-activated PBMC. Correspondingly, immunocytochemical studies showed the disruption of tight junctional complexes. Combination of biochemical and biophysical techniques provides a promising tool to study changes in the blood-brain barrier function associated with cerebral malaria. Moreover, it is shown that the porcine endothelial model is able to respond to human inflammatory cells.     

Western blot diagnosis of vivax malaria with multiple stage-specific antigens of the parasite.

Western blot analysis was performed to diagnose vivax malaria using stage-specific recombinant antigens. Genomic DNA from the whole blood of a malaria patient was used as templates to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), merozoite surface protein (MSP-1), apical merozoite antigen (AMA-1), serine repeat antigen (SERA), and exported antigen (EXP-1) of Plasmodium vivax. Each amplified DNA fragment was inserted into a pGEX-4T plasmid to induce the expression of GST fusion protein in Escherichia coli by IPTG. The bacterial cell extracts were separated on 10% SDS-PAGE followed by western blot analysis with patient sera which was confirmed by blood smear examination. When applied with patient sera, 147 (91.9%) out of 160 vivax malaria, 12 (92.3%) out of 13 falciparum malaria, and all 9 vivax/falciparum mixed malaria reacted with at least one antigen, while no reactions occurred with 20 normal uninfected sera. In the case of vivax malaria, CSP-1 reacted with 128 (80.0%) sera, MSP-1 with 102 (63.8%), AMA-1 with 128 (80.0%), SERA with 115 (71.9%), and EXP-1 with 89 (55.6%), respectively. We obtained higher detection rates when using 5 antigens (91.9%) rather than using each antigen solely (55.6-80%), a combination of 2 (76.3-87.5%), 3 (85.6-90.6%), or 4 antigens (89.4-91.3%). This method can be applied to serological diagnosis, mass screening in endemic regions, or safety test in transfusion of prevalent vivax malaria.     

Defective production of and response to IL-2 in acute human falciparum malaria

Patients with acute Plasmodium falciparum malaria have defective cell-mediated immune responses to malaria-specific Ag (MA). This immunologic defect may partially explain the difficulty with which natural immunity to falciparum malaria develops and may have important implications for the efficacy of potential malaria vaccines in endemic areas. To investigate the basis of this immune defect, we have examined the capacity of PBMC from patients with acute falciparum malaria to produce IL-2 and to express I1-2R in response to Ag stimulation. The effect of exogenous IL-1 and IL-2 on lymphocyte proliferation was studied. Soluble IL-2R levels were measured in acute and convalescent sera. Our results showed that no detectable IL-2 was produced and no IL-2R were expressed by PBMC in response to MA during the acute infection. IL-2 production and IL-2R expression were also depressed when PBMC were exposed to streptococcal Ag. The specific immune defect was not reconstituted by the addition of graded doses of purified human IL-1 or IL-2 and could not be attributed to suppressor adherent cells. In contrast to the absence of IL-2 and cell-bound IL-2R, circulating soluble IL-2R was elevated in acute sera. These findings suggest that the lack of IL-2, through either a defect in its production or inhibition of its activity, may be the basis of the Ag-specific immune unresponsiveness in acute P. falciparum malaria.     

Mycobacterium leprae-specific T cells from a tuberculoid leprosy patient suppress HLA-DR3-restricted T cell responses to an immunodominant epitope on 65-kDa hsp of mycobacteria.

The polar tuberculoid type (TT) of leprosy, characterized by high T cell reactivity to Mycobacterium leprae, is associated with HLA-DR3. Surprisingly, DR3-restricted low T cell responsiveness to M. leprae was found in HLA-DR3-positive TT leprosy patients. This low responsiveness was specifically induced by M. leprae but not by M. tuberculosis and was seen only in patients and not in healthy controls. We studied this patient-specific, M. leprae-induced, DR3-restricted low T cell responsiveness in depth in one representative HLA-DR3-positive TT leprosy patient by using T cell clones. From this patient two types of T cell clones were obtained: one type was cross-reactive with M. tuberculosis and recognized an immunodominant epitope (amino acids 3 to 13) on the 65-kDa heat shock protein (hsp) the other type was M. leprae specific and reacted to a protein other than the 65-kDa one. To examine whether these M. leprae-specific T cell clones were responsible for the DR3-restricted low responsiveness to M. leprae, we tested them for the ability to suppress the proliferation of the DR3-restricted, 65-kDa, hsp-reactive clones. The DR3-restricted, M. leprae-specific T cells completely suppressed the proliferative responses of DR3-restricted, cross-reactive T cell clones to the 65-kDa hsp from the same patient as well as from other individuals. Also, DR3-restricted responses to an irrelevant Ag were suppressed by the M. leprae-specific T cell clones. However, no suppression of non-DR3-restricted T cell responses was seen. Although the mechanism must still be elucidated, this M. leprae-induced, DR3-restricted immunosuppression may at least partly explain the observed DR3-associated low T cell responsiveness in TT leprosy patients.     

A systematic molecular analysis of the T cell-stimulating antigens from Mycobacterium leprae with T cell clones of leprosy patients. Identification of a novel M. leprae HSP 70 fragment by M. leprae-specific T cells

Both protective immunity and immunopathology induced by mycobacteria are dependent on Ag-specific, CD4+ MHC class II-restricted T lymphocytes. The identification of Ag recognized by T cells is fundamental to the understanding of protective and pathologic immunity as well as to the design of effective immunoprophylaxis and immunotherapy strategies. Although some T cell clones are known to respond to recombinant mycobacterial heat shock proteins (hsp) like hsp3 65, the specificity of most T cells has remained unknown. We therefore have undertaken a specificity analysis of 48 well defined Mycobacterium leprae- and/or Mycobacterium tuberculosis-reactive (Th-1-like) T cell clones. Most clones (n = 44) were derived from different leprosy patients, and the remainder from one healthy control. Their HLA restriction molecules were DR2, DR3, DR4, DR5, DR7, DQ, or DP. T cell clones were stimulated with large numbers (n = 20 to 40) of mycobacterial SDS-PAGE-separated fractions bound to nitrocellulose. Each clone recognized a single fraction or peak with a particular Mr range. Some of the clones (n = 7) recognized the fraction that contained the hsp 65 as confirmed with the recombinant Ag. Most clones (n = 41), however, responded to Ag other than the hsp 65. Nine clones responded to a 67- to 80-kDa fraction. Five of them responded also to an ATP-purified, 70-kDa M. leprae protein, but only one of these five (that was HLA-DR2 restricted and cross-reactive with M. tuberculosis) recognized the recombinant C-terminal half (amino acids 278-621) of the M. leprae hsp 70 molecule and also recognized the recombinant M. tuberculosis hsp 70. We therefore have used the 5' part of the M. leprae hsp 70 gene that we have cloned recently. This fragment (that encodes amino acids 6-279) was indeed recognized by the other four M. leprae-specific T cells that were all HLA-DR3 restricted and did not cross-react with the highly homologous (95%) M. tuberculosis hsp 70. These results suggest that this novel fragment is a relevant T cell-stimulating Ag for leprosy patients. A panel of other recombinant Ag, including hsp 18 was tested. The majority of T cell clones appeared to recognize antigenic fractions distinct from hsp. In conclusion, T cells of leprosy patients see a large variety of different Ag including non-hsp, and one newly recognized moiety is the N-terminal M. leprae hsp 70 fragment.(ABSTRACT TRUNCATED AT 400 WORDS)