Intrinsic immunogenicity of liposomes for tuberculosis vaccines: Effect of cationic lipid and cholesterol.

Tuberculosis (TB) is still among the deadliest infectious diseases, hence there is a pressing need for more effective TB vaccines. Cationic liposome subunit vaccines are excellent vaccine candidates offering effective protection with a better safety profile than live vaccines. In this study, we aim to explore intrinsic adjuvant properties of cationic liposomes to maximize immune activation while minimizing aspecific cytotoxicity. To achieve this, we developed a rational strategy to select liposomal formulation compositions and assessed their physicochemical and immunological properties in vitro models using human monocyte-derived dendritic cells (MDDCs). A broad selection of commercially available cationic compounds was tested to prepare liposomes containing Ag85B-ESAT6-Rv2034 (AER) fusion protein antigen. 1,2-Dioleoyl-sn­glycero-3-ethylphosphocholine (EPC)-based liposomes exhibited the most advantageous activation profile in MDDCs as assessed by cell surface activation markers, cellular uptake, antigen-specific T-cell activation, cytokine production, and cellular viability. The addition of cholesterol to 20 mol% improved the performance of the tested formulations compared to those without it; however, when its concentration was doubled there was no further benefit, resulting in reduced cell viability. This study provides new insights into the role of cationic lipids and cholesterol in liposomal subunit vaccines.

Inflammatory responses in SARS-CoV-2 associated Multisystem Inflammatory Syndrome and Kawasaki Disease in children: An observational study.

Multisystem Inflammatory Syndrome in Children (MIS-C) is a severe inflammatory disease in children related to SARS-CoV-2 with multisystem involvement including marked cardiac dysfunction and clinical symptoms that can resemble Kawasaki Disease (KD). We hypothesized that MIS-C and KD might have commonalities as well as unique inflammatory responses and studied these responses in both diseases. In total, fourteen children with MIS-C (n=8) and KD (n=6) were included in the period of March-June 2020. Clinical and routine blood parameters, cardiac follow-up, SARS-CoV-2-specific antibodies and CD4+ T-cell responses, and cytokine-profiles were determined in both groups. In contrast to KD patients, all MIS-C patients had positive Spike protein-specific CD3+CD4+ T-cell responses. MIS-C and KD patients displayed marked hyper-inflammation with high expression of serum cytokines, including the drug-targetable interleukin (IL)-6 and IFN-γ associated chemokines CXCL9, 10 and 11, which decreased at follow-up. No statistical differences were observed between groups. Clinical outcomes were all favourable without cardiac sequelae at 6 months follow-up. In conclusion, MIS-C and KD-patients both displayed cytokine-associated hyper-inflammation with several high levels of drug-targetable cytokines.

Evolutionary conservation of major histocompatibility complex-DR/peptide/T cell interactions in primates.

Many major histocompatibility complex (MHC) polymorphisms originate from ancient structures that predate speciation. As a consequence, members of the Mhc-DRB1*03 allelic lineage are not only present in humans but in chimpanzees and rhesus macaques as well. This emphasizes that Mhc-DRB1*03 members must have been present in a common ancestor of these primate species that lived about 30 million years ago. Due to the accumulation of genetic variation, however, alleles of the Mhc-DRB1*03 lineage exhibit species-unique sequences. To investigate the biological importance of such conservation and variation, we have studied both the binding and antigen presentation capacity of various trans-species Mhc-DRB1*03 lineage members. Here we show that p3-13 of the 65-kD heat-shock protein (hsp65) of Mycobacterium leprae and M. tuberculosis binds not only to HLA-DR17(3) but also to some chimpanzee and rhesus macaque class II-positive cells. Comparison of the corresponding human, chimpanzee, and rhesus macaque Mhc-DRB1*03 lineage members revealed the presence of uniquely shared amino acid residues, at positions 9-13 and 26-31, of the antigen-binding site that are critical for p3-13 binding. In addition it is shown that several nonhuman primate antigen-presenting cells that bind p3-13 can activate HLA-DR17-restricted T cells. Certain amino acid replacements, however, in Mhc-DRB1*03 lineage members did not influence peptide binding or T cell recognition. Therefore, these studies demonstrate that some polymorphic amino acid residues (motifs) within the antigen-binding site of MHC class II molecules that are crucial for peptide binding and recognition by the T cell receptor have been conserved for over 30 million years.

HLA-DO is a negative modulator of HLA-DM-mediated MHC class II peptide loading.

BACKGROUND: Class II molecules of the major histocompatibility complex become loaded with antigenic peptides after dissociation of invariant chainderived peptides (CLIP) from the peptide-binding groove. The human leukocyte antigen (HLA)-DM is a prerequisite for this process, which takes place in specialised intracellular compartments. HLA-DM catalyses the peptide-exchange process, simultaneously functioning as a peptide 'editor', favouring the presentation of stably binding peptides. Recently, HLA-DO, an unconventional class II molecule, has been found associated with HLA-DM in B cells, yet its function has remained elusive. RESULTS: The function of the HLA-DO complex was investigated by expression of both chains of the HLA-DO heterodimer (either alone or fused to green fluorescent protein) in human Mel JuSo cells. Expression of HLA-DO resulted in greatly enhanced surface expression of CLIP via HLA-DR3, the conversion of class II complexes to the SDS-unstable phenotype and reduced antigen presentation to T-cell clones. Analysis of peptides eluted from HLA-DR3 demonstrated that CLIP was the major peptide bound to class II in the HLA-DO transfectants. Peptide exchange assays in vitro revealed that HLA-DO functions directly at the level of class II peptide loading by inhibiting the catalytic action of HLA-DM. CONCLUSIONS: HLA-DO is a negative modulator of HLA-DM. By stably associating with HLA-DM, the catalytic action of HLA-DM on class II peptide loading is inhibited. HLA-DO thus affects the peptide repertoire that is eventually presented to the immune system by MHC class II molecules.

Plasma granulysin levels and cellular interferon-gamma production correlate with curative host responses in tuberculosis, while plasma interferon-gamma levels correlate with tuberculosis disease activity in adults.

Granulysin is a recently identified cytolytic protein which is expressed by human cytotoxic T-lymphocytes and natural killer (NK)-cells, and has broad antimicrobial and tumoricidal activity. Circulating granulysin levels are associated with T- and NK-cell activity, and may thus reflect protection-associated cellular immune responses. In a case-control study in Indonesia, a highly tuberculosis (TB)-endemic country, we therefore determined plasma granulysin levels in adults with active pulmonary TB before, during, and after TB treatment, both in mild/moderate-TB and advanced-TB patients, and compared these to healthy neighbourhood controls. Adults with active pulmonary TB had significantly lower plasma granulysin levels compared to controls. After 2 months of anti-TB therapy, levels in TB patients had significantly increased, reaching values similar to those in controls. Plasma granulysin levels further increased after completion of TB therapy, being significantly higher than those in controls. Plasma granulysin levels correlated inversely with TB disease activity but not with TB disease severity. In contrast, plasma interferon-gamma (IFN-gamma) levels were significantly higher in active TB cases than in controls, normalised during treatment and correlated with both TB disease activity and TB disease severity. At the cellular level, granulysin and IFN-gamma expression both correlated inversely with disease activity. Interestingly, granulysin was predominantly expressed by IFN-gamma negative T-cells, suggesting that the cellular sources of IFN-gamma and granulysin in TB are partly non-overlapping. The observation that plasma granulysin levels and cellular IFN-gamma production correlate with curative host responses in pulmonary tuberculosis points to a potentially important role of granulysin, next to IFN-gamma, in host defence against M. tuberculosis.

HLA-DR binding analysis of peptides from islet antigens in IDDM.

HLA molecules are essential for thymic education and HLA restriction of T-cell responses. We therefore analyzed the HLA-DR binding affinities of synthetic peptides covering the entire sequences of GAD65, islet cell antigen 69 (ICA69), and (pro)insulin, which are candidate antigens in the autoimmune process of T-cell-mediated destruction of the pancreatic beta-cells. Subsequently, peptide HLA-DR binding was correlated to peptide antigenicity by comparing known T-cell epitopes with their HLA-binding affinities defined in this study. The results demonstrate the following. 1) (Pro)insulin peptides display a strong binding affinity for HLA-DR2, which is associated with negative genetic predisposition to IDDM, whereas poor binding was observed for HLA-DR molecules neutrally or positively associated with IDDM. This suggests that the absence of insulin-reactive T-cells in DR2+ individuals may be explained by negative selection on high-affinity DR2 binding insulin peptides. 2) Most autoantigenic peptides display promiscuous HLA-DR binding patterns. This promiscuity in itself is not sufficient to explain the genetic association of HLA-DR with development of IDDM. 3) HLA-DR3 binding of autoantigenic GAD65 peptides is relatively weak compared with that of other known T-cell epitopes. 4) All peptide epitopes recognized by HLA-DR-restricted T-cells from either IDDM patients or GAD65-immunized HLA-DR transgenic mice bind with high affinity to their HLA-DR restriction molecule (P < 0.0006). In contrast, T-cell epitopes recognized by nondiabetic controls bind DR molecules with weak or undetectable affinity. These results thus indicate a strong correlation between antigenicity and HLA-DR binding affinity of GAD65 peptides in IDDM. Furthermore, negative thymic selection of insulin peptides in low-risk (HLA-DR2 expressing) subjects may explain the lack of autoreactivity to insulin in such individuals.

Clip binds to HLA class II using methionine-based, allele-dependent motifs as well as allele-independent supermotifs.

The invariant chain (Ii) region that interacts with class II and inhibits premature peptide binding has been mapped to amino acids 82-107, known as CLIP. It is unclear whether CLIP binds directly to the class II peptide binding groove and thus competitively blocks binding of other peptides, or whether it binds to conserved class II sites and indirectly inhibits peptide binding by inducing conformational changes in class II. Here we show evidence that strongly suggests that CLIP binds within the peptide binding groove, as CLIP binds to various HLA-DR alleles using allele-dependent as well as allele-independent, methionine-based binding motifs. First, a core sequence of 12 amino acids was identified within CLIP which is required for optimal binding to DR1, DR2, DR3(17) and DR7. This sequence is composed of CLIP p88-99 (SKMRMATPLLMQ). By substitution analysis, all three methionine residues appeared to control CLIP binding to HLA-DR. However, whereas M90 controlled binding to all four alleles, M92 and M98 were of different importance for the various alleles: M92 is involved in CLIP binding to DR1 and DR3(17) but not to DR2 or DR7, and M98 controls CLIP binding to DR2, DR3(17) and DR7 but not DR1. Also, CLIP competes with known immunogenic peptides for class II binding in a manner indistinguishable from regular, class II binding competitor peptides. Finally, the dissociation rates of CLIP-class II complexed are similar to those of antigenic peptide-class II complexes. Thus, CLIP most likely binds to the class II peptide binding groove, since most allelic class II differences are clustered here. CLIP uses unconventional methionine anchor residues representing an allele-independent supermotif (M90) as well as allele-dependent motifs (M92 and M98).

A sensitive fluorometric assay for quantitatively measuring specific peptide binding to HLA class I and class II molecules.

A sensitive, highly reproducible assay was developed for measuring binding of peptides to various HLA class I and II alleles. The assay is based on competition for binding to HLA between a peptide of interest and a fluorescent labelled standard peptide. This mixture is incubated with HLA to obtain equilibrium binding, and subsequently separated on an HPLC size-exclusion column in (i) a protein fraction containing HLA and bound peptide and (ii) a free peptide fraction. Each assay uses only 100 fmol labelled peptide and approximately 10 pmol of HLA. The analytical system contains an autosampler that samples from 96-well microtiter plates. Injections and data recording/evaluation is fully automated. Typical analysis time is 10-12 min per sample. The fluorescence in the HLA-bound peptide and free peptide containing fractions is measured on-line. The ratios of fluorescence signal in protein and peptide fractions at various concentrations of the peptide of interest are determined. IC50 values are calculated from the binding curve as obtained by curve fitting of the data. Here we show results for peptide binding to HLA-DR1 and -DR17 molecules purified from detergent solubilized cell lysates. and for recombinant HLA-A*0201 and HLA-A*0301 expressed in E. coli. The assay reported is sensitive and reproducible. It is non-radioactive and is non-labor intensive due to the high degree of automation.

The biologic importance of conserved major histocompatibility complex class II motifs in primates.

Phylogenetic comparisons of polymorphic second-exon sequences of MHC class II DRB genes showed that equivalents of the HLA-DRB1*03 alleles are present in various nonhuman primate species such as chimpanzees, gorillas, and rhesus macaques. These alleles must root from ancestral structure(s) that were once present in a progenitor species that lived about 35 million years ago. Due to accumulation of genetic variation, however, sequences that cluster into a lineage are generally unique to a species. To investigate the biologic importance of such conservation and variation, the peptide-binding capacity of various Mhc-DRB1*03 lineage members was studied. Primate Mhc-DRB1*03 lineage members successfully binding the p3-13 peptide of the 65-kD heat-shock protein of Mycobacterium tuberculosis/leprae share a motif that maps to the floor of the peptide-binding site. Apart from that, some rhesus macaque MHC class-II-positive cells were able to present the p3-13 peptide to HLA-DR17-restricted T cells whereas cells obtained from great ape species failed to do so. Therefore, these studies open ways to understand which MHC polymorphisms have been maintained in evolution and which MHC residues are essential for peptide binding and T-cell recognition.

Flexibility in T-cell receptor ligand repertoires depends on MHC and T-cell receptor clonotype.

T-cell receptors (TCR) recognize peptides complexed to self-major histocompatibility complex (MHC) molecules. Recognition of peptide/MHC ligands by the TCR is highly peptide specific. However, certain TCRs can also recognize sequence-related and -unrelated ('mimicry') epitopes presented by homologous MHC molecules. Using two human, human leucocyte antigen-DR1 (HLA-DR1)-restricted T-cell clones specific for HA p307-319, we identified several diverse combinations of peptide-MHC complexes that are functionally equivalent in their ability to trigger T-cell stimulation. These findings demonstrate that a single TCR can productively interact with different peptides complexed to self- as well as non-self-MHC molecules. This extended reactivity is human leucocyte antigen (HLA) allele and TCR clonotype dependent, as the peptide repertoire recognized depends on the presenting HLA-DR molecule and varies among different TCRs that both recognize the HA p307-319/DR1 complex. Importantly, certain peptide analogues can completely change the HLA-restriction pattern of the TCR: T-cell recognition of the wild-type peptide that was absent in the context of a non-self HLA-DR molecule, was restored by complementing substitutions in altered peptide ligands, that could not be presented by the original restriction element. This mechanism may play an important role in allorecognition.

Bronchoalveolar lavage cells from sarcoidosis patients and healthy controls can efficiently present antigens.

OBJECTIVES: The interaction between antigen-presenting cells (APC) and T lymphocytes, that recognize the antigen-HLA complex using its T cell-receptor for antigen, is of crucial importance for a subsequent specific immune response. In patients with pulmonary sarcoidosis, the local antigen-presenting capacity in the lungs has been suggested to be abnormally enhanced, and implicated in the immunopathogenesis of the disease. This study was aimed at increasing the understanding of the capacity to present antigens by APC in the lung compartment. DESIGN AND SUBJECTS: We used bronchoalveolar lavage (BAL) cells and paired peripheral blood mononuclear cells (PBMC) of six sarcoidosis patients and two healthy controls to stimulate in total eight well characterized T-cell clones with known HLA and antigen specificities. All subjects were HLA typed. RESULTS: BAL cells of sarcoidosis patients as well as of healthy controls efficiently induced proliferation of the relevant T-cell clone in an HLA-restricted manner when adding either intact antigen or antigenic peptides. CONCLUSIONS: BAL cells have the capacity to process and present antigens adequately, irrespective of whether they are derived from healthy individuals or from patients with sarcoidosis, implying the alveolar space as an important location for active immune reactions.

Altered peptide ligands of islet autoantigen Imogen 38 inhibit antigen specific T cell reactivity in human type-1 diabetes.

Type 1 diabetes, insulin-dependent diabetes mellitus (IDDM) results from autoimmune T cell-dependent destruction of insulin producing beta-cells in the pancreatic islets of Langerhans. T cells from recent-onset IDDM patients specifically proliferate to beta cell membrane Ag enriched fractions, containing the mitochondrial 38 kD islet antigen (Imogen). Recently, we identified a peptide epitope (Imogen p55-70) that is recognized by a 38 kD-specific, Th1 clone from an IDDM patient. In animal models of autoimmune diseases, altered self peptide ligands (APL) have been used effectively in peptide-based immune prevention or therapy. No such APL, however, have been reported so far that can modulate autoreactive T-cell responses in IDDM. Here, we have designed APL of p55-70. These APL efficiently downregulate in vitro activation of the 38 kD-specific Th1 clone induced by either p55-70 or by native beta cell autoantigens. Self peptide reactive T-cell proliferation could be inhibited only when APL and the self peptide were present on the same APC. Unrelated peptides with equal HLA-DR binding affinity were not effective, excluding simple MHC competition as the mechanism for T-cell modulation. APL triggered upregulation of CD69 and CD25 expression, but not T-cell proliferation, TCR down-modulation or T-cell anergy. Thus, the p55-70 APL inhibit beta cell autoantigen-induced activation of an Imogen-reactive T-cell clone derived from an IDDM patient, by acting as partial TCR agonists that inhibit TCR down-modulation.

Glucocorticoids transform CD40-triggering of dendritic cells into an alternative activation pathway resulting in antigen-presenting cells that secrete IL-10.

Dendritic cell (DC) activation through CD40-CD40 ligand interactions is a key regulatory step for the development of protective T-cell immunity and also plays an important role in the initiation of T-cell responses involved in autoimmune diseases and allograft rejection. In contrast to previous reports, we show that the immunosuppressive drug dexamethasone (DEX) redirects rather than simply blocks this DC activation process. We found that DCs triggered through CD40 in the presence of DEX were unable to acquire high levels of costimulatory, adhesion, and major histocompatibility complex class I and II molecules and failed to express the maturation marker CD83, whereas antigen uptake was not affected. Moreover, DEX strikingly modified the CD40-activated DC cytokine secretion profile by suppressing the production of the proinflammatory cytokine interleukin (IL)-12 and potentiating the secretion of the anti-inflammatory cytokine IL-10. Accordingly, DEX-exposed CD40-triggered DCs displayed a decreased T-cell allostimulatory potential and a dramatically impaired ability to activate cloned CD4(+) T helper 1 (Th1) cells. Moreover, interaction between Th1 cells and these DCs rendered the T cells hyporesponsive to further antigen-specific restimulation. Collectively, our results demonstrate that DEX profoundly modulates CD40-dependent DC activation and suggest that the resulting alternatively activated DCs can be exploited for suppression of unwanted T-cell responses in vivo.

HLA-DR/DQ transgenic, class II deficient mice as a novel model to select for HSP T cell epitopes with immunotherapeutic or preventative vaccine potential.

Protective immunity against mycobacteria is dependent on antigen/MHC class II specific, CD4+ Th1 cells. HLA-DR3-restricted Th1 cells respond to a subset of mycobacterial antigens, including the immunodominant hsp65, and recognize a single epitope in hsp65, notably p1-20. Altered peptide ligands (APL) of p1-20 can inhibit p1-20/hsp65-induced proliferation of DR3-restricted T cells in an allele specific manner in vitro. In order to develop a preclinical model in which p1-20 APL can be tested in vivo in the context of HLA, we have used murine class II deficient, HLA transgenic (Ab0) mice, in which all CD4+ T cells are restricted by the tg HLA molecule. BCG-immunized DR3.Ab0 and DQ8.Ab0 mice both responded well to hsp65. Furthermore, DR3.Ab0 mice recognized precisely the same p1-20 epitope as DR3-restricted human T cells, whereas DQ8.Ab0 mice responded to a different set of hsp65 peptides. This shows that (i) the same immunodominant protein and peptide epitope are recognized by T cells from DR3.Ab0 mice and DR3+ humans and (ii) indicates the major role of HLA-polymorphism in controlling the human T cell response to mycobacterial antigens. Thus, HLA-transgenic, Ab0 mice provide a novel, preclinical model system to analyze APL and vaccines in the context of HLA polymorphism.

A DR17-restricted T cell epitope from a secreted Mycobacterium tuberculosis antigen only binds to DR17 molecules at neutral pH.

The assembly of peptide-major histocompatability class II complexes in vitro is accelerated at low pH, comparable to that found in the intracellular compartments of metabolically active antigen-presenting cells (APC). Mycobacteria such as Mycobacterium tuberculosis reside in phagosomes with only mildly acidic pH. Therefore, we investigated the pH dependency of peptide-HLA-DR binding for several T cell epitopes of mycobacterial proteins, focussing particularly on well-defined, immunodominant HLA-DR17(3)-restricted T cell epitopes: peptide (p) 3-13 from the cytoplasmic 65-kDa heat shock protein of M. tuberculosis/M. leprae, and peptide 56-65 from the secreted 30/31-kDa protein from M. tuberculosis/M. leprae. p3-13 bound to purified, cell-free DR17 under both acidic and neutral conditions. Four other, unrelated DR17-binding peptides showed the same pH-dependent binding characteristics as p3-13. p56-65, however, only bound to purified DR17 at pH 7 but not at all at pH 4.5. These DR17 peptide binding data were confirmed in cell-bound DR17, in T cell stimulation assays in which fixed APC were peptide-pulsed at acidic or neutral pH before addition of peptide-specific DR17-restricted T cells. As far as we are aware, p56-65 is the only human T cell epitope binding to HLA exclusively at neutral pH. The binding characteristics of p56-65 may reflect dominant processing in alternative, less acidic vacuolar compartments specifically related to the generation of epitopes from (secreted) mycobacterial proteins. The observation that p56-65 is an immunodominant epitope for anti-mycobacterial T cells suggests the relevance of such novel processing compartments in T cell-mediated immunity.

T cell receptor and peptide-contacting residues in the HLA-DR17(3) beta 1 chain.

Previously, we have proposed that the beta 1 residues 9-13, 26, 28 and 86 in HLA-DR17, the most common subtype of DR3, might be critical for the binding of an immunodominant, mycobacterial epitope (peptide 3-13 of the 65-kDa heat shock protein). In order to examine directly (i) which DR17 residues are involved in peptide binding, (ii) whether the same or other DR17 residues are involved in the binding of different peptides, and (iii) whether subtle differences in the mode of peptide binding can influence T cell stimulation, we have now systematically mutated 15 highly polymorphic DR17 beta 1 residues, located in the proposed peptide binding groove of DR17, and examined the effect thereof on binding and presentation of two peptides, hsp65 p3-13 and p56-65 of the 30/31-kDa secreted mycobacterial protein. Mutations in residues 28 (D-->H) and 86 (V-->G) completely eliminated binding of p3-13 and significantly reduced binding of p56-65. A mutation in residue 26 (Y-->F) decreased binding of p3-13 but did not affect binding of p56-65. Substitutions of amino acid residues 28, 67, 71 and 86 in the DR17 beta 1 chain abrogated peptide-specific stimulation of both the p3-13- and the p56-65-specific T cell clones, while specific stimulation by only one peptide was eliminated by substitution at positions 26 and 74 (p3-13) and by substitution of residues 11 and 37 (p56-65). The observation that substitution of several other peptide-contacting DR17 beta 1 chain residues does not significantly affect peptide binding but does affect T cell stimulation, suggests that these substitutions alter the conformation of the bound peptide.

Immunological crossreactivity of the Mycobacterium leprae CFP-10 with its homologue in Mycobacterium tuberculosis.

Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) (Rv3874) is considered a promising antigen for the immunodiagnosis of tuberculosis (TB) together with early secreted antigens of M. tuberculosis (ESAT-6). Both ESAT-6 and CFP-10 are encoded by the RD1 region that is deleted from all tested M. bovis bacille Calmette-Guérin (BCG) strains but present in M. leprae, M. tuberculosis, M. bovis, M. kansasii, M. africanum and M. marinum. In this study, the homologue of CFP-10 in M. leprae (ML0050) is identified and characterized. Interferon-gamma production in response to this homologue by T cells from leprosy patients, TB patients and unexposed controls shows that CFP-10 of M. leprae is a potent antigen that crossreacts with CFP-10 of M. tuberculosis at the T-cell level. This crossreactivity has implications for the use of CFP-10 of these mycobacterial species as diagnostic tool in areas endemic for both the diseases.

Functional analysis of DR17(DR3)-restricted mycobacterial T cell epitopes reveals DR17-binding motif and enables the design of allele-specific competitor peptides.

We have previously shown that p3-13 (KTIAY-DEEARR) of the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis and Mycobacterium leprae is selected as an important T cell epitope in HLA-DR17+ individuals, by selectively binding to (a pocket in) DR17 molecules, the major subset of the DR3 specificity. We have now further studied the interaction between p3-13, HLA-DR17 and four different TCR (V beta 5.1, V beta 1, and V beta 4) by using T cell stimulation assays, direct peptide-DR binding assays, and a large panel (n = 240) of single amino acid substitution analogs of p3-13. We find that residues 5(I) and 8(D) of p3-13 are important DR17 binding residues, whereas the residues that interact with the TCR vary slightly for each DR17-restricted clone. By using N- and C-terminal truncated derivatives of p2-20 we defined the minimal peptide length for both HLA-DR17 binding and T cell activation: the minimal peptide that bound to DR17 was seven amino acids long whereas the minimal peptide that activated T cell proliferation was eight amino acids in length. Furthermore, two new DR17-restricted epitopes were identified on hsp70 and hsp18 of M. leprae. Alignment of the critical DR17-binding residues 5(I) and 8(D) of p3-13 with these two novel epitopes and two other DR17-binding peptides revealed the presence of highly conserved amino acids at positions n and n + 3 with I, L, and V at position n and D and E at position n + 3. D and E are particularly likely to interact with the DR17-specific, positively charged pocket that we have defined earlier. Based on these results, a set of single amino acid substituted analogs that failed to activate these T cell clones but still bound specifically to DR17 was defined and tested for their ability to inhibit T cell activation by p3-13 or other DR17-restricted epitopes. Those peptides were able to inhibit the response to p3-13 as well as other DR17-restricted mycobacterial epitopes in an allele-specific manner, and are anticipated to be of potential use for immunotherapeutic and vaccine design strategies.

Antigenic equivalence of human T-cell responses to Mycobacterium tuberculosis-specific RD1-encoded protein antigens ESAT-6 and culture filtrate protein 10 and to mixtures of synthetic peptides.

The early secreted antigenic target 6-kDa protein (ESAT-6) and culture filtrate protein 10 (CFP-10) are promising antigens for reliable immunodiagnosis of tuberculosis. Both antigens are encoded by RD1, a genomic region present in all strains of Mycobacterium tuberculosis and M. bovis but lacking in all M. bovis bacillus Calmette-Guérin vaccine strains. Production and purification of recombinant antigens are laborious and costly, precluding rapid and large-scale testing. Aiming to develop alternative diagnostic reagents, we have investigated whether recombinant ESAT-6 (rESAT-6) and recombinant CFP-10 (rCFP-10) can be replaced with corresponding mixtures of overlapping peptides spanning the complete amino acid sequence of each antigen. Proliferation of M. tuberculosis-specific human T-cell lines in response to rESAT-6 and rCFP-10 and that in response to the corresponding peptide mixtures were almost completely correlated (r = 0.96, P < 0.0001 for ESAT-6; r = 0.98, P < 0.0001 for CFP-10). More importantly, the same was found when gamma interferon production by peripheral blood mononuclear cells in response to these stimuli was analyzed (r = 0.89, P < 0.0001 for ESAT-6; r = 0.89, P < 0.0001 for CFP-10). Whole protein antigens and the peptide mixtures resulted in identical sensitivity and specificity for detection of infection with M. tuberculosis. The peptides in each mixture contributing to the overall response varied between individuals with different HLA-DR types. Interestingly, responses to CFP-10 were significantly higher in the presence of HLA-DR15, which is the major subtype of DR2. These results show that mixtures of synthetic overlapping peptides have potency equivalent to that of whole ESAT-6 and CFP-10 for sensitive and specific detection of infection with M. tuberculosis, and peptides have the advantage of faster production at lower cost.

Detection of active tuberculosis infection by T cell responses to early-secreted antigenic target 6-kDa protein and culture filtrate protein 10.

The purified protein derivative (PPD) skin test has no predictive value for tuberculosis (TB) in Mycobacterium bovis bacillus Calmette-Guérin (BCG)-vaccinated individuals because of cross-reactive responses to nonspecific constituents of PPD. T cell responses to early-secreted antigenic target 6-kDa protein (ESAT-6) and the newly identified culture filtrate protein 10 (CFP-10), 2 proteins specifically expressed by M. tuberculosis (MTB) but not by BCG strains, were evaluated. Most TB patients responded to ESAT-6 (92%) or CFP-10 (89%). A minority of BCG-vaccinated individuals responded to both ESAT-6 and CFP-10, their history being consistent with latent infection with MTB in the presence of protective immunity. No responses were found in PPD-negative controls. The sensitivity and specificity of the assay were 84% and 100%, respectively, at a cutoff of 300 pg of interferon-gamma/mL. These data indicate that ESAT-6 and CFP-10 are promising antigens for highly specific immunodiagnosis of TB, even in BCG-vaccinated individuals.