Abu Salim Mustafa

Human T Cell Responses to the ESAT-6 Antigen from Mycobacterium tuberculosis

Human T cell responses to ESAT-6 and eight synthetic overlapping peptides were investigated in tuberculosis (TB) patients and control subjects from regions of high and low endemicity for TB. ESAT-6 was recognized by 65% of all tuberculin purified protein derivative-responsive TB patients, whereas only 2 of 29 bacille Calmette-Guérin-vaccinated Danish healthy donors recognized this molecule. In Ethiopia, a high frequency (58%) of healthy contacts of TB patients recognized ESAT-6. All of the peptides were recognized by some donors, indicating that the molecule holds multiple epitopes. Danish and Ethiopian patients differed in the fine specificity of their peptide responses. Recognition of the C-terminal region (aa 72-95) was predominant in Danish patients, whereas recognition of aa 42-75 was predominant in Ethiopia. The relationship of these differences to the distribution of HLA types in the two populations is discussed. This study demonstrates that ESAT-6 is frequently recognized during early infection and holds potential as a component of a future TB-specific diagnostic reagent.

Seminested PCR for Diagnosis of Candidemia: Comparison with Culture, Antigen Detection, and Biochemical Methods for Species Identification

ABSTRACT The rapid detection and identification of Candida species in clinical laboratories are extremely important for the management of patients with hematogenous candidiasis. The presently available culture and biochemical methods for detection and species identification of Candida are time-consuming and lack the required sensitivity and specificity. In this study, we have established a seminested PCR (snPCR) using universal and species-specific primers for detection of Candida species in serum specimens. The universal outer primers amplified the 3′ end of 5.8S ribosomal DNA (rDNA) and the 5′ end of 28S rDNA, including the internally transcribed spacer 2 (ITS2), generating 350- to 410-bp fragments from the four commonly encountered Candida species, viz., C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis. The species-specific primers, complementary to unique sequences within the ITS2 of each test species, amplified species-specific DNA in the reamplification step of the snPCR. The sensitivity of Candida detection by snPCR in spiked serum specimens was close to 1 organism/ml. Evaluation of snPCR for specific identification of Candida species with 76 clinical Candida isolates showed 99% concordant results with the Vitek and/or ID32C yeast identification system. Further evaluation of snPCR for detection of Candida species in sera from culture-proven (n = 12), suspected (n = 16), and superficially colonized (n = 10) patients and healthy subjects (n = 12) showed that snPCR results were consistently negative with sera from healthy individuals and colonized patients. In culture-proven candidemia patients, the snPCR results were in full agreement with blood culture results with respect to both positivity and species identity. In addition, snPCR detected candidemia due to two Candida species in five patients, compared to three by blood culture. In the category of suspected candidemia with negative blood cultures for Candida, nine patients (56%) were positive by snPCR; two of them had dual infection with C. albicans and either C. tropicalis or C. glabrata. In conclusion, the snPCR developed in this study is specific and more sensitive than culture for the detection of Candida species in serum specimens. Moreover, the improved detection of cases of candidemia caused by more than one Candida species is an additional advantage.

Elevated levels of IL-8 in dengue hemorrhagic fever.

Dengue virus causes dengue fever, a mild febrile illness, and at times dengue hemorrhagic fever (DHF), a severe illness the pathogenesis of which is not fully understood. Given the crucial roles played by interleukin‐8 (IL‐8) as a chemoattractant cytokine and in inflammatory processes, levels of circulating IL‐8 in the sera and IL‐8 mRNA in the peripheral blood mononuclear cells (PBMC) were measured in 99 patients of a recent dengue epidemic that occurred in India in 1996 and in 21 normal healthy controls. Twenty‐six of the patients had dengue fever (DF) and the remaining 73 were diagnosed as having different grades of DHF. All the control normal sera were negative for IL‐8, so were their PBMC for IL‐8 mRNA. Increased levels of IL‐8 in the sera and IL‐8 mRNA in their PBMC were observed in patients with severe illness of DHF grades III and IV. Only two out of 26 patients of DF and one out of 10 DHF grade I patient were positive for IL‐8 and all three deteriorated to DHF grade IV within 24 hr. All six patients of DHF grade IV who died had higher serum level of IL‐8 above 200 pg/ml, the highest being 5,568 pg/ml in one patient; the presence of mRNA for IL‐8 was very high in all patients. A striking correlation was observed between increased levels of IL‐8 and severe DHF, with greater levels in patients with increased grade of the disease and death. These results suggest that IL‐8 may have an important role and may be an indicator of increasing severity of the disease and death. J. Med. Virol. 56:280–285, 1998.

Comparison of Antigen-Specific T-Cell Responses of Tuberculosis Patients using Complex or Single Antigens of Mycobacterium tuberculosis

We have screened peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients for proliferative reactivity and interferon‐γ (IFN‐γ) secretion against a panel of purified recombinant (r) and natural (n) culture filtrate (rESAT‐6, nMPT59, nMPT64 and nMPB70) and somatic‐derived (rGroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis. The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole‐cell M. tuberculosis, M. tuberculosis culture filtrate (MT‐CF) and cell wall antigens, as well as the vaccine strain, Mycobacterium bovis bacillus Calmette–Guérin (BCG). In addition, M. tuberculosis and MT‐CF‐induced T‐cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T‐cell lines tested for antigen‐induced proliferation and IFN‐γ secretion showed that the most frequently recognized antigen was ESAT‐6, followed by MPT59, GroES, MPB70, MPT64, DnaK, GroEL and PstS. The frequency of ESAT‐6 responders, as measured both by proliferation (18/19) and secretion of IFN‐γ (16/19) was comparable to the results obtained with whole‐cell M. tuberculosis, MT‐CF and M. bovis BCG. We also observed that most of the high responders to complex antigens recognized all of the antigens tested (covariation), demonstrating that the repertoire of human T‐cell specificities induced by natural infection is directed towards several unrelated culture filtrate as well as somatic‐derived protein antigens. In conclusion, the results obtained suggest that the cellular immune response in humans is directed against several important target antigens of M. tuberculosis and that some antigens, such as ESAT‐6, are recognized by a high number of individuals. Such antigens represent candidates to be used for development of specific diagnostic reagents or in subunit vaccines.

Comparative evaluation of (1, 3)-β-D-glucan, mannan and anti-mannan antibodies, and Candida species-specific snPCR in patients with candidemia

BackgroundCandidemia is a major infectious complication of seriously immunocompromised patients. In the absence of specific signs and symptoms, there is a need to evolve an appropriate diagnostic approach. A number of methods based on the detection of Candida mannan, nucleic acid and (1,3)-beta- D- glucan (BDG) have been used with varying specificities and sensitivities. In this retrospective study, attention has been focused to evaluate the usefulness of two or more disease markers in the diagnosis of candidemia.MethodsDiagnostic usefulness of Platelia Candida Ag for the detection of mannan, Platelia Candida Ab for the detection of anti-mannan antibodies, Fungitell for the detection of BDG, and of a semi-nested PCR (snPCR) for the detection Candida species-specific DNA have been retrospectively evaluated using 32 sera from 27 patients with culture-proven candidemia, 51 sera from 39 patients with clinically suspected candidemia, sera of 10 women with C. albicans vaginitis, and sera of 16 healthy controls.ResultsUsing cut-off values recommended by the manufacturers, the sensitivity of the assays for candidemia patients were as follows: Candida snPCR 88%, BDG 47%, mannan 41%, anti-mannan antibodies 47%, respectively. snPCR detected 5 patients who had candidemia due to more than one Candida species. The sensitivities of the combined tests were as follows: Candida mannan and anti-mannan antibodies 75%, and Candida mannan and BDG 56%. Addition of snPCR data improved the sensitivity further to 88%, thus adding 10 sera that were negative by BDG and/or mannan. In clinically suspected, blood culture negative patients; the positivities of the tests were as follows: Candida DNA was positive in 53%, BDG in 29%, mannan in 16%, and anti-mannan antibodies in 29%. The combined detection of mannan and BDG, and mannan, BDG and Candida DNA enhanced the positivity to 36% and 54%, respectively. None of the sera from Candida vaginitis patients and healthy subjects were positive for Candida DNA and mannan.ConclusionThe observations made in this study reinforce the diagnostic value of snPCR in the sensitive and specific diagnosis of candidemia and detection of more than one Candida species in a given patient. Additionally, in the absence of a positive blood culture, snPCR detected Candida DNA in sera of more than half of the clinically suspected patients. While detection of BDG, mannan and anti-mannan antibodies singly or in combination could help enhancing sensitivity and eliminating false positive tests, a more extensive evaluation of these assays in sequentially collected serum samples is required to assess their value in the early diagnosis of candidemia.

Development of new vaccines and diagnostic reagents against tuberculosis.

Tuberculosis (TB) is a major infectious disease problem with one-third of the world population infected, 8 million people developing the active disease and 2 million dying of TB each year. The attenuated Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the only available vaccine against TB. However, the trials conducted in different parts of the world have shown that this vaccine doe not provide consistent protection against TB. The purified protein derivative (PPD) of Mycobacterium tuberculosis is the commonly used reagent for the diagnosis of TB. However, PPD lacks specificity because of the presence of antigens crossreactive with M. bovis BCG and other mycobacteria. The studies to identify M. tuberculosis antigens and epitopes as candidates for new protective vaccines and specific diagnostic reagents against TB have led to the identification and characterization of several major antigens of M. tuberculosis including heat shock proteins (hsp) and secreted antigens present in the culture filtrate (CF) of M. tuberculosis. Some of these antigens have shown promise as new candidate vaccines (hsp60, Ag85 and ESAT-6, etc.) and specific diagnostic reagents (ESAT-6 and CFP10, etc.) for TB. Moreover, in the mouse model of TB, vaccination with DNA-hsp60 has immunotheraputic effects and helps in eradication of persisters. In addition, identification of proper adjuvant and delivery systems has shown the promise to overcome the problem of poor immunogenicity associated with subunit and peptide based vaccines. More recently, the comparison of the genome sequence of M. tuberculosis with M. bovis BCG and other mycobacteria has led to the identification of M. tuberculosis-specific genomic regions. Evaluation of these regions for encoding proteins with immunological reactivity can lead to the identification of additional antigens of M. tuberculosis useful as new vaccines and reagents for specific diagnosis of TB.

Identification and HLA Restriction of Naturally Derived Th1-Cell Epitopes from the Secreted Mycobacterium tuberculosis Antigen 85B Recognized by Antigen-Specific Human CD4+ T-Cell Lines

ABSTRACT Antigen 85B (Ag85B/MPT59) is a major secreted protein fromMycobacterium tuberculosis which is a promising candidate antigen for inclusion in novel subunit vaccines against tuberculosis (TB). The present study was undertaken to map naturally derived T-cell epitopes from M. tuberculosis Ag85B in relation to major histocompatibility complex (MHC) class II restriction. Antigen-specific CD4+ T-cell lines were established from HLA-typed TB patients and Mycobacterium bovis BCG vaccinees by stimulation of peripheral blood mononuclear cells with purified Ag85B in vitro. The established T-cell lines were then tested for proliferation and gamma interferon (IFN-γ) secretion in response to 31 overlapping synthetic peptides (18-mers) covering the entire sequence of the mature protein. The results showed that the epitopes recognized by T-cell lines from TB patients were scattered throughout the Ag85B sequence whereas the epitopes recognized by T-cell lines from BCG vaccinees were located toward the N-terminal part of the antigen. The T-cell epitopes represented by peptides p2 (amino acids [aa] 10 to 27), p3 (aa 19 to 36), and p11 (aa 91 to 108) were frequently recognized by antigen-specific T-cell lines from BCG vaccinees in both proliferation and IFN-γ assays. MHC restriction analysis demonstrated that individual T-cell lines specifically recognized the complete Ag85B either in association with one of the self HLA-DRB1,DRB3, or DRB4 gene products or nonspecifically in a promiscuous manner. At the epitope level, panel studies showed that peptides p2, p3, and p11 were presented to T cells by HLA-DR-matched as well as mismatched allogeneic antigen-presenting cells, thus representing promiscuous epitopes. The identification of naturally derived peptide epitopes from the M. tuberculosisAg85B presented to Th1 cells in the context of multiple HLA-DR molecules strongly supports the relevance of this antigen to subunit vaccine design.

Restoration of mycobacterial antigen‐induced proliferation and interferon‐γ responses in peripheral blood mononuclear cells of tuberculosis patients upon effective chemotherapy

Peripheral blood mononuclear cells (PBMC) were obtained from culture-proven tuberculosis (TB) patients before and after 2 and 6 months of chemotherapy with a multi-drug regimen. PBMC were tested for cellular responses in antigen-induced proliferation and interferon-gamma (IFN-gamma) assays in response to complex mycobacterial antigens (whole cell Mycobacterium bovis BCG and M. tuberculosis, cell walls and short-term culture filtrate [ST-CF] of M. tuberculosis), fractionated ST-CF antigens (fractions F1-F10) and ESAT-6. The responses in TB patients before anti-TB treatment were low (median stimulation index (SI)=1-7, median delta IFN-gamma=0-12 U ml(-1), and percent responders=13-67%) to all the antigenic preparations. Following the administration of anti-TB chemotherapy for 2 months, there were significant (P<0.05) improvements in the cellular responses (median SI=9-76, median delta IFN-gamma=3-70 U ml(-1), and percent responders=33-100%) to most of the antigenic preparations tested. However, concanavalin A-induced proliferation responses of PBMC from the same patients before and after 2 months of chemotherapy were high and comparable (median SI=101 and 114, respectively, P>0.05, 100% responders). A further increase in IFN-gamma responses (median delta IFN-gamma=14-250 U ml(-1) and percent responders=43-100%) to mycobacterial antigens was observed in patients receiving chemotherapy for 6 months. Among the ST-CF fractions, F1 and F2 containing low molecular mass proteins resulted in the highest responses, whereas ESAT-6 showed responses comparable to these fractions only in a minority of the patients. HLA-DR typing of these patients showed heterogeneity in the expression of molecules encoded by HLA-DRB genes. These results show that effective chemotherapy restores cellular responses of TB patients to a large number of M. tuberculosis antigens, which could be useful in monitoring the efficacy of anti-TB treatment.

Immunogenicity of Mycobacterium tuberculosis RD1 region gene products in infected cattle

Current immuno‐diagnostic tests for the detection of Mycobacterium bovis infection in cattle rely on the use of tuberculin PPD as antigens. However, the use of a cattle vaccine is effectively prohibited because BCG, the only potentially available cattle TB vaccine, compromises the current tuberculin test. The main objective of this study was to identify specific antigens, which could increase the test sensitivity to levels achieved with tuberculin. Our approach utilized the availability of the genome sequences of Mycobactereium tuberculosis and BCG by applying principles of comparative genomics to the identification of species‐specific antigens. Eight open‐reading frames (Rv3871 to Rv3878) encoding for putative antigens in the RD1 region of the M. tuberculosis genome, which is deleted in all strains of BCG, were selected and screened in the form of pools of synthetic peptides for immunological reactivity (antigen induced proliferation and IFN‐γ secretion) with peripheral blood mononuclear cells from cattle experimentally infected with M. bovis. Our results confirm the immunodominant role of two RD1 region products, CFP‐10 (Rv3874) and ESAT‐6 (Rv3875). In addition, we were able to identify 3 more antigens (Rv3871, Rv3872 and Rv3873), which induced immunological reactivity in PBMC from more than 50%M. bovis of infected cattle.

Efficient Testing of Large Pools of Mycobacterium tuberculosis RD1 Peptides and Identification of Major Antigens and Immunodominant Peptides Recognized by Human Th1 Cells

ABSTRACT Comparative genomics has identified several regions of difference (RDs) of Mycobacterium tuberculosis that are deleted or absent in Mycobacterium bovis BCG vaccines. To determine their relevance for diagnostic and vaccine applications, it is imperative that efficient methods are developed to test the encoded proteins for immunological reactivity. In this study, we have used 220 synthetic peptides covering sequences of 12 open reading frames (ORFs) of RD1 and tested them as a single pool (RD1pool) with peripheral blood mononuclear cells obtained from pulmonary tuberculosis (TB) patients and M. bovis BCG-vaccinated healthy subjects in Th1 cell assays that measure antigen-induced proliferation and IFN-γ secretion. The results showed that RD1pool induced strong responses in both TB patients and BCG-vaccinated healthy subjects. The subsequent testing of peptide pools of individual ORFs revealed that all ORFs induced positive responses in a portion of donors, but PPE68, CFP10, and ESAT6 induced strong responses in TB patients and PPE68 induced strong responses in BCG-vaccinated healthy subjects. In addition, HLA-DR and -DQ typing of donors and HLA-DR binding prediction analysis of proteins suggested HLA-promiscuous presentation of PPE68, CFP10, and ESAT6. Further testing of individual peptides showed that a single peptide of PPE68 (121-VLTATNFFGINTIPIALTEMDYFIR-145) was immunodominant. The search for sequence homology revealed that a part of this peptide, 124-ATNFFGINTIPIAL-137, was present in several PPE family proteins of M. tuberculosis and M. bovis BCG vaccines. Further experiments limited the promiscuous and immunodominant epitope region to the 10-amino-acid cross-reactive sequence 127-FFGINTIPIA-136.