Epitope-specific antibody levels demonstrate recognition of new epitopes and changes in titer but not affinity during treatment of tuberculosis

Antibody levels rise during treatment of tuberculosis. This study examined when this rise occurred, whether there was recognition of new antigen binding sites (epitopes) on the same or different antigens, and how long specific antibody persisted. Forty patients with smear-positive pulmonary tuberculosis provided serum before and during treatment. Antibody levels were measured using a monoclonal antibody competition assay to epitopes restricted to the Mycobacterium tuberculosis complex and an enzyme-linked immunosorbent assay for lipoarabinomannan. Significant increases in antibody levels were apparent after 7 days of treatment. Five samples (12.5%) had positive titers to all epitopes at the start of treatment, and this increased to 23 (58%) during treatment. Antibody to epitopes with the poorest sensitivity (the TB23 epitope of the 19-kDa antigen and the TB78 epitope of hsp65) showed the greatest increases after treatment. Antibody to these two epitopes was also absent in some patients with relapsed tuberculosis until after treatment. Antibody titers showed a biphasic response, with a fall at 2 to 3 months of treatment. Sera from two patients showed changes in the affinity of epitope-specific antibody during treatment, whereas the majority did not. Those infected with isoniazid-resistant strains of M. tuberculosis showed a late rise in antibody. Antibody to the TB68 epitope of the 16-kDa alpha-crystallin homolog was short-lived, but it recurred with bacteriological relapse during treatment. Positive antibody titers persisted for at least 3 to 18 months after treatment. Diagnostic tests for tuberculosis should be evaluated using only pretreatment sera. Delayed antigenic recognition could be due to active suppression and/or failure to engage internal antigens of M. tuberculosis.     

Changes in Avidity and Level of Immunoglobulin G Antibodies to Mycobacterium tuberculosis in Sera of Patients Undergoing Treatment for Pulmonary Tuberculosis

Much is known about specific antibodies and their titers in patients with tuberculosis. However, little is known about the avidity of these antibodies or whether changes in avidity occur during the progression of the disease or during treatment. The aims of this study were to determine the avidity of antibodies to Mycobacterium tuberculosis in patients with pulmonary tuberculosis, to explore the value of avidity determination for the diagnosis of tuberculosis, and to study changes in levels of antibodies and their avidity during treatment. Antibody avidity was measured by an enzyme-linked immunosorbent assay with thiocyanate elution. Avidity indices and serum levels of immunoglobulin G to M. tuberculosis were determined for 22 patients with pulmonary tuberculosis before and during treatment and for 24 patients with other pulmonary diseases. Antibody levels and avidity were both significantly higher in untreated tuberculosis patients than in the controls. Avidity determination had more diagnostic potential than determination of the antibody levels. Tuberculosis patients with a long duration of symptoms had higher antibody avidity than those with a recent onset of symptoms, indicating affinity maturation of specific antibodies during active disease. In the early phase of treatment, a decrease in antibody avidity was observed for 73% of all tuberculosis patients, accompanied by an initial increase in antibody levels in 36% of these patients. These phenomena could be explained by an intense stimulation of the humoral response by antigens released from killed bacteria, reflecting early bactericidal activity of antituberculous drugs leading to the production of low-affinity antibodies against these released antigens.     

Heterogeneous Antibody Responses in Tuberculosis

Antibody responses during tuberculosis were analyzed by an enzyme-linked immunosorbent assay with a panel of 10 protein antigens of Mycobacterium tuberculosis. It was shown that serum immunoglobulin G antibodies were produced against a variety of M. tuberculosis antigens and that the vast majority of sera from tuberculosis patients contained antibodies against one or more M. tuberculosis antigens. The number and the species of serologically reactive antigens varied greatly from individual to individual. In a given serum, the level of specific antibodies also varied with the antigen irrespective of the total number of antigens recognized by that particular serum. These findings indicate that person-to-person heterogeneity of antigen recognition, rather than recognition of particular antigens, is a key attribute of the antibody response in tuberculosis.     

Levels of antibody to defined antigens of Mycobacterium tuberculosis in tuberculous meningitis.

The aim of the study was to develop an antibody immunoassay for tuberculous meningitis with a sensitivity greater than that of the initial bacterial smear while maintaining a 100% specificity. Antibody titers to five purified antigens of Mycobacterium tuberculosis were measured in cerebrospinal fluid by using an enzyme-linked immunosorbent assay technique. Seventy-four patients with tuberculous meningitis (26 culture positive) were compared with 26 patients with purulent meningitis, 69 patients with suspected but excluded tuberculous meningitis, and 29 patients with other neurological diseases. Antibody responses to both the 14-kilodalton (kDa) antigen and lipoarabinomannan were immunodominant, accounting for 74% of those with any antibody titer to a soluble extract of M. tuberculosis. A sensitivity of 61% was achieved (77% for the culture-positive samples) by using immunoglobulin G titers to lipoarabinomannan and the 14-, 19-, and 38-kDa antigens. Thus, estimation of levels of antibody in cerebrospinal fluid to selected mycobacterial antigens would be valuable in the diagnosis of tuberculous meningitis. The possible reasons for the immunodominance of the 14-kDa antigen and lipoarabinomannan in the pathogenesis of tuberculous meningitis are discussed.     

Immunological activity of a 38-kilodalton protein purified from Mycobacterium tuberculosis.

A 38-kilodalton (kDa) protein antigen from Mycobacterium tuberculosis was purified by monoclonal antibody TB71-based affinity chromatography. This molecule carries two nonoverlapping epitopes recognized by monoclonal antibodies TB71 and TB72, which are expressed substantially more strongly by M. tuberculosis than by Mycobacterium bovis. However, cross-reactive determinants between these two species were revealed on the 38-kDa protein by a rabbit anti-BCG serum. An immunoradiometric assay based on the TB71 and TB72 antibody pair specifically determined 38-kDa-antigen concentrations in mycobacterial extracts. Antibodies in sera from tuberculosis patients estimated by binding to 38-kDa-antigen-coated microtiter plates were positively correlated with TB72 competing titers. Unlike antibodies, T-cell proliferative responses to the 38-kDa protein were expressed equally by 60% of tuberculosis patients and healthy BCG-vaccinated subjects. Similarly, delayed-type hypersensitivity skin reactions were elicited in both M. tuberculosis- and M. bovis-sensitized guinea pigs. The results suggest the immunodominance of the species-specific B-cell and cross-reactive T-cell stimulatory epitopes.     

Humoral response to defined epitopes of tubercle bacilli in adult pulmonary and child tuberculosis

In order to investigate the humoral response to tuberculosis in different categories of patients, serum antibody levels to six epitopes ofMycobacterium tuberculosis in adult pulmonary and child tuberculosis were determined. Serum antibody titres were determined by competitive inhibition with radio-labelled murine monoclonal antibodies in 67 adults and 85 children with tuberculosis and in 79 age-matched controls. BCG vaccination (n=39) and self-healed tuberculosis (n=11) in adults gave rise to higher antibody titres to TB68, TB23 and TB72 epitopes (all p<0.003) when compared to non-vaccinated controls (n=18). TB68 titres were higher (p=0.006) in self-healed than in vaccinated adults. Adult sputum-negative patients (n=15) had higher titres to TB71 (p=0.015) and ML34 (p=0.02) epitopes compared to BCG-vaccinated healthy controls, while sputum-positive patients (n=41) had higher titres to all epitopes tested (all p<10^–4). The diagnostic sensitivity, with a 95 % specificity, was best with the combination of probes TB23, TB68, TB72 for sputum-positive (85 %) and TB78, ML34 (53 %) for sputum-negative patients. Antibody titres in children with tuberculosis were lower than in adult patients; diagnostic sensitivity in histologically or microbiologically proven cases (n=18) was only 44 %, while that in mediastinal lymphadenitis (n=67) was 13.5 %. This study suggests that the magnitude and specificity of the humoral response to tubercle bacilli varies with site and severity of infection; the implications for pathogenesis or protective immunity are discussed.     

Serodiagnostic value of the 19 kilodalton antigen ofMycobacterium tuberculosis in indian patients

Previous studies in UK subjects suggested that the 19 kDa protein antigen ofMycobacterium tuberculosis might be valuable in the serodiagnosis of paucibacillary tuberculosis. In this study, antibody titres for the 19 kDa antigen were higher in healthy controls in India than in the UK. Consequently, the diagnostic sensitivity of this antigen and its TB23 epitope was negligible in Indian patients with tuberculosis. However, a diagnostic sensitivity of 50 % was achieved in patients with skin tuberculosis on the basis of a high ratio between antibody titres for the whole antigen and its TB23 epitope.     

Comparison of Antibody Responses to a Potential Combination of Specific Glycolipids and Proteins for Test Sensitivity Improvement in Tuberculosis Serodiagnosis

The humoral response to different proteinaceous antigens of Mycobacterium tuberculosis is heterogeneous among patients with active disease, and this has originated in the proposal to use a combination of several specific antigens to find an efficient serodiagnostic test for tuberculosis (TB). However, to date, comparisons of antibody responses to several antigens in the same population have been carried out without consideration of antigenic cell wall glycolipids. In the present study the presence of immunoglobulin G (IgG), IgM, and IgA antibodies to M. tuberculosis glycolipids (sulfolipid I, diacyltrehaloses, triacyltrehaloses, and cord factor) was compared with the response to four commercially available tests based on the 38-kDa protein mixed with the 16-kDa protein or lipoarabinomannan. Fifty-two serum samples from TB patients and 83 serum samples from control individuals (48 healthy individuals and 35 non-TB pneumonia patients) were studied. Three relevant results were obtained. (i) Smear-negative TB patients presented low humoral responses, but the sera which did react principally showed IgA antibodies to some glycolipidic antigens. (ii) TB patients exhibit heterogeneous humoral responses against glycolipidic antigens. (iii) Finally, test sensitivity is improved (from 23 to 62%) when IgG and IgA antibodies are detected together in tests based on different antigens (proteins and glycolipids). We conclude that it is possible to include glycolipidic antigens in a cocktail of specific antigens from M. tuberculosis to develop a serodiagnostic test.     

Mycobacterium tuberculosis Region of Difference (RD) 2 Antigen Rv1985c and RD11 Antigen Rv3425 Have the Promising Potential To Distinguish Patients with Active Tuberculosis from M. bovis BCG-Vaccinated Individuals

Antigens encoded in the region of difference (RD) of Mycobacterium tuberculosis constitute a potential source of specific immunodiagnostic antigens for distinguishing tuberculosis (TB) infection from BCG vaccination. We evaluated the diagnostic potential of specific T-cell epitopes selected from two immunodominant antigens, Rv1985c and Rv3425, from RD2 and RD11, respectively, on the basis of epitope mapping, in TB patients and BCG-vaccinated healthy individuals. Using a whole-blood gamma interferon release assay, a wide array of epitopes was recognized on both Rv1985c and Rv3425 in TB patients. Those epitopes that could specifically discriminate TB infection from BCG vaccination were carefully selected, and the most promising peptide pools from Rv1985c showed a sensitivity of 53.9% and a specificity of 95.5%. When the novel specific peptides from Rv1985c joined the diagnostic antigens in the QuantiFERON-TB Gold In-Tube (QFT-IT) assay, the sensitivity was increased from 86.4% to 96.2%, with no drop in specificity. These results indicate that the peptide pools selected from Rv1985c and Rv3425 have the potential to diagnose TB infection by a method that may be routinely used in clinical laboratories.     

Use of Multiepitope Polyproteins in Serodiagnosis of Active Tuberculosis

Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of ~98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of ~93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection.     

Humoral Immune Responses against the Mycobacterium tuberculosis 38-Kilodalton,MTB48, and CFP-10/ESAT-6 Antigens in Tuberculosis

The diagnosis of smear-negative and culture-negative patients with active tuberculosis (TB) is challenging. The detection of Mycobacterium tuberculosis-specific antibodies in human sera has been an important diagnostic aid. However, detection of antibody responses to a single antigen usually has a low sensitivity for diagnosis of TB. In this study, humoral immune responses against recombinant M. tuberculosis 38-kDa, MTB48, and CFP-10/ESAT-6 (culture filtrate protein 10/6-kDa early secreted antigen target of M. tuberculosis) antigens in 250 Chinese TB patients and 260 healthy subjects were evaluated by an enzyme-linked immunosorbent assay (ELISA). The levels of antibodies against those antigens in TB patients, even in bacterium-negative ones, were significantly higher than those in healthy subjects (P < 0.001). The serodiagnostic sensitivities to detect antibodies against individual antigens, i.e., recombinant M. tuberculosis 38-kDa, MTB48, and CFP-10/ESAT-6 antigens, in TB patients were 73.6%, 73.2%, and 60.4%, respectively, with specificities of 85.4%, 77.7%, and 73.8%, respectively. Importantly, the sensitivity to positively detect humoral responses to one of the antigens increased further. Our data suggest that the humoral immune responses to M. tuberculosis antigens in TB patients are heterogeneous. The 38-kDa, MTB48, and CFP-10/ESAT-6 antigens can be used as the cocktail antigens in the serodiagnosis of active TB, especially for smear- or culture-negative TB cases.The control of tuberculosis (TB) remains challenging in China (18). Currently, the diagnosis of active TB mainly relies on clinical symptoms, radiologic findings, and the detection of Mycobacterium tuberculosis in clinical samples using smear staining and mycobacterial culture. However, the diagnosis of TB in smear- and culture-negative TB patients is difficult. The detection of M. tuberculosis-specific antibodies in human sera has been an important aid in diagnosis of TB. Notably, several antigens have been demonstrated to have merit in TB diagnosis, including the 38-kDa protein, which is commonly used in serodiagnostic tests (4, 5, 8, 13, 19, 22, 23). Previous studies suggest that the antibody responses to M. tuberculosis antigens are heterogeneous among individuals (17) so that the detection of antibodies against a single antigen usually has a low sensitivity for diagnosis of TB, especially for bacterium-negative cases. Therefore, it may be valuable to evaluate antibodies against the 38-kDa antigen and other major antigens for the diagnosis of active TB (14, 15).Notably, the MTB48, CFP-10 (culture filtrate protein 10), and ESAT-6 (6-kDa early secreted antigen target of M. tuberculosis) genes are conserved in M. tuberculosis and Mycobacterium bovis isolates but partially deleted or absent in M. bovis BCG as well as in most nontuberculous mycobacteria (NTM) (1-3, 10, 16). Importantly, the proteins encoded by these genes are immunogenic (7, 9, 12, 16). In this study, we cloned the 38-kDa, MTB48, CFP-10, and ESAT-6 genes and generated recombinant 38-kDa, MTB48, and CFP-10/ESAT-6 fusion proteins in Escherichia coli. Subsequently, we developed an enzyme-linked immunosorbent assay (ELISA) for the characterization of serum antibodies against 38-kDa, MTB48, and CFP-10/ESAT-6 antigens in a population of 250 active TB patients and 260 healthy subjects. We found that characterization of antibodies against multiple M. tuberculosis antigens were valuable for the diagnosis of active TB.     

Evaluation of Antigen-Specific Immunoglobulin G Responses in Pulmonary Tuberculosis Patients and Contacts

This study aimed to evaluate the serodiagnostic potential of immunoglobulin G (IgG) responses to Mycobacterium tuberculosis antigens in pulmonary tuberculosis (TB) patients, recent TB contacts with latent TB infection (LTBI), and healthy subjects. Infections were assessed using tuberculin skin tests, QuantiFERON-TB Gold In-Tube tests, drug susceptibility testing, and molecular genotyping of clinical isolates. Serum IgG responses to selective M. tuberculosis antigens, including the 38-kDa and 16-kDa antigens, lipoarabinomannan (LAM), and recombinant early secreted antigen target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP-10), were determined. We found that the serum IgG responses to all antigens might differentiate between active TB and LTBI, with LAM having the highest diagnostic value (area under the curve [AUC] of 0.7756, P < 0.001). Recurrent TB cases showed significantly higher IgG responses to 38 kDa, CFP-10 (P < 0.01), and LAM (P < 0.05) than new cases, and male patients had higher levels of antigen-specific IgG than females (P < 0.05). Conversely, drug resistance and patient body mass index did not affect IgG responses (P > 0.05). LAM-specific IgG responses differentiated between acid-fast bacillus (AFB) smear-positive and -negative patients (P < 0.01), whereas antigen-specific IgG responses did not vary with the M. tuberculosis genotype (P > 0.05). Significantly higher IgG responses to 38 kDa and 16 kDa were observed in AFB smear-negative patients than in controls. These results suggest that assessment of serum IgG responses to selective purified M. tuberculosis antigens may help improve the diagnosis of active TB, particularly for sputum smear-negative patients or recurrent cases, and these may also help to differentiate between active TB and LTBI.     

Control by H-2 genes of murine antibody responses to protein antigens of Mycobacterium tuberculosis.

The genetic control of antibody responses after immunization with Mycobacterium tuberculosis soluble antigens was examined in inbred and H-2 congenic mouse strains. Antibody levels to five distinct epitopes were determined by a competitive inhibition test using radiolabelled murine monoclonal probes. High or low responder antibody levels were associated with either the H-2b (TB23, TB71 and TB72 specificities) or the H-2k (TB68) allele on both B10 and BALB backgrounds. The high response of TB78 specificity associated with the H-2k on the BALB but with H-2b haplotype on B10 background. The phenotype of (C57BL/6 X CBA)F1 hybrids reflected the high response for four and the low or intermediary response for one (TB68) of the tested paratopes. This is the first demonstration of immune response gene control in respect of defined mycobacterial protein epitopes. The implications towards the analysis of pathogenic or protective mechanisms during mycobacterial infection are briefly outlined.     

Serodiagnostic Potential of Culture Filtrate Antigens of Mycobacterium tuberculosis

Our studies of the humoral responses of tuberculosis (TB) patients have defined the repertoire of culture filtrate antigens of Mycobacterium tuberculosis that are recognized by antibodies from cavitary and noncavitary TB patients and demonstrated that the profile of antigens recognized changes with disease progression (K. Samanich et al., J. Infect. Dis. 178:1534–1538, 1998). We have identified several antigens with strong serodiagnostic potential. In the present study we have evaluated the reactivity of cohorts of human immunodeficiency virus (HIV)-negative, smear-positive; HIV-negative, smear-negative; and HIV-infected TB patients, with three of the candidate antigens, an 88-kDa protein, antigen (Ag) 85C, and MPT32, and compared the reactivity of the same patient cohort with the 38-kDa antigen and Ag 85A. We have also compared the reactivity of native Ag 85C and MPT32 with their recombinant counterparts. The evaluation of the reactivity was done by a modified enzyme-linked immunosorbent assay described earlier (S. Laal et al., Clin. Diag. Lab. Immunol. 4:49–56, 1997), in which all sera are preadsorbed against Escherichia coli lysates to reduce the levels of cross-reactive antibodies. Our results demonstrate that (i) antigens identified on the basis of their reactivity with TB patients' sera provide high sensitivities for serodiagnosis, (ii) recombinant Ag 85C and MPT32, expressed in E. coli, show reduced reactivity with human TB sera, and (iii) of the panel of antigens tested, the 88-kDa protein is the most promising candidate for serodiagnosis of TB in HIV-infected individuals. Moreover, these results reaffirm that both the extent of the disease and the bacterial load may play a role in determining the antigen profile recognized by antibodies.     

Antigens of Mycobacterium tuberculosis Recognized by Antibodies during Incipient, Subclinical Tuberculosis

Serum samples obtained from human immunodeficiency virus (HIV)-infected tuberculosis (TB) patients months prior to clinical TB were used to delineate the profile of Mycobacterium tuberculosis culture filtrate proteins recognized during subclinical TB. A subset of ~12 antigens was recognized by antibodies in these serum samples. Antibodies to two of these antigens (81 [88]-kDa malate synthase [GlcB] and MPT51) were present in serum samples obtained during incipient subclinical TB in 19 (~90%) of the 21 HIV-infected TB patients tested. These antigens will be useful for devising diagnostic tests that can identify HIV-positive individuals who are at a high risk for developing clinical TB.     

Specificity of antibodies to immunodominant mycobacterial antigens in pulmonary tuberculosis.

A serological survey was performed in groups of patients with active sputum smear-positive or smear-negative pulmonary tuberculosis, healthy household contacts, and controls. Sera were tested for titers of antibodies which bound to each of five purified mycobacterial antigens by enzyme immunoassay and for competition of binding to single epitopes, using six radiolabeled monoclonal antibodies directed toward corresponding molecules. The evaluation of diagnostic specificity was based on a positive score represented by titers above the cutoff point of 2 standard deviations above the mean titer of a control group. For smear-positive samples, the best sensitivity (83%) was achieved by exclusive use of the 38-kilodalton (kDa) antigen or its corresponding monoclonal antibodies. For smear-negative samples, levels of antibodies binding to the 19-kDa antigen showed a lower sensitivity of 62% compared with the control group or 38% compared with the contact group. Titers of antibody binding to the 14-kDa antigen were raised in Mycobacterium bovis BCG-vaccinated contacts, indicating that the greatest potential of this antigen may be in the detection of infection in a population for which tuberculin testing is unreliable. The results demonstrated the differing antibody responses to each of the tested antigens and distinct associations with the stage of infection or disease.     

Profiling antibodies to Mycobacterium tuberculosis by multiplex microbead suspension arrays for serodiagnosis of tuberculosis

Tuberculosis (TB) is a serious global disease. The fatality rate attributed to TB is among the highest of infectious diseases, with approximately 2 million deaths occurring per year worldwide. Identification of individuals infected with Mycobacterium tuberculosis and screening of their immediate contacts is crucial for controlling the spread of TB. Current methods for detection of M. tuberculosis infection are not efficient, in particular, for testing large numbers of samples. We report a novel and efficient multiplex microbead immunoassay (MMIA), based on Luminex technology, for profiling antibodies to M. tuberculosis. Microbead sets identifiable by unique fluorescence were individually coated with each of several M. tuberculosis antigens and tested in multiplex format for antibody detection in the experimental nonhuman primate model of TB. Certain M. tuberculosis antigens, e.g., ESAT-6, CFP-10, and HspX, were included to enhance the specificity of the MMIA, because these antigens are absent in nontuberculous mycobacteria and the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin. The MMIA enabled simultaneous detection of multiple M. tuberculosis plasma antibodies in several cohorts of macaques representing different stages of infection and/or disease. Antibody profiles were defined in early and latent/chronic infection. These proof-of-concept findings demonstrate the potential clinical use of the MMIA. In addition, the MMIA serodetection system has a potential for mining M. tuberculosis open reading frames (about 4,000) to discover novel target proteins for the development of more-comprehensive TB serodiagnostic tests.     

Monoclonal antibodies produced in BALB.B10 mice define new antigenic determinants in culture filtrate preparations of Mycobacterium tuberculosis.

A panel of monoclonal antibodies were derived from BALB.B10 mice immunized with a culture filtrate from Mycobacterium tuberculosis H37Rv. Of these antibodies, 10 were examined more closely for antigen specificity and interspecies reactivity. Six antibodies were used as immunosorbents for affinity purification of their corresponding antigens. Two monoclonal antibodies (HBT 2 and HBT 11) reacted with a 17-kilodalton antigen, and a competition assay showed that these antibodies are directed against the same epitope or against epitopes that are sterically very close to each other. Monoclonal antibody HBT 12 reacted with the same molecule with which a previously described 38-kilodalton reactive antibody reacted but was directed against a different epitope. Antibody HBT 10 reacted with a culture filtrate of M. tuberculosis but not of Mycobacterium bovis BCG. This latter finding was further studied by testing different preparations of M. tuberculosis H37Rv antigens and, additionally, culture filtrates of four M. tuberculosis and two BCG strains. Interspecies reactivity was assayed by immunoblotting and revealed that the majority of the monoclonal antibodies were specific to M. tuberculosis complex.     

Serum Concentrations of Antibodies against Outer Membrane Protein P6, Protein D,and T- and B-Cell Combined Antigenic Epitopes of Nontypeable Haemophilus influenzae in Children and Adults of Different Ages

Nontypeable Haemophilus influenzae (NTHi) is one of the most common etiologies of acute otitis media, rhinosinusitis, and pneumonia. Outer membrane proteins (OMPs) are the main focus in new vaccine development against NTHi, as the H. influenzae type b (Hib) vaccine does not cover noncapsulated NTHi. The OMPs P6 and protein D are the most promising candidate antigens for an NTHi vaccine, and low antibody levels against them in serum may be correlated with infection caused by NTHi. In the current study, we measured the antibody titers against P6, protein D, and their T- and B-cell combined peptide epitopes in healthy individuals of different ages. We found that children <1 month old had the lowest antibody levels against NTHi P6, protein D, and their T- and B-cell combined antigenic epitopes. Antibody titers increased at ages 1 to 6 months, peaked at 7 months to 3 years, and remained high at 4 to 6 years. The antibody titers started to decrease after 6 years and were the lowest in the 21- to 30-year group. The geometric mean titers (GMTs) of T- and B-cell combined antigenic epitopes in P6 and protein D were positively correlated with those of the protein antigens. Among 12 peptides tested, P6-61, P6-123, and protein D-167 epitopes were better recognized than others in human serum. These findings might contribute to the development of an effective serotype-independent vaccine for H. influenzae.