Changes in serum immunomolecules during antibiotic therapy for Mycobacterium avium complex lung disease.

Little information is available regarding changes in immune status for patients with Mycobacterium avium complex (MAC) lung disease during antibiotic therapy. Serum immunomolecules from 42 patients with MAC lung disease were assayed comparatively using an array-based system according to (i) patients with MAC lung disease at the time of diagnosis versus healthy controls and (ii) alterations after 12 months of antibiotic therapy in the MAC lung disease group. In addition, cytokine analyses were performed to determine whether cytokine responses were associated specifically with the disease phenotype, treatment outcome and aetiological agent. Notably, the serum concentrations of type 1 cytokine-associated molecules, such as CD40L, interferon (IFN)-γ, interleukin (IL)-8 and IL-23, were decreased significantly in patients at the time of diagnosis, suggesting that these molecules may serve as indicators of host susceptibility to MAC disease. Although the overall serum level of T helper type 1 (Th1)-related molecules, such as CD40L and IFN-γ, was restored after treatment, Th17-related cytokines, such as IL-17 and IL-23, were down-regulated significantly at 12 months post-treatment compared to pretreatment. Furthermore, these cytokine patterns differed among patient subgroups. Decreased serum concentrations of IL-17 and/or IL-23 were associated with failure of sputum conversion, the fibrocavitary disease phenotype and M. intracellulare lung disease. Thus, the reciprocal balance between Th1 and Th17 immunity during antibiotic therapy for MAC lung disease is critical for dictating the treatment response. In conclusion, a low level of Th1-related immunomolecules may perpetuate MAC lung disease, and the serum concentrations of Th17-related cytokines can reflect the treatment outcome, disease phenotype and aetiological agent.

Mtb32 is a promising tuberculosis antigen for DNA vaccination in pre- and post-exposure mouse models.

Identification of antigens that provide protective immunity via prophylactic and therapeutic vaccination against Mycobacterium tuberculosis is critical for the development of subunit vaccines for tuberculosis (TB). In this study, we performed a head-to-head comparison of seven well-known TB antigens delivered by DNA vaccine, and evaluated their respective immunogenicities and protective efficacies in pre- and post-exposure mouse models. All TB antigens were designed as a chimeric fusion with Flt3-L to enhance antigen-specific T-cell immunity upon vaccination. Prophylactic vaccination with the Flt3L (F)-Mtb32 DNA vaccine elicited significant protection in both the spleen and lungs against M. tuberculosis challenge, comparable to the Bacillus Calmette-Guerin vaccine. F-Ag85A and F-Mtb32 DNA vaccines, in combination with chemotherapy, reduced the bacterial burden to undetectable levels in the lungs of all mice infected with M. tuberculosis. These data collectively indicate that the F-Mtb32 DNA vaccine confers the most efficient protective immunity that suppresses bacterial growth in the active or latent status of M. tuberculosis.

The responses of multiple cytokines following incubation of whole blood from TB patients, latently infected individuals and controls with the TB antigens ESAT-6, CFP-10 and TB7.7.

The development of clinically relevant biomarkers is important for diagnosing latent tuberculosis infection (LTBI) and active tuberculosis (TB) and predicting their prognoses. This study examined whether the responses of multiple cytokines can be used as a biomarker to distinguish the TB infection status and mycobacterial load. We analysed the responses of multiple cytokines (IFN-γ, IL-2, IL-10, IL-13, IL-17 and TNF-α) in the supernatant from the QuantiFERON-TB Gold In-Tube assay following stimulation of whole blood from the TB group (n = 32), LTBI group (n = 19) and healthy controls (n = 30) with TB antigens (ESAT-6, CFP-10 and TB7.7). The median responses of IFN-γ, IL-2, IL-10 and IL-13 were higher in the LTBI and active TB groups than in the non-TB control group (IFN-γ, P < 0.001; IL-2, P < 0.001; IL-10, P = 0.012; IL-13, P < 0.001). The median IL-2/IFN-γ ratio of the LTBI group was higher than that of the active TB group (P = 0.014) and differed significantly between patients with LTBI, patients with smear-negative TB and patients with smear-positive TB (P = 0.027). This difference was especially evident between the patients with LTBI and patients with smear-positive TB (P = 0.047). In conclusion, IFN-γ, IL-2, IL-10 and IL-13 can serve as biomarkers for distinguishing TB infection. In addition, the IL-2/IFN-γ ratio appears to be a biomarker for diagnosing LTBI and may be useful as a prognostic factor and for evaluating treatment responses.

Identification of Rv2041c, a novel immunogenic antigen from Mycobacterium tuberculosis with serodiagnostic potential.

Novel immunogenic antigens are continually required for the improvement of diagnostic techniques for Mycobacterium tuberculosis infection. Some proteins with serodiagnostic value are not expressed under normal culture conditions, but may be induced under specific conditions such as gradual oxygen depletion and low pH, and from inside macrophages. Using a customized amplification library, we previously found that Rv2041c from M. tuberculosis H37Rv was highly expressed in vitro under conditions of low pH and hypoxia. In this study, recombinant (r)Rv2041c was produced in Escherichia coli to examine its role in immune responses. Increased Rv2041c expression in vitro during dormancy and during infection in human macrophages was confirmed by Western blotting and reverse transcription polymerase chain reaction, respectively. Interestingly, positive antibody responses to rRv2041c were detected only in those patients with active tuberculosis (TB) and in mice infected with M. tuberculosis H37Rv. Finally, Rv2041c was used successfully in the serodiagnosis of active M. tuberculosis infection in Korean patients in conjunction with other M. tuberculosis proteins, including Ag85 complex, 38 kDa, rESAT-6, rHSP-X and rCFP-10. Our Rv2041c-ELISA had comparable diagnostic sensitivity and equivalent specificity to the use of an M. tuberculosis H37Rv cellular extract. In addition, seven of 46 serum samples collected from TB patients (15.28%) showed positive antibody responses to Rv2041c, but not to the other proteins. These results suggest that Rv2041c can be used to increase assay sensitivity alongside well-known antigens for the serodiagnosis of M. tuberculosis infection.

Tissue-specific changes in mRNA expression of Abc and Slc transporters in murine pulmonary tuberculosis.

A pulmonary tuberculosis mouse model was used to assess the pharmacodynamic and pharmacokinetic characteristics of tuberculosis therapeutics. While membrane transporters play important roles in drug disposition and physiological homeostasis, their expressional changes and contribution have never been analysed in a tuberculosis animal model. The mRNA expression level of 20 Abc family transporters and 32 Slc family transporters in tuberculosis-infected mice were compared with those in naïve uninfected mice using real-time polymerase chain reaction (PCR). Mycobacterium tuberculosis infection induced many dramatic expression changes of families of both Abc transporters and Slc transporters at 4 and 8 weeks, as observed in the livers, kidneys, and intestines of test mice--and in a different mode, in the lungs and spleens as well. These changes were dependent on the tuberculosis progression with the tissue-specific manner, that is, in the lungs, the number of transporters of which the expression level changed due to M. tuberculosis infection had increased, and the magnitude of change also greater at 8 weeks, while in the spleen, the transcription of most transporters except Mrps had not changed or had recovered back to the same level of naïve transcription at 8 weeks. Understanding the expression changes of transporters will assist in setting up rational preclinical dosing plans through the ability to predict the pharmacokinetics of new anti-tuberculosis chemotherapeutics and, furthermore, will assist in the design of safer and more efficient drug regimens.

Protection of mice against Mycobacterium tuberculosis infection by immunization with aqueous fraction of Triton X-100-soluble cell wall proteins.

The aqueous fraction of Triton X-100-soluble proteins (TSP-Aq) of Mycobacterium tuberculosis cell wall was reported to stimulate T-cell responses in peripheral blood monocytes from tuberculosis (TB) patients and to induce Th1 cytokines, suggesting presence of protective antigens. In this study, therefore, we examined the protective efficacy of TSP-Aq against M. tuberculosis infection in a mouse model. C57BL/6 mice were immunized with TSP-Aq or culture filtrate proteins (CFP) mixed with incomplete Freund's adjuvant or with BCG followed by i.v. challenge with M. tuberculosis H37Rv. TSP-Aq induced strong interferon-gamma production by spleen cells, and mice immunized with TSP-Aq antigens gave a significant reduction in M. tuberculosis CFU counts by 1.17-1.32 log10 CFU in the lungs and 1.31-2.08 log10 CFU in the spleen from 6 to 28 weeks. The degree of protection offered by TSP-Aq was comparable to that of CFP and of the BCG vaccine. The results demonstrated that the TSP-Aq antigens confer a significant level of protection against the growth of the organism in the lungs and spleen in a mouse model of TB and indicate that TSP contains major protective antigens of M. tuberculosis.

The impact of social conditions on patient adherence to pulmonary tuberculosis treatment.

SETTING: Tuberculosis (TB) remains one of the main concerns in global health. One of the main threats to treatment success is patient non-adherence to anti-tuberculosis treatment. OBJECTIVE: To identify the relation between social conditions and treatment adherence in a prospective cohort setting in an intermediate TB burden country. DESIGN: To identify associations between poor adherence and social conditions, including educational level, type of residence and occupation, we constructed hierarchical logistic regression models. RESULTS: A total of 551 participants were included in the study. Low educational levels, poor housing and occupations in the construction and manufacturing industries and service sectors were associated with poor adherence; this association was likely to be differentiated by previous history of anti-tuberculosis treatment. CONCLUSION: Policy making should focus on improving the social conditions of patients by working towards better housing conditions and providing health promoting working conditions to enable treatment adherence.

Safety and efficacy of tuberculin skin testing with microneedle MicronJet600™ in healthy adults.

SETTING: Intradermal injection using a syringe and needle is generally accepted as the most accurate method for the tuberculin skin test (TST). However, the Mantoux technique using a conventional needle is often difficult to perform reliably, affecting testing results and safety. OBJECTIVE: We evaluated the efficacy and safety of a novel intradermal injection device, the MicronJet600(TM) microneedle, compared with conventional injection in terms of skin reactivity to the TST. DESIGN: A prospective, open-label clinical study was conducted. The TST was administered by both methods in the same subject. For pain assessment, participants filled in a visual analogue scale (VAS) after each TST. Any side effects due to TST or injections were observed. RESULTS: TST reaction rates (cut-off ⩾5 mm) from microneedles and needles were respectively 44.0% and 47.2%, with no significant difference between the two. Furthermore, agreement of positivity between the two methods was excellent with both 5 mm and 10 mm cut-off values. However, the level of pain experienced when microneedles were used for TST was significantly lower than with conventional needles. No adverse effects were attributed to the MicronJet device. CONCLUSION: The novel microneedle device used for TST in this study was effective, safe and less painful in healthy adult volunteers.

Conventional and real-time PCR targeting 16S ribosomal RNA for the detection of Mycobacterium tuberculosis complex.

SETTING: Conventional diagnostic methods for tuberculosis (TB) have limited sensitivity and specificity or are time-consuming. OBJECTIVE: 16S rDNA and 16S rRNA of Mycobacterium tuberculosis complex (MTC) were used as targets to develop sensitive and specific polymerase chain reactions (PCRs) to improve the diagnosis of MTC. DESIGN: We developed conventional and real-time PCRs targeting 16S rDNA and rRNA of MTC. RESULTS: PCRs targeting 16S rRNA had a 10-100 times lower limit of detection for M. tuberculosis than PCRs targeting 16S rDNA. The sensitivities of the 16S rDNA PCR, 16S rRNA reverse transcription PCR (RT-PCR), 16S rDNA real-time PCR and 16S rRNA real-time RT-PCR for sputum specimens were respectively 92%, 94.6%, 96% and 100%. Real-time PCR showed no cross-reactivity, but conventional PCR had cross-reactivity to M. avium, M. gastri and M. nonchromogenicum. CONCLUSION: PCRs targeting the 16S rRNA of MTC were more sensitive than those targeting 16S rDNA; 16S rRNA real-time RT-PCR showed the highest sensitivity and specificity for MTC.

Anti-T antibodies and peanut-agglutinin-binding glycoproteins in sera of patients with gastric cancer.

Agglutinating antibodies to neuraminidase-treated red blood cells (anti-T agglutinins) are known to be reduced in patients with gastric cancer. The antigenic determinant of anti-T agglutinin is known to have a disaccharide structure [Gal(beta1-3)GalNAc], the same specificity as peanut agglutinin (PNA). We examined sera of 27 patients with gastric cancer and 30 controls for anti-T agglutinins, anti-T antibodies and PNA-binding glycoproteins. Anti-T agglutinins were titrated by a microtiter hemagglutination method. Levels of anti-T antibodies were determined by enzyme immunoassay using synthetic glycoconjugate [Gal(beta1-3)GalNAc O-alpha-linked to human serum albumin] as an antigen. Levels of PNA-binding glycoproteins in sera were measured by sandwich enzyme-linked lectin assay using wheat germ agglutinin and peroxidase-conjugated PNA. Titers of anti-T agglutinins were significantly lower in patients with gastric cancer than in controls (P = 0.041). Levels of anti-T antibodies were not significantly different in patients with gastric cancer and controls; however, decreased levels of anti-T antibodies were more frequent in patients with gastric cancer than in controls (P = 0. 001). Levels of PNA-binding glycoproteins were significantly higher in sera of patients with gastric cancer than in controls (P = 0.001). The levels of anti-T antibodies inversely correlated with the levels of PNA-binding glycoproteins in sera of patients with gastric cancer (r = -0.44, P = 0.021). These results suggest that the decrease in anti-T antibodies in sera of patients with gastric cancer might be due to immune complex formation between circulating PNA-binding glycoproteins and anti-T antibodies.

A simplified serological test for leprosy based on a 3,6-di-O-methylglucose-containing synthetic antigen.

The recent advent of synthetic antigens containing the Mycobacterium leprae-specific epitope, 3,6-di-O-methyl-beta-D-glucopyranoside, has allowed the development of simple specific serological tests for leprosy. The incorporation of one such product, 8-carbonyloctyl O-[4-O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-alpha-L- rhamnopyranoside]-BSA, into a simple "spot" test, diffusion-in-gel enzyme-linked immunosorbent assay (ELISA), allowed an over 90% detection rate of untreated lepromatous leprosy, and the results showed good concordance with conventional ELISA based on the native phenolic glycolipid I.

The response to chemotherapy of serum Mycobacterium leprae-specific antigen in multibacillary leprosy patients.

We have examined the Mycobacterium leprae phenolic glycolipid-I (PG-I) antigen levels in the sera of 45 multibacillary leprosy patients commencing chemotherapy. The PG-I antigen levels correlated with the bacterial and morphological indices, but not with the serum IgM anti-PG-I antibody levels. Antigen levels were significantly higher in patients with diffuse skin infiltration, but did not vary significantly with other parameters reflecting the duration and extent of untreated disease. The PG-I antigen levels in 27 patients examined serially decreased consistently over the first year of multidrug therapy.

Exclusive mutations related to isoniazid and ethionamide resistance among Mycobacterium tuberculosis isolates from Korea.

SETTING: The single base change at the 94th codon of inhA has been referred to as the event that confers resistance on the drugs isoniazid (INH) and ethionamide (ETH) in Mycobacterium smegmatis and M. bovis. From this observation, it has been anticipated that some of the INH-resistant clinical isolates of M. tuberculosis would carry missense mutations in the same region of the gene. However, few polymorphisms have been identified in this region among INH-resistant isolates. OBJECTIVE: To understand the molecular basis for M. tuberculosis resistance to INH and ETH. DESIGN: The sequence polymorphism at the 94th codon of inhA among M. tuberculosis isolates from Korea was analyzed by polymerase chain reaction (PCR) cloning and sequence analysis. RESULTS: No nucleotide change at the 94th codon of inhA was detected in any of the 24 INH-resistant isolates analyzed in this study. On the other hand, a point mutation was found exclusively at the regulatory region flanking a putative ribosome-binding site of the inhA locus in 14 isolates. Interestingly, all the mutations were of the same kind, which substitutes C to T. Among 14 isolates, 12 were resistant to INH as well as to ETH, while two were resistant to INH only. DISCUSSION: It seems that mutations previously found at the 94th codon of inhA have no particular relationship with the mechanism involved in the resistance of M. tuberculosis to INH and/or ETH. On the other hand, the resistance mechanism of M. tuberculosis to INH/ETH may involve an altered level of InhA, an expression which may have been influenced by the sequence change in the regulatory region of the inhA locus.

Molecular analysis of rifampin-resistant Mycobacterium tuberculosis isolated from Korea by polymerase chain reaction-single strand conformation polymorphism sequence analysis.

OBJECTIVE: To assess the molecular mechanism of rifampin (RMP) resistance in clinical strains of Mycobacterium tuberculosis. DESIGN: The molecular nature of a part of the rpoB gene in 77 M. tuberculosis clinical strains isolated in Korea was analyzed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and PCR-sequence analysis. RESULTS: Among 67 RMP-resistant isolates, 50 showed SSCP profiles different from that of an RMP-sensitive control strain, M. tuberculosis H37Rv, indicating the possible existence of a sequence alteration in this region of the rpoB gene, while 17 resistant isolates displayed SSCP profiles indistinguishable from that of the sensitive control strain. Subsequently, 17 clinical isolates whose SSCP profiles were difficult to distinguish from the control strain were subjected to sequence analysis. The analysis revealed that all 17 isolates did indeed contain mutations in the 81 bp region of the rpoB gene, which is associated with RMP resistance. CONCLUSION: The results from our study clearly indicate that the molecular mechanism of RMP resistance in M. tuberculosis isolates from Korea involves alterations in the rpoB gene. In addition, this study suggests that PCR-direct sequence analysis works more efficiently and accurately than PCR-SSCP analysis for rapid screening of RMP-resistant M. tuberculosis clinical isolates.

Chemical synthesis and seroreactivity of O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl- alpha-L-rhamnopyranosyl)-(1----9)-oxynonanoyl-bovine serum albumin--the leprosy-specific, natural disaccharide-octyl-neoglycoprotein.

The outer disaccharide segment, namely, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3-di-O-methyl-alpha -L-rhamnopyranose, of the trisaccharide-containing, leprosy-specific, phenolic glycolipid I has been synthesized as the 8-(methoxycarbonyl)octyl glycoside in high yield and absolute stereospecificity by a series of modified Koenigs-Knorr and Helferich reactions. A particular feature of the synthetic pathway involves methylation of the 2-hydroxyl group of the rhamnose moiety under neutral conditions, after first preparing the 8-(methoxycarbonyl)octyl glycoside as the alpha anomer via the 1,2-orthoacetate, and thus precluding the possible formation of an anomeric mixture. The 8-(methoxycarbonyl)octyl O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3-di-O-methyl-alpha -L-rhamnopyranoside was converted into the crystalline hydrazide, and this was coupled to bovine serum albumin (BSA), via intermediate acyl-azide formation, to produce the corresponding neoglycoprotein, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)- (1----4)-O-(2,3-di-O-methyl-alpha-L-rhamnopyranosyl)- (1----9)-oxynonanoyl-BSA, the so-called natural disaccharide-octyl-BSA. Extensive serological testing of this product against sera from leprosy patients and control populations, and comparison with the native glycolipid and previously synthesized neoglycoproteins, have shown that it is unparalleled in terms of sensitivity and specificity, and highly suited to replace the native glycolipid for the serodiagnosis of worldwide lepromatous leprosy.

Synthesis and immunoreactivity of neoglycoproteins containing the trisaccharide unit of phenolic glycolipid I of Mycobacterium leprae.

The trisaccharide segment, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl- alpha-L-rhamnopyranosyl)-(1----2)-3-O-methyl-L-rhamnopyranose, of the Mycobacterium leprae-specific phenolic glycolipid I has been synthesized as its 8-(methoxycarbonyl)octyl glycoside and coupled to a carrier protein, to produce a leprosy-specific neoglycoprotein, the so-called natural trisaccharide-octyl-bovine serum albumin (NT-O-BSA). Special features of the synthetic strategy were the use of silver trifluoromethanesulfonate (triflate) to promote glycosylation, resulting in the rhamnobiose in high yield and absolute stereospecificity. The terminal 3,6-di-O-methyl-D-glucopyranosyl group was introduced after O-deallylation of the rhamnobiose. Removal of protecting groups yielded the trisaccharide hapten suitable for coupling to carrier protein. Poly(acrylamide)-gel electrophoresis of the neoglycoprotein demonstrated its purity, and subsequent immunoblotting with a monoclonal antibody directed to the terminal 3,6-di-O-methyl-beta-D-glucopyranosyl epitope of the native glycolipid demonstrated its antigenicity. Comparative serological testing in enzyme-linked immunosorbent assays of NT-O-BSA, the corresponding disaccharide-containing products, and another trisaccharide-containing neoglycoprotein, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O- methyl-alpha-L-rhamnopyranosyl)-(1----2)-(3-O-methyl-alpha-L-rhamnopy ran osyl)- (1----4')-oxy-(3-phenylpropanoyl)-BSA (NT-P-BSA) [Fujiwara et al., Agric. Biol. Chem., 51 (1987) 2539-2547] against sera from leprosy patients and control populations showed concordance; the presence of the innermost sugar did not contribute significantly to sensitivity or specificity. The di- and tri-saccharide-containing neoantigens, on account of ready availability and solubility, provide greater flexibility than the native glycolipid for the serodiagnosis of leprosy.

Detection of IgA anti-PGL-I specific antigen to Mycobacterium leprae in mangabey monkeys inoculated with M. leprae.

Using sera from 4 pairs of mangabey monkeys inoculated with titrated doses of Mycobacterium leprae we demonstrated that IgA antibodies against M. leprae specific PGL-I antigen were present in 75% of inoculated monkey's sera. High IgA antibody was detected in 50% (3/6) of infected animals and all three developed lepromatous leprosy (LL). Antibody titers correlated with PGL-I antigen in serum. The highest IgA peak appeared late and corresponded to the beginning of treatment, and in two of them appeared shortly after or corresponded with neurological damage. Low IgA response was found in the other 3 monkeys (50%-3/6), two of which developed indeterminate leprosy (I) and the other one LL. Low IgA levels appeared late after IgG and IgM, and shortly after neurologic signs. Both I monkeys were negative for PGL-I in serum. The remaining 2 monkeys (25%-2/8) did not show an IgA response; one of them developed LL but the disease regressed to I. IgM seemed to correspond to the appearance of PGL-I in serum. The other animal did not develop clinical symptoms of leprosy, and PGL-I in serum was negative. Although there was no clear relation between the development of anti-PGL-I IgA and experimental leprosy, the finding of a high IgA response in some animals suggests that further studies are needed to evaluate the role of antigen-specific IgA in the disease process.

Prevalence of IgM antibodies to phenolic glycolipid I among household contacts and controls in Korea and the Philippines.

Phenolic glycolipid I (PGL-I) is a Mycobacterium leprae-specific antigen and the antibodies to the antigen may suggest an M. leprae infection. To compare the M. leprae transmission among the populations, we compared the prevalence of anti-PGL-I IgM antibodies among household contacts and controls between Korea and the Philippines. In Korea (prevalence of leprosy--0.04: 1000), the prevalence of anti-PGL-I antibodies were 4.8% among controls and 8.0% among contacts, respectively. On the other hand, the seroprevalence rate was 10.8% among controls and 13.4% among contacts in the Philippines (prevalence of leprosy--0.70: 1000). Interestingly, a marked difference was noted in the prevalance of anti-PGL-I antibodies among children between the countries; 10-14% among children under 10 years old and 15-18% among those aged between 10 and 19 in the Philippines compared to 0% and 2.9-6.4% in Korea, respectively. This study, therefore suggests that a high prevalance of anti-PGL-I IgM antibodies among children may indicate an active transmission of M. leprae, resulting in a higher incidence of leprosy in the population.

Experimental leprosy in monkeys. II. Longitudinal serological observations in sooty mangabey monkeys.

In this study, 11 SMM were grouped and inoculated with differing doses of SMM-origin Mycobacterium leprae (ML) between 4.5 x 10(8) and 1 x 10(9) by either combined IV/IC routes or by IV or IC route alone. The combined route was the most effective in eliciting progressive, disseminated LL leprosy. In all, 6 of 7 SMM inoculated by the combined routes developed leprosy requiring treatment at some point. Only 1 of 4 inoculated by a single route developed persisting leprosy requiring chemotherapy. Either no disease or spontaneous regression of initial disease occurred in the other 3 animals inoculated by a single route. Doses in excess of 1 x 10(9) ML were more effective than lesser doses. An association was observed between the development of IgG anti-PGL-I ELISA OD values and resistance to leprosy and between IgM anti-PGL-I and leprosy progression or susceptibility. Serum PGL-I antigen levels, determined by dot ELISA, paralleled disease severity longitudinally. High positive OD values of anti-LAM IgG prior to ML inoculation were observed in the majority of leprosy-susceptible SMM in contrast to negative levels in more resistant animals. Anti-LAM IgG OD values exceeded the positive cut-off point after inoculation in 5 of 11 SMM; 3 of these 5 had concurrent detectable serum levels of PGL-I antigen.

Protective immunization of monkeys with BCG or BCG plus heat-killed Mycobacterium leprae: clinical results.

Rhesus and sooty mangabey monkeys (RM and SMM) were vaccinated and boosted with BCG or BCG + low dose (LD) or high dose (HD) heat-killed Mycobacterium leprae (HKML). One group was not vaccinated. Except for a group of controls, all monkeys were challenged with live M. leprae. All animals were studied longitudinally to determine antileprosy protective efficacy. BCG reduced the numbers of RM with histopathologically-diagnosed leprosy by 70% and slowed and ameliorated the appearance of symptoms. BCG + LDHKML reduced the number of RM with leprosy by 89% and BCG + HDHKML by 78%. BCG did not protect SMM from developing leprosy, but disease progress was slowed; disease in SMM was exacerbated by the addition of HKML to the vaccine. RM, as a species, are prone to paucibacillary (PB) forms of leprosy, whereas SMM are prone to multibacillary (MB) forms. Thus, BCG vaccination offers significant protection from clinical disease and slows/ameliorates the rate of progression/degree of disease at the PB end and appears to at least ameliorate symptoms at the MB end of the leprosy spectrum. BCG + HKML protects at the PB end and exacerbates disease progress at the MB end of the leprosy spectrum.