Induction of lepromin positivity and immunoprophylaxis in household contacts of multibacillary leprosy patients: a pilot study with a candidate vaccine, Mycobacterium w.

We screened 487 household contacts of multibacillary (MB) patients for evidence of disease and their lepromin status. From the 444 results available, 302 (68.02%) were lepromin positive and 142 (31.98%) were lepromin negative on initial testing. The initial lepromin status as assessed in the group of 54 contacts having disease at the outset showed 24 out of 46 (52.2%) to be lepromin positive and 22 of 46 (47.8%) to be lepromin negative. In the same group, among 24 lepromin positives, 22 (91.7%) had paucibacillary (PB) and 2 (8.3%) had multibacillary (MB) disease; among the lepromin negatives, 12 (54.5%) had PB and 10 (45.5%) had MB disease. Out of 72 initially lepromin-negative contacts administered Mycobacterium w vaccine and followed up, the cumulative percentages show that 53 (73.6%) converted to positivity after a single dose, 10 (87.5%) after a second dose and 67 (93.1%) after the third dose. The incidence of new cases with leprosy was 8 out of 231 (3.46%) among lepromin-positive contacts and 5 out of 93 (5.38%) among lepromin-negative contacts administered Mycobacterium w vaccine. Among 231 lepromin-positive contacts, the new cases occurred in those with a 1+ and 2+ lepromin response only, and no case occurred among 51 contacts with a 3+ lepromin response. The incidence among lepromin-positive contacts in this study (3.46%) was similar to the observations in two other studies: 3.2% by Dharmendra, et al. and 6.9% by Chaudhary, et al. However, the incidence among lepromin-negative contacts administered Mycobacterium w vaccine was significantly lower than that observed among lepromin-negative contacts not administered any vaccination in the other two studies (14.1% by Dharmendra, et al. and 29.0% by Chaudhary, et al.). To conclude, although a study of small sample size, the preliminary evaluation indicates that administration of Mycobacterium w vaccine seems to have the potential to reduce the incidence of leprosy among household contacts of leprosy patients. More explicit results about the vaccine will be available from the ongoing field trials in Kanpur Dehat in the near future.

Reactional states and neuritis in multibacillary leprosy patients following MDT with/without immunotherapy with Mycobacterium w antileprosy vaccine.

A vaccine based on autoclaved Mycobacterium w was administered, in addition to standard multidrug therapy (MDT), to 157 untreated, bacteriologically positive, lepromin negative multibacillary leprosy patients, supported by a well matched control group of 147 patients with similar type of disease, who received a placebo injection in addition to MDT. The MDT was given for a minimum period of 2 years and continued until skin smear negativity, while the vaccine/placebo was given at 3-monthly intervals up to a maximum of eight doses. The incidence of type 2 reaction and neuritis during treatment and follow-up showed no statistically significant difference in the vaccine and placebo groups. The incidence of type 1 reaction (mild in most cases), however, was higher in the vaccine group (P = 0.041, relative risk ratio 1.79), considering LL, BL and BB leprosy types together, and considerably higher (P = 0.009) in LL type, probably because of confounding due to higher number of patients with previous history of reaction in this group. The occurrence of reactions and neuritis in terms of single or multiple episodes was similar in the vaccine and placebo groups. The association of neuritis and reactions, as well as their timing of occurrence (during MDT or follow-up), was also similar in the two groups, with more than 90% of occurrences taking place during MDT. The incidence of reversal reaction was significantly higher among the males in the vaccine group (34.5% versus 8.3%, P = 0.019). Patients with high initial BI (4.1-6.0) showed higher incidence of reactions (70.3%) as compared to those with medium (2.1-4.0) and low (0.3-2.0) BI where the reactions were observed with a frequency of 56.1% and 38.8%, respectively. However, unlike reactions, neuritis incidence did not seem to be affected by initial BI to the same extent in the vaccine group, with frequencies of 35.3%, 36.3% and 25.9% in the three mentioned BI ranges. Overall, the vaccine did not precipitate reactional states and neuritis over and above that observed with MDT alone.

Mycobacterium w vaccine, a useful adjuvant to multidrug therapy in multibacillary leprosy: a report on hospital based immunotherapeutic clinical trials with a follow-up of 1-7 years after treatment.

A vaccine based on autoclaved Mycobacterium w was administered, in addition to standard multidrug therapy (MDT), to 156 bacteriologically positive, lepromin negative multibacillary leprosy patients compared to a well matched control group of 145 patients with a similar type of disease who received a placebo injection in addition to MDT. The MDT was given for a minimum period of 2 years and continued until skin smear negativity, while the vaccine was given at 3-month intervals up to a maximum of eight doses. The fall in clinical scores and bacteriological indices was significantly more rapid in vaccinated patients, from 6 months onward until years 2 or 3 of therapy. However, no difference was observed in the fall in bacteriological index in the two groups from year 4 onwards. The number of LL and BL patients released from therapy (RFT) following attainment of skin smear negativity, after 24-29 months of treatment was 84/133 (63.1%) in vaccinated and 30/120 (25.0%) in the placebo group; the difference was highly statistically significant (P < 0.0001). In all, 90.2% patients (146/162) converted from lepromin negativity to positivity in the vaccine group, as against 37.9% (56/148) in the placebo group. The average duration of lepromin positivity maintained following eight doses of vaccine administered over 2 years was 3.016 years in the vaccine and 0.920 years in the placebo group. Histological upgrading after 2 years of treatment in the LL type was observed in 34/84 (40.5%) cases in the vaccine and 5/85 (5.9%) cases in the placebo group, the difference being statistically significant (P < 0.001). The incidence of type 1 reactions was significantly higher (30.5%) in the vaccine group than (19.7%) in the placebo group (P = 0.0413); the difference was mainly observed in LL type (P = 0.009). The incidence of type 2 reactions was similar (31.8 and 34.6%) in vaccine and placebo groups. The vaccine did not precipitate neuritis or impairments over and above that encountered with MDT alone. After 5 years of follow-up following RFT, no incidence of bacteriological or clinical relapses was observed in both groups.

Disabilities in multibacillary leprosy following multidrug therapy with and without immunotherapy with Mycobacterium w antileprosy vaccine.

A vaccine based on autoclaved Mycobacterium w was administered, in addition to standard multidrug therapy (MDT), to 157 bacteriologically positive, lepromin-negative, multibacillary leprosy patients supported by a well-matched control group of 147 patients with similar type of disease who received a placebo injection in addition to MDT. The MDT was given for a minimum period of 2 years and continued until skin-smear negativity, while the vaccine/placebo was given at 3-month intervals up to a maximum of 8 doses in the initial 2 years. The overall incidence of type 1 and type 2 reactions and neuritis during treatment and follow up was nearly equal in the patients in the vaccine and placebo groups; the differences were not statistically significant. The occurrence of disabilities, such as anesthesia, trophic ulcers, claw hand and grade 3 deformities, were not different statistically in the vaccine and placebo groups, an observation valid both for deformities present at induction and for those which developed during the course of therapy and surveillance. A statistically significant difference was observed in the recovery of newly developed trophic ulcers; recovery was quicker in the vaccine group. The recovery rate for motor deformities was marginally higher in the vaccine group, although not significant (p = 0.068) statistically. There was a statistically significant reduction in the incidence of grade 3 deformities following MDT with and without immunotherapy. To conclude, the addition of vaccine to MDT did not precipitate neuritis or deformities over and above that encountered with MDT alone, although it did accelerate bacteriological clearance, histopathological upgrading, conversion to lepromin positivity, and clinical improvement.

Induction of lepromin positivity following immuno-chemotherapy with Mycobacterium w vaccine and multidrug therapy and its impact on bacteriological clearance in multibacillary leprosy: report on a hospital-based clinical trial with the candidate antileprosy vaccine.

A vaccine based on autoclaved Mycobacterium w was administered, in addition to standard multidrug therapy (MDT), to 157 bacteriologically positive, lepromin-negative, multibacillary (LL, BL and BB) leprosy patients. The vaccinees were supported by a well-matched control group of 147 patients with similar type of disease who received a placebo injection in addition to MDT. The MDT was given for a minimum period of 2 years and continued until skin-smear negativity, while the vaccine was given at 3-month intervals up to a maximum of 8 doses. The lepromin response evaluated in terms of percentage of subjects converting to positivity status, measurement in millimeters, and duration of lepromin positivity sustained, reflected a statistically significant better outcome in the vaccine group patients (especially LL and BL leprosy) in comparison to those in the placebo group. The data indicate that lepromin-positivity status seems to have an impact on accelerating the bacteriological clearance, as is evident by the statistically significant accelerated decline in the BI of those patients who converted to lepromin positivity as compared to those remaining lepromin negative throughout therapy and post-therapy follow up. To conclude, the addition of the Mycobacterium w vaccine to standard MDT induces a lepromin response of a statistically significant higher magnitude than that observed with MDT alone.

Diversity of HLA-DR2 in North Indians: the changed scenario after the discovery of DRB1*1506.

DRB1*1506, a new allele of DR2, differs from DRB1*1501 only at codon 50 in the second exon, where the nucleotide sequence has changed from GTG to GCG resulting in an amino acid substitution from valine to alanine in DRB1*1506. Since codon 50 was considered non-polymorphic until the discovery of this new allele by sequence-based typing, it became necessary to study what fraction of subjects thought to have DRB1*1501 actually had DRB1*1506. For this purpose, 116 DNA samples with DR2 coming from normal healthy individuals, leprosy patients and childhood tuberculosis patients were amplified using PCR and hybridized with 32P-labeled probes specific for DRB1*1501, DRB1*1502, DRB1*1503, DRB1*1506, DRB1*1601 and DRB1*1602. The oligonucleotide probe for DRB1*1506 was designed to span codons 47-52 based on the published nucleotide sequence. DRB5, DQA1 and DQB1-specific amplifications and hybridizations were also carried out to study the diversity of DR2 haplotypes. It was found that 21% of the samples identified previously as DRB1*1501 were actually DRB1*1506. DRB1*1506 was found to be associated with DQB1*0502 and DQB1*0601. Haplotypes of DRB1*1501, DRB1*1502, DRB1*1506 and DRB1*1602 showed a marked heterogeneity. Besides the rare haplotypes which have not yet been reported in any other populations, haplotypes characteristic of different ethnic groups, such as Croatians, South Chinese and Gypsies, were also found in the North Indians, suggesting the extent of racial admixture and migrations to and from India.

Antisphingolipid antibodies in the sera of leprosy patients.

Earlier we reported the presence of significant levels of antigalactocerebroside (GalC) antibodies in the sera of leprosy patients. This study corroborates the above result and also gives evidence for the presence of antibodies to the nonpolar ceramide (Cer) moiety of GalC. AntiCer antibody titres were higher as compared to antiGalC antibodies in all categories of leprosy. The specificity of antibodies directed to the Cer moiety was confirmed using Lactosyl-BSA and neutralization assays. Statistically significant and positive correlations were observed between antiGalC and antiCer antibodies. Responsiveness factors were computed using natural logarithmic transformation of the variables.

Addition of immunotherapy with Mycobacterium w vaccine to multi-drug therapy benefits multibacillary leprosy patients.

Immunotherapy with a vaccine consisting of autoclaved Mycobacterium w, was given in addition to standard chemotherapy (multidrug therapy (MDT)) to 93 multibacillary (MB) leprosy patients. One hundred and seven patients with similar types of disease served as controls and received MDT + placebo injections. The study was a double-blind randomised trial. On opening the codes, results obtained were in concordance with those in a single-blind trial which has been extensively reported. Bacteriological clearances were significantly more rapid in vaccinated patients (p < 0.03). Thirty-five LL or BL patients with a high bacterial index (BI) of 6 were completely cleared of acid-fast bacilli (AFB) after eight doses of vaccine. Only 8 patients in the control group became bacteriologically negative in the same time period. They all had BIs < 4. Associated with decreasing BI was accelerated clinical regression of lesions after vaccination and lepromin conversion rates of 100% for BB, 71% for BL and 70% for LL. A significant number of immunised patients showed histological improvement (p < 0.004). Thirty-six showed a complete disappearance of dermal granulomas and a picture of non-specific infiltration. The vaccine did not precipitate neuritis or deformities; episodes were noted in vaccinated patients as were incidences of Type 2 reaction. The overall improvement was reflected by a shorter duration of treatment and faster release of vaccinated patients.

Identification of myelin basic proteins in circulating immune complexes associated with lepromatous leprosy.

Circulating immune complexes (CIC) were first measured in lepromatous patients (LL) by the 125I-C1q binding assay and the polyethylene glycol (PEG) precipitation test. High levels were found by both methods (95 and 90% of positives, respectively). LL-CIC were investigated for the presence of neural antigens. CIC were precipitated in 3.5% PEG, filtered through protein A-Sepharose affinity chromatography, eluted with glycine-HCl, pH 2.8, and washed with PBS; fractions after CIC dissociation were studied by SDS-PAGE and Western blotting. The LL-CIC PEG precipitates and the glycine-HCl eluates were positive in 76 and 71% respectively against anti-myelin basic proteins (MBP) monoclonal antibody, showing a single band at 15-25 kDa similar to the one obtained incubating MBP with anti-MBP. No reaction was detected with CIC-PBS fractions; strips were incubated with other anti-neural antibodies such as anti-glial fibrillary acidic proteins, anti-S-100, and anti-neurofilaments, without any reactivity. Our results demonstrate that LL-CIC contain MBP as an antigen; its significance could be related to the pathogenesis of leprosy since the liberation of MBP after Mycobacterium leprae nerve damage may elicit anti-MBP autoantibodies to myelin breakdown, which reacts with peripheral nerve MBP inducing CIC formation. This mechanism may be important in demyelination and destruction of nerve in leprosy.

Localized lepromatous leprosy and its response to chemo-immunotherapy.

BACKGROUND: This is an unusual presentation of lepromatous leprosy (LL) in a young boy, 12 years of age. The study forms part of a large scale immunotherapeutic trial with Mycobacterium w (M.w) antileprosy vaccine. The trial is being conducted in two major hospitals in New Delhi, India. MATERIALS AND METHODS: This patient presented with three lesions: one on each forearm and the third on the left leg. He was classified initially as borderline tuberculoid leprosy. Slit-skin smears and histopathology from the lesions proved the diagnosis to be lepromatous leprosy with a bacterial index (BI) 6+. The initial lepromin test was negative. The patient was treated with chemo-immunotherapy (standard multidrug therapy and immunotherapy with Mycobacterium w vaccine). RESULTS: Investigations after 1 year (15 months) of multi-drug therapy and three doses of vaccine, showed a remarkable fall in the BI from 6 to 0 in the lesions, a lepromin positivity of 5 mm, and a histological upgrading from lepromatous leprosy to borderline tuberculoid. Immunologic studies at 15 months revealed a good LTT response and high levels of cytokines, specifically IL-2 and IFN-gamma. CONCLUSIONS: This report presents an LL patient with disease limited to a few sites. It stresses the importance of slit-smear and biopsy in all patients of leprosy, and it highlights the upgrading observed on administration of chemo-immunotherapy.

Field trials on the use of Mycobacterium w vaccine in conjunction with multidrug therapy in leprosy patients for immunotherapeutic and immunoprophylactic purposes.

A double blind field trial was started with a candidate anti-leprosy vaccine, Mycobacterium w as an immunotherapeutic and immunoprophylactic agent against leprosy in a highly endemic region with a prevalence rate of over 18 per 1000 population. By 31 August 1992, 224 villages have been surveyed, covering a population of 307,981 (1981 census). A total of 979 MB patients and 2801 PB patients have been registered. A total of 19,453 household contacts of leprosy patients have been examined for clinical signs of disease, of which 16,519 have received the initial dose while 10,434 have also received the booster dose of vaccine/placebo. The aims and objectives, study design of the trial, present status as well as the socio-cultural aspect involved are highlighted in this paper.

An immunodominant 30-kDa antigen of a candidate anti-leprosy vaccine, Mycobacterium w, shares T and B cell determinants with M. leprae and M. tuberculosis.

Earlier we reported that vaccination of leprosy patients with Mycobacterium w induces an immune response directed predominantly against low molecular weight antigens. One of these antigens, with a molecular mass of 30-kDa, was recognized by a majority of the vaccinated subjects as well as the tuberculoid leprosy patients and healthy contacts. In the present communication we report further characterization of this antigen. Immunofluorescence and Western blot studies with antibodies raised against this antigen demonstrate that it is associated with the cell surface and has homologues present in M. leprae and M. tuberculosis. Delayed-type hypersensitivity studies carried out in guinea pigs immunized with the 30-kDa antigen show that in addition to sharing B cell determinants, this immunodominant antigen of M. w also shares T cell determinants with M. leprae and M. tuberculosis.

Reversal reaction in multibacillary leprosy patients following MDT with and without immunotherapy with a candidate for an antileprosy vaccine, Mycobacterium w.

Immunotherapy with a candidate for an antileprosy vaccine, Mycobacterium w, was given in addition to standard multidrug therapy (MDT) to 53 multibacillary lepromin negative patients belonging to BB, BL and LL types of leprosy (vaccine group). An equal control group received MDT and injections of micronized starch as placebo. Both the vaccine and placebo were administered intradermally every 3 months. The patients were evaluated at determined intervals by clinical, bacteriological and histopathological parameters and lepromin testing. Reactional episodes were analysed with reference to incidence, onset, frequency and severity during and after release from treatment (RFT). Incidence of reversal reaction (RR) was marginally higher in the vaccine group (22.6% vaccine group vs 15% control group). All cases with a history of downgrading type 1 reaction developed RR during therapy. Most episodes occurred within the 1st year of the commencement of therapy--50% developing within 3 months. Late reversal reaction (after RFT) were observed in 3.8% of cases in both groups, and 50% of the reactors in the control group and 33% in the vaccine group had repeated reactional episodes. Incidence of neuritis associated with RR as well as isolated neuritis was similar in both groups.

Immunotherapy with Mycobacterium w vaccine decreases the incidence and severity of type 2 (ENL) reactions.

Immunotherapy with Mycobacterium w (M.w) vaccine was given to 45 patients with multibacillary (MB) leprosy; 41 similarly classified patients served as controls. All patients received standard multidrug therapy (MDT). Incidence, severity and frequency of type 2 (ENL) reactional episodes were monitored in both groups in a follow-up extending up to 4 years. Reactions were seen in fewer vaccinated (10/37) BL and LL patients than in the control group (12/34). A total of 20 episodes were recorded in the vaccine group as against 29 in the controls, 75% of reactions were mild in vaccinated and 51.72% were mild in the control group patients, and 3 patients in the control group had more than 3 reactional episodes. None of the vaccinated patients showed this. No additional incidence of neuritis were seen among vaccinated individuals during reactional episodes.

Combined multidrug and Mycobacterium w vaccine therapy in patients with multibacillary leprosy.

Immunotherapy with Mycobacterium w vaccine was attempted in patients with borderline-borderline, borderline lepromatous (BL), or lepromatous leprosy (LL) to determine whether immunization can hasten recovery and reduce treatment time by invigorating cell-mediated immunity. Mycobacterium w, a nonpathogenic, rapidly growing, atypical mycobacterium, shares a number of common B and T cell determinants with Mycobacterium leprae and Mycobacterium tuberculosis. Patients receiving the vaccine had rapid clinical improvement and accelerated bacteriologic clearance. After treatment with vaccine for 2 years, 13 of 31 BL and LL patients were bacteriologically negative as were 5 of 25 controls. Vaccinated patients had one of two distinct histologic features, either an upgrading in the disease spectrum or complete clearance of granuloma. Some 80% of lepromin conversions were in BL and LL patients who received vaccine versus none and 14.3% of BL and LL controls, respectively. Thirteen of 17 vaccinated LL patients were released from treatment after 2 years in contrast to 2 of 15 controls.

Induction of lepromin positivity by a candidate anti-leprosy vaccine Mycobacterium w in lepromin negative healthy contacts of multibacillary leprosy patients.

In a hospital based study, 362 household contacts of multibacillary leprosy patients were screened for evidence of leprosy and 54 (14.9%) were found to be having leprosy. The remaining 308 apparently healthy contacts were lepromin tested and 109 (35.4%) were observed to be negative to Mitsuda lepromin. M.w vaccine was administered intradermally to 95 of these 109 lepromin negative contacts. Sixty eight of them could be retested for lepromin A reactivity. Fifty six (82.35%) manifested lepromin conversion. The twelve subjects who did not show lepromin conversion, received a second dose of the vaccine, and eleven subsequently became lepromin positive. The overall lepromin conversion rate was thus 98.5% (67 out of 68). Follow-up of these contacts upto a period of 30 months did not demonstrate reversion of lepromin positivity back to negativity status. No untoward effects of vaccination were observed except for local ulceration at the site of vaccine administration.

Do human leukocyte antigens have a role to play in differential manifestation of multibacillary leprosy: a study on multibacillary leprosy patients from north India.

118 multibacillary leprosy patients with differential manifestations were studied for the antigens they expressed at MHC loci to investigate the role of human leukocyte antigens in the differential response to the same causative agent. While the lepromatous leprosy (LL) patients showed a significant increase of Bw60, DR2, DRw8 and DQw1, borderline lepromatous (BL) patients had Bw52, DR9 and DQw7 significantly more often as compared to the normal controls. A comparison of LL, BL and mid-borderline (BB) patients showed a significantly higher frequency of Bw60 in LL patients as compared to the BL. However, Bw52, Bw53, DR9 and DQw7 were found significantly more often in the BL patients as compared to the LL patients but the difference failed to reach significance after pc. A comparison of HLA antigens in BB patients with those of either the LL or BL patients did not show any significant differences.