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

Vaccine, Vol. 13, No. 12. pp. 1102-1110, 1995 Cowrioht 0 1995 Elsevier Science Ltd

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Addition of immunotherapy with

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Mycobacterium w vaccine to multi-drug therapy benefits multibacillary leprosy patients

S.A. Zaheer*, K.R. Beena*, H.K. Kar’f, A.K. Sharmaf-, R.S. Misral, A. Mukherjees, R. Mukherjee*, H. Kaur*, R.M. Pandey*, R. Walia*, A. Mukhopadhyay” and G.P. Talwarii

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 sign$cantIy more rapid in vaccinated patients (~~0.03). Thirty-Jive LL or BL patients with a high bacterial index (BZ) of 6 were completely cleured of acid-fast bacilli (AFB) after eight doses of vaccine. Only 8 patients in the control group became bacteriologically negative in the same time period. They all had BZs <4. Associated with decreasing BZ was accelerated clinical regression of lesions after vaccination and lepromin conversion rates of 100% for BB, 71% for BL and 70% for LL. A signljicant number of immunisedpatients showed histological improvement (p<O.O04). Thirty-six showed a complete disappearance of dermal granulomas and a picture of non-spec$c 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.

Keywords: Combined chemotherapy and immunotherapy: bacteriological clearance; lepromin conversion; histological upgrading; release from treatment (RFT); double blind

Leprosy is a chronic, systemic, infectious and debili- tating disease of man caused by Mycobacterium leprae. It is among six priority diseases identified in the WHO Tropical Diseases Research Programme. There are an estimated lo-12 million people suffering from this malady. One-quarter of them are in India alone’.

The disease is classified as a spectrum which recog- nises the tuberculoid (TT)/lepromatous (LL) polarity to be, in essence, an indication of the immune status of a patient. In between the two poles: LL and TT are the borderline forms. In the center is the borderline-

*National institute of Immunology, New Delhi-l 10067, India. TDepartment of Dermatology, Venereology and Leprology, Dr Ram Manohar Lohia Hospital, New Delhi-l 10001, India. $Department of Dermatology and Leprology, Safdarjung Hospital, New Delhi-l 10029, India. slnstitute of Pathology, Indian Council of Medical Research, Safdarjung Hospital Campus, New Delhi-l 10029, India. IlTo whom correspon- dence should be addressed. (Received 30 June 1993; revised 27 July 1994; accepted 25 October 1994)

borderline type (BB) with two subsidiary forms on each side of BB: borderline tuberculoid (BT) and borderline lepromatous (BL)‘.

Multibacillary (MB) leprosy encompasses BB, BL or LL forms of leprosy’. These are patients with pronounced deficiency of cell-mediated immune (CMI) responses, bacteriological

P ositivity in slit skin smears,

and lepromin negativity-. The immunomodulatory effects of boosting CM1 responses by a vaccine can thus be critically assessed in this group of patients.

Mvcobacterium w (M. w) vaccine, the immunotherapeutic effects of which we are assessing, is currently in Phase III clinical trials at two major hospitals in Delhi and in a rural leprosy control community block in Kanpur Dehat along with the National Leprosy Eradi- cation Programme (NLEP) and U.P. Health Director- ate. The vaccine is based on an atypical, cultivable and rapidly growing mycobacterium”J. It resembles bacilli included in Runyon’s group IV, but differs in one respect or another from bacilli presently in that group4.5. The background studies relating to its selection as a

1102 Vaccine 1995 Volume 13 Number 12

Chemo-immunotherapy in leprosy: S.A. Zaheer et al.

candidate anti-leprosy vaccine have been reported earlier&lo. Recent data, based on the sequence of bases in a polymorphic region of 65 kDa gene, indicate Mycobacterium w is a new species”. Phase I clinical trials conducted in 1983 at Calcutta have been published”.

Phase II/III trials had two series. The initial trial was a single-blind study, where the nature of the vaccine was known to the Head of the clinic but not to the attending Doctors or Investigators. This study had 120 patients. Extensive results on these trials have been published’ 3p20. Similar studies conducted at the Indian Council of Medical Research, JALMA Institute of Leprosy, have confirmed the immunotherapeutic effects of M. w~‘,~~.

The second series of trials was a double-blind one. The codes, which were maintained by the biostatistician, were opened in 1992 and this communication reports results on 200 patients in this study.

In 1990, the vaccine trials were extended to large scale field studies on 414 000 inhabitants in Kanpur Dehat of Uttar Pradesh state of North India. Twenty thousand household contacts and 1100 multibacillary patients have already been enrolled2’. M.w vaccine also forms part of a comparative immunoprophylactic trial being conducted at Chingelput-District (Tamil Nadu State of South India) by the ICMR24. Fifteen thousand subjects of the general population have already been vaccinated with M. w13.

MATERIALS AND METHODS

Vaccine

The vaccine is a suspension of killed Mycobacterium w in physiological saline at 10” bacilli per ml. The details of vaccine preparation have been given previously13. Briefly, M. w from master seed stock was initially grown in Lowenstein-Jensen (L-J) medium. Seed lots were expanded in Middlebrook medium (Difco Laboratories, Detroit, MI) with bovine albumin, dextrose and casein (ADC) enrichment. The bacteria were harvested during the log phase on days 8-9 of culture. The pellet was centrifuged and washed thrice with normal saline (0.085% NaCl). The purity of the preparation was assessed in nutrient agar. The bacilli suspended in saline were autoclaved for 15 min at 15 lb inch - ’ pressure. Complete killing was assessed by lack of growth of the autoclaved specimens in L-J medium after 2 weeks of culture. The sterility of the vaccine preparation was tested in thioglycolate and soya bean casein digest medium. Thiomersal was added to a final concentration of 0.01% as a preservative.

Placebo

One gram of micronised starch (Sarabhai Chemicals, Baroda, India) was dissolved in 100 ml of distilled water. This was autoclaved at 15 lb inch _ ’ pressure for 15 min and dispensed in sterile vials.

Lepromin

Armadillo lepromin containing 40 x lo6 killed bacilli ml-’ was kindly made available by WHO.

Vaccine dose, administration, regimen

The first dose of vaccine was 1 x lo9 autoclaved bacilli in 0.1 ml physiological saline (0.85% NaCl). Subsequent doses contained half the number of bacilli. The vaccine was administered intradermally in the deltoid region. A total of 8 doses were given at 3-month intervals.

Multidrug therapy (MDT)

MDT consisted of 2 weeks of intensive therapy with 600 mg rifampicin, 100 mg clofazimine and 100 mg dapsone daily. Subsequently, theZgatients received for 2 years the WHO recommended regimen of 600 mg rifampicin and 300 mg clofazimine once a month, super- vised, plus 100 mg of dapsone and 50 mg of clofazimine daily, self-administered.

Subjects and design of study

Permission of Drugs Controller of India, Institutional Ethics Committees and consent of subjects enrolled was obtained before initiating the study. Multibacillary patients, belonging to LL, BL or BB forms of leprosy on the basis of clinical status and histopathology were enrolled. All were initially negative to lepromin-A (armadillo-derived). On ethical grounds, the protocol envisaged administration of standard multidrug therapy to all patients. However, one group received, in addition, the vaccine, whereas the other group was given placebo injection of micronised starch.

The study was a double-blind one in which neither the evaluating agency nor the patients were aware of whether the M. w vaccine or micronised starch was injected. Clinical scoring was done by the attending physician and a record of these was maintained in the form of clinical photographs taken at fixed intervals in addition to complete body charts. Furthermore, slit smear examination for BI as well as histopathological examination were carried out by experts, who at no time during the study period had the opportunity to see the patients or know their clinical details. Individual code numbers of patients was the only form of identification provided to the evaluating pathologists.

Clinical scores, bacteriological status, lepromin testing, histological examination

Details have been described13. For purposes of clinical assessment Ramu’s scor-

ing33.34 method was followed. In this system a score of 1 is given to predominantly macular lesions, 2 to diffuse infiltration, 3 to few papules or plaques and 4 to predominantly papulonodular lesions. The body is divided into seven regions, i.e. face, head and neck, right and left upper limbs, chest and abdomen, back and buttock and right and left lower limbs. Each region is scored independently from 1 to 4. The maximum score recordable is 28.

RESULTS

Table I gives the total number of patients belonging to different types of leprosy enrolled in either vaccine or control groups. All patients listed had completed a minimum of 24 months of treatment.

Vaccine 1995 Volume 13 Number 12 1103


Table 1 Changes in clinical scores (mean f S.E.M.)

Type of No. of disease Group patients Initial 6 months 12 months 18 months 24 months

LL Vaccine 53 13.6kO.9 10.3+0.7 7.5kO.6 6.OkO.6 @X0.03) 3.8kO.4 (p.zO.001) Control 68 13.5i0.6 10.8iO.6 8.3kO.5 7.2kO.5 6.3kO.4

BL Vaccine 31 10.9+1.0 7.0i0.8 5.6kO.8 3.8kO.4 2.1 io.3 (~0.001) Control 30 10.4+0.9 7.9i0.6 5.4kO.5 4.6kO.5 4.4i0.6

BB Vaccine 9 8.6k1.7 4.8i0.8 4.6kO.6 3.8zt0.7 2.7k1.2 Q~0.02) Control 9 9.7i0.8 5.8+0.4 5.1il.O 4.6il .l 4.6+1 .O

Statistical analyses were done using Mann-Whitney U-test (non-parametric). For the significant cases, p values are indicated in parentheses

Table 2 Histological changes in patients with LL type leprosy

A. Leprosy type

No. of 12 months

Group patients LL BL BT NSI

24 months

LL BL BB NSI

LL Vaccine Control

53 38 11 2 2 33 11 1 9 68 65 2 - 1 64 3 - 3

B. Leprosy type BL BT Ind NSI BL BT Ind NSI

BL Vaccine Control

31 15 1 2 13 8 - 3 20 30 26 2 - 2 20 1 1 8

C. Leprosy type Histological diagnosis BB BT Ind NSI BB BT Ind NSI

BB Vaccine Ind-1 - 2 1 5 - - 1 7 BT-1 BT/BB-1 BB-5

Control Ind-1 1 1 5 1 1 1 1 5 BT-2

Two patients S-129 and S-159, clinically BB, with macular lesions, showed no histological evidence of leprosy, hence are excluded from analysis. Ind, indeterminate disease. NSI, non-specific infiltration

Local reactions to vaccine

The vaccine was extremely well tolerated. Erythema and induration was seen within a week of vaccination. In the majority of patients, by 2 weeks, there was formation of a well defined ulcer whose size ranged from 3 mm x 3 mm to 9 mm x 9 mm. The ulcer healed spontaneously in 34 weeks with scar formation.

Clinical improvement

As was our experience in the single-blind study’“, patients in the double-blind study receiving the M. w vaccine showed marked clinical improvement by 6 months. This was evident as rapid regression and flattening of papulonodular lesions and plaques and disappearance of hypopigmented lesions as compared to patients in the control group (Figures 1 and 2). Table I gives the changes in Ramu’s clinical scores (mean & S.E.M.) in the two groups. Decline in scores in BB, BL and LL types of leprosy receiving vaccine was statistically significantly greater than the control group (~~0.02).

Bacteriological changes

Changes in bacterial index (BI) were noted every 3 months. Figure 3a, b, c are bar diagrams depicting mean

BI changes f S.E.M. in the vaccine (93) and control (107) subjects. The fall in BI in LL patients receiving M.w vaccine was significant from 6 months onwards @<O.OOl). This was from 3.64 (& 0.17 S.E.M.) to 1.07 ( f 0.16 S.E.M.) for the vaccine and 4.09 (f 0.13) to 2.46 ( f 0.15 S.E.M.) for the control group. In fact, the decrease in BI after two doses of vaccine (6 months) was lower in the vaccine than that achieved in the control group at 24 months. In BL leprosy patients, initial BIs in the two groups were very similar (Figure 3b) and significant decreases were seen from 12 months onwards in the vaccine group (pcO.03). The BI at 12 months in the vaccine group (0.771 f 0.16 S.E.M.) was lower than that in the control group at 24 months (0.84 f 0.14 S.E.M.). In BB leprosy patients, all patients receiving M. w became BI=O at 18 months. This was not so in the control group patients (Figure 3~).

Lepromin conversion

At induction all patients were negative in their late reaction (3-4 weeks) to lepromin-A (armadillo- derived). With progressive immunisation, there was a gradual increase in number of patients showing


Chemo-immunotherapy in ieprosy: S.A. Zaheer et al.

Figure 1 Clinical changes in patients after administration of M.w vaccine. Upper panel illustrates clinical improvement in an LL patient. Face before (a) and after (b) eight doses of vaccine (24 months) demonstrating clearing of the nodular lesions. Middle panel represents the back of a patient with LL type of leprosy. Before vaccination (c) and after (d) four doses of vaccine (12 months) showing marked decrease in papulonodular lesions. Lower panel presents the back of a patient with BL leprosy. The plaques (e) cleared after four doses of vaccine (12 months) (f)

lepromin conversion. Figure 4u-c depicts the per- Histological changes centage of conversion with subsequent vaccine/ Histological monitoring in the double-blind series of placebo doses. BB patients showed 100% conver- patients revealed similar trends as seen in the single- sion, BL patients 71% and LL patients 70% after 2 blind series13,‘6%‘9. Though skin biopsies were taken years of chemo-immunotherapy. At 2 years, conversion every 6 months, Table ZA-C gives analysis of histologi- rates in vaccinated patients were highly significant cal changes after 4 and 8 doses of vaccine (12 and 24 (JKO.005). months, respectively). Histological improvement was


Chemo-immunotherapy in leprosy: S.A. i’aheer et al.

Figure 2 Clinical changes in patients in the control group (MDT + placebo). Upper panel illustrates the back of a patient with LL leprosy (a) initially and (b) after eight doses of placebo (24 months), showing partial clearing of nodular lesions. Middle panel represents the buttocks of an LL patient with multiple nodules (c) and after eight doses of placebo (24 months). Lesions, although fewer, still persist (d). Lower panel demonstrates the changes on the upper back of a BL patient (e) before and (f) after eight doses of placebo (24 months)

seen in -40% vaccinated LL patients at 24 months, as compared to ~6% in the control group. Of these, nine vaccinated patients showed a complete disappearance of dermal granuloma giving a picture of non-specific infiltration (NSI) (Table ZA). This was more evocatively demonstrated in BL vaccinated patients, where 20 of 31 (64.5%) showed NSI (Tab/e 2B). Eleven vaccinated LL patients upgraded to BL and one to BB at 2 years. At 12 months, 2LL patients had upgraded to BT (four doses of vaccine) (Table 2A). Using the chi-square test, histological improvement for vaccine group LL patients (p~O.004) and BL patients (p<O.O04) was found to be highly significant. BL patients receiving just four doses of vaccine (12 months) showed a better histopathological improvement than control group patients at 24

months (Table 2s). At 12 months, 13 patients had cleared granulomas from the dermis (NSI); two showed features of indeterminate disease and one upgraded to BT (Table 2B).

Patients clinically classified as BB had varied initial histological features of indeterminate disease, BT, BT/BB or BB. A follow-up of these patients for a period of 2 years did not reveal any appreciable changes in the two groups. Seven of eight vaccinated and five of eight control group patients showed features of NSI (Table 2A).

Incidence of reactions and neuritis

Type 1 reactions in leprosy are delayed hypersensitivity reactions associated with rapidly changing



I 6

1

i 6

6 I2 18 24 Treatment months

Treatment months Treatment months

n Vaccine q Control n Vaccine 0 Control

n Vaccine 0 Control

(A) W CC)

Figure 3 Changes in BI of leprosy patients in course of treatment with MDT + vaccine or MDT + placebo. (a) Changes in BI of LL patients (53 vaccine; 68 control). Values are mean f S.E.M. The difference between the vaccine and control groups at 6, 12, 18 and 24 months was highly significant, pcO.001. (b) Bacterial Indices in BL patients (31 vaccine; 30 control). Bars give the mean BI + S.E.M. The p value was co.03 between the vaccine and control group at 12 months and ~0.001 at 18 and 24 months. (c) Changes in BI of BB patients. All patients (9) in vaccine group became bacteriologically negative at 18 months

80 t

0 6 12 18 24 Treatment months

- Vaccine - - - Control

@I

Treatment months

- Vaccine - - - Control - Vaccine . - - Control

(4 0

Figure 4 Lepromin conversion in LL (a), BL (b) and BB (c) patients given the vaccine or placebo

cell-mediated immune (CMI) responses”. Type 2 reac- (Table 3A). Using the chi-square test it was revealed tions (ENL or erythema nodosum leprosum) are im- that control group patients had significantly more mune complex reactions associated with deposition of immune complexes at specific target organ sidez5.

episodes of Type 2 reactions (p~O.01). With regards to incidence of neuritis, vaccine group patients had

Higher incidence of upgrading type 1 reactions were less episodes of neuritis than controls (vaccine 68 seen in vaccine (51 episodes in 33 patients) compared episodes in 29 patients; control 91 in 39 patients) to control group (32 episodes in 19 patients). This was (Table 3B). Using the chi-square test it was seen that largely due to the 38 episodes seen in 24 LL patients the control group had significantly more episodes of against only 11 episodes in 7 patients in LL patients neuritis (~~0.04). The ulnar nerve was the most receiving MDT+placebo (Table 3A). Contrary to inci- commonly affected nerve. Fifteen vaccinated dence of Type 1 reactions were incidence of Type 2 patients had deformities during the 2 year follow-up reactions. Only 64 episodes were noted among vacci- against 25 in the control group (Table 3B, Table 4). nated patients vs 109 in the control group. Thirty- Ulnar claw was the commonest deformity followed by seven patients in the vaccine group and 46 in the foot drop. No statistically significant differences were control group showed evidence of Type 2 reactions seen in the two groups.



Table 3 DISCUSSION

A. Reactional episodes during treatment

Type I reaction

Group BB BL LL

Type II reaction

BL LL

Vaccine 1 (1) ‘2 (8) 38 (24) 11 (7) 53 (30) Control 5 (3) ‘6 (9) ‘1 (7) 10 (5) 99 (41)

B. Incidence of neuritis and deformities

Group Type of leprosy

No. in study

No. of episodes of neuritis

No. with deformities

Vaccine BB 9 0 1

BL 31 14 (8) LL 53 54 (21) z

Control BB 9 1 (1) 3 BL 30 20 (20) 7 LL 68 70 (70) 15

Figures in parentheses give number of patients experiencing reactional episodes (A) or incidence of neuritis (B)

Table 4 Patients released from treatment (RFT) after administration of MDT + M.w vaccine (vaccine group) and MDT + placebo (control group)

No. of Treatment months Type of patients Total

Group leprosy in study 24-26 27-36 37-48 >48 RFT

Vaccine LL 53 19 (35.84) 11 2 - 32 BL 31 23(74.19) 4 - - 27 BB 9 9 (100) - - - 9

Control LL 68 2 (2.94) BL 30 5 (16.66) z BB 9 4 (44.44) 1

Figures in parentheses indicate percent of patients RFT

Release from treatment

Patients were released from treatment (RFT) after the disease became clinically inactive and three consecutive slit smears were negative for acid-fast bacilli (AFB)‘6. At the time of writing, 32 (-60%) of 53 LL patients given vaccine have been RFT; 19 after 2 years of treatment; 11 between 2 and 3 years and 2 beyond 3 years. Fourteen ( 2: 20%) of 68 control group LL patients have been RFT; 2 after 2 years of treatment, 5 between 2 and 3 years and 7 beyond 3 years. Twenty-seven of 31 (2: 87%) (77.4%) patients with BL leprosy, who received M.w vaccine, have been RFT: 23 within 2 years and 4 between 2 and 3 years. In the control BL group, 18 of 30 (60%) have been RFT: 5 within 2 years; 9 between 2 and 3 years and 4 beyond 3 years. In the BB group given vaccine, all 9 patients were released within the mandatory 2 years of treatment. Six of nine control group patients have been RFT: 4 in 2 years; 1 between 2 and 3 years and 1 beyond 3 years (Table 4). Using the chi-square test, the number of patients RFT in BL and LL vaccine group was found to be highly significant (p<O.OOOl) for BL; p<O.OOOl for LL). Fischer’s exact test was used for BB patients as frequencies were small. Patients RFT was again significant for the vaccine group (pcO.04).

Developed in 1 978’7 and currently in advanced Phase III immunotherapeutic trials in Delhi, Mycobacterium w

vaccine has shown definite immunotherapeutic effects in multibacillary (MB) leprosy patients. Reports on results on the first series of patients1”.‘5*‘9 and results reported here on the second series are consistent and in concur- rence with each other. The vaccine has been extremely well tolerated both in hospital-based trials as well as in field trials at several centres.

Attempts are also being made in several laboratories around the world to develop a vaccine for leprosy. These candidate vaccines are at different stages of clinical trials. They are being tested both for immunotherapeutic and immunoprophylactic potential. They are (i) killed M. leprae and live BCG, originally proposed by Convitt e? a1.28; (ii) Indian C ancer Research Centre strain (ICRC)‘9; and (iii) M. vaccae, proposed by Stanford”‘. Two cytokines are also being tested for their immunomodulatory effects in LL patients. These are gamma interferon3’ and interleukin-23’. Trials with A4.1~ are unique in several respects. They are the first immunotherapeutic trials where active MB patients have been critically assessed by clinical, immunological, bacteriological and histological parameters. These trials have a well matched control group receiving MDT+placebo. Numbers enrolled have been higher than those in any other study, and all patients have received a minimum of 2 years of treatment.

Patients receiving M. w vaccine have shown marked reduction in infiltration and rapid clearing of papulonodular lesions. Using Ramu’s clinical scores33.34, vaccinated patients showed a significant fall in scores in all three forms of leprosy (~~0.02).

With MDT alone, bacterial index (BI) decreases are stated to be between 0.6 and 1 .O units yeai’ (Ref. 35). In our study, rapid BI decreases were seen among vaccinated patients. Thirty-five BL or LL patients with high BI attained bacteriological negativity within 2628 months of treatment with M.w vaccine + MDT. This was associated with lepromin conversion and/or histological improvement. Two patients with a high BI of 6 (Index Nos 225 and 81) were rendered BI negative in the 2-year time period. This was associated, in both, with a lepromin conversion to positivity status. In the control group, receiving MDT+placebo, only eight BL or LL patients becoming BI=O could be enumerated. Most of them had initial BIs of ~4. It was interesting to note that in all three forms of the disease, BI values in vaccinated patients at 12 months were lower than that achieved by control group patients at 2 years. BI decreases were also statistically significant in vaccinated patients (pcO.03).

Shifts in cell-mediated immune (CMI) status were reflected by the progressive lepromin conversion from negativity to positivity in vaccinated patients. LL and BL patients showed ~70% conversion; BB patients 100% conversion. These are the highest conversion rates attained by any immunomodulator in leprosy.

Histological improvement was significantly more in vaccinated BL and LL patients QcO.04). Forty-four of them showed histological improvement, of which 29 showed a complete disappearance of dermal granuloma resulting in a histological picture of non-specific infiltration (NSI). BB patients, in either vaccine or control


groups, histologically did not show any appreciable differences.

That the vaccine upgraded CM1 responses was also evident by the higher incidence of Type 1 reactions in vaccinated patients. In fact, Type 1 reactions are not commonly encountered among LL patients. In our study, we have noted these reactions in 24 patients in vaccine as compared to 7 in the control group. Type 2 reactions occur in the presence of M. leprae antigen?. Far fewer episodes of Type 2 reactions were observed among vaccinated patients (vaccine 64 and control 109), possibly due to rapid clearance of antigen. These obser- vations were also seen in the single-blind series and have been reported18.

It is imperative to monitor incidence of reactions and deformities when using immunomodulators in leprosy. It was reassuring to note that there was a lower incidence of neuritis and deformities in the vaccine group and that A4. w vaccine did not aggravate either neuritis or associated motor disturbances. This was also observed histopathologically, where vaccination did not precipitate inflammation of dermal nerve twigs16.

The overall clinical, bacteriological and histological improvement among vaccinated patients was reflected by a shorter treatment duration and a significant number being released from treatment (p~O.0001). Thus vaccinated patients had less morbidity, were rendered BI negative faster and ceased to be sources of infection to the community more quickly and there was a reduction in costs of drugs and medical care.

Several studies on Mycobacterium w have shown that it shares a number of B36 and T37 cell epitopes with M. leprae. CD4+ clones from subjects immunised with BCG+M. leprae react with M. w37. Peripheral blood mononuclear cells from TT patients and healthy contacts, not previously exposed to M.w proliferate in vitro in response to its antigens38. An immunodominant 28-31 kDa antigen, in M. w, carrying B and T cell determinants, has been reported38. Monoclonal antibodies hitherto considered specific to M. leprae or M. tuberculosis react with antigens of Mycobacterium w in Western blots36. Additional studies with this strain have shown that it stimulates T helper-l subsets in murine models with enhanced production of IL-2 and gamma interferon”‘. The vaccine also imparts protection in mice and guinea pigs to challenge with live M. tuberculosis H,,Rv. This effect is seen in both BCG “responder”, and “non-responder” genetic strains of micea.

The precise mechanism of action of the vaccine is difficult to state, however, based on the above studies and results of clinical trials, certain assumptions can be put forward. Talwar” has used the approach of hetero- immunisation, in contrast to the commonly used approach of immunisation with a homologous antigen. The reason is simple. Multibacillary (MB) leprosy patients are devoid of autoregressive potentials as they carry both dead and live acid-fast bacillli AFB for several years and these antigens do not evoke a regres- sive immune response. Moreover, it is our experience (in the control group), that repeated lepromin injections (which are like mini homologous vaccines) do not or rarely alleviate the immune-deficit in polar multibacillary patients. Thus there exists a unique situation, in relation to M. leprae and the host, which demands an


overriding of immunological non-responsiveness to the bacillary antigens. Mycobacterium w is able to achieve this by generation of cross reactive T,l type of clones. Gamma interferon seems to be one of the cytokines secreted by antigen stimulated lymphocytes that acti- vates macrophages39. This is associated with enhanced secretion of reactive oxygen intermediates (02 -, H,OJ in the macrophages which leads to a rapid killing and clearance of M. leprae4’. The rapid clearance of AFB from tissues after vaccination, may partially be explained by this mechanism.

Thus, based on our experience with Mycobacterium w vaccine in this study and other studies at several centres, of rapid BI clearance, quicker clinical recovery, shorten- ing of duration of treatment and overall recovery of patients, we would recommend its addition as an adjunct to multidrug therapy in MB leprosy patients.

ACKNOWLEDGEMENTS

The trials are supported from a grant from the Depart- ment of Biotechnology, Government of India (No. BT/ 15/09/006/89 dated 22.10.90). The technical assistance of Mr Dinesh Negi, Mr Amarnath Prasad, Mr Anil Bobin, Mrs Namita Manocha and Mr Kishan Pal is gratefully acknowledged.

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