Phase III, placebo-controlled, randomized, double-blind trial of tableted, therapeutic TB vaccine (V7) containing heat-killed M. vaccae administered daily for one month.

OBJECTIVE: Immunotherapy of tuberculosis (TB) to shorten treatment duration represents an unmet medical need. Orally delivered, tableted TB vaccine (V7) containing heat-killed Mycobacterium vaccae (NCTC 11659) has been demonstrated in prior clinical studies to be safe and fast-acting immune adjunct. METHODS: The outcome of Phase III trial of V7 containing 10 µg of hydrolyzed M. vaccae was evaluated in 152 patients randomized at 2:1 ratio: V7 (N = 100), placebo (N = 52). Both arms received conventional 1st or 2nd line TB drugs co-administered with daily pill of V7 or placebo. RESULTS: After one month mycobacterial clearance was observed in 68% (P < 0.0001) and 23.1% (P = 0.04) of patients on V7 and placebo. Stratified conversion rates in V7 recipients with drug-sensitive and multidrug-resistant TB were 86.7% and 55.6% vs 27.2% and 15% in placebo. Patients on V7 gained on average 2.4 kg (P < 0.0001) vs 0.3 kg (P = 0.18) in placebo. Improvements in hemoglobin levels, erythrocyte sedimentation rate and leukocyte counts were significantly better than in controls. Liver function tests revealed that V7 can prevent chemotherapy-induced hepatic damage. CONCLUSION: Oral M. vaccae is safe, can overcome TB-associated weight loss and inflammation, reduce hepatotoxicity of TB drugs, improve sputum conversion three-fold OR 3.15; 95%CI (2.3,4.6), and cut treatment length by at least six-fold. Longer follow-up studies might be needed to further substantiate our findings (Clinicaltrials.gov: NCT01977768).

Leprosy Associated with Atypical Cutaneous Leishmaniasis in Nicaragua and Honduras.

In Central America, few cases of leprosy have been reported, but the disease may be unrecognized. Diagnosis is based on clinical criteria and histology. Preliminary field work in Nicaragua and Honduras found patients, including many children, with skin lesions clinically suggestive of atypical cutaneous leishmaniasis or indeterminate leprosy. Histology could not distinguish these diseases although acid-fast organisms were visible in a few biopsies. Lesions healed after standard antimicrobial therapy for leprosy. In the present study, patients, family members, and other community members were skin-tested and provided nasal swabs and blood samples. Biopsies were taken from a subgroup of patients with clinical signs of infection. Two laboratories analyzed samples, using local in-house techniques. Mycobacterium leprae, Leishmania spp. and Leishmania infantum were detected using polymerase chain reactions. Mycobacterium leprae DNA was detected in blood samples and nasal swabs, including some cases where leprosy was not clinically suspected. Leishmania spp. were also detected in blood and nasal swabs. Most biopsies contained Leishmania DNA and coinfection of Leishmania spp. with M. leprae occurred in 33% of cases. Mycobacterium leprae DNA was also detected and sequenced from Nicaraguan and Honduran environmental samples. In conclusion, leprosy and leishmaniasis are present in both regions, and leprosy appears to be widespread. The nature of any relationship between these two pathogens and the epidemiology of these infections need to be elucidated.

Beneficial effects of immunotherapy with extracts derived from Actinomycetales on rats with spontaneous obesity and diabetes.

AIMS: To determine whether immunotherapy with heat-killed, selected Actinomycetales species could influence the progression of spontaneous Type 2 diabetes mellitus and obesity in a rat model. MATERIALS & METHODS: Preparations of either Gordonia bronchialis, Tsukamurella inchonensis or a saline placebo were given by three subcutaneous injections, 30 days apart, starting when rats were aged 120 days, just before development of Type 2 diabetes mellitus, and at day 440, when the disease was well established. Bodyweight, blood sugar, cholesterol, triglycerides and insulin levels were measured to determine the effects and at the end of the experiments, animals were subjected to necropsy. RESULTS: The development of Type 2 diabetes mellitus was prevented by both reagents, most effectively by T. inchonensis. In the treatment experiment, the effects of the disease were reduced by both treatments, markedly so by T. inchonensis. In both experiments obesity was reduced in treated animals. The possible mechanisms of action are discussed. CONCLUSION: Our findings suggest that Type 2 diabetes mellitus in the studied rats is associated with obesity, and that both diabetes and obesity can be prevented or improved by treatment with Actinomycetales immune modulators.

Successful immunotherapy of canine flea allergy with injected Actinomycetales preparations.

AIMS: Can heat-killed, borate-buffered suspensions of Gordonia bronchialis, Rhodococcus coprophilus or Tsukamurella inchonensis be used to treat canine flea allergy? MATERIALS & METHODS: Organisms cultured on Sauton's medium into stationary phase were autoclaved in borate-buffered saline and stored at 10 mg wet weight/ml. Intradermal injections of 0.1 ml containing 1 mg of bacilli were administered on the first and 20th days of the study. G. bronchialis and R. coprophilus were most effective in a pilot study of a small number of dogs with flea allergy. A larger number of affected dogs were then randomized to receive placebo or either of the two selected reagents. The extent and severity of allergic signs and symptoms were scored and blood samples were collected just before the first injection and 28 days after the second. RESULTS: Both selected reagents reduced the extent and severity of lesions (p < 0.001) and reduced scratching. Eosinophil numbers were reduced (p < 0.0001) between the first and second assessment. CONCLUSIONS: Injections of G. bronchialis or R. coprophilus effectively reduce the signs and symptoms of flea allergy in dogs.

Immunological basis for the introduction of immunotherapy with Mycobacterium vaccae into the routine treatment of TB.

An account is given of the immunological investigations carried out in Rosario (Argentina) to identify suitable methods for the assessment of the efficacy of immunotherapy for TB. Some of these were then applied to three small studies: one of a single injected dose of heat-killed, borate-buffered Mycobacterium vaccae administered early in treatment, another of three such doses administered at monthly intervals from the start of treatment, and the third of ten oral doses at frequent intervals throughout short-course chemotherapy. All three displayed better clearance of bacilli from the sputum, faster improvement in clinical symptoms, better radiological resolution of lesions and a return of most immunological parameters towards those of healthy persons. In principle, the immune change achieved is an increase in Th1 mechanisms, notably IL-2 and -12 with downregulation of the tissue damaging aspects of Th2. As an addition to chemotherapy for drug-susceptible or drug-resistant TB, with or without concomitant HIV infection, this immunotherapy offers a safe and effective improvement.

Immunotherapy with oral, heat-killed, Mycobacterium vaccae in patients with moderate to advanced pulmonary tuberculosis.

A research investigation to evaluate the potential of an oral preparation of Mycobacterium vaccae SRL172 (NCTC 11659) as an immunotherapeutic has been carried out in ten patients with moderate to advanced pulmonary tuberculosis at Carrasco Hospital, Argentina. Comparison was made between oral and injected M.vaccae sharing a mutual control group. Clinical, bacteriological, hematological, radiological and immunological assessments all showed comparable benefits for both injected and oral treatment over those achieved with chemotherapy alone. The only significant difference between results of injected and oral M.vaccae was the failure of the latter to reduce TNF-alpha production by cultured mononuclear cells. A more intensive regime for the oral preparation was used, which as an addition to the directly observed therapy, short-course, treatment should improve results in both drug susceptible and drug-resistant cases. A Phase II Good Clinical Practice trial is now required.

The common mycobacterial antigens and their importance in the treatment of disease.

The mycobacteria are one of a number of genera making up the aerobic Actinomycetales. Their antigens demonstrable by immuno-precipitation methods can be divided into four groups. The group i antigens, common to all mycobacterial species, cross-react with their counterparts in animal cells, largely derived from mitochondria. Notable amongst these antigens are the heat-shock, or stress, proteins and possibly bacterial sugars. Tests of cell-mediated immunity show that people can be separated by their responsiveness in skin-test, or lymphocyte proliferation techniques, into four categories of responders. Category 1 individuals respond to all mycobacterial reagents through recognition of the group i antigens. Many chronic diseases are associated with a lack of cell-mediated responsiveness to the group i antigens, and have a raised antibody titre to them. This reflects a predominance of T helper 2 activity and reduced T helper 1 responsiveness as part of the pathogenesis of their diseases, which include chronic bacterial, viral and parasitic infections, allergies, auto-immunities and neoplasms. Packaged together, the group i antigens and the cell-wall adjuvants of selected aerobic Actinomycetales make potent immuno-modulatory reagents. An example is heat-killed Mycobacterium vaccae, useful in both prevention and treatment of disease. Treatment with such reagents results in alleviation of disease, restoration of cellular responsiveness to the common mycobacterial antigens and a decrease in antibody titres to them. This new approach to treatment for such a wide range of diseases has few disadvantageous side effects and can accompany other non-immunosuppressive therapies.

Potential for immunotherapy with heat-killed Mycobacterium vaccae in respiratory medicine.

Immunotherapy with Mycobacterium vaccae has been shown to be beneficial as part of the treatment for a wide range of diseases. In the respiratory system, the late airway response in bronchial asthma is modified by a single dose and bronchial aspects of hayfever are reduced allowing a major reduction in the use of bronchial dilators. In studies of advanced adenocarcinoma of the lung survival is increased by an average of 4 months when up to five doses of M. vaccae are added to the course of chemotherapy. The quality of life of cancer patients receiving immunotherapy with M. vaccae is improved, even if survival is not increased. It is suggested that the mechanism of action of immunotherapy with heat-killed, borate-buffered M. vaccae is likely to be very similar in all these diseases for which human pulmonary tuberculosis provides a model. In this study, additional immunological data are reported from material stored from an earlier study of immunotherapy for pulmonary tuberculosis to help complete the information on the way that treatment with three monthly injections of heat-killed, borate-buffered M. vaccae (SRL172) may act.

Immunological consequences of three doses of heat-killed Mycobacterium vaccae in the immunotherapy of tuberculosis.

We report the first study of triple-dose immunotherapy with heat-killed Mycobacterium vaccae (SRL 172) combined with short-course, directly observed chemotherapy in newly diagnosed pulmonary tuberculosis patients. The study was carried out in Rosario, Argentina, where single-dose immunotherapy with M. vaccae has previously been shown effective. Twenty-two HIV seronegative patients, sputum-positive for tubercle bacilli, entered a randomised and partly blinded trial. Twelve patients received injections of SRL 172 and 10 patients received placebo on days 1, 30 and 60 of chemotherapy. All patients were followed up clinically, by sputum bacteriology, chest radiography and haematology. Patients receiving SRL 172 showed faster and more complete clinical improvement, accelerated disappearance of bacilli from sputum, better radiological clearance and a more rapid fall in ESR, than did those receiving placebo. Follow-up continued for a year after therapy and no patient failed treatment or relapsed. Special investigations included longitudinal assessments of respiratory bursts and expression of CD11b on separated polymorphonuclear and mononuclear leukocytes. Tumour necrosis factor alpha (TNF-alpha) was measured in the supernates of cultured cells and both TNF-alpha and interleukin-4 (IL-4) were measured in serum samples. Immunotherapy recipients showed a significantly faster return towards normal values in all the immunological parameters, than did placebo recipients. The results are consistent with a regulatory activity on cellular immunity, reducing the influence of Th2 and enhancing Th1 to the benefit of the patients. This could allow a reduced period of chemotherapy without loss of efficacy and help to prevent the development of multi-drug resistance.

Immunotherapy with Mycobacterium vaccae in the treatment of tuberculosis.

All the trials of immunotherapy of tuberculosis with killed Mycobacterium vaccae, published or not, that are known to the authors are reviewed here. Following an introduction giving a brief account of some earlier immunotherapies for tuberculosis, the origins of the concept of immunotherapy with M.vaccae are considered. Progress is traced from the early work with irradiation-killed organisms in leprosy to the study in London of modulation of tuberculin skin-test responses, and the first comparative trials in The Gambia and Kuwait. In the last of these studies, dosages and different preparations were compared. As a result of this subsequent studies have used 109 heat-killed organisms, equivalent to 1mg wet-weight of bacilli, as a standard dose. A series of small trials in Argentina, India, Nigeria, Romania, South Africa and Vietnam have pioneered the way forward, disclosing geographic variability, with South Africa as the only country where almost no effects were recorded. Together the studies have shown that a single dose may not be sufficient. These studies have confirmed the mode of action of M.vaccae to be regulation of cell-mediated immunity with enhancement of Th1 and down-regulation of Th2, and they have shown benefits in faster bacteriological conversion, reduction in ESR, recovery of body weight and resolution of radiological opacities, leading to better recovery from the disease even when given to patients receiving directly observed therapy, short-course (DOTS). Three major randomised, placebo-controlled and partly blinded trials have been carried out in Africa. The first, in South Africa showed no M.vaccae-related effects. The second trial, in Uganda, confirmed the observations made in the earlier studies of faster sputum conversion and better radiological clearance. The third trial, in Zambia and Malawi, showed a trend towards benefits in the treatment of HIV seronegative patients but failed to show beneficial effects in HIV seropositive patients. Studies in patients with multi-drug-resistant tuberculosis have shown that multiple doses of immunotherapy are required in most cases, and that these markedly improve cure-rates for these patients. This is especially so when they are also treated with chemotherapy tailored to the resistance pattern of their infecting organisms. A small study has just commenced in which repeated doses of M.vaccae are being administered to a group of patients who have failed treatment with DOTS-Plus (directly observed therapy with drugs selected on the basis of drug susceptibility profiles). Late in the investigation came publications from China supporting and confirming the data in both drug-sensitive and drug-resistant disease, by the use of multiple injections of their own different preparation of M.vaccae. The trial that is now beginning in Vietnam of 3 doses of M.vaccae in the treatment of newly diagnosed pulmonary tuberculosis, is accompanied by a chemotherapeutic regimen with a shortened continuation phase. If this important study is successful, immunotherapy with killed M.vaccae should be introduced into the treatment regimens for tuberculosis worldwide.

Skin test studies on close contacts of leprosy patients in India

Skin-test studies with a series of tuberculins have been carried out in close contacts of multibacillary (MB) leprosy patients around three leprosy centers in India, and casual contacts of the disease around two centers. The results show that the rate of acquisition of leprosin A positivity is associated with age and the closeness of contact with MB leprosy. At the age of 15 years, the differences between the two types of contact were highly significant (p less than 0.00001). Many responses to leprosin A are directed toward the group iv species-specific, antigens of the leprosy bacillus, and the significance of positivity is discussed in relation to protective immunity from leprosy. The differences from Iran show that positivity to leprosin A is not solely the effect of the degree of contact with the disease, but must also have a genetic or environmental element, the latter being favored. The results from Miraj show that the high levels of tuberculin, scrofulin, and vaccin positivity seen in Fathimanagar, and to a lesser extent in Karigiri, are not a consequence of contact with leprosy. BCG vaccination made little difference to the leprosin A positivity of close contacts of leprosy patients, although it significantly enhanced positivity among casual contacts around Miraj (p less than 0.002). BCG vaccination significantly increased tuberculin positivity in Miraj and Karigri, and in those under 11 years of age in Fathimanagar. It made no difference to the already high level of positivity found in older persons around Fathimanagar

Vaccination and skin test studies on the children of leprosy patients

The purpose of this study carried out in Iranian Azerbaijan was to determine the pattern of skin-test positivity to mycobacterial antigens in children living in the valley, and to assess the effect on this of a series of vaccines against mycobacterial disease. Set up in 1978, 1707 tuberculin-negative children without scars of previous BCG vaccination were vaccinated with BCG Glaxo alone (vaccine A) or with the addition of a suspension of killed Mycobacterium vaccae (vaccine B). One hundred children were vaccinated with BCG Glaxo plus a suspension of M. leprae (vaccine C). Eight to 10 years later about half of the children were found for follow up. At this time further children were skin tested, and the results obtained were related to whether or not they had scars of vaccination with BCG Pasteur (Teheran) given by the local health authorities. Between setting up the study and the first follow up, cases of leprosy or tuberculosis had occurred in some of the villages, although not among those we had vaccinated. Differences between the effects of the vaccines were only found in villages with cases of leprosy. In these villages positivity to leprosin A was significantly greater after vaccine B (49%) than after vaccine A (36%; p less than 0.04). The results for scrofulin and vaccine were the same after both vaccines, and significantly lower than in the villages without cases of leprosy. The general reduction in skin-test positivity in the villages with leprosy cases was mainly due to a loss of category 1 responders to group i, common mycobacterial, antigens. It was concluded that where casual contact with cases of leprosy occurs the combination of BCG with killed M. vaccae is likely to be a better vaccine for leprosy than is BCG alone. Although few children received the combination with M. leprae, the results obtained were not particularly promising

Vaccination and skin test studies on children living in villages with differing endemicity for leprosy and tuberculosis

The purpose of this study carried out in Iranian Azerbaijan was to determine the pattern of skin-test positivity to mycobacterial antigens in children living in the valley, and to assess the effect on this of a series of vaccines against mycobacterial disease. Set up in 1978, 1707 tuberculin-negative children without scars of previous BCG vaccination were vaccinated with BCG Glaxo alone (vaccine A) or with the addition of a suspension of killed Mycobacterium vaccae (vaccine B). One hundred children were vaccinated with BCG Glaxo plus a suspension of M. leprae (vaccine C). Eight to 10 years later about half of the children were found for follow up. At this time further children were skin tested, and the results obtained were related to whether or not they had scars of vaccination with BCG Pasteur (Teheran) given by the local health authorities. Between setting up the study and the first follow up, cases of leprosy or tuberculosis had occurred in some of the villages, although not among those we had vaccinated. Differences between the effects of the vaccines were only found in villages with cases of leprosy. In these villages positivity to leprosin A was significantly greater after vaccine B (49%) than after vaccine A (36%; p less than 0.04). The results for scrofulin and vaccine were the same after both vaccines, and significantly lower than in the villages without cases of leprosy. The general reduction in skin-test positivity in the villages with leprosy cases was mainly due to a loss of category 1 responders to group i, common mycobacterial, antigens. It was concluded that where casual contact with cases of leprosy occurs the combination of BCG with killed M. vaccae is likely to be a better vaccine for leprosy than is BCG alone. Although few children received the combination with M. leprae, the results obtained were not particularly promising