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Research into the immunology of TB has had the task of discovering why Koch’s old tuberculin had two opposing effects, namely “cure” or catastrophe. Recent interest has grown since the discoveries that BCG is not universally protective and of HIV. Thus many questions have been raised about mechanisms of immune protection and immunopathology. This complex subject is summarised from recent publications (Coovadia and Benatar 1991; Porter and McAdam 1994; Roitt, Brostoff et al. 1996; Rook and Hemandez-Pando 1996).

The Koch phenomenon

Four to six weeks after infection with M.tuberculosis, or following inoculation with the organism in guinea pigs, an intradermal challenge with whole organisms or culture filtrate sometimes causes necrosis locally and in any distant tuberculous site. This is due to delayed cell-mediated hypersensitivity. Koch noted this could resolve a cutaneous lupus lesion and he hoped the same would occur in systemic TB. Unfortunately deep necrosis in the spine or lung caused sloughing, extensive tissue damage and facilitated spread o f fulminating infection. Necrosis is not inevitable, however, as it does not occur in normal BCG recipients or in tuberculoid leprosy patients.

Immune protection following tuberculosis infection

Live M tuberculosis is intrinsically toxic to certain tissue cells. Human macrophages that ingest more than about five organisms die. This is in contrast to M.avium which can multiply to large numbers within cells without killing them. The reason for this is not known. M. tuberculosis is a facultative intracellular parasite requiring aerobic conditions for growth. The first cells that the organisms encounter once inhaled or ingested are resident neutrophils and naive non-activated macrophages. Following phagocytosis they are transported to local lymph nodes. Some organisms remain viable intracellularly and replicate whilst others are destroyed and broken up within phago-lyso somes. When the proliferation at the primary site reaches a critical threshold a specific acquired immune response results:

i) Certain polypeptides are released from the phago-lysosomes, transported to the surfaces o f macrophages and presented together with MHC (major histocompatibility complex) molecules determined by the HLA (human leukocyte antigen system) alleles o f the host cell. This combination of parasite antigen and host HLA determinant is recognised by circulating T (thymus-derived) lymphocytes.

ii) Once a T cell recognises the presented antigen (its combining site being complimentary to the antigen-presenting cell) various cytokines (circulating peptide mediators) are released. The main ones are ILl (interleukin-1) fi-om the macrophage and IL2 by the T cell. The next stage depends upon whether the type of activated T cell is “TH l" or “TH2" and whether or not it is “CD4" or “CD8". The “CD” (cluster designation) system represents cell markers identified by monoclonal antibodies and are broadly concerned with three roles - lineage of T cell lines (eg. CD3), maturation (eg. CD l), and activation (eg.CD25). The CD3 complex is subdivided into T cell antigen receptor groups: TCR-1 (10%, mucosal) and TCR-2 (90%). The TCR-2 group is further differentiated into CD4 (TH or T-helper, antigen recognition by MHC class II molecules) and CDS (TC or T-cytotoxic, antigen recognition by MHC class I molecules). The CD4 TH cytokines are designated THl (cytotoxic to intracellular pathogens and associated with the local inflammatory response) or TH2 (stimulate B cell humoral antibody production mainly against free-living microorganisms). The mechanism of selection between THl and TH2 type cells is not clear but may be dose-dependent, with low doses favouring THl responses.

iii) The key cytokines secreted by THl cells are EL2 and ylFN (gamma interferon) which in turn can activate macrophages to ingest and digest mycobacteria and also secrete other cytokines such as IL8 and TNFa (tumour necrosis factor alpha) which activate the acute phase inflammatory response. TNFa produced by infected macrophages is induced by a tubercle bacillus cell wall constituent called “LAM” (lipo-arabino-mannan) and has a key role in granuloma formation by attracting monocytes, encouraging their transformation into epitheliod and giant cells. Granulomata physically contain the bacilli and provide an anaerobic environment in which the bacilli cannot thrive.

TH2 cells secrete IL’s 4,5,6 and 10. The present evidence is that immunity to tuberculosis involves a mixture of THl and TH2 and TNFa cytokines. The precise mechanism for killing M tuberculosis is unknown but is considered to be dependent on reactive oxygen metabolites within phago-lysosomes (eg, hydrogen peroxide and nitric oxide). Possible factors regulating a TH1-TH2 switch to pathology include active vitamin D3 metabolites and adrenal steroids. l,25(OH)2 cholecalciferol inhibits production of ylFN and IL2 but increases IL4 and 115 whilst cortisol reduces macrophage activation and THl T cell activity and synergises TF12 functions.

iv) The role of CDS cytotoxic cells is less clear, but they have the ability to recognise and kill infected macrophages. M. tuberculosis may be destroyed with lysis of their host cells, or may be released into the extracellular environment to be engulfed by other activated macrophages.

v) B cell humoral immunity is considered to have a more minor role in protective immunity, yet to be elucidated. One such antibody is agalactosyl IgG.

The complex network of responses also includes natural killer cells, acute phase proteins and vitamin D derivatives. Other possible components include platelet activating factor, endothelium-leucocyte adhesion molecules and granulocyte/macrophage colony stimulating factor.

Immune pathology in tuberculosis disease

Most infected individuals do not experience recognisable ill-effects. However bacilli remain viable within granulomata or in regional lymph nodes for many years. Some cases proceed to more substantial tissue damage and clinical disease, sometimes by haematogenous spread. An alternative situation is where the granuloma cannot contain local proliferation o f bacilli, initiating cellular hypersensitivity. This leads to inflammation, release of large amounts of TN Fa, death of infected macrophages and other cells, release o f bacilli, their ingestion by new activated macrophages and accumulation of necrotic caseous debris within the grooving granuloma. However ill the patient at this stage he or she is not usually infectious. To become so requires breakdown of a localised lesion or reinfection when host resistance has weakened, eventually leading to cavity formation where there is an abundant oxygen supply for further proliferation and bacilli-laden sputum. This may occur with advancing age, malignancy, immunosuppressive therapy, malnutrition, alcoholism or other insults to the cell- mediated immune system. Tuberculosis associated with HIV and AIDS is thought to be mainly due to reactivation of latent infection and a declining CD4 count. HIV preferentially infects T lymphocytes via the CD4 receptor and then progressively destroys these cells, the dominant cell type in protection against TB. It is estimated that the progression from infection to tuberculous disease is 30% with HTV/TB dual infection compared with the single infection risk of 5%.

Research into improving BCG efficacy

Current research to improve on BCG alone consists of three approaches. The first is to seek identification of protective antigens, if they exist. The second is utilising M. vaccae as an immunotherapeutic tool encouraging a TH2-TH1 cytokine switch. The third idea is to improve on BCG as it exists; for instance reduce the dose on the premise it may invoke a THl predominant response or, alternatively, administer multiple doses of BCG (Fine in (Porter and McAdam 1994)).

The mycobacterial persister

Mycobacteria are known to remain viable for long periods, not replicating, in the sediment of liquid cultures. And yet six to 12 months of bactericidal isoniazid chemotherapy is able to eliminate a high percentage o f persisters in healthy infected individuals. Possible explanations include a steady state equilibrium of replication and immune destruction within

tissues, or minute forms without cell walls that have been overlooked in view of their much smaller size (sometimes termed Much’s granules). The mystery endures (Grange 1992).

3.5 M vaccae IMMUNOTHERAPY

Mycobacterium vaccae NCTC 11659 was selected from soil in the region of Uganda where BCG had been shown to be very effective against leprosy (Stanford and Stanford 1996b). The interest in M. vaccae began with the finding outlined above that it is rich in common mycobacterial antigen whilst remaining non-pathogenic (Pozniak, Stanford et al. 1987). The common epitopes are capable of initiating protective responses and so the loss of dermal reactivity to them in the disease state creates the possibility that they can be used therapeutically. Tuberculous patients differ from matched healthy controls in their inability to recognise common antigens (Stanford, Bahr et al. 1990a). When used at the optimal THl-inducing doseM vaccae has demonstrated significant improvements in survival o f mice infected w ithM tuberculosis (Rook and Hemandez-Pando 1996). Adding W M .vaccae to BCG enhances skin reactivity to casually encountered mycobacteria,as does 10^ M. vaccae alone. Further skin tests with a mixture of mycobacteria including Vaccin (or similar reagents from made from other certain fast-growing mycobacterial species) produced smaller, less tissue damaging, reactions than if a mixture without Vaccin was administered; this regulation of the skin-test response induced at the injection site by the complete mixture on one arm exhibits the same regulation around the site of an injection o f a mixture without Vaccin in the other arm. This led to the concept of an intradermal injection being capable of regulating immune responsiveness around pulmonary (or deep) lesions; hence the practical application immunotherapy (Stanford, Stanford et al 1994; Stanford and Stanford 1996). The aim is to aid standard chemotherapy in eradicating persisting dormant tubercle bacilli and possibly to then shorten the course of chemotherapy further. The desire is to produce type 1 cytokines IL-2, IL-12, ylFN and to regulate (or switch ofi) type 2 cytokines IL-4, IL-5 and IL-10 leading to destruction of intracellular bacilli; the regulation may be under the control of adrenal cortical hormones (IBID). Chemotherapy patient compliance varies from 40% in parts o f Africa and India to nearly 100% in the UK; immunotherapy could restore protective antibacterial immunity, augmenting initial chemotherapy bactericidal activity (IBID). Pilot studies have been encouraging and M. vaccae is currently undergoing Phase 3 Good Clinical Practice efficacy trials in South Africa and Uganda.

Published studies

Published trials to date have taken place in Kuwait (Stanford, Bahr et al 1990b), The Gambia (Corrah 1994), Nigeria (Onyebujoh 1994, Onyebujoh, Abdulmumini et al. 1995), Iran (Farid, Etem adi et al. 1993, Farid, Etemadi et al. 1994), South India (Faizy 1996) and Romania (Corlan, Marica et al. 1997a; Corlan, Marica et al. 1997b). The key features and outcomes o f these studies are shown in Table 3.1. The summary shows the year each study was published. Only the Kuwait and Gambia studies were carried out as double blind placebo clinical trials. The dilemma for researchers is to balance best possible blindness with the opportunity to carry out the trial according to the protocol. This can be a particular problem when funding is limited.

Table 3.1 Summary findings of published M vaccae case-control trials Si lid)

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I rial type u/lotal ii=acti\e Chemotherapy c r-n ml (weeks) 1 ollow-up (weeks) Siguilieanl fmdiiigs of immuiiolherap\ group K uw ait (1 9 9 0 ) D BF 47 112 2 S H R /7 H R 4 16 Tweiglit gain T l\in p h o c> te d i anges G am bia (1 9 9 4 ) D BF 115 251 2SH7., 16H Z, 2 H R Z t:,4 H R , 6 76 T cutlu re cu re if B C G -ve i lale m ortality 18m d ie m o Iran M D R -T B (1 9 9 4 )

eonlrolled 4 1 ? I'n k n o w n 0. 2 4 .3 2 . 40 40 T cultu re cure

N igeria (1 9 9 5 ) SBF (* non-rand). 90 180 (+ 5 6 56) ISH RZ, ? (lui know n) 1 - 3 64 ^ m o rtality T m icroscopy clearan ce tw e ig lu gain ÎE S R fall Tagalactosyl Ig fa ll S. bidia (1 9 9 6 ) ?SBF 70 136 2H R Z E ,4H R < 0 ^ 4-8 24 Rom ania new F I B (1 9 9 7 )

SBF 9 7 206 2S H R Z , 4H R , 4 104 TESR fall; Tw eight gam Tradiological clearan ce ire la p s e R om ania d iro n ic F T B ( 1 9 9 7 ) non-random placebo 56 102 2 S H R Z /4 H R , 4 102 T cu b u re c u re at 2m . 12m TE SR fall Tradiological clearan ce D B F = do u b le blind placebo; SB P = single blind placebo

The table also shows the number of patients in each study as a total and the number that were identified to receive active immunotherapy. The total number studied varies from 102 to 251. The Nigerian data comprises 180 cases published in the paper cited and an additional 56 included in Onyebujoh’s Ph D thesis. The chemotherapy regimes are also shown. These range from six to 18 month regimens. The chemotherapy-immunotherapy interval varies between studies; the optimum timing and frequency of doses o f M. vaccae are still under investigation. The interval varies from one to eight weeks and only the South Indian trial included a second dose. All used saline as the placebo injection. The measurement and outcome criteria were varied as discussed below. As a synopsis the table shows those findings reaching statistical significance. As will be seen the Gambian and Nigerian work report a reduction in mortality. Three trials showed a significant gain in weight whilst two demonstrated a notable fall in ESR. Improved bacteriological clearance or cure was evident in four o f the studies shown. The Romanian papers showed greater radiological clearance and also reduced relapse of disease. The Kuwait and Nigeria trials included immunological parameters, finding meaningful changes in lymphocyte transformation studies and agalactosyl immunoglobulin.

In order to appraise the trials further each will be considered in terms of study design and completion of protocol, chemotherapy compliance, assessment criteria and data analysis.

Area of study: Kuwait (Stanford, Bahr et al. 1990b)

Study design/protocol: 47 of the 112 patients in this trial received M vaccae; patient selection and method of randomisation are not discussed. Other matching data is not given as the paper concentrates on its findings. One of the authors administered the injections but it is not apparent if he/she took part in other areas of the study as this would affect the level of blindness used.

Chemotherapy compliance: not stated.

Assessment criteria: these were wide-ranging comprising clinical examination (including weight), bacteriology (sputum culture drug sensitivities were sent to Cardiff, UK - mode not given), biochemistry (20 parameters, unidentified), haematology (haemoglobin, white cell count, erythrocyte sedimentation rate), immunology (quadruple skin tests with new

tuberculins, IgA/IgG/IgM antibodies to five mycobacterial species and Streptococcus pyogenes and Candida albicans, lymphocyte transformation studies or ‘"LTT” to four mycobacterial species and interleukin-2) and radiology (numerical scheme). Each of these parameters were studied at monthly intervals for four months, except the Ig and LTT estimations which were studied at two unstated times. Not all the active group patients were skin tested (41/47) or had LTT carried out (38/47) for unknown reasons.

Data analysis; the vast majority of the data is not shown as no difference between treatment and placebo groups was found. However weight gain was 6.1kg (S.D. 3.0) in the active group, significantly more than the 4.5kg (S.D. 3.4) in the placebo group. Much is made of the immunology data in terms of stimulation indices and LTT studies though these parameters are not explained. The conclusion is drawn that the immunological profile shows a partial return to recognition of group (i) mycobacterial antigens. Mortality and relapse data is not given, presumably due to the short follow-up period.

Area of study: Gambia (Corrah 1994)

Study design/protocol: this thesis presents data on three aspects of tuberculosis patients - differences between six and 18 month chemotherapy regimens, differences between M. vaccae and saline groups, and differences between HIV positive and negative patients. Of the original 285 cases eight were lost and 13 died in the first six weeks before the injection was given; another 13 subsequently lost to follow-up have been excluded from analysis. 251 remain, 115 M vaccae and 136 saline recipients; in the outcome analysis 13 reappear. The two treatment arms were matched for gender and age, but not initial chest X-ray (widespread disease 27% active, 39% placebo); severity of disease clinically is not stated. An independent nurse was employed to administer the injections; the method of randomisation is not stated. Injection reactions were measured at three and seven days after they were administered. It was intended to exclude foreigners as difficulties were envisaged in obtaining follow-up data given their unpredictable movements; however some Senagalese managed to be included - their number and possible influence on bias are not stated. For the criteria mentioned the immunotherapy and placebo groups were matched but for significantly greater wasting and worse chest x-rays in the placebo group.

Chemotherapy compliance; patients were encouraged to take their drugs regularly and taxi fares were provided to assist in follow-up assessments. The success of chemotherapy compliance is not documented.

Assessment criteria: these entailed clinical examinations, quadruple mycobacterial antigen skin tests, chest x-rays, bacteriology, haematology, biochemistry, HIV status, immunology and virology; data is presented for 251 patients receiving courses of six or 18-month chemotherapy. The only data given for M vaccae and saline groups is for outcome, stratified by BCG status and chemotherapy regimen. Although three and five follow-up appointments were scheduled for the six and 18-month chemotherapy groups respectively no weight measurements or other follow-up data are recorded, even though they were intended. No data is given at the interim follow-up points.

Data analysis: with the longer 18 month chemotherapy there were no late (6-18 months) deaths (0/33) in th e M vaccae group compared with 6/42 in the placebo group (p=0.03). Otherwise there was no significant difference between active and placebo groups. There were 35 deaths in all - 13 before the six week injection, 16 between six weeks and six months

(6 active, 10 placebo) and the six late deaths mentioned. Of the 22 deaths six occurred in the six-month chemotherapy regimen group (2/60 immunotherapy, 4/63 placebo) whilst 16 befell the 18-month chemotherapy group (4/59 immunotherapy, 12/82 placebo). Cause of death is not stated as being from TB or other reasons. Overall placebo mortality was 16/136 (12%). Interestingly there were no deaths amongst BCG+ve cases for either group, and there was a significant reduction in death in the immunotherapy group for BCG-ve status (6/78 active, 16/101 placebo). BCG would appear to be highly effective in this area and M vaccae beneficial where evidence of prior BCG immunoprophylaxis is absent. BCG scars were noted in 35% active cases and 32% placebo cases. Possible confounding factors include younger age and higher socio-economic status o f BCG recipients. 102/135 (75.6%) cures and 11/135 (8.1%) relapses occurred on short chemotherapy with corresponding figures of 93/150 (62.0%) and 23/150 (15.3%) on long term therapy (p<0.08); cure was defined as being sputum-negative plus weight gain plus an improved chest X-ray. Combined immunotherapy cure was 84% with 73% placebo cure after both chemotherapy regimens.

Area of study: Iran (Farid, Etemadi et al. 1994)

Study design/protocol: This small study is the only M. vaccae paper specifically dealing with MDR-TB. Information given in the journal is brief. 41 patients entered the trial; no details o f controls, randomisation or blinding are provided. Four injections were given: 41/41 received the first, 29/41 a second, 25/41 a third and 9/41 a fourth. Chemotherapy regimens are not stated but five were resistant to two drugs, 10 to three drugs, seven to four drugs and

16 to five drugs.

Chemotherapy compliance: not stated.

Assessment criteria: it is inferred fi-om the discussion that sputum culture, weight gain, ESR