Phase IIb randomized trial of adjunct immunotherapy in patients with first-diagnosed tuberculosis, relapsed and multi-drug-resistant (MDR) TB

Tuberculosis (TB) is a re-emerging global public health problem, especially in developing countries. Ukraine is a representative country as it concerns the TB epidemic. In 1961, the incidence of TB in Ukraine was 155 cases per 100,000 individuals, which then declined to 32 cases per 100,000 in 1991. However this trend reversed and by 2008 the incidence became 81 per 100,000. The mortality doubled from 10.2/100,000 to 21.6/100,000 between 1990 and 2001 [1]. In addition the Ukraine experiences the worsening epidemic of drug resistant TB. Isoniazid and rifampicin resistance, which defines the MDR-TB, has been found in 44% and 32.9% of TB isolates [2]. The success rates of TB therapy in Ukraine are below average when compared with other regions of the world [3]. Despite availability of TB drugs the situation is far from ideal and it is clear that better therapeutic interventions are needed to reverse the current trend.

Oral therapeutic vaccine V5 was originally developed for the management of chronic hepatitis B and C [4–6]. The preparation is derived from pooled blood of HBV- and HCV-positive donors, which following chemical- and heat inactivation was formulated into an oral pill according to proprietary technology developed by us [6]. It is well known that one third of people carry M. tuberculosis without showing symptoms of the disease. Therefore V5 inherently contains circulating M. tuberculosis antigens. During hepatitis C trial in 20 patients who happened to have pulmonary TB with HIV-co-infection, V5 produced mycobacterial clearance in sputum smears of 94.4% of patients within one month [7]. Subsequent, one-month, placebo-controlled trial in 55 patients confirmed the anti-TB property of V5 revealing negative smear conversion in 96.3% of treated subjects [8]. Our study was, thus, aimed to verify these findings in an independent clinical setting at our TB hospital in Kharkov. The advantage of adding V5 to standard ATT was compared to a treatment regimen consisting of ATT and placebo.

This placebo-controlled, comparative study involving 34 patients with first-diagnosed TB, relapsed TB, and MDR-TB reveals that when conventional or individualized TB drug regimens are combined with V5, the combination can produce significant improvement in clinical endpoints and clearance of M. tuberculosis at higher rate than in patients on placebo. Biochemistry and hematological analysis of blood samples support favorable adjunctive effect of V5, which has not shown any adverse effects throughout the study duration.

Our findings support two earlier clinical trials of V5 demonstrating favorable outcome in TB patients [7, 8]. These studies reported a range of beneficial effects including better quality of life, body weight gain, reversal of ATT-associated hepatotoxicity, reduced inflammation, faster deffervescence, and higher clearance rate of M. tuberculosis in sputum smears. Furthermore, the adjunct immunotherapy resulted in much shorter duration of treatment than among those who received standard ATT. Both prior studies were conducted at the Lisichansk Regional TB Dispensary but these results are now confirmed by the present study performed independently at our TB hospital based in Kharkov.

The normalization of inflammatory indices is considered to have favorable effect on the course of the TB disease [10, 11]. There were earlier indications that V5 exhibits the anti-inflammatory activity [4–8]. These observations are supported by this study. Contrary to the outcome in placebo group the markers of inflammation such as elevated leukocyte counts and prolonged erythrocyte sedimentation rate have been significantly reduced in V5 recipients. A favorable change in the blood picture is supported by the increase in total lymphocyte percentage among V5 recipients, but not in the control group. The restoration of suppressed lymphocyte counts and decrease in leukocyte counts is associated with positive treatment outcome [12]. Thus, changes in relative and absolute lymphocyte and leukocyte numbers may hold promise as surrogate markers of treatment response. Further studies aimed at evaluating specific immune phenotypes of peripheral blood cell subsets, including their functional characterization are needed.

The hepatotoxicity induced by anti-TB drugs has serious adverse consequences to treated patients and imposes limitations on treatment options [13]. Addition of V5 appeared to reduce baseline bilirubin and ALT levels, as well as the abnormal liver size when compared to placebo regimen. These observations confirm prior studies in which V5 has been shown to counter the hepatotoxicity of TB drugs [8, 9]. For this reason the use of V5 in combination with ATT is advisable to prevent or reverse iatrogenic liver damage.

The immunotherapy has shown clear benefit in reversing body weight loss. The average gain in V5 and placebo groups was 3.5 kg and 0.9 kg which is almost identical to the results of placebo controlled trial involving a comparable group of 55 TB patients from the Lisichansk TB dispensary, i.e., 3.4 kg (59.7 ± 8 vs 63.1 ± 9 kg; P = 5.7E-007) and 1.07 kg (59.1 ± 10 vs 60.1 ± 10.4 kg; P = 0.003) respectively [8]. In contrast, the mean weight gain observed in the earlier conducted, open-label trial involving 20 patients with HIV-TB was 7.7 kg (P = 4.6E-007) [7]. This almost two-fold discrepancy is perhaps due to the fact that all these patients had HIV infection - a condition that is associated with worsened wasting. Nevertheless, there appears to be the strong relationship between body weight gain and favorable outcome, which needs to be confirmed in a larger cohort of patients in independent clinical settings [14, 15].

Conversion of sputum smear from positive to negative is a main indicator of the efficacy of anti-TB intervention. We have observed that V5 accelerates and significantly enhances bacillary clearance as compared to control group on ATT (Figs. 1 and 2). This observation supports earlier studies at Lisichansk TB dispensary which reported similar results albeit at higher conversion rate [7, 8]. The difference in outcome between placebo and V5 recipients was also significant. In the prior placebo-controlled study the conversion in placebo arm was seen in 25% of patients while in our hands 0% converted. We do not know what the reason for such a discrepancy is. In Arjanova et al., study the duration of ATT prior to study initiation 4 ± 3/3 and 3.4 ± 2.7/3 months respectively [8]. In our study mean ± SD/median duration of ATT prior to V5 and placebo administration was shorter and uneven 1.6 ± 2.5/1 versus 0.5 ± 0.7/0 months. This discrepancy was mainly due to the presence of outlier patients in V5 group. Particularly, five patients #4, #6, #10, #18, and #21 were on TB drugs for 3, 3, 3, 9, and 4 months respectively (Table 1). Nevertheless, while difference in time on TB drugs can account to some degree for discrepancy in obtained results, we do not think that this was the main reason. This is supported by stratified analysis of patients on V5. If we exclude outlier patients we have 19 individuals who were on TB drugs for an average 0.57 months, which is same as in the placebo group (P = 0.76; by unpaired t-test). In this subgroup of patients 15 out of 19 (78.9%) patients had converted after one month, which is identical to the original conversion rate 78.3% we have observed for V5 group as a whole. Thus, while longer pre-treatment with TB drugs may contribute to higher conversion rate as shown by Lisichansk study, in our case this contribution was negligible, since the conversion rate was still drastically different between V5 and placebo, regardless how long they were treated with TB drugs. Nevertheless, additional studies are needed to assess the effect of the length of prior exposure to ATT in relation to the conversion rate induced by immunotherapy.

V5 contains heat-inactivated M. tuberculosis antigens which are commonly present in the blood of people who do not have active TB disease [16]. In this sense V5 resembles therapeutic vaccine RUTI which contains detoxified M. tuberculosis antigens [17]. There are several other immune adjuncts of bacterial origin which enhance sputum conversion when used together with ATT. These include M. vaccae [18], M. phlei [19], M. w [20], and Likopid [21]. Even attenuated M. bovis preparation, known as BCG vaccine since 1921, has been occasionally used as a therapeutic modality. For example, in Chinese army trial involving 360 patients with MDR-TB the negative sputum conversion rate in BCG recipients was 98.3% and 97.2% in chemotherapy control. While this difference was not significant the recurrence of TB after 5 years was 2.3% in the BCG group, but 6.9% in the control group [22]. The authors of this study, however, did not mention whether use of BCG was accompanied by reactivation of TB or aggravation of disease symptoms - a phenomenon first observed by Robert Koch - the discoverer of Mycobacterium tuberculosis.

Koch's phenomenon of TB reactivation has been thwarting the development of safe and effective therapeutic vaccine since 1890 [23]. While the risk of inducing deleterious Koch-like reaction appears to be relatively low in humans [24], extreme caution is still needed as it occurs commonly in animal models of post-exposure vaccination when various TB vaccine candidates, including BCG, were tested and produced occasionally lethal outcomes [25–30]. Recent BCG revaccination trial in the area of high TB endemicity with increased probability of occult disease has produced worrisome outcome, which might be interpreted as a fatal Koch-like TB reactivation. The trial carried out in Guinea-Bissau on 2871 children was stopped prematurely because of a cluster of deaths in the BCG arm of the study [31]. In our case we have not seen any reactivation of the disease; quite contrary we have consistently observed the reduction of inflammation signs such as ESR, leukocytosis and fever. This, we believe, is largely due to the well-known fact that orally delivered antigens induce immune tolerance instead of immune activation, e.g., Koch's reaction [32]. An immunomodulator we have studied extensively in the past is a multiherbal extract Dzherelo (Immunoxel) which has shown similar properties as V5 on the conversion rate in drug-sensitive as well as MDR-TB and reduction of inflammation [11, 33]. Contrary to TB drugs the efficacy of immune-based therapy on drug-resistant TB is considered to be the same as against drug-sensitive strains [34]. In our hands V5 seemed to be equally effective against both forms of TB, as no statistical difference was observed when we compared the conversion outcome between first-diagnosed TB and relapsed/MDR-TB subsets of patients.

V5 is shown to be safe and capable of reversing ATT-associated hepatotoxicity. In addition, V5 reduces the inflammation as evidenced by several hematological and biochemical markers. Weight gain and sputum smear conversion rate have been significantly enhanced as compared to conventional ATT. As evidenced by obtained improvements the combination of V5 and ATT can shorten substantially the duration of treatment. Our preliminary findings need to be explored further in a larger population of patients followed for longer periods of time to establish the usefulness of V5 as a therapeutic TB vaccine.

Considering that 2 billion people are latently infected with M. tuberculosis, it is likely that two different types of prophylactic TB vaccines will be needed: one is so called pre-exposure vaccine to prevent mycobacterial infection in naïve individuals and second, post-exposure vaccine, to prevent TB disease in tubercle bacilli carriers without exacerbating disease manifestations. Up to now, the majority of vaccine candidates are in the first category and only M. vaccae and RUTI are considered to be in the second category [35]. Studying V5 as post-exposure vaccine can contribute to better understanding of the immunopathogenesis of TB resulting in the design of effective vaccines and accelerate evaluation of their efficacy. In addition, V5 studies can yield important insights into correlates of immune protection, which are still poorly understood [36].