In 1958, Indian Cancer Research Centre (ICRC) bacilli were grown in vitro culture in a modified Dubos medium, supplemented with non-essential amino acids and 5% human serum [1] . This strain of ICRC was noted to inhibit M. leprae growth in mouse foot pad [2] . Later, inoculation of ICRC bacilli (10⁹) was shown to convert lepromatous individuals from lepromin negativity to positivity along with induction of histopathological regression of lepromatous lesions. Considering the above observations, ICRC bacillus was considered for a trial as a candidate vaccine for leprosy [3] [4] . Intradermal inoculation of lepromatous patients with the C-44 vaccine strain of ICRC resulted in lepromin conversion from negativity to positivity in 57.7% and 91% of lepromatous leprosy (LL) and borderline lepromatous (BL) patients respectively. The above mentioned encouraging results led research workers to conduct field trials to find out its protective efficacy against leprosy [5] .

ICRC vaccine [ICRC bacilli suspended in phosphate buffered saline (PBS), pH 7.2] is administered by intra-dermal inoculation of 0.1 ml (10⁹ bacilli) in the deltoid region of the arm. This vaccine has been reported to evoke local reaction at the inoculated site in the form of pustules, ulcers [1] and enlargement of regional lymph nodes [1] [3] [6] between 3rd and 8th week of the post-vaccination period. Although pustules and sometimes ulcerations ultimately healed without consequences, the reason for the occurrence of such pustules and ulcerations and enlargement of draining lymph nodes induced by this vaccine has not been explained by the investigators. The present study was undertaken to find out whether ICRC strain which was used in the vaccine had any other biological component which might have been responsible for induction of pustules, ulcerations and lymph node enlargement in the vaccinated subjects.

Both ICRC antigen and human IgG reacted with anti-ICRC rabbit serum in Ouchterlony’s gel diffusion showing a line of identity (Figure 1(a)). An additional precipitin reaction line against ICRC antigen was also noted (Figure 1(a)). After absorption of anti ICRC rabbit serum with human IgG the antibody activity with human IgG was abolished keeping the reactivity with ICRC antigen only (Figure 1(b)).

ICRC bacilli from the vaccine vial subjected to direct IF staining using FITC-GAHIgG were found to fluoresce with yellowish green coloration under the fluorescent microscope (Figure 2(a)). On the other hand, when IgG was digested by treating the smears with CCl₄ and trypsin solution ICRC bacilli did not show any fluorescence (Figure 2(b)).

It has been recently reported that ICRC vaccine provided maximum protection against leprosy out of all candidate vaccines which were evaluated for their efficacies in a South Indian endemic normal population, in a field area at Avadi. Further, out of all these vaccines, ICRC vaccine evoked hard skin indurations [1] [3] , often leading to pustule formation [3] [6] , ulceration [1] and lymphadenitis [1] [3] . These reactions indicated that ICRC bacilli must have recruited more immunoreactive cells liberating more interleukins which might have caused extensive granuloma formation. Out of four vaccines under trial BCG, M. w is grown in bacteriological media and is purified as pure bacilli in the vaccine vial. M. leprae from armadillo tissue is purified by a WHO protocol and it does not contain any significant amount of foreign protein [9] . On the other hand, ICRC is grown in a human serum containing medium and is washed in PBS by centrifugation and is dispersed in aqueous suspension to contain 10⁹ bacilli/ml before irradiation. In the present study it was noted that ICRC bacilli could evoke antibody response not only to ICRC but also to human IgG when injected in rabbit as identified by precipitin reaction developed by gel diffusion (Figure 1(a)). However, earlier Rao et al. [10] were also able to detect only antibodies to human serum in rabbit sensitized with ICRC and not against any antigenic components of ICRC. Failure to detect antibody against antigens of ICRC [6] might be due to the use of suboptimal dose of ICRC antigen for immunization of rabbit. As the present study has hyper immunized rabbits using an optimal dose of ICRC protein, it could detect antibody against ICRC antigens and as well as human IgG.

Further, it was proved by direct immunofluorescence with FITC-GAHIgG that ICRC bacilli in the vaccine vial were coated with human IgG (Figure 2(a)). This was presumably due to coating of ICRC bacilli with cross reactive mycobacterial antibody present in the human serum which was in the culture medium. The treatment of bacilli with trypsin and CCl₄ destroyed the IgG present on the bacilli as revealed by the negative reaction to direct binding of FITC-GAHIgG (Figure 2(b)).

The presence of ICRC bacilli along with its antibody (human IgG) is akin to the presence of molecules of antigen and antibody, i.e., immune complexes (ICs). These 10⁹ units of ICs might have evoked stronger and more prolonged immunogenic reactions in patients than those induced by other candidate vaccines which are simple suspensions of purified bacilli. Immune complexes are known to regulate the antigen uptake, processing and presentation to T cells [11] . Also internalization of immune complex was reported to have role in dendritic cell maturation and antigen presentation [12] . Thus, ICRC along with its antibody (human IgG) might have caused more local cellular infiltration with enlargement of draining lymph nodes.

The present study, therefore, indicates that the ICRC bacilli (C-44) in the vaccine are coated with human IgG. Therefore, ICRC vaccine might have evoked strong immune reaction in the individuals than other candidate vaccines due to the presentation of the antigens in the form of ICs. However, as human serum is incorporated in the culture medium, a close watch should be kept to screen human serum for the presence of any infectious agents specially hepatitis and AIDS viruses.

The authors thanks to Dr. C. V. Bapat, Emeritus Medical Scientist, Indian Council of Medical Research, New Delhi, India for providing ICRC vaccine vials for experiments.

The authors declare that they have no conflict of interests.

U Sengupta conceptualized the study. U Sengupta and KK Mohanty designed the study protocol. AR Yadav performed the experiments. All authors have contributed for writing the manuscript and editing it.

Yadav, A.R., Mohanty, K.K. and Sengupta, U. (2017) ICRC Bacillus a Vaccine Candidate Strain (C-44) Is Coated with Human IgG. Open Journal of Immunology, 7, 45-50. https://doi.org/10.4236/oji.2017.73004