ICRC Bacillus a Vaccine Candidate Strain ( C-44 ) Is Coated with Human IgG

Indian Cancer Research Centre (ICRC) bacillus strain (C-44), a candidate vaccine against leprosy is cultured in vitro in Dubos medium enriched with amino acids and human serum. The study was conducted to find out whether ICRC bacilli obtained from these cultures are coated with anti mycobacterial antibody. Anti-ICRC antibody raised by intradermal inoculation of sonicated ICRC bacilli in rabbits reacted with both human immunoglobulin G (IgG) and antigens of ICRC. Further, ICRC bacilli could also be fluoresced directly with FITC labelled anti human IgG. Positive fluorescence of ICRC could be abolished by digestion of human IgG with trypsin and carbon tetrachloride (CCL4). It is concluded that ICRC bacilli present in the vaccine are coated with human IgG.


Introduction
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 (10 9 ) 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]  tients 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 (10 9 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.

Preparation of ICRC Antigen
ICRC vaccine (C-44) vials (human serum used for this batch was negative for both HIV and hepatitis B) were obtained from Dr. C.V. Bapat, Emeritus Medical Scientist, Indian Council of Medical Research, New Delhi, India.ICRC bacilli suspended in PBS were centrifuged at 8000 g to precipitate the organisms.After decanting the supernatant, the deposit of bacilli was washed thrice in PBS.ICRC organisms were then sonicated for 15 minutes at 100 watts on ice.The sonicated antigen was stored at 4˚C and was used as soluble antigen of ICRC.

Raising Antibodies against ICRC (C-44) in Rabbit
Anti-ICRC antibodies were raised in a rabbit (out bred New Zealand strain rabbit) by hyper immunizing the animals with a total of 4 intra-dermal doses [each dose of 1.0 ml emulsion of equal volumes of ICRC soluble protein (100 μg) and Freund's incomplete adjuvant (Sigma, USA)] at weekly intervals.Hyper immunized serum was collected after 7 days of the 4 th injection.

Ouchterlony's Gel Diffusion
Hyper-immunized rabbit anti-ICRC antibody was tested against ICRC soluble antigens by agarose gel diffusion method [7].Anti-ICRC rabbit antibody absorbed with anti human IgG was also tested against soluble antigens of ICRC and human IgG by the agarose gel diffusion method.

Direct Immunofluorescence (IF) Staining
Smears were made from ICRC bacillary suspension obtained from the vaccine Open Journal of Immunology vial on microscopic glass slides.After fixation of the smears in formalin vapour atmosphere the slides were washed thrice with PBS and were used for direct immunofluorescence (IF).
A set of slides with smears were rinsed in carbon tetra chloride (CCL 4 ) for 10 minutes at room temperature followed by digestion of the smears with 0.1% solution of trypsin in Tris-HCl buffer, pH 8.0 for one hour [8].After rinsing the slides (both untreated and CCL4 treated-trypsin digested smears) in PBS for 15 minutes, the wet preparation of the slides were subjected to direct IF by overlaying the smears with fluorescent isothiocyanate conjugated Goat anti-human IgG (FITC-GAHIgG) followed by incubation for 1 hr.at 37˚C in a bread box with wet filter paper and kept at 37˚C incubator (WISWO BOD, Thermotech L-700).
The wet preparations of smears were examined under an inverted fluorescence microscope (Leitz GmbH & Co. Germany).

Results
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

Discussion
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 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 [4].Intradermal inoculation of lepromatous pa-How to cite this paper: 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.2017.73004Open Journal of Immunology (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 CCl 4 and trypsin solution ICRC bacilli did not show any fluorescence (Figure 2(b)).

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[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.