The Evaluation Of Protective Efficacy Of Tuberculosis Subunit Vaccines EAMM And MH Using Rabbit Skin Model

Objective:Using rabbit skin liquefaction model to test the protective efficacy of subunit vaccine candidates EAMM (ESAT6-Ag85B-MPT64190-198-Mtb8.4) and MH (Mtb10.4-HspX), in order to lay the foundation for application of the two vaccines and provide a visual and convenient animal model to evaluate protective efficacy of TB vaccines.Methods:Rabbits were divided into five groups with three animals in each group. Three groups were vaccinated three times at3-week intervals with subunit vaccines and injected with MH, EAMM, MH plus EAMM respectively. One group received BCG at5*106CFU dissolved in200μl saline at the time of the first subunit vaccination. The last group received the same volume of saline as the control. All the rabbits were injected subcutaneously.6weeks after the final immunization, enzyme-linked immunosorbent assay was used to test the levels of antigen-specific IgG antibodies in rabbit serum. After the antibody detection, all animals were injected intradermally at two sites on each flank with100μl BCG containing5×106CFU. The lesions were observed every day on their sizes and the liquefaction and healing process. We recorded the time when the lesions formed granulomas, time of ulceration, time of liquefaction peak, onset of healing and time of the lesions healed. When the lesions at the stage of liquefaction peak, the number of tubercle bacilli within liquefied caseum was determined. The lesions and normal skin tissues nearby were collected when the lesions healed and were made for pathological sections and subjected to HE staining.Results:1. Tuberculosis subunit vaccines could generate high antigen-specific IgG antibodies. The antibody titers in saline control group were negative. ESAT6-specific, Ag85B-specific IgG antibody titers in EAMM group and HspX-specific IgG antibody titers in MH group were higher than those in BCG group (P<0.01). Animals receiving EAMM plus MH subunit vaccine also generated higher levels of ESAT6-specific, Ag85B-specific, HspX-specific IgG antibodies than BCG group (P<0.01).2. Tuberculosis subunit vaccines could accelerate the liquefaction and healing process of tubercle following BCG challenge. In group BCG and group EAMM plus MH, the BCG-induced skin lesions came to the stages of ulceration and the liquefaction peak earlier than single subunit vaccine groups and the control. The beginning of the liquefaction in group BCG and group EAMM plus MH appeared at the8th day after BCG challenge. The ulceration appeared at the11th day after BCG challenge. The lesions in the two groups reached their liquefaction peak at day13. We found that the maximum size of the lesion was at the time of liquefaction peak. The volume of the liquefied lesions in BCG group was the largest in all groups, the lesion size at the peak of liquefaction was679.67±62.31mm3, was significantly larger than the size of saline control group (544.78±64.89mm3)(P<0.05). The volume of the liquefied lesions in combined vaccine group (EAMM+MH) was the smallest in all groups, the lesion size at the peak of liquefaction was only380.15±53.85mm3, was significantly smaller than the size of saline control group (544.78±64.89mm3)(P<0.05). The lesions in group BCG and group EAMM plus MH came to healing more quickly than that of the other three groups, the onset of healing appeared at the29th day after BCG challenge.In MH and EAMM single protein vaccine groups, lesions reached their maximum sizes at day15and the saline control group was the last group to reach the liquefaction peak at day17. The lesion volume was504.35±65.46mm3in EAMM group and544.05±63.60mm3in MH group at the liquefaction peak. They were both significantly smaller than the size of BCG group (679.67±62.31mm3)(P<0.05) and larger than the size of combined vaccine group (380.15±53.85mm3)(P<0.05).3. Tuberculosis subunit vaccines could reduce the bacterial load in liquefied caseum at the peak of liquefaction. The CFU count in the combined vaccine group was the lowest (1.38×106CFU/g) in all groups. It was significantly lower than the CFU count in the saline control group (5.34×106CFU/g)(P<0.05). The CFU count at peak of liquefaction in BCG group (3.20×106CFU/g) was only lower than the CFU count in the saline control group (P<0.05). The CFU count in EAMM group (2.04×106CFU/g) and in MH group (2.13×106CFU/g) lay between BCG and combined vaccine groups.4. Pathological biopsy of skin lesions in each groups (HE staining) suggested that the pathological manifestations were induced by BCG but not by other contaminating bacteria. By observing the pathological biopsy of the skin lesions which were collected when the lesions healed, we found epithelioid cells, monocytes and lymphocytes infiltrated. Langhans giant cells were seen along with unabsorbed liquefied material and necrosis.Conclusion:Tuberculosis subunit vaccines EAMM and MH showed protective efficacy in rabbit skin model. EAMM and MH generated high level of serum antigen-specific IgG antibodies, accelerated the pathological process of tubercle, and reduced the bacteria load in liquefied caseum at the time of liquefication peak. The combination of EAMM and MH showed the best protective efficacy among all groups. Meanwhile, the pathological process of rabbit skin model is similar to human beings. Therefore, the rabbit skin model can be used as a visual and convenient model to evaluate the protective efficacy of TB vaccines.