Preliminary Evaluation Of A Subunit Vaccine Based On Fusion Proteins For Its Therapeutic Efficacy In Mycobacterium Tuberculosis Infected Guinea Pig And Mouse Model

BACKGOUND:Tuberculosis (TB) is the most deadly infectious disease worldwide, which mainlycaused by Mycobacterium tuberculosis aerosol transmission. The only vaccine fortuberculosis, Bacillus Calmette Guerin (BCG), only has prophylactic effect in children,but cannot prevent TB infection and latent tuberculosis infection (LTBI) reactivation inadults. As the vaccine mechanism and the pathogenesis of TB remain unclear, it is muchdifficult to explore new safe and effective prophylactic vaccine. Drugs such as Isoniazidacid hydrazide (INH) and Rifampicin (RFP) used in TB chemotherapy are effective withthe vegetative bacteria, but have less effect on dormant bacilli which is characterized bylow growth and metabolism. The inevitable side effects and long treatment course of TBchemical drug get rise to poor patient compliance, and contributes to the prevalence ofmultiple drug-resistant strains. Thus it is urgent to research and development new drug and strategy for TB control. The therapeutic vaccine can induce specific immuneresponse which help host recognize and clear the persistent and dormant bacilli andshorten the course of treatment. Nowadays the study of therapeutic vaccine is becoming aresearch hotspot in TB field.OBJECT:To establish guinea pig model of M. tuberculosis infection and treat it with subunitvaccine of Ag85B-ESAT-6+Hsp65-hIL-2with adjuvant MPLA or combined withchemotherapy, evaluate the vaccine therapeutic efficacy and ability to inhibit therecurrence of M tuberculosis after immunosuppression. To establish mouse model ofinfection and treat it with this subunit vaccine with or without chemotherapy, evaluate thevaccine therapeutic efficacy of M. tuberculosis infection.METHODS&RESULTS:1. Immunological characteristics of fusion Ag85B-ESAT-6proteinMethods: The fusion Ag85B-ESAT-6and Hsp65-hIL-2proteins were expressed byprokaryotic expression system and purified by Ni2+affinity chromatography. Guinea pigswere subcutaneously immunized with different doses (20μg,50μg,100μg,200μg,600μg) of fusion Ag85B-ESAT-6protein with50μg adjuvant MPLA for3times, with3weeks interval. The titer of IgG, IgG1and IgG2antibody against M. tuberculosis in thesera of guinea pigs were measured by ELISA two weeks after each immunization.Guinea pigs were sacrificed and the proportion of CD4+and CD8+T cell in peripheralblood of guinea pig were measured by flowcytometry.Results: Recombinant proteins Ag85B-ESAT-6and Hsp65-hIL-2were purified byaffinity chromatography. High level of specific IgG were detected in sera of guinea pigsafter twice immunization of20μg Ag85B-ESAT-6with MPLA. The proportion of CD4+and CD8+T cell significantly decreased in Ag85B-ESAT-6immunized guinea pigs after3times immunization.2. Evaluation of the subunit vaccine for its therapeutic efficacy in M. tuberculosisinfected guinea pig modelMethods: Guinea pigs were subcutaneously injected with105CFU M. tuberculosis, 6weeks post infection the bacterial loads in lung and spleen of guinea pig were measuredby plate count method to confirm the infection. Then guinea pigs were grouped intountreated (UN), immunotherapy (IM), chemotherapy (CH) and chemotherapy+immunotherapy (CH+IM), uninfected guinea pigs are for naive control (Naive).6postinfection, drinking water with108.5mg/L INH and105mg/L RFP were given5daysweekly for11weeks as chemotherapy.10weeks post infection,10μg Ag85B-ESAT-6＋10μg Hsp65-hIL-2＋5μg MPLA were injected subcutaneously twice with1dayinterval, and the second immunization were given3weeks later following same protocol.After treatment,1mg dexamethasone were injected3times a week for4weeks to inducethe recurrence of MTB infection. The body weight of guinea pigs were recorded weekly.Levels of anti-M. tuberculosis IgG, IgG1and IgG2antibodies in the sera of guinea pigswere measured by ELISA. The pathological change of lungs were observed by HEstaining. CD4+, CD8+T lymphocytes and B lymphocytes subpopulation in peripheralblood and spleen were measured by flowcytometry. The bacteria loads in spleen and lungwere measured by plate counting after the therapy and immunosuppression.Results: Tubercles were observed in the lung and spleen post6weeks infection andthe bacteria loads were5.82±0.29lgCFU and6.53±0.33lgCFU respectively.10weekspost infection, the mean body weight of UN group stopped increasing. Afterimmunosuppression, guinea pigs in IM group started to lose weight, all IM group diedbefore21weeks post infection. The anti-M. tuberculosis IgG levels in UN group andCH+IM group were significantly higher than UN group (P<0.001, P<0.001), whichdominated with IgG2subclass. The HE staining of lungs shows the UN group had a moresevere inflammation and pathological change compared with naive control, whereas twogroups with chemotherapy had a milder inflammation change compared with UN group.IM and CH+IM group had a higher level of T lymphocytes in blood and spleen, with asignificantly higher CD8+T cell. After treatment the bacteria loads in spleen from IM,CH and CH+IM group were significant lower than UN group (P<0.05, P<0.05, P<0.01),and the bacteria loads in lung CH and CH+IM group were significant lower than UNgroup (P<0.01, P<0.01). CH+IM group showed a significant lower bacteria loads in lungs than UN group after immunosuppression (P<0.05).3. Evaluation of the subunit vaccine for its therapeutic efficacy in M. tuberculosisinfected mouse modelMethods:105CFU M. tuberculosis H37Rv were injected in caudal vein ofC57BL/6J mice.4weeks post infection the bacterial loads in lung and spleen of micewere measured to confirm the infection. Then mice were grouped into untreated (UN),immunotherapy (IM), short-term chemotherapy (SC) and short-term chemotherapy+immunotherapy (SC+IM), long-term chemotherapy (LC) and long-term chemotherapy+immunotherapy (LC+IM), uninfected mice are for naive control (Naive).4weeks postinfection, the chemotherapy was given as same method in guinea pig experiment,4-weektreatment for short-term chemotherapy groups and11-week treatment for long-termchemotherapy groups.8weeks post infection, immunotherapy was implemented bysubcutaneous injections of5μg Ag85B-ESAT-6＋5μg Hsp65-hIL-2＋5μg MPLA,thesecond shot were given3weeks later. The SC course of treatment lasted4weeks and theLC course until the end of the experiment. The splenic lymphocytes were separatedsterilely and cultured with M. tuberculosis antigens. After treatment, the proportion ofCD4+and CD8+T cells expressing IFN-γ, IL-2, IL-4and TNF-α were measured byflowcytometry. The bacteria loads in spleen and lung of mice were measured by platecounting.Results:4weeks post infection, bacteria loads in spleen and lung of mice were3.67±0.10lgCFU and3.54±0.14respectively. The UN and IM group showed a higher levelof CD4+cells expressing IFN-γ in splenocytes than Naive group (P<0.001, P<0.001), aswell as a higher level of CD4+T cells expressing IL-2(P<0.01, P<0.01). Levels of CD4+T cells expressing IL-2in SC+IM and LC+IM group were significantly higher than SCand LC group respectively (P<0.001, P<0.001). All infected groups showed a decreasedtrendency in CD4+T cells expressing IL-4compared with Naive group. LC+IM groupshowed a lower level of CD4+T cells expressing TNF-α and a higher level CD4+Texpressing TNF-α cells in splenocytes compared with LC group (P<0.001, P<0.001). Asto the level of CD8+T cells expressing IFN-γ in splenocytes, no significant difference among7groups. Levels of CD8+T expressing IL-2cells in UN and IM group weresignificantly higher than Naive group (P<0.001, P<0.001) and SC+IM and LC+IMgroup higher than SC and LC group respectively (P<0.001, P<0.01). Levels of CD8+Tcells expressing IL-4in UN and IM group were higher than Naive group (P<0.001，P<0.001). Moreover, SC+IM group had a higher level of CD8+T cells expressing IL-4compared with SC group (P<0.05). SC+IM and LC+IM group showed a higher level ofCD8+T cells expressing TNF-α than Naive group (P<0.05，P<0.001), LC+IM groupwere higher than that of LC group(P<0.001). Chemotherapy significantly reducedbacteria loads in lung and spleen. Unfortunately, immunotherapy, either alone orcombined with chemotherapy, showed no impact on lung and spleen bacteria loads in M.tuberculosis infected mice.CONCLUSION:The subunit vaccine of Ag85B-ESAT-6and Hsp65-hIL-2with adjuvant MPLAeffectively induced guinea pigs to produce M. tuberculosis specific IgG antibody,enhanced the proportion of T cells in peripheral blood and spleen and reduce the bacterialoads in the lung of infected guinea pigs. When combined with chemotherapy,immunotherapy showed significant efficacy in inhibiting the recurrence of M.tuberculosis after immunosuppression.The subunit vaccine reimbursed the decreasing of CD4+and CD8+T cells secretingIL-2after chemotherapy in M. tuberculosis infected mice. Immunotherapy combinedwith chemotherapy reduced the proportion of CD4+T cells expressing TNF-α, whileinduced the CD8+T cells. Though immunotherapy, either alone or combined withchemotherapy, showed no significant impact on lung and spleen bacteria loads in M.tuberculosis infected mice in this study, thus further study need to be carried out todetermine its immunotherapeutic efficacy in mouse model.