Investigation Of The Effectiveness Of New Vaccines For Precise Prevention Of Tuberculosis And The Reaction Mechanism Of A New Screening Reagent Ec

Tuberculosis(TB),caused by Mycobacterium tuberculosis(MTB),is a chronic infectious disease transmitted predominantly via the respiratory route.TB is the major global public health challenges.Individuals newly infected with MTB and those with latent tuberculosis infection(LTBI)are the main sources of TB patients.Bacillus Calmette–Guérin(BCG),the only vaccine currently available for TB prevention,has a good protective effect in infants and young children.However,its efficacy decreases with age and cannot prevent the development of TB in those with LTBI.The vaccination status,the presence of sufficient protection after vaccination with BCG,and the occurrence of LTBI are the key factors that determine the TB prevention needs of different populations.Therefore,for TB prevention,our research group proposed that the population should be categorized into three types based on the differences in MTB infection and BCG immune status:LTBI population,BCG-unvaccinated or BCG-immunized individuals with immune memory turned negative(referred to as BCG immune-negative population),and BCG-immunized individuals with sustained immune memory(referred to as BCG immune-positive population).Various TB vaccines are being developed for different populations to formulate a precise immune prevention strategy for the entire Chinese population.Aiming at this strategy,our research group studied two new TB vaccines for population-level prevention:the AEC/BC02 vaccine containing innovative adjuvants for the prevention of TB in LTBI populations and the monoclonal BCG(BCG-S_Ⅲ)for TB prevention in BCG immune-negative populations.Preliminary studies have confirmed that AEC/BC02 can effectively inhibit the proliferation of MTB in animals with LTBI and have entered PhaseⅠclinical studies.The development of PhaseⅡandⅢclinical studies necessitates the understanding of clinical immunogenicity and potential risks of the vaccine.BCG-S_Ⅲvaccine has been shown to offer good protection in animals infected with sensitive MTB,but its protective effect against different drug-resistant and different genotypes of MTB remains uncertain.In addition,to resolve the screening problem of the three types of individuals in the above strategy,our research group devised the MTB-specific antigen ESAT6-CFP10(EC)skin test method,which can effectively identify MTB infection and BCG immunity.However,these studies have observed that the EC skin test reaction is mainly erythema,which is different from the conventionally purified protein derivate(PPD)skin test reaction,which is chiefly induration.Owing to the unclear mechanism of the clinical manifestations of this innovative product,doubts remain regarding the scientific validity of using erythema as a positive judgment criterion.The use of this test in large-scale screening for TB prevention in target populations is therefore limited.Based on the current research progress and the unresolved scientific issues of our research group,studies were conducted on the following three aspects:the immunogenicity and safety of the AEC/BC02 vaccine,the protective effect of the BCG-SⅢvaccine against MTB with different drug resistance and different genotypes,and the mechanism of EC skin test reaction.1.Studies on the immunogenicity and safety of the AEC/BC02 vaccineThe AEC/BC02 vaccine is the first recombinant protein vaccine for TB to enter clinical research in China and is also one of the 16 TB vaccines to enter clinical research globally.PhaseⅠclinical immunogenicity studies on the vaccine were conducted to provide a basis for formulating subsequent PhaseⅡandⅢclinical research plans.The results indicated that 5μg of AEC/BC02 vaccine can induce a certain level of cellular immune response,mainly Th1 type(IFN-γand IL-2)CD4~+T cell immune response,in healthy subjects when injected into the deltoid muscle of the upper arm.Moreover,the vaccine elicited a high level of humoral immune response and induced the secretion of antigen-specific Ig G and Ig G1 antibodies.PhaseⅡandⅢclinical studies on the AEC/BC02 vaccine will be conducted in the LTBI population.One of the biggest risks of preventive vaccine research in this population is that the vaccine causes severe Koch reactions.This risk is also the main reason for the termination of several international clinical trials on LTBI vaccines.For our independent innovative AEC/BC02 vaccine,avoiding this risk is a key challenge in PhaseⅡandⅢclinical research.This study intends to use the LTBI guinea pig model to examine a combination scheme for inhibiting the Koch reaction.This approach is expected to provide a scientific basis for the formulation of PhaseⅡandⅢclinical protocols for the vaccine.The findings showed that isoniazid(INH)and rifampicin(RFP)dual anti-TB drug(INH-RFP,IR)administered orally for 4 weeks,combined with three or six injections of the AEC/BC02 vaccine,can effectively inhibit Koch reaction and exert an enhanced protective effect without producing drug-resistant MTB.The immunogenicity and safety of the research have laid a scientific basis for the formulation of PhaseⅡandⅢclinical research plans for the vaccine.2.Studies on the protective effect of the BCG-SⅢvaccine against MTB with different drug resistance and different genotypesIn the early stage,our research group screened out a monoclonal BCG(BCG-S_Ⅲ)with a good protective effect against sensitive MTB from different early freeze-dried BCG strains available globally.This study further evaluated the immunogenicity of BCG-S_Ⅲvaccine and its protective effect against MTB strains with different drug resistance and different genotypes.The results established that immunization with BCG-S_Ⅲvaccine can elicit obvious Th1-type cellular immune responses,a certain degree of Th2-type humoral immune responses,and Th17-type immune responses.The BCG-S_Ⅲvaccine exerted a protective effect against multidrug-resistance,polydrug-resistant and RFP mono-resistant MTB strains,as well as strains with different genotypes such as Beijing family,T family and EAI family.The vaccine significantly reduced the spleen and lung bacterial load and the degree of organ lesions in animals infected with different strains.This study is the first to demonstrate the broad-spectrum protective effect of BCG-S_Ⅲvaccine against MTB strains with different drug resistance and different genotypes.3.Mechanism studies on the skin test reactions of TB precision prevention new screening reagent ECThe EC skin test,a novel diagnostic tool for MTB infection,provides an effective and convenient screening method for precise immune prevention strategies against TB.Clinical studies have observed that while the conventional PPD skin test causes mainly induration,the EC skin test can produce induration,but is chiefly erythema.If induration is simply used as an indicator of EC,misdiagnosis and missed diagnosis may occur,thereby reducing the sensitivity of the diagnosis.Owing to the unclear mechanism of EC erythema in the diagnosis of MTB infection,misgivings prevail regarding the clinical application of this innovative product.In this study,systematic comparative research was performed on the mechanisms of EC erythema and PPD induration using the MTB-sensitized guinea pig model.The findings signified that compared with PPD induration,EC erythema was consistent with the characteristics of delayed-type hypersensitivity(DTH).The major immune pathways were similar for both reactions,indicating that the erythema produced by EC has the main DTH immune characteristic of PPD induration.The diagnostic significance of EC erythema was mechanistically proven,which offered theoretical support for its clinical application.In summary,this study targeted individuals with LTBI using the AEC/BC02vaccine.The PhaseⅠclinical research confirmed that the vaccine displayed good immunogenicity in healthy individuals.The LTBI guinea pig model confirmed that IR combined with AEC/BC02 vaccine had a good safety profile.These results provide a firm scientific basis for the formulation of PhaseⅡandⅢclinical research plans for the vaccine.BCG-S_Ⅲvaccine was used for the prevention of the BCG immune-negative population.The study showed that the BCG-S_Ⅲvaccine had good immunogenicity and a broad-spectrum protective against MTB with different drug resistance and different genotypes.The clinical response characteristics of the new screening reagent for the precise prevention of TB are different from that of conventional reagent.Systematic comparative studies showed that the main immune mechanism of the erythema produced by EC was similar to that of the induration-induced DTH produced by conventional PPD.This observation provides a strong scientific basis for using EC erythema as a clinical diagnostic indicator and justifies the application of this method for the screening of MTB-infected individuals for the precise prevention of TB.