Tuberculosis (TB) is a chronic wasting infectious disease caused by Mycobacterium tuberculosis infection that poses a serious threat to human health and life. The World Health Organization (WHO) estimates that if TB is not effectively controlled, 1 billion people will be infected with Mycobacterium tuberculosis between 2000 and 2020, 200 million people will develop TB disease, and 35 million people will die from the disease. High levels of latent infection with Mycobacterium tuberculosis and co-infection with HIV make prevention, diagnosis, and treatment of tuberculosis very difficult.
BCG is a live attenuated vaccine developed by French scientists Camille Guerin and Albert Calmette by growing a strong strain of Mycobacterium tuberculosis in vitro for a long period of time, and has been used in humans since 1921, with more than 3 billion people vaccinated. Currently, BCG is still widely used in developing countries as the only TB vaccine. Although BCG has made important contributions to the prevention of tuberculosis, such as effectively helping to prevent infectious diseases such as tuberculosis in children, there are many problems, such as the large controversy over the immune protection of BCG against tuberculosis in adults, with a protection rate of 0-80% and generally 40% to 60%. Other studies have shown that BCG is blocked by the body’s pre-existing immune response in populations with prior exposure to environmental mycobacteria or BCG vaccination, resulting in immune failure; therefore, BCG is only effective in populations that have neither been infected with mycobacteria nor vaccinated with BCG.
Therefore, the impact of Mycobacterium tuberculosis on human health and the many problems of BCG vaccine make the development of a new vaccine for tuberculosis very urgent.
I. Research strategies and progress of tuberculosis vaccines
Currently, there are two main directions of tuberculosis vaccine research, one is to modify the existing BCG vaccine, such as developing recombinant BCG vaccine; the other is to develop effective new vaccines, such as constructing subunit vaccines (including protein peptide and DNA vaccines) and virus-based vaccines.
A single immunization with a TB vaccine alone is usually not sufficient to produce good immune protection. Therefore, on the basis of the initial immunization, a booster immunization with the same or a different vaccine is used for the purpose of enhancing immunity, i.e., an initial booster immunization strategy. This strategy is important for improving the immune efficacy of TB vaccines, especially for improving immune protection in adults with TB. This strategy can be divided into a homologous initial-booster immunization strategy (initial and booster immunization with the same vaccine) and a heterologous initial-booster immunization strategy (initial and booster immunization with different vaccines). The heterologous initial-booster immunization strategy has been reported to elicit a more efficient cellular immune response than the homologous initial-booster immunization strategy and is likely to be the most desirable approach for future TB vaccine immunization. There are two main TB vaccine options applied in the heterologous initial booster immunization strategy: initial immunization with DNA vaccine and booster immunization with viral vector vaccine; and initial immunization with BCG vaccine and booster immunization with subunit vaccine. The main TB vaccines studied so far are recombinant BCG vaccine, protein peptide vaccine, DNA vaccine and live vector vaccine.
1. Recombinant BCG vaccine: Although BCG is safe and effective in children, immune protection in adults is more controversial. With the goal of improving the immune protection of BCG in adults, researchers have done a lot of work, such as introducing genes encoding TB immunodominant antigens or immunostimulatory cytokines into BCG to construct recombinant BCG vaccines, which effectively enhance the immune protection of BCG. In addition, since the inoculated BCG vaccine is easily persisted in phagosomes after phagocytosis and is inactive, it weakens its stimulatory effect on immune cells, thus affecting the intensity of immune response. Therefore, the construction of recombinant BCG vaccine that induces the release of BCG from phagosomes is also a measure to improve immunity. A recombinant BCG vaccine expressing PfoA has been constructed, and PfoA can induce the disintegration of phagosomes and release recombinant BCG into the cytoplasm to enhance the stimulatory effect on CD8+ T lymphocytes, thus improving the level of cellular immune response.
However, the study of recombinant BCG faces a challenge that the general bacterial expression system cannot function well for gene expression in BCG, which poses a great difficulty for constructing expression vectors for BCG. To solve this problem, many studies have been done on the transcriptional signatures of Mycobacterium tuberculosis to provide a good basis for understanding the transcription and expression of tuberculosis genes and the differences between the promoters of Mycobacterium tuberculosis and other bacteria as well as to solve the expression problem. Recombinant BCG vaccines constructed with expression vectors containing the Mycobacterium tuberculosis promoter can efficiently express some immunodominant antigens, such as Ag85A, Ag85B, Ag85C, and 6×103 early secretory target antigen protein (ESAT6). In addition, recombinant expression of cytokines such as IFN and IL-12 in BCG can increase the level of immune response, but there are also findings that too high local cytokines can instead cause pathological damage to the organism.
The recombinant BCG vaccines currently entering clinical trials are recombinant expression of Ag85A vaccine (r BCG 30) and recombinant expression of Listeria monocytogenes lysate, urease deficient BCG (VPM 1002)c9]. Although the r BCG vaccine VPM 1002 has completed phase I clinical trials in the United States, and the developers have indicated that the vaccine has good immune tolerance and immunogenicity, no encouraging results have yet emerged.
2. Protein peptide vaccine: Protein peptide vaccine not only has good safety, but also can specifically induce the activation of CD4+ Th1 cells and CD8+ cytotoxic T lymphocytes, which is one of the more ideal vaccine forms. The choice of antigen and the application of adjuvant may play a decisive role in its development.
The proteins or peptides used as vaccine candidates are selected from immunodominant antigens containing human T lymphocyte recognition epitopes, mainly secreted proteins and cell wall components of Mycobacterium tuberculosis, such as ESAT-6, Ag85, MTB39 and MTB32. Single antigens are not sufficient to elicit effective immune protection due to their narrow antigenic spectrum; therefore, mixing or fusion expression of multiple antigens is a measure to improve vaccine immune protection. Studies have shown that fusion proteins induce immune protection more effectively than mixtures of multiple antigens, but the mechanism is unclear and may be related to antigen stability as well as uptake and delivery efficiency. Other studies have shown that there may be competition between multiple antigens, thereby reducing the immunogenicity and immunoprotection of the vaccine. Therefore, it is necessary to examine the immune effects of single and mixed antigens separately when designing multi-component antigen vaccines.
Protein peptide vaccines require effective adjuvant adjuvant to induce the desired immune response, and different adjuvants can elicit different types of immune responses; therefore, finding suitable adjuvants is an important issue to be addressed in the development of protein peptide vaccines. Some progress has been made in related research, such as bacterial toxins, cytokines, liposomes and CpG DNA have shown good immune enhancing effects in experimental animals.
The peptide vaccines currently in clinical trials are, Ag85B and ESAT-6 mixed peptide (Hybrid 1-IC31) with IC31 as adjuvant, Ag85B and ESAT-6 mixed peptide (Hybrid1-CAF01) with CAF01 as adjuvant, MTB39 and MTB32 fusion protein (M72) and Ag85B and TB10.4 (Hybrid 4/AERAS 404-IC31), all of which are used as booster vaccines, and the first two are also expected to be the first doses of vaccine, with the combination of adjuvants enhancing the immune effect of these vaccines. preliminary results from clinical trials of the M72 vaccine suggest that it is safe and effective in adults, with mild adverse effects and an immune response that lasts up to 6 The immune response can last for 6 months.
3. DNA vaccines: DNA vaccines are recombinant plasmid vectors encoding an antigenic protein that are introduced into the host to synthesize the antigenic protein through the expression system of the host cells, thereby enabling the host to acquire immunity to that antigen for the purpose of disease prevention and treatment. DNA vaccines can induce immune protection against a variety of viruses, bacteria, and parasites, among other pathogens, as well as against tuberculosis in animal models. CD4+ and CD8+ T lymphocyte immune responses.
The advantages of this vaccine are.
(i) the intracellularly generated antigen is easily presented by major histocompatibility complex (MHC) class I and II molecules, which activate CTL and Th;
②The immune response is more durable;
③Stable, easy to prepare and store;
④Does not induce an immune response against the vector and can be used for repeated booster immunization;
⑤ Safe for use in immunodeficient hosts. In addition, the DNA vaccine provides excellent protection in CD4+ T lymphocyte-deficient mice, which provides a reference for immunization strategies in HIV-positive TB-susceptible high acenocouple populations.
Although DNA vaccines can be administered without adjuvants, adjuvants can significantly improve the immune efficacy of DNA vaccines and reduce the amount and frequency of vaccine administration, and almost all adjuvants used in protein peptide vaccines have been tried for DNA vaccines with some progress.
Several TB DNA vaccines have been used to induce effective immune protection in mice, such as ESAT-6, Ag85A/B, Mtb 8.4, Mtb 41, MPT 39, MPT 63, MPT 64, MPT 83, and hsp65. However, the protective effect of DNA vaccine is generally only 50% to 80% of that of BCG vaccine under the same experimental conditions, and its protective ability can be improved by combining multiple DNA vaccines or constructing expression vectors for multiple antigens. In addition, the immunogenicity of DNA vaccines against large mammals is generally poor, probably due to the low efficiency of plasmid transfection in vivo. The immunogenicity can be enhanced by using methods such as in vivo electroshock, non-toxic bacteria or viruses as vectors.
4, live vector vaccine: live vector vaccine uses microorganisms that are safe for the body as carriers and continuously expresses TB-specific antigens in vivo, which has the advantages of economy and high efficiency, in addition, live vectors have certain adjuvant effects. Commonly used live vectors are poxvirus vectors, adenovirus vectors, etc. These vaccines are usually used in booster immunizations to enhance the T-lymphocyte immune response. The disadvantage of live vector vaccines is that the body’s pre-existing immune response can prevent the immune antigenic response, thus weakening the efficacy of the vaccine.
Currently, live virus vector vaccines entering clinical trials include the recombinantly modified Ankara poxvirus vector vaccine A expressing Ag85 (OxfordMVA85A/AERAS 485), the replication-deficient adenovirus vector 35 vaccine expressing Ag85A, Ag85B and TBlO.4 (AERAS 402/CrucellAd35) and the The replication-deficient adenovirus vector 5 vaccine expressing Ag85A (AdAg85A), all designed as booster vaccines for BCG and recombinant BCG.Oxford MVA85A/AERAS 485 has been shown to have good safety and immunogenicity in different populations.AERAS 402/Crucell Ad35 induces CD4+ and CD8+ T-lymphocyte response without serious adverse effects.
5. Other tuberculosis vaccines: heat-inactivated Mycobacterium mare (M-vaccae) is a vaccine derived from environmental mycobacteria and contains many antigens of Mycobacterium tuberculosis. Phase III clinical trials have been completed in BCG-vaccinated HIV-infected patients in Tanzania. The M. smegmatis vaccine is a whole cell extract of M. smegmatis that shares antigens with Mycobacterium tuberculosis and was developed by the Chinese Institute for the Control of Pharmaceutical and Biological Products.
In addition, the BCG mutant strain has a certain effect on inhibiting the maturation of phagosomes and can perform antigen processing and presentation more effectively to enhance immune protection. Callewaert et al. constructed a BCG mutant strain, which has better immune protection than BCG in mice and has higher safety.
II. Problems of TB vaccine development
1. Lack of detection indicators related to immune protection: The clinical protection of most marketed vaccines can be tested by alternative endpoints, for example, by detecting the level of vaccine-induced antibodies to determine the immune protection of the vaccine, but no relevant indicators were found for the immune protection of TB vaccines. Although studies have confirmed that CD4+ and CD8+ and other T lymphocytes can produce different types of cytokines after TB infection and immunization, and new immunoassay techniques have been used in recent years to identify and quantify T lymphocyte responses that secrete specific cytokines, these studies have not found a correlation between a specific cell or cytokine response and natural infection or immune protection.
Lack of suitable animal models: The evaluation of the same TB vaccine candidate varies between animal models, and Reed et al. found that a TB-specific antigen achieved good immunity in mice and guinea pigs, but failed in non-human primates. Therefore, the existing animal models confirm that an immunoprotective TB vaccine does not guarantee good immunoprotection in humans.
3. Lack of economic, sensitive and specific diagnostic methods for tuberculosis: The effectiveness of tuberculosis vaccines is tested based on clinical endpoints. Currently, the widely used clinical diagnostic methods for TB include tuberculin skin test and bacterial culture, but these methods have the disadvantages of poor specificity and inability to detect latent infection; the diagnostic method of fluorescent quantitative PCR is sensitive but cannot detect latent infection; the IFN-γ in vitro release test can effectively solve the problem, but its promotion is limited by the high cost of TB, which is mainly prevalent in developing countries. Therefore, the construction of an economic, sensitive and specific diagnostic method for TB is necessary to evaluate the clinical protection of the vaccine.
4. HIV infection: An important issue facing the development of all vaccines is the safety and efficacy in HIV-infected populations, which are also at high risk for TB. WHO estimates that at least 6 million people worldwide have both TB and AIDS, and in some African countries, TB is the leading cause of death in AIDS patients. Vaccination with BCG vaccine can cause their infection and there is a great insecurity, therefore, it becomes another major challenge for the development of new TB vaccines.
III. Outlook
The incidence and mortality rates of tuberculosis have rebounded dramatically in recent years due to HIV infection, widespread use of immunosuppressive drugs, the prevalence of multidrug-resistant tuberculosis, and an aging population, making prevention, diagnosis, and treatment of tuberculosis very difficult. The successful eradication of smallpox suggests that a vaccine is an effective way to completely eradicate an infectious disease, and WHO has set a goal of complete elimination of TB worldwide (less than 1 case per million people) by 2050 based on progress in research on new TB vaccines. At least six TB vaccine candidates completed phase I clinical trials in 2009, and three vaccines are currently in phase II clinical trials, most of which are being developed as booster vaccines for BCG. WHO estimates that a new TB vaccine will be approved for marketing between 2014 and 2015, and hopes to have sufficient financial resources and products in reserve to meet the goal of achieving high coverage rapidly once the vaccine is available. With the development of genomics as well as proteomics, the understanding of the pathogenesis of Mycobacterium tuberculosis and the immune response against infection is also improving, providing a theoretical basis for the research of a more cost-effective TB vaccine to deal with TB, which is a serious threat to human health.