Enhancement of mucosal immune response against HIV-1 Gag by DNA immunization

In order to examine the feasibility of Gag-expression DNA as a potential candidate for HIV vaccine using a mouse model, we injected DNA into mice either intramuscularly or by using a gene gun. Both methods induced a low level of antibody production. However, after booster immunization with p24 protein emulsified with complete Freund's adjuvant via a footpad, we found that only the preceding intramuscular DNA immunization induced an anti-Gag Th1-type (IgG(2a)) antibody response, in addition to the enhancement of a Th2-type (IgG(1)) antibody response. Importantly, when mice were boosted intranasally with p24 and cholera toxin, intramuscular DNA injection was found to enhance both systemic and mucosal Gag-specific immune responses. These results indicate that intramuscular DNA immunization confers the inducibility of memory cells, which circulate around various mucosal tissues. Therefore, intramuscular DNA priming, followed by a mucosal booster immunization, could be considered as a regimen applicable to HIV vaccine.     

DNA immunization of mice with a plasmid encoding Neisseria gonorrhea PorB protein by intramuscular injection and epidermal particle bombardment

Immunogenicity of a DNA vaccine encoding PorB from Neisseria gonorrhoeae strain FA1090 was analyzed in BALB/C mice immunized by intramuscular needle injection or epidermal gene gun bombardment. Both delivery routes generated measurable specific antibodies although the gene gun response was slower. Antibody isotypes were indicative of Th2 activation following gene gun immunization and of Th1 activation following intramuscular injection. In both immunization protocols, boosting with either renatured recombinant (rr) PorB protein or PorB expressed from Venezuelan equine encephalitis virus replicon particles (VRPs) significantly increased anti-PorB antibody levels. Boosting with rrPorB protein had little effect on antibody isotypes, while boosting with VRPs expressing PorB-enhanced a Th1 type response. Whole cell binding experiments showed that a portion of the antibodies recognized the surface of the homologous N. gonorrhoeae strain. Serum from groups with high antibody levels showed some opsonization of the homologous strain using human neutrophils. These results showed the potential of DNA vaccination for the purpose of priming an antibody response against PorB of N. gonorrhoeae. When combined with a protein or VRP boost, DNA priming resulted in high-titer and long-lasting responses. Based on different prime-boost protocols, we could polarize immune responses to predominantly Th1 or Th2, which should enable future studies of the types of immune responses that are protective in mouse models of gonorrhea.     

Leishmania mexicana Gp63 cDNA Using Gene Gun Induced Higher Immunity to L. mexicana Infection Compared to Soluble Leishmania Antigen in BALB/C

Background

Leishmaniasis is a worldwide disease prevalent in tropical and sub tropical countries. Many attempts have been made and different strategies have been approached to develop a potent vaccine against Leishmania. DNA immunisation is a method, which is shown to be effective in Leishmania vaccination. Leishmania Soluble Antigen (SLA) has also recently been used Leishmania vaccination.

Methods

The immunity generated by SLA and L. mexicana gp63 cDNA was compared in groups of 6 mice, which were statistically analysed by student t- test with the P-value of 0.05. SLA was administered by two different methods; intramuscular injection and injection of dendritic cells (DCs) loaded with SLA. L. mexicana gp63 cDNA was administered by the gene gun.

Results

Immunisation of BALB/c mice with L. mexicana gp63 resulted in high levels of Th1-type immune response and cytotoxic T lymphocytes (CTL) activity, which were accompanied with protection induced by the immunisation against L. mexicana infection. In contrast, administration of SLA, produced a mixed Th1/Th2-type immune responses as well as a high level of CTL activity but did not protect mice from the infection.

Conclusion

The results indicate higher protection by DNA immunisation using L. mexicana gp63 cDNA compared to SLA, which is accompanied by a high level of Th1 immune response. However, the CTL activity does not necessarily correlate with the protection induced by the vaccine. Also, gene gun immunisation is a potential approach in Leishmania vaccination. These findings would be helpful in opening new windows in Leishmania vaccine research.     

All-trans retinoic acid biases immune response induced by DNA vaccine in a Th2 direction

Vitamin A deficiency diminishes Th2-mediated Ab responses. Providing Vitamin A or its active metabolites reverses this defect. All-trans retinoic acid (ATRA), an acid derivation of Vitamin A, regulates the balance of immune response induced by TR421-hCGbeta DNA vaccine. Compared to DNA vaccine alone or treatment with vehicle, significantly higher level of antibody against the protein encoded by DNA vaccine was observed in mice 6 weeks after the first immunization. The IgG2a/IgG1 ratio was lower in mice treated with ATRA. We also found that treatment with ATRA also diminishes specific cellular immune response induced by gene immunization by measuring the marker of cellular immune response. We conclude that ATRA biases the immune response to Th2 direction induced by DNA vaccine and acts as a candidate adjuvant and immunomodulatory molecule.     

Gene gun DNA vaccination with Rev-independent synthetic HIV-1 gp160 envelope gene using mammalian codons.

DNA immunization with HIV envelope plasmids induce only moderate levels of specific antibodies which may in part be due to limitations in expression influenced by a species-specific and biased HIV codon usage. We compared antibody levels, Th1/Th2 type and CTL responses induced by synthetic genes encoding membrane bound gp160 versus secreted gp120 using optimized codons and the efficient gene gun immunization method. The in vitro expression of syn.gp160 as gp120 + gp41 was Rev independent and much higher than a classical wt.gp160 plasmid. Mice immunized with syn.gp160 and wt.gp160 generated low and inconsistent ELISA antibody titres whereas the secreted gp120 consistently induced faster seroconversion and higher antibody titres. Due to a higher C + G content the numbers of putative CpG immune (Th1) stimulatory motifs were highest in the synthetic gp160 gene. However, both synthetic genes induced an equally strong and more pronounced Th2 response with higher IgG1/IgG2a and IFNgamma/IL-4 ratios than the wt.gp160 gene. As for induction of CTL, synthetic genes induced a somewhat earlier response but did not offer any advantage over wild type genes at a later time point. Thus, optimizing codon usage has the advantage of rendering the structural HIV genes Rev independent. For induction of antibodies the level of expression, while important, seems less critical than optimal contact with antigen presenting cells at locations reached by the secreted gp120 protein. A proposed Th1 adjuvant effect of the higher numbers of CpG motifs in the synthetic genes was not seen using gene gun immunization which may be due to the low amount of DNA used.     

Effective induction of HIV-specific CTL by multi-epitope using gene gun in a combined vaccination regime

Reliable and effective induction of cytotoxic T-lymphocytes (CTL) is one of the prime objectives of vaccine research. Previously, novel HIV vaccine candidates were constructed as a string of CTL epitopes (20 human, 3 macaque and 1 mouse) delivered using a DNA vector [Hanke T, Schneider J, Gilbert SG, Hill AVS, McMichael A. DNA multi-CTL epitope vaccines for HIV and Plasmodium falciparum: immunogenicity in mice. Vaccine 1998;16:426-435.] or modified vaccinia Ankara (MVA [Hanke T, Blanchard TJ, Schneider J, Ogg GS, Tan R, Becker MSC, Gilbert SG, Hill AVS, Smith GL, McMichael A. Immunogenicities of intravenous and intramuscular administrations of MVA-based multi-CTL epitope vaccine for HIV in mice. J Gen Virol 1998;79:83-90.]), i.e. vaccine vehicles acceptable for use in humans. In mice, a single intramuscular (i.m.) needle injection of either vaccine alone elicited good CTL responses. Here, it is demonstrated that the multi-epitope DNA also induced CTL when delivered intradermally using the Accell gene gun. The CTL responses increased after re-immunization and after three deliveries were comparable to those induced by a single i.m. injection. Recent evidence indicates that combining routes and vaccine vehicles enhances the immunogenicity of vaccine-delivered or -encoded antigens. Here, it is shown that administration of DNA by an i.m. priming/gene gun boosting more efficiently induced CTL than gene gun priming/i.m. boosting. A similar increment was obtained by sequential vaccinations using a gene gun-delivered DNA followed by recombinant MVA. Thus particular sequences of routes or vaccine vehicles rather than simple prime-boost delivery of a single vaccine is critical for an effective elicitation of CTL.     

Modulation of Th1/Th2 immune responses to HIV-1 Tat by new pro-GSH molecules

We have previously demonstrated that in Ova-immunized mice the increase in intra-macrophage thiol pool induced by pro-GSH molecules modulates the Th1/Th2 balance in favour of a Th1-type immune response. We show now that the same molecules can support a Th1-type over Th2-type immunity against Tat, which is an early HIV-1 regulatory protein and a Th1 polarizing immunomodulator that is increasingly considered in new anti-HIV vaccination strategies. Our results indicate that Tat-immunized mice pre-treated with the C4 (n-butanoyl) derivative of reduced glutathione (GSH-C4) or a pro-drug of N-acetylcysteine (NAC) and beta-mercaptoethylamine (MEA) (I-152), have decreased levels of anti-Tat IgG1 as well as increased levels of anti-Tat IgG2a and IgG2b isotypes suggesting a Th1-type response. Moreover, Th1-(IFN-γ and IL-2) Ag-specific cellular responses were detected by ELISPOT assay in splenocytes of the same animals as well as an increase of IL-12 levels in the plasma. These findings suggest that the Th1 immune response to HIV-1 Tat could be further polarized by these molecules. These results together with those previously reported suggest that pro-GSH molecules could be used to modulate the immune response towards different antigens and may be further exploited for inducing specific Th1 immune responses against other HIV antigens as well as other intracellular pathogens in new Tat-based vaccination protocols.     

Efficacy of DNA-hsp65 vaccination for tuberculosis varies with method of DNA introduction in vivo

A DNA vaccine codifying the mycobacterial hsp65 can prevent infection with Mycobacterium tuberculosis in a prophylactic setting and also therapeutically reduce the number of bacteria in infected mice. The protective mechanism is thought to be related to Th1-mediated events that result in bacterial killing. To determine the best method of hsp65 introduction for vaccination efficacy against tuberculosis (TB), we evaluated the immunogenicity and protection of DNA-hsp65 administered by gene gun bombardment or intramuscular (i.m.) injection of naked DNA. Immunization by gene gun induced immune response with plasmid doses 100-fold lower than those required for intramuscular immunization. However, in contrast to intramuscular immunization, which was protective in these studies, gene gun immunization did not protect BALB/c mice against challenge infection.     

Potent, long lasting systemic antibody levels and mixed Th1/Th2 immune response after nasal immunization with malaria antigen loaded PLGA microparticles

The immunogenicity of the synthetic malaria vaccine SPf66 has been recently improved by the application of new adjuvants as QS-21 saponin or poly-D,L-lactide-co-glycolide (PLGA) polymers. The search for less invasive administration routes made us test the immunogenicity of SPf66-loaded microparticles by the nasal route in Balb/c mice. We report here that the intranasal administration of the adequate PLGA vaccine formulations greatly improves and maintains higher antibody levels compared to the conventional alum adjuvant and to the administration of the particles by other routes (subcutaneous, oral). Systemic immune responses were characterized as mixed Th1/Th2-type: IFN-gamma and IgG2a isotype were found as signs of Th1 activation, whilst IgE and IgG1 secretions indicate Th2 response. Since both types of response have been associated to protective immunity in malaria, we postulate that this new approach supposes an advantage over the traditional adjuvants and routes.     

Prototype Alzheimer's disease epitope vaccine induced strong Th2-type anti-Abeta antibody response with Alum to Quil A adjuvant switch

Beta-amyloid (Abeta) peptide has been proposed to be a causal factor in Alzheimer's disease (AD). Currently being investigated, active and passive Abeta-immunotherapy significantly reduce Abeta plaque deposition, neuritic dystrophy, and astrogliosis in the brains of APP transgenic (APP/Tg) mice. Immunization with Abeta42 formulated in the Th1-type adjuvant QS21 was beneficial for AD patients with significant titers of anti-Abeta antibodies, however, 6% of participants developed meningoencephalitis, likely due to anti-Abeta-specific autoimmune Th1 cells. Thus, successful Abeta vaccination requires the development of strong antibody responses without Th1-type cellular immunity. In this study, we compared the induction of humoral immune responses with Th1-type (Quil A) and Th2-type (Alum) adjuvants singly and in combination, using our novel epitope vaccine composed of self B cell epitope Abeta(1-15) and foreign T cell epitope PADRE (PADRE-Abeta(1-15)-MAP). Formulated in Quil A, this vaccine resulted in significantly higher anti-Abeta antibody responses in both BALB/c (H-2d) and C57BL/6 (H-2b) mice, compared with Alum. Anti-Abeta antibodies induced by Alum were predominantly IgG1 type accompanied by lower levels of IgG2a and IgG2b. Quil A induced robust and almost equal titers of anti-Abeta antibodies of IgG1 and IgG2a isotypes and slightly lower levels of IgG2b. Switching adjuvants from Alum to Quil A induced higher concentrations of antibodies than injections with Alum only, however slightly lower than Quil A only. Switching both adjuvants did not change the profile of antibody responses generated by the initial adjuvant injected. These results suggest that switching from Alum to Quil A would be beneficial for AD patients because anti-Abeta antibody production was enhanced without changing the initially generated and likely beneficial Th2-type humoral response.     

Multivalent DNA vaccine protects mice against pulmonary infection caused by Pseudomonas aeruginosa

For efficacious vaccine development against Pseudomonas aeruginosa (P. aeruginosa), the immunogenicity of multivalent DNA vaccine was evaluated. Three different plasmids each targeting a fusion of outer membrane proteins (OprF/OprI), a protein regulating type III secretion system (PcrV), or an appendage (PilA) were prepared and mice were immunized with single (monovalent) or a combination of these plasmids (multivalent) via intramuscular electroporation (imEPT) or gene gun. Immunization with multivalent DNA vaccine via imEPT induced the most potent protection against lethal pneumonia. Although the serum levels of IgG binding to whole bacteria cells were comparable between groups, the strongest immune protection was associated with the serum levels of Th1-dominated multivalent IgG, the bronchoalveolar levels of macrophage inflammatory protein 2 (MIP-2) and IFN-gamma, and the number of neutrophils and macrophages in the bronchoalveolar lavage following intranasal challenge. These results implied the possible clinical application of multivalent DNA vaccine against P. aeruginosa.     

Respiratory syncytial virus infection of gene gun vaccinated mice induces Th2-driven pulmonary eosinophilia even in the absence of sensitisation to the fusion (F) or attachment (G) protein

Complete protection against respiratory syncytial virus (RSV) infection was induced in mice vaccinated on two occasions with 2.5 microg of DNA, encoding the fusion (F) protein of RSV, precipitated onto gold microbeads. In contrast, immunisation with DNA encoding the attachment (G) protein of RSV resulted in a significant reduction in viral load following infection, but did not afford complete protection. Gene gun delivery of DNA-F elicited a T helper-2 (Th2) biased immune response that could not be modulated by the co-delivery of plasmids encoding IL-2, IL-12 or IFNgamma. Similarly gene gun delivery of DNA-G primed a Th2 response. Thus, all gene gun vaccinated mice produced a predominant Th2 biased pulmonary immune response characterised by the production of IL-4 and IL-5 with little IFNgamma following RSV challenge. Analysis of bronchoalveolar lavage (BAL) cells, 5 days post challenge, indicated that there was only a two-fold increase in the number of inflammatory cells in vaccinated compared with control animals. Despite the strong Th2 cytokine bias of lung lymphocytes and the predominant recruitment of CD4(+) T cells, following challenge, there was not a marked pulmonary eosinophilic response (range from 2 to 7% of BAL). In contrast, the BAL from mice vaccinated with control plasmid contained significantly more eosinophils than any other group.     

Th1-type epitopes-based cocktail PDDV attenuates hepatic fibrosis in C57BL/6 mice with chronic Schistosoma japonicum infection

Schistosomiasis is one of the world's major public health problems in terms of morbidity and mortality, which is characterized by a marked egg-induced CD4⁺ T-cell programmed granulomatous inflammation and cumulative fibrosis. Here PDDV (peptide–DNA dual vaccine), a widely used non-viral gene delivery system, was applied. The cocktail PDDV, based on four Th1-type epitope peptides identified from Schistosoma japonicum vaccine candidates and CpG ODN1826, could induce dominant Th1-type response in C57BL/6J mice (P < 0.05). The histopathological staging and collagen assessment for fibrosis showed that the cocktail PDDV presented an obvious down-regulation effect on hepatic fibrosis caused by chronic S. japonicum infection (P < 0.05), and IFN-γ, IL-4 and IL-13 mRNAs in liver detected by RT-PCR also showed that the cocktail PDDV represented the ability to up-regulate Th1-type responses, which paralleled with a decrease expression of α-SMA (P < 0.05) and the up-regulated MMP9/TIMP1 balance (P < 0.05) when compared to the control groups. Therefore, it is indicated that the cocktail PDDV can significantly attenuate hepatic fibrosis, in parallel with the decreased HSCs activation and the up-regulated MMP9/TIMP1 balance in favor of matrix degradation, which may be partially dependent on the increased Th1 response to restore the Th1/Th2 balance.     

Interleukin-18 enhances Th1 immunity and tumor protection of a DNA vaccine

DNA vaccines show efficacy in many preclinical models, but these results have not yet translated to consistent clinical efficacy. Co-administration of molecularly encoded adjuvants is one approach that may enable DNA vaccines to achieve enhanced immune response induction in humans. Interleukin-18 (IL-18) is a Th1-type cytokine that has been shown to augment the activity of DNA vaccines in some preclinical models. A prostate-specific antigen (PSA) DNA vaccine was tested in a mouse tumor model system to explore the impact of co-administration of a pIL-18 plasmid. Low doses of the pPSA vaccine were not capable of inducing tumor protection, but when pIL-18 was co-administered, complete tumor protection was observed in all mice. Tumor protection was mediated by both CD4(+) and CD8(+) T cells. Detailed analysis of the immune response in mice immunized with either pPSA or pPSA/pIL-18 demonstrated that pIL-18 skewed the PSA-specific immune response toward Th1. More importantly, stronger CD4(+) and CD8(+) T cell responses developed in the pPSA/pIL-18-immunized mice, with faster kinetics. These results suggest that IL-18 is a powerful adjuvant molecule that can enhance the development of antigen-specific immunity and vaccine efficacy.     

Vaccination of mice with a cocktail DNA vaccine induces a Th1-type immune response and partial protection against Schistosoma japonicum infection.

Several defined vaccine candidate antigens of Schistosoma japonicum have shown promise in large animal vaccination experiments. However, vaccination of mice in the laboratory with either single recombinant antigens or DNA encoding forms of the individual antigens has so far failed to induce significant protection against S. japonicum cercarial challenge infection as judged by worm reduction, although specific antibodies were generated. This is in contrast to the results achieved using radiation-attenuated vaccines which are highly protective. Even in large animal vaccination experiments, the protection levels obtained with single defined antigens were far below those achieved using the attenuated vaccines. One possible interpretation is that the immune responses induced by single antigen vaccination may not be strong enough to combat the challenging infection. We, therefore, carried out mouse vaccination experiments using a cocktail DNA vaccine comprising four DNA plasmids encoding four different S. japonicum antigens, Sj62, Sj28, Sj23 and Sj14-3-3, respectively. We, also investigated whether co-injection of the mouse IL-12 encoding plasmid with the cocktail DNA vaccine was able to enhance the Th1 responses and hence the protective immunity. Three intramuscular injections of the cocktail DNA vaccine induced a significant Th1-type cellular response with high level of IFN-gamma production by splenocytes upon in vitro stimulation with recombinant antigens. Importantly, significant IgG antibody responses were also induced against crude worm antigens. In two out of three experiments, significant resistance (34-37 and 44-45%, respectively) was demonstrated while another experiment did not show any protection against S. japonicum cercarial challenge infection. Co-injection of the IL-12 encoding DNA did not further enhance these responses, nor the level of resistance, compared with the cocktail DNA alone.     

Innate IL-10 promotes the induction of Th2 responses with plasmid DNA expressing HIV gp120

Like most DNA vaccines, intramuscular immunization with plasmid DNA coding for influenza virus haemagglutinin (HApDNA) induced Th1 responses and IgG2a antibodies in mice. However, plasmid DNA coding for HIV gp120 (gp120pDNA) induced Th2-biased responses and predominantly IgG1 antibodies. Responses to gp120pDNA switched to a Th1-type in IL-10-defective mice and to exclusively IgG2a antibodies in IL-4-defective mice. Conversely, antigen-specific IFN-gamma production induced by gp120pDNA or HApDNA was reduced in IL-12-defective mice, whereas addition of plasmid DNA coding for IL-12 enhanced Th1 responses. Plasmid DNA stimulated IL-10 and IL-12 production by macrophages and dendritic cells (DCs) in vitro and anti-IL-10 antibodies enhanced IL-12 production and DC maturation in response to gp120pDNA. Our findings suggest that T cell responses induced by DNA vaccines is influenced by the nature of the antigen, and that the induction of Th2-biased responses with gp120pDNA is mediated in part through the stimulation of innate IL-10, which inhibits activation of DCs that direct the induction of Th1 cells.     

一种CpG佐剂增强HIV-1DNA疫苗免疫原性的研究

目的以一种CpG寡聚核苷酸为HIV-1DNA疫苗候选佐剂,研究该CpG佐剂增强DNA疫苗免疫原性,体外促进DC细胞成熟等特点。方法在Balb/c小鼠模型上连续3次联合免疫HIV-1DNA疫苗及CpG佐剂,通过IFN-γ、IL-2ELISPOT及ELISA检测HIV特异性细胞免疫反应及体液免疫应答强度;体外制备小鼠骨髓来源的树突状细胞,通过FACS技术、高通量细胞因子检测等方法评价CpG佐剂刺激活化DC的能力。结果 CpG能够增强HIV-1DNA疫苗诱导的特异性细胞免疫反应水平,降低DNA疫苗使用剂量;CpG体外刺激原代小鼠骨髓来源的树突状细胞(BMDC),能显著上调CD40、CD80、CD86等BMDC表面共刺激分子的表达,活化BMDC并分泌各型细胞因子IL-5、IL-12p70,促炎症因子IL-1α、IL-1β、IL-6、IL-10、MIP-2、KC、MIG、Eotaxin、GM-CSF等以发挥佐剂效应。结论综合体内体外实验数据,证实该型CpG能够充分活化BMDC,显著提高HIV-1DNA疫苗免疫原性,降低疫苗使用剂量,可成为HIV-1DNA疫苗临床试验用候选佐剂。     

Both immunisation with a formalin-inactivated respiratory syncytial virus (RSV) vaccine and a mock antigen vaccine induce severe lung pathology and a Th2 cytokine profile in RSV-challenged mice

Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children. Immunopathology may play a role in RSV-induced disease and a severe RSV infection may also be associated with an increased risk of developing asthma. Vaccination with formalin-inactivated RSV (FI-RSV) prior to infection resulted both in human and in the mouse model in extensive lung pathology. In the mouse model, it has been shown that this aggravation of disease was associated with a shift in the balance between Th1 and Th2 cytokines towards a Th2-type response. The aim of the present study was to characterise the immunological and inflammatory responses in BALB/c mice upon RSV infection with or without prior vaccination with aluminium-adjuvanted FI-RSV or control antigens (FI-Mock). As previously reported by others, we also observed that a primary RSV infection in BALB/c mice resulted in a predominant Th1-type cytokine response, which was associated with slight bronchiolitis and alveolitis. FI-RSV vaccination prior to RSV challenge prevented virus replication and was associated with an aggravation of pulmonary histopathology and a shift towards a Th2-type response. Vaccination with FI-Mock did not prevent RSV replication in the lung but resulted in an even more pronounced Th2 response after infection while these mice were not sensitised to specific viral antigens. Thus, viral replication in a Th2 responding animal (induced by aluminium-adjuvanted mock vaccine) appears to boost the Th2 response upon RSV infection.     

Improved immunogenicity of a tuberculosis DNA vaccine encoding ESAT6 by DNA priming and protein boosting

The study evaluated the immune response elicited by a DNA vaccine encoding ESAT6 protein of Mycobacterium tuberculosis by DNA prime-protein boost protocol. BALB/c mice were respectively vaccinated with plasmid DNA encoding ESAT6 protein, with ESAT6 protein in IFA adjuvant, or a combined DNA prime-protein boost regimen. While DNA immunization induced Th1-polarized immune response, protein-in-adjuvant vaccination elicited a Th2-dominant response. When animals were primed with DNA and boost with protein, both antibodies and Th-cell proliferative response were significantly enhanced. Moreover, production of Th1-type cytokine (IFN-gamma) was increased significantly by DNA priming-protein boosting. This protocol also resulted in an increased relative ratio of IgG2a to IgG1 and the cytotoxicity of T cells. Thus, this study demonstrated that the formation of ESAT6 DNA prime-protein boost inoculation could improved antigen-specific cellular immune responses, which are important for protection against TB infection.