The expression and purification of LpxA of Chlamydia trachomatis and preparation of its polyclonal antibody.

The purpose of this study is to purify the LpxA protein of Chlamydia trachomatis (Ct) and prepare the polyclonal antibody against LpxA protein, so as to lay a foundation for studying the function of LpxA protein. The LpxA gene was amplified by PCR. The expression plasmid pET28a-LpxA was constructed by using pET28a as the vector. The fusion protein containing 6 histidine tag was induced by IPTG and purified by Ni2+ chromatography gel. The purified His-LpxA protein was used as an immunogen to immunize New Zealand rabbits subcutaneously through the back to prepare polyclonal antibody. Immunoblotting was used to detect the reaction between the antibody and His-LpxA. The determination of polyclonal antibody titer was detected by ELISA. The relative molecular weight of His-LpxA was 32.8 kDa, and it could be expressed in Escherichia coli. The purity of the purified protein was about 95%. After immunizing New Zealand rabbits, the antiserum was able to recognize the recombinant His-LpxA protein with a titer greater than 1:10240. In this study, LpxA protein was successfully purified and antiserum was prepared, which provided an experimental basis for studying the function of LpxA protein.

Enhanced delivery of Mycobacterium tuberculosis antigens to antigen presenting cells using RVG peptide.

Among the various strategies to improve vaccines against infectious diseases, targeting of antigens to dendritic cells (DCs), which are professional antigen presenting cells (APCs), has received increased attention in recent years. Here, we investigated whether a synthetic peptide region named RVG, originated from Rabies Virus Glycoprotein that binds to the α-7 subunit of the nicotinic acetylcholine receptors (AchR-α7) of APCs, could be used for the delivery of Mycobacterium tuberculosis (Mtb) peptide antigens to DCs and macrophages. Mouse bone marrow derived DCs (BMDCs) and human THP-1 macrophages stimulated with RVG fused peptide epitopes 85B241 and 85B96 (represent Ag85B241-256 and Ag85B96-111, respectively) from antigen 85B (Ag85B) of Mtb showed enhanced antigen presentation as compared to unfused peptide epitopes and BCG. Further, BMDCs stimulated with RVG fused 85B241 showed higher levels of IL-12 positive cells. Consistent with in vitro data, splenocytes of mice immunized with RVG-85B241 showed increased number of antigen specific IFN-γ, IL-2, and TNF-α producing cells in relation to splenocytes from mice immunized with 85B241 alone. These results suggest that RVG may be a promising tool to develop effective alternate vaccines against tuberculosis (TB).

Evaluation of a temperature-restricted, mucosal tuberculosis vaccine in guinea pigs.

Tuberculosis (TB) is currently the leading cause of death in humans by a single infectious agent, Mycobacterium tuberculosis. The Bacillus Calmette-Guérin (BCG) vaccine prevents pulmonary TB with variable efficacy, but can cause life-threatening systemic infection in HIV-infected infants. In this study, TBvac85, a derivative of Mycobacterium shottsii expressing M. tuberculosis Antigen 85B, was examined as a safer alternative to BCG. Intranasal vaccination of guinea pigs with TBvac85, a naturally temperature-restricted species, resulted in serum Ag85B-specific IgG antibodies. Delivery of the vaccine by this route also induced protection equivalent to intradermal BCG based on organ bacterial burdens and lung pathology six weeks after aerosol challenge with M. tuberculosis strain Erdman. These results support the potential of TBvac85 as the basis of an effective TB vaccine. Next-generation derivatives expressing multiple M. tuberculosis immunogens are in development.

In vivo electroporation of a codon-optimized BERopt DNA vaccine protects mice from pathogenic Mycobacterium tuberculosis aerosol challenge.

DNA vaccines have been extensively studied as preventative and therapeutic interventions for various infectious diseases such as tuberculosis, HIV/AIDS and influenza. Despite promising progresses made, improving the immunogenicity of DNA vaccine remains a technical challenge for clinical development. In this study, we investigated a tuberculosis DNA vaccine BERopt, which contained a codon-optimized fusion immunogen Ag85B-ESAT-6-Rv2660c for enhanced mammalian cell expression and immunogenicity. BERopt immunization through in vivo electroporation in BALB/c mice induced surprisingly high frequencies of Ag85B tetramer+ CD8+ T cells in peripheral blood and IFN-γ-secreting CD8+ T cells in splenocytes. Meanwhile, the BERopt vaccine-induced long-lasting T cell immunity protected BALB/c mice from high dose viral challenge using a modified vaccinia virus Tiantan strain expressing mature Ag85B protein (MVTT-m85B) and the virulent M. tb H37Rv aerosol challenge. Since the BERopt DNA vaccine does not induce anti-vector immunity, the strong immunogenicity and protective efficacy of this novel DNA vaccine warrant its future development for M. tb prevention and immunotherapy to alleviate the global TB burden.

Proof of concept in utilizing in-trans surface display system of Lactobacillus plantarum as mucosal tuberculosis vaccine via oral administration in mice.

BACKGROUND: Tuberculosis is one of the most common and deadliest infectious diseases worldwide affecting almost a third of the world's population. Although this disease is being prevented and controlled by the Bacille Calmette Guérin (BCG) vaccine, the protective efficacy is highly variable and substandard (0-80%) in adults. Therefore, novel and effective tuberculosis vaccine that can overcome the limitations from BCG vaccine need to be developed. RESULTS: A novel approach of utilizing an in-trans protein surface display system of Lactobacillus plantarum carrying and displaying combination of Mycobacterium tuberculosis subunit epitope antigens (Ag85B, CFP-10, ESAT-6, Rv0475 and Rv2031c) fused with LysM anchor motif designated as ACERL was constructed, cloned and expressed in Esherichia coli Rossetta expression host. Subsequently the binding capability of ACERL to the cell wall of L. plantarum was examined via the immunofluorescence microscopy and whole cell ELISA where successful attachment and consistent stability of cell wall binding up to 4 days was determined. The immunization of the developed vaccine of L. plantarum surface displaying ACERL (Lp ACERL) via the oral route was studied in mice for its immunogenicity effects. Lp ACERL immunization was able to invoke significant immune responses that favor the Th1 type cytokine response of IFN-γ, IL-12 and IL-2 as indicated by the outcome from the cytokine profiling of spleen, lung, gastrointestinal tract (GIT), and the re-stimulation of the splenocytes from the immunized mice. Co-administration of an adjuvant consisting of Lactococcus lactis secreting mouse IL-12 (LcIL-12) with Lp ACERL was also investigated. It was shown that the addition of LcIL-12 was able to further generate significant Th1 type cytokines immune responses, similar or better than that of Lp ACERL alone which can be observed from the cytokine profiling of the immunized mice's spleen, lung and GIT. CONCLUSIONS: This study represents a proof of concept in the development of L. plantarum as a carrier for a non-genetically modified organism (GMO) tuberculosis vaccine, which may be the strategy in the future for tuberculosis vaccine development.

Adjuvant Potential of Poly-α-l-Glutamine from the Cell Wall of Mycobacterium tuberculosis.

Novel adjuvants are in demand for improving the efficacy of human vaccines. The immunomodulatory properties of Mycobacterium tuberculosis cell wall components have been highlighted in the formulation of complete Freund's adjuvant (CFA). We have explored the adjuvant potential of poly-α-l-glutamine (PLG), a lesser-known constituent of the pathogenic mycobacterial cell wall. Immune parameters indicated that the adjuvant potency of PLG was statistically comparable to that of CFA and better than that of alum in the context of H1 antigen (Ag85B and ESAT-6 fusion). At 1 mg/dose, PLG augmented the immune response of Ag85B, BP26, and protective antigen (PA) by increasing serum antibodies and cytokines in the culture supernatant of antigen-stimulated splenocytes. PLG modulated the humoral response of vaccine candidate ESAT-6, eliciting significantly higher levels of total IgG and isotypes (IgG1, IgG2a, and IgG2b). Additionally, the splenocytes from PLG-adjuvanted mice displayed a robust increase in the Th1-specific gamma interferon, tumor necrosis factor alpha, interleukin-2 (IL-2), Th2-specific IL-6 and IL-10, and Th17-specific IL-17A cytokines upon antigenic stimulation. PLG improved the protective efficacy of ESAT-6 by reducing bacillary load in the lung and spleen as well as granuloma formation, and it helped in maintaining vital health parameters of mice challenged with M. tuberculosis The median survival time of PLG-adjuvanted mice was 205 days, compared to 146 days for dimethyl-dioctadecyl ammonium bromide-monophosphoryl lipid A (DDA-MPL)-vaccinated groups and 224 days for Mycobacterium bovis BCG-vaccinated groups. PLG enhanced the efficiency of the ESAT-6 vaccine to the level of BCG and better than that of DDA-MPL (P < 0.05), with no ill effect in C57BL/6J mice. Our results propose that PLG is a promising adjuvant candidate for advanced experimentation.

Ag85A-specific CD4+ T cell lines derived after boosting BCG-vaccinated cattle with Ad5-85A possess both mycobacterial growth inhibition and anti-inflammatory properties.

There is a need to improve the efficacy of the BCG vaccine against human and bovine tuberculosis. Previous data showed that boosting bacilli Calmette-Guerin (BCG)-vaccinated cattle with a recombinant attenuated human type 5 adenovirally vectored subunit vaccine (Ad5-85A) increased BCG protection and was associated with increased frequency of Ag85A-specific CD4+ T cells post-boosting. Here, the capacity of Ag85A-specific CD4+ T cell lines - derived before and after viral boosting - to interact with BCG-infected macrophages was evaluated. No difference before and after boosting was found in the capacity of these Ag85A-specific CD4+ T cell lines to restrict mycobacterial growth, but the secretion of IL-10 in vitro post-boost increased significantly. Furthermore, cell lines derived post-boost had no statistically significant difference in the secretion of pro-inflammatory cytokines (IL-1ß, IL-12, IFNγ or TNFα) compared to pre-boost lines. In conclusion, the protection associated with the increased number of Ag85A-specific CD4+ T cells restricting mycobacterial growth may be associated with anti-inflammatory properties to limit immune-pathology.

T follicular helper and T follicular regulatory cells have different TCR specificity.

Immunization leads to the formation of germinal centres (GCs) that contain both T follicular helper (Tfh) and T follicular regulatory (Tfr) cells. Whether T-cell receptor (TCR) specificity defines the differential functions of Tfh and Tfr cells is unclear. Here we show that antigen-specific T cells after immunization are preferentially recruited to the GC to become Tfh cells, but not Tfr cells. Tfh cells, but not Tfr cells, also proliferate efficiently on restimulation with the same immunizing antigen in vitro. Ex vivo TCR repertoire analysis shows that immunization induces oligoclonal expansion of Tfh cells. By contrast, the Tfr pool has a TCR repertoire that more closely resembles that of regulatory T (Treg) cells. Our data thus indicate that the GC Tfh and Tfr pools are generated from distinct TCR repertoires, with Tfh cells expressing antigen-responsive TCRs to promote antibody responses, and Tfr cells expressing potentially autoreactive TCRs to suppress autoimmunity.

Safety and immunogenicity of the novel H4:IC31 tuberculosis vaccine candidate in BCG-vaccinated adults: Two phase I dose escalation trials.

BACKGROUND: Novel vaccine strategies are required to provide protective immunity in tuberculosis (TB) and prevent development of active disease. We investigated the safety and immunogenicity of a novel TB vaccine candidate, H4:IC31 (AERAS-404) that is composed of a fusion protein of M. tuberculosis antigens Ag85B and TB10.4 combined with an IC31® adjuvant. METHODS: BCG-vaccinated healthy subjects were immunized with various antigen (5, 15, 50, 150µg) and adjuvant (0, 100, 500nmol) doses of the H4:IC31 vaccine (n=106) or placebo (n=18) in two randomized, double-blind, placebo-controlled phase I studies conducted in a low TB endemic setting in Sweden and Finland. The subjects were followed for adverse events and CD4+ T cell responses. RESULTS: H4:IC31 vaccination was well tolerated with a safety profile consisting of mostly mild to moderate self-limited injection site pain, myalgia, arthralgia, fever and post-vaccination inflammatory reaction at the screening tuberculin skin test injection site. The H4:IC31 vaccine elicited antigen-specific CD4+ T cell proliferation and cytokine production that persisted 18weeks after the last vaccination. CD4+ T cell expansion, IFN-γ production and multifunctional CD4+ Th1 responses were most prominent after two doses of H4:IC31 containing 5, 15, or 50µg of H4 in combination with the 500nmol IC31 adjuvant dose. CONCLUSIONS: The novel TB vaccine candidate, H4:IC31, demonstrated an acceptable safety profile and was immunogenic, capable of triggering multifunctional CD4+ T cell responses in previously BCG-vaccinated healthy individuals. These dose-escalation trials provided evidence that the optimal antigen-adjuvant dose combinations are 5, 15, or 50µg of H4 and 500nmol of IC31. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02066428 and NCT02074956.

Recombinant bacille Calmette-Guerin coexpressing Ag85b, CFP10, and interleukin-12 elicits effective protection against Mycobacterium tuberculosis.

BACKGROUND: The tuberculosis (TB) pandemic remains a leading cause of human morbidity and mortality, despite widespread use of the only licensed anti-TB vaccine, bacille Calmette-Guerin (BCG). The protective efficacy of BCG in preventing pulmonary TB is highly variable; therefore, an effective new vaccine is urgently required. METHODS: In the present study, we assessed the ability of novel recombinant BCG vaccine (rBCG) against Mycobacterium tuberculosis by using modern immunological methods. RESULTS: Enzyme-linked immunospot assays demonstrated that the rBCG vaccine, which coexpresses two mycobacterial antigens (Ag85B and CFP10) and human interleukin (IL)-12 (rBCG2) elicits greater interferon-γ (IFN-γ) release in the mouse lung and spleen, compared to the parental BCG. In addition, rBCG2 triggers a Th1-polarized response. Our results also showed that rBCG2 vaccination significantly limits M. tuberculosis H37Rv multiplication in macrophages. The rBCG2 vaccine surprisingly induces significantly higher tumor necrosis factor-α (TNF-α) production by peripheral blood mononuclear cells that were exposed to a nonmycobacterial stimulus, compared to the parental BCG. CONCLUSION: In this study, we demonstrated that the novel rBCG2 vaccine may be a promising candidate vaccine against M. tuberculosis infection.

Recombinant Bacillus subtilis spores for the delivery of Mycobacterium tuberculosis Ag85B-CFP10 secretory antigens.

Tuberculosis continues to be a great cause of morbidity and mortality in different parts of the world. Unfortunately, the current BCG vaccine being administered is not fully protective against tuberculosis; therefore, there is a great need for alternate vaccines. With an aim to develop such vaccines, we have analyzed the utility of Bacillus subtilis spores for the expression of two major immunodominant antigens of Mycobacterium tuberculosis, Ag85B and CFP10. We created three recombinant B. subtilis strains to express a truncated fusion of Ag85B191-325 and CFP101-70 antigens (T85BCFP), either on the spore coat (MTAG1 strain) or in the cytosol of B. subtilis (MTAG 2 and MTAG 3 strains). Examination of spores isolated from these strains revealed successful expression of T85BCFP antigens on the spore coat of MTAG1 as well as in the cytosol of vegetatively grown cells of MTAG2 and MTAG3, indicating that spores can indeed express M. tuberculosis antigens. In vitro antigen presentation assays with spore-infected mouse bone marrow derived macrophages (BMDM) showed that all three recombinant spores could deliver these antigens to antigen presenting cells (APCs). Mice immunized with recombinant spores displayed significantly higher levels of Ag85B specific IFN-γ producing cells in the spleen than in mice immunized with wild-type (non-recombinant) spores. In addition, these mice showed relatively higher levels of Ag85B specific IgG antibodies in the serum in comparison to mice immunized with non-recombinant spores, thus providing additional evidence that recombinant spores can deliver these antigens in vivo. These results suggest that B. subtilis spores are ideal vehicles for antigen delivery and have great potential in the development of primary and booster vaccines against tuberculosis.

Improved protection against tuberculosis after boosting the BCG-primed mice with subunit Ag 85a delivered through intact skin with deformable vesicles.

To improve vaccination against tuberculosis (TBC) with Bacillus Calmette-Guerin (BCG), we introduce novel, non-invasive, secondary immunisations relying on epicutaneous (e.c.) applications of the TBC subunit antigen, Ag 85a, associated with deformable carrier vesicles. Immuno-boosting with such antigen-vesicles recruits more CD11c positive cells into the draining murine lymph nodes, and typically stimulates, especially the proximal, immune cells more than immunogen injections. Non-invasive antigen application also protects mice better against an infection with TBC. Subcutaneous injections of vesicular Ag 85a into BCG-primed mice mainly yield IgG1 and IgG2a, indicative of a mixed Th1 and Th2 response. Conversely, transcutaneous immuno-boosts of such mice with a deformable vesicle-Ag 85a combination mainly generate serum IgA and IgG2a, indicative of an IgA facilitated, Th1-mediated, immune response. The Ag 85a specific antibody titres are generally low, but T lymphocytes also proliferate in the immunised mice. The new, partially non-invasive, vaccination method lowers the burden of pulmonary infection with M. tuberculosis. In mice immunised with Ag85a associated with deformable vesicles we measured 116× (e.c.) to 51× (s.c.) lower colony forming units number in spleen and 9× (e.c.) to 3× (s.c.) lower such number in lungs.

Using a prime and pull approach, lentivector vaccines expressing Ag85A induce immunogenicity but fail to induce protection against Mycobacterium bovis bacillus Calmette-Guérin challenge in mice.

Although bacillus Calmette-Guérin (BCG) is an established vaccine with excellent efficacy against disseminated Mycobacterium tuberculosis infection in young children, efficacy in adults suffering from respiratory tuberculosis (TB) is suboptimal. Prime-boost viral vectored vaccines have been shown to induce effective immune responses and lentivectors (LV) have been shown to improve mucosal immunity in the lung. A mucosal boost to induce local immunogenicity is also referred to as a 'pull' in a prime and pull approach, which has been found to be a promising vaccine strategy. The majority of infants worldwide receive BCG immunization through current vaccine protocols. We therefore aimed to investigate the role of a boost (or pull) immunization with an LV vaccine expressing the promising TB antigen (Ag85A). We immunized BALB/c mice subcutaneously with BCG or an LV vaccine expressing a nuclear factor-κB activator vFLIP together with Ag85A (LV vF/85A), then boosted with intranasal LV vF/85A. Prime and pull immunization with LV85A induced significantly enhanced CD8(+) and CD4(+) T-cell responses in the lung, but did not protect against intranasal BCG challenge. In contrast, little T-cell response in the lung was seen when the prime vaccine was BCG, and intranasal vF/85A provided no additional protection against mucosal BCG infection. Our study demonstrates that not all LV prime and pull approaches may be successful against TB in man and careful antigen and immune activator selection is therefore required.

First-in-human trial of the post-exposure tuberculosis vaccine H56:IC31 in Mycobacterium tuberculosis infected and non-infected healthy adults.

BACKGROUND: H56:IC31 is a candidate tuberculosis vaccine comprising a fusion protein of Ag85B, ESAT-6 and Rv2660c, formulated in IC31 adjuvant. This first-in-human, open label phase I trial assessed the safety and immunogenicity of H56:IC31 in healthy adults without or with Mycobacterium tuberculosis (M.tb) infection. METHODS: Low dose (15 µg H56 protein in 500 nmol IC31) or high dose (50 µg H56, 500 nmol IC31) vaccine was administered intramuscularly thrice, at 56-day intervals. Antigen-specific T cell responses were measured by intracellular cytokine staining and antibody responses by ELISA. RESULTS: One hundred and twenty-six subjects were screened and 25 enrolled and vaccinated. No serious adverse events were reported. Nine subjects (36%) presented with transient cardiovascular adverse events. The H56:IC31 vaccine induced antigen-specific IgG responses and Th1 cytokine-expressing CD4(+) T cells. M.tb-infected vaccinees had higher frequencies of H56-induced CD4(+) T cells than uninfected vaccinees. Low dose vaccination induced more polyfunctional (IFN-γ(+)TNF-α(+)IL-2(+)) and higher frequencies of H56-specific CD4(+) T cells compared with high dose vaccination. A striking increase in IFN-γ-only-expressing CD4(+) T cells, displaying a CD45RA(-)CCR7(-) effector memory phenotype, emerged after the second high-dose vaccination in M.tb-infected vaccinees. TNF-α(+)IL-2(+) H56-specific memory CD4(+) T cells were detected mostly after low-dose H56 vaccination in M.tb-infected vaccinees, and predominantly expressed a CD45RA(-)CCR7(+) central memory phenotype. Our results support further clinical testing of H56:IC31.

[Preparation of TAT-Ag85B protein vaccine and evaluation of its anti-Myobacterium tuberculosis effect].

OBJECTIVE: To obtain a new Ag85B protein fused with protein transduction domain (PTD) produced by a HIV-trans-activating transduction domain (TAT-PTD) expression system and investigate its protective effect against Myobacterium tuberculosis as a subunit vaccine. METHODS: The pET28a-Ag85B and pET28a-TAT-Ag85B plasmids were established and transformed into E.coli BL21(DE3) strains for recombinant protein expression and purification. Then three groups of BALB/c mice were subcutaneously vaccinated three times with Ag85B protein, TAT-Ag85B protein and PBS, respectively. One week after the last immunization, 5 mice in each group were sacrificed for detecting serum specific anti-Ag85B and IFN-γ/IL-2 produced by spleen cells using ELISA. Simultaneously, the levels of CD80 and CD86 on macrophages which were stimulated by Ag85B or TAT-Ag85B protein were measured using flow cytometry. Subsequently, the rest of the mice were intravenously injected with virulent Myobacterium tuberculosis H37Rv and their bacterial loads in the lung and spleen were determined 1, 2, 4 and 8 weeks after infection. Moreover, pulmonary pathological changes were observed by HE staining at 8 weeks after infection. RESULTS: Ag85B and TAT-Ag85B proteins were obtained successfully. Compared with Ag85B, higher titers of IgG antibodies and the levels of IFN-γ and IL-2 were induced by TAT-Ag85B. Lower bacterial loads in the lung and spleen and smaller scale of pulmonary lesion were found in mice immunized with TAT-Ag85B than those in Ag85B-treated mice. In addition, TAT-Ag85B stimulated higher CD80 and CD86 expressions on macrophages. CONCLUSION: TAT-Ag85B protein is an efficient vaccine that induces a strong Th1 immune response and provides a good protection against Myobacterium tuberculosis infection. The mechanism of the subunit vaccine may be partially explained as the enhanced capability of antigen-presentation of macrophages.

Enhanced anti-tuberculosis immunity by a TAT-Ag85B protein vaccine in a murine tuberculosis model.

BACKGROUND: The development of more effective anti-tuberculosis vaccines would contribute to the control of the global problem of infection with Mycobacterium tuberculosis (MTB). Recently, increasing evidences showed that HIV-Tat protein transduction domain is implicated in promotion of vaccines by inducing cellular immuno-response. However, it is rare known about the role of TAT in vaccines against MTB. METHODS: In this study, we expressed recombinant protein-fused Ag85B with TAT (TAT-Ag85B) which was used as a vaccine to inoculate mice infected with MTB. RESULTS: As s result, both IgG2a in serum and IFN-γ or TNFα produced by spleen cells were all increased significantly in the mice inoculated by TAT-Ag85B. Furthermore, consistently, TAT-Ag85B inoculation significantly reduced MTB loads both in lung and spleen. CONCLUSIONS: These findings demonstrate that a novel protein vaccine of TAT-Ag85B enhances immune response both in humoral and cellular immunity, and contributes to protective efficacy against MTB.

Development and characterization of guar gum nanoparticles for oral immunization against tuberculosis.

The main aim of this study was to develop an effective carrier system containing Ag85A-loaded guar gum nanoparticles for oral vaccination against tuberculosis. Nanoparticles were prepared by Nanoprecipitation method. The developed particles with mean diameter 895.5 ± 14.73 nm and high antigen entrapment seem to be optimum for oral vaccine delivery. The acid protection assay, Peyer's patch uptake study and in-vitro antigen study confirmed that the developed formulations can protect the antigen from harsh gastric environment and can safely deliver the antigen to the intestinal region. In vivo studies data indicated that the developed nanocarriers can induce a strong mucosal as well as systemic immune response. Therefore, the experimental evidence suggests that guar-gum nanoparticle findings indicated that the guar gum nanoparticles can be utilized for safe and effective vaccine delivery via oral route.

Prime-boost bacillus Calmette-Guérin vaccination with lentivirus-vectored and DNA-based vaccines expressing antigens Ag85B and Rv3425 improves protective efficacy against Mycobacterium tuberculosis in mice.

To prevent the global spread of tuberculosis (TB), more effective vaccines and vaccination strategies are urgently needed. As a result of the success of bacillus Calmette-Guérin (BCG) in protecting children against miliary and meningeal TB, the majority of individuals will have been vaccinated with BCG; hence, boosting BCG-primed immunity will probably be a key component of future vaccine strategies. In this study, we compared the ability of DNA-, protein- and lentiviral vector-based vaccines that express the antigens Ag85B and Rv3425 to boost the effects of BCG in the context of immunity and protection against Mycobacterium tuberculosis in C57BL/6 mice. Our results demonstrated that prime-boost BCG vaccination with a lentiviral vector expressing the antigens Ag85B and Rv3425 significantly enhanced immune responses, including T helper type 1 and CD8(+) cytotoxic T lymphocyte responses, compared with DNA- and protein-based vaccines. However, lentivirus-vectored and DNA-based vaccines greatly improved the protective efficacy of BCG against M. tuberculosis, as indicated by a lack of weight loss and significantly reduced bacterial loads and histological damage in the lung. Our study suggests that the use of lentiviral or DNA vaccines containing the antigens Ag85B and Rv3425 to boost BCG is a good choice for the rational design of an efficient vaccination strategy against TB.

Immunization with different formulations of Mycobacterium tuberculosis antigen 85A induces immune responses with different specificity and protective efficacy.

To test the relative efficacy of CD4 and CD8T cells in mediating protective immunity to Mycobacterium tuberculosis (Mtb), we compared three immunization regimes designed to induce preferentially each subset. BALB/c mice were immunized intranasally (i.n.) or parenterally with antigen 85A either in a recombinant Adenoviral vector (Ad85A), as recombinant protein (r85A) or as a set of overlapping 15mer peptides (p85A). For the first time we show that i.n. immunization with overlapping 85A synthetic peptides as well as Ad85A or r85A can provide protection against Mtb challenge. For all forms of the antigen, i.n. induces greater protection against Mtb challenge than parenteral immunization. Ad85A induces a predominantly CD8T cell response against the 85A(70-78) epitope, r85A a CD4 response to 85A(99-118) and p85A a balanced CD4/CD8 response to the CD4 85A(99-118 )and CD8 85A(145-152) epitopes. Immune responses to CD4 85A(99-118) and CD8 85A(70-78) but not CD8 85A(145-152) are protective. Although Ad85A induces a strong response to the protective CD8 85A(70-78) epitope, we could not induce any response to this epitope by peptide immunization. These results show that although peptide immunization can induce protective immunity to Mtb challenge, it can also induce a response to a non-protective epitope in antigen 85A, indicating that the specificity of an immune response may be more important for protection against Mtb than its magnitude. These findings have important implications for the application of such vaccines in humans.

Mucosal and systemic immune responses to Mycobacterium tuberculosis antigen 85A following its co-delivery with CpG, MPLA or LTB to the lungs in mice.

Pulmonary vaccination is a promising route for immunization against tuberculosis because the lung is the natural site of infection with Mycobacterium tuberculosis. Yet, adjuvants with a suitable safety profile need to be found to enhance mucosal immunity to recombinant antigens. The aim of this study was to evaluate the immunogenicity, the safety and the protective efficacy of a subunit vaccine composed of the immunodominant mycolyl-transferase antigen 85A (Ag85A) and one of three powerful mucosal adjuvants: the oligodeoxynucleotide containing unmethylated cytosine-phosphate-guanine motifs (CpG), the monophosphoryl lipid A of Salmonella minnesota (MPLA) or the B subunit of heat-labile enterotoxin of Escherichia coli (LTB). BALB/c mice were vaccinated in the deep lungs. Our results showed that lung administration of these adjuvants could specifically induce different types of T cell immunity. Both CpG and MPLA induced a Th-1 type immune response with significant antigen-specific IFN-γ production by spleen mononuclear cells in vitro and a tendency of increased IFN-γ in the lungs. Moreover, MPLA triggered a Th-17 response reflected by high IL-17A levels in the spleen and lungs. By contrast, LTB promoted a Th-2 biased immune response, with a production of IL-5 but not IFN-γ by spleen mononuclear cells in vitro. CpG did not induce inflammation in the lungs while LTB and MPLA showed a transient inflammation including a neutrophil influx one day after pulmonary administration. Pulmonary vaccination with Ag85A without or with MPLA or LTB tended to decrease bacterial counts in the spleen and lungs following a virulent challenge with M. tuberculosis H37Rv. In conclusion, CpG and MPLA were found to be potential adjuvants for pulmonary vaccination against tuberculosis, providing Th-1 and Th-17 immune responses and a good safety profile.