Memory CD73+IgM+ B cells protect against Plasmodium yoelii infection and express Granzyme B.

To better understand anti-malaria protective immune responses, we examined the cellular mechanisms that govern protective immunity in a murine Plasmodium yoelii 17X NL (PyNL) re-infection model. Initially, we confirmed that immune B cells generated during a primary PyNL infection were largely responsible for protection from a second PyNL infection. Using the previously identified memory B cell markers CD80, PD-L2, and CD73, we found an increase in the frequency of CD80-PD-L2-CD73+ B cells up to 55 days after a primary PyNL infection and at 4-6 days following a second PyNL infection. Moreover, injection of enriched immune CD19+CD73+ B cells into nonimmune mice were significantly more protective against a PyNL infection than CD73- B cells. Interestingly, a substantial fraction of these CD73+ B cells also expressed IgM and granzyme B, a biomolecule that has been increasingly associated with protective responses against malaria.

Artificially induced MAIT cells inhibit M. bovis BCG but not M. tuberculosis during in vivo pulmonary infection.

There is significant interest in targeting MAIT cells with immunostimulatory agents to enhance immune responses. Mycobacterium tuberculosis (M. tb.) is a pervasive respiratory disease that could benefit from treatments that augment immunity. Here we investigate the role of MAIT cells in M. tb. infection and the potential for MAIT cell-targeted immunotherapy to control bacterial burdens. We find that MAIT cells fail to substantially accumulate in the lungs during murine pulmonary M. bovis BCG and M. tb. infections but this defect is overcome by intranasal installation of a TLR2/6 agonist and a MAIT cell antigen. Although artificially induced MAIT cells produce important cytokines in both infections, they control BCG but not M. tb. growth in the lungs. Correspondingly, M. tb.-infected mouse macrophages are relatively resistant to MAIT cell antimicrobial activities in vitro. Thus, MAIT cell antigen-mediated immunotherapy for M. tb. presents a complex challenge.

TACI Contributes to Plasmodium yoelii Host Resistance by Controlling T Follicular Helper Cell Response and Germinal Center Formation.

The delay in parasite-specific B cell development leaves people in malaria endemic areas vulnerable to repeated Plasmodium infections. Here, we investigated the role of transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), a molecule involved in the generation of antigen-specific antibody secreting cells, in host response to non-lethal Plasmodium yoelii infection. We found that TACI deficiency not only resulted in higher peak parasitemia levels in P. yoelii challenged mice, but also led to a delay in parasite clearance and anti-P. yoelii Merozoite Surface Protein 1(C-terminal 19-kDa fragment [rMSP-119]) protein and anti-rMSP-119 and anti-P. yoelii IgG antibody development. There was also a delay in the generation of splenic high affinity antibody secreting cells that recognize rMSP-119 protein as compared to wild-type mice. Interestingly, coinciding with the delay in parasite clearance there was a delay in the resolution of T follicular helper (TFH) cell and germinal center (GC) B cell responses in TACI -/- mice. The persistence of TFH and GC B cells is likely a result of enhanced interaction between TFH and GC B cells because inducible costimulator ligand (ICOSL) expression was significantly higher on TACI -/- GC B cells than wild-type cells. The difference in the kinetics of GC reaction appeared to also impact the emergence of plasma cells (PC) because there was a delay in the generation of TACI -/- mice PC. Nevertheless, following the recovery from P. yoelii infection, TACI -/- and wild-type mice were both protected from a rechallenge infection. Establishment of protective B cell response was responsible for the resolution of parasitemia because B cells purified from recovered TACI -/- or wild-type mice were equally protective when introduced to naïve wild-type mice prior to P. yoelii challenge. Thus, despite the increased susceptibility of TACI -/- mice to P. yoelii infection and a delay in the development of protective antibody levels, TACI -/- mice are able to clear the infection and resist rechallenge infection.

Trained Immunity and Susceptibility to HIV.

In this issue of Clinical and Vaccine Immunology, K. Jensen et al. (Clin Vaccine Immunol 24:e00360-16, 2017, https://doi.org/10.1128/CVI.00360-16) describe a dual-purpose attenuated Mycobacterium tuberculosis-simian immunodeficiency virus vaccine (AMTB-SIV). Interestingly, immunized infant macaques required fewer oral exposures to SIV to become infected relative to nonimmunized animals. The authors hypothesized that augmented susceptibility to SIV was due to activation of CD4+ T cells through trained immunity. This commentary explores the possible relationship between trained immunity, enhanced CD4 T cell responses, and increased susceptibility to human immunodeficiency virus (HIV).

A simplified mycobacterial growth inhibition assay (MGIA) using direct infection of mouse splenocytes and the MGIT system.

We describe a simplified Mycobacterial Growth Inhibition Assay (MGIA) for pre-clinical assessment of vaccine-mediated protection in mice. The assay is accomplished by directly infecting splenocytes from vaccinated mice with Mycobacterium tuberculosis and quantifying mycobacteria using Mycobacterial Growth Indicator Tubes (MGIT). Vaccine-mediated immunogenicity detected by this assay correlated with protection.

Induction of Unconventional T Cells by a Mutant Mycobacterium bovis BCG Strain Formulated in Cationic Liposomes Correlates with Protection against Mycobacterium tuberculosis Infections of Immunocompromised Mice.

Earlier studies aimed at defining protective immunity induced by Mycobacterium bovis BCG immunization have largely focused on the induction of antituberculosis CD4(+) and CD8(+) T cell responses. Here we describe a vaccine consisting of a BCGΔmmaA4 deletion mutant formulated in dimethyl dioctadecyl-ammonium bromide (DDA) with d-(+)-trehalose 6,6'-dibehenate (TDB) (DDA/TDB) adjuvant (A4/Adj) that protected TCRδ(-/-) mice depleted of CD4(+), CD8(+), and NK1.1(+) T cells against an aerosol challenge with M. tuberculosis These mice were significantly protected relative to mice immunized with a nonadjuvanted BCGΔmmaA4 (BCG-A4) mutant and nonvaccinated controls at 2 months and 9 months postvaccination. In the absence of all T cells following treatment with anti-Thy1.2 antibody, the immunized mice lost the ability to control the infection. These results indicate that an unconventional T cell population was mediating protection in the absence of CD4(+), CD8(+), NK1.1(+), and TCRγδ T cells and could exhibit memory. Focusing on CD4(-) CD8(-) double-negative (DN) T cells, we found that these cells accumulated in the lungs postchallenge significantly more in A4/Adj-immunized mice and induced significantly greater frequencies of pulmonary gamma interferon (IFN-γ)-producing cells than were seen in the nonvaccinated or nonadjuvanted BCG control groups. Moreover, pulmonary DN T cells from the A4/Adj group exhibited significantly higher IFN-γ integrated median fluorescence intensity (iMFI) values than were seen in the control groups. We also showed that enriched DN T cells from mice immunized with A4/Adj could control mycobacterial growth in vitro significantly better than naive whole-spleen cells. These results suggest that formulating BCG in DDA/TDB adjuvant confers superior protection in immunocompromised mice and likely involves the induction of long-lived memory DN T cells.

Co-expression of Interleukin-15 Enhances the Protective Immune Responses Induced by Immunization with a Murine Malaria MVA-Based Vaccine Encoding the Circumsporozoite Protein.

Malaria remains a major global public health problem with an estimated 200 million cases detected in 2012. Although the most advanced candidate malaria vaccine (RTS,S) has shown promise in clinical trials, its modest efficacy and durability have created uncertainty about the impact of RTS,S immunization (when used alone) on global malaria transmission. Here we describe the development and characterization of a novel modified vaccinia virus Ankara (MVA)-based malaria vaccine which co-expresses the Plasmodium yoelii circumsporozoite protein (CSP) and IL-15. Vaccination/challenge studies showed that C57BL/6 mice immunized with the MVA-CSP/IL15 vaccine were protected significantly better against a P. yoelii 17XNL sporozoite challenge than either mice immunized with an MVA vaccine expressing only CSP or naïve controls. Importantly, the levels of total anti-CSP IgG were elevated about 100-fold for the MVA-CSP/IL15 immunized group compared to mice immunized with the MVA-CSP construct that does not express IL-15. Among the IgG subtypes, the IL-15 expressing MVA-CSP vaccine induced levels of IgG1 (8 fold) and IgG2b (80 fold) higher than the MVA-CSP construct. The significantly enhanced humoral responses and protection detected after immunization with the MVA-CSP/IL15 vaccine suggest that this IL-15 expressing MVA construct could be considered in the development of future malaria immunization strategies.

Effect of cationic liposomes on BCG trafficking and vaccine-induced immune responses following a subcutaneous immunization in mice.

While formulating Mycobacterium bovis BCG in lipid-based adjuvants has been shown to increase the vaccine's protective immunity, the biological mechanisms responsible for the enhanced potency of lipid encapsulated BCG are unknown. To assess whether mixing BCG in adjuvant increases its immunogenicity by altering post-vaccination organ distribution and persistence, mice were immunized subcutaneously with conventional BCG Pasteur or BCG formulated in DDA/TDB adjuvant and the bio-distribution of BCG bacilli was evaluated in mouse lungs, spleens, lymph nodes, and livers for up to 1 year. Although BCG was rarely detected in mouse livers, mycobacteria were found in mouse lungs, spleens, and lymph nodes for at least 1 year post-vaccination. However, at various time points during the 1 year study, the frequency of lung and spleen infections and the number of mycobacteria in infected organs of individual mice were highly variable. In contrast, mycobacteria were nearly always detected in the lymph nodes of vaccinated mice. While the frequency and extent of lymph node infections generally were not significantly different between mice vaccinated with adjuvanted or nonadjuvanted BCG preparations, multiparameter flow cytometry analysis of lymph node cells showed significantly higher frequencies of CD4+ and CD8+ T cells expressing IFN-γ and IFN-γ/TNF-α in mice immunized with adjuvanted BCG. Overall, our data suggest that the relationship between lymph node infection and the generation of anti-tuberculosis protective responses following BCG vaccination should be further investigated.

Intranasal administration of Mycobacterium bovis BCG induces superior protection against aerosol infection with Mycobacterium tuberculosis in mice.

Despite the widespread use of Mycobacterium bovis BCG, the only licensed vaccine against tuberculosis (TB), TB remains a global epidemic. To assess whether more direct targeting of the lung mucosa by respiratory immunization would enhance the potency and longevity of BCG-induced anti-TB protective immunity, the long-term impact of intranasal (i.n.) BCG vaccination was compared to conventional subcutaneous (s.c.) immunization by using a mouse model of pulmonary tuberculosis. Although significantly improved protection in the lung was seen at early time points (2 and 4 months postvaccination) in i.n. BCG-immunized mice, no differences in pulmonary protection were seen 8 and 10 months postvaccination. In contrast, in all of the study periods, i.n. BCG vaccination induced significantly elevated protective splenic responses relative to s.c. immunization. At five of nine time points, we observed a splenic protective response exceeding 1.9 log10 protection relative to the s.c. route. Furthermore, higher frequencies of CD4 T cells expressing gamma interferon (IFN-γ) and IFN-γ/tumor necrosis factor alpha, as well as CD8 T cells expressing IFN-γ, were detected in the spleens of i.n. vaccinated mice. Using PCR arrays, significantly elevated levels of IFN-γ, interleukin-9 (IL-9), IL-11, and IL-21 expression were also seen in the spleen at 8 months after respiratory BCG immunization. Overall, while i.n. BCG vaccination provided short-term enhancement of protection in the lung relative to s.c. immunization, potent and extremely persistent splenic protective responses were seen for at least 10 months following respiratory immunization.

Host-directed therapy of tuberculosis based on interleukin-1 and type I interferon crosstalk.

Tuberculosis remains second only to HIV/AIDS as the leading cause of mortality worldwide due to a single infectious agent. Despite chemotherapy, the global tuberculosis epidemic has intensified because of HIV co-infection, the lack of an effective vaccine and the emergence of multi-drug-resistant bacteria. Alternative host-directed strategies could be exploited to improve treatment efficacy and outcome, contain drug-resistant strains and reduce disease severity and mortality. The innate inflammatory response elicited by Mycobacterium tuberculosis (Mtb) represents a logical host target. Here we demonstrate that interleukin-1 (IL-1) confers host resistance through the induction of eicosanoids that limit excessive type I interferon (IFN) production and foster bacterial containment. We further show that, in infected mice and patients, reduced IL-1 responses and/or excessive type I IFN induction are linked to an eicosanoid imbalance associated with disease exacerbation. Host-directed immunotherapy with clinically approved drugs that augment prostaglandin E2 levels in these settings prevented acute mortality of Mtb-infected mice. Thus, IL-1 and type I IFNs represent two major counter-regulatory classes of inflammatory cytokines that control the outcome of Mtb infection and are functionally linked via eicosanoids. Our findings establish proof of concept for host-directed treatment strategies that manipulate the host eicosanoid network and represent feasible alternatives to conventional chemotherapy.

The transcription factor T-bet regulates parasitemia and promotes pathogenesis during Plasmodium berghei ANKA murine malaria.

The pathogenesis of experimental cerebral malaria (ECM) is an immunologic process, mediated in part by Th1 CD4(+) T cells. However, the role of the Th1 CD4(+) T cell differentiation program on the ability to control parasitemia and susceptibility to ECM disease during blood stage malaria has never been assessed directly. Using the Plasmodium berghei ANKA murine model of ECM and mice deficient for the transcription factor T-bet (the master regulator of Th1 cells) on the susceptible C57BL/6 background, we demonstrate that although T-bet plays a role in the regulation of parasite burden, it also promotes the pathogenesis of ECM. T-bet-deficient (Tbx21(-/-)) mice had higher parasitemia than wild type controls did during the ECM phase of disease (17.7 ± 3.1% versus 10.9 ± 1.5%). In addition, although 100% (10/10) of wild type mice developed ECM by day 9 after infection, only 30% (3/10) of Tbx21(-/-) mice succumbed to disease during the cerebral phase of infection. Resistance to ECM in Tbx21(-/-) mice was associated with diminished numbers of IFN-γ-producing CD4(+) T cells in the spleen and a lower accumulation of CD4(+) and CD8(+) T cells in the brain. An augmented Th2 immune response characterized by enhanced production of activated GATA-3(+) CD4(+) T cells and elevated levels of the eotaxin, MCP-1, and G-CSF cytokines was observed in the absence of T-bet. Our results suggest that in virulent malarias, immune modulation or therapy resulting in an early shift toward a Th2 response may help to ameliorate the most severe consequences of malaria immunopathogenesis and the prospect of host survival.

Immunogenicity and protective efficacy of novel Mycobacterium tuberculosis antigens.

With tuberculosis continuing to be a major cause of global morbidity and mortality, a new vaccine is urgently needed. Tuberculosis subunit vaccines have been shown to induce robust immune responses in humans and are a potentially safer alternative to BCG for use in HIV-endemic areas. In this study, we investigated the protective efficacy of 16 different novel Mycobacterium tuberculosis antigens using an aerogenic mouse model of pulmonary tuberculosis. These antigens were tested as subunit vaccines formulated in dimethyl dioctadecyl ammonium bromide (DDA) - D(+) with trehalose 6,6 dibenenate (TDB) (DDA/TDB) adjuvant administered alone as monovalent vaccines or in combination. Six of these antigens (Rv1626, Rv1735, Rv1789, Rv2032, Rv2220, and Rv3478) were shown to consistently and significantly reduce bacterial burdens in the lungs of mice relative to nonvaccinated controls. Three of these six (Rv1789, Rv2220, and Rv3478) induced levels of protective immunity that were essentially equivalent to protection induced by the highly immunogenic antigen 85B (>0.5 log10CFU reduction in the lungs relative to naïve mice). Importantly, when these three antigens were combined, protection essentially equivalent to that mediated by BCG was observed. When either Rv1626 or Rv2032 were combined with the highly protective E6-85 fusion protein (antigen 85B fused to ESAT-6), the protection observed was equivalent to BCG-induced protection at one and three months post-aerosol infection and was significantly greater than the protection observed when E6-85 was administered alone at 3 months post-infection. Using multiparameter flow cytometry, monofunctional IFNγ CD4T cells and different multifunctional CD4T cell subsets capable of secreting multiple cytokines (IFNγ, TNFα and/or IL-2) were shown to be induced by the three most protective antigens with splenocyte CD4T cell frequencies significantly greater than observed in naïve controls. The identification of these highly immunogenic TB antigens and antigen combinations should allow for improved immunization strategies against tuberculosis.

Molecular analysis of non-specific protection against murine malaria induced by BCG vaccination.

Although the effectiveness of BCG vaccination in preventing adult pulmonary tuberculosis (TB) has been highly variable, epidemiologic studies have suggested that BCG provides other general health benefits to vaccinees including reducing the impact of asthma, leprosy, and possibly malaria. To further evaluate whether BCG immunization protects against malarial parasitemia and to define molecular correlates of this non-specific immunity, mice were vaccinated with BCG and then challenged 2 months later with asexual blood stage Plasmodium yoelii 17XNL (PyNL) parasites. Following challenge with PyNL, significant decreases in parasitemia were observed in BCG vaccinated mice relative to naïve controls. To identify immune molecules that may be associated with the BCG-induced protection, gene expression was evaluated by RT-PCR in i) naïve controls, ii) BCG-vaccinated mice, iii) PyNL infected mice and iv) BCG vaccinated/PyNL infected mice at 0, 1, 5, and 9 days after the P. yoelii infection. The expression results showed that i) BCG immunization induces the expression of at least 18 genes including the anti-microbial molecules lactoferrin, eosinophil peroxidase, eosinophil major basic protein and the cathelicidin-related antimicrobial peptide (CRAMP); ii) an active PyNL infection suppresses the expression of important immune response molecules; and iii) the extent of PyNL-induced suppression of specific genes is reduced in BCG-vaccinated/PyNL infected mice. To validate the gene expression data, we demonstrated that pre-treatment of malaria parasites with lactoferrin or the cathelicidin LL-37 peptide decreases the level of PyNL parasitemias in mice. Overall, our study suggests that BCG vaccination induces the expression of non-specific immune molecules including antimicrobial peptides which may provide an overall benefit to vaccinees by limiting infections of unrelated pathogens such as Plasmodium parasites.

Nonclinical Development of BCG Replacement Vaccine Candidates.

The failure of current Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccines, given to neonates to protect against adult tuberculosis and the risk of using these live vaccines in HIV-infected infants, has emphasized the need for generating new, more efficacious and safer replacement vaccines. With the availability of genetic techniques for constructing recombinant BCG (rBCG) strains containing well-defined gene deletions or insertions, new vaccine candidates are under evaluation at both the preclinical and clinical stages of development. Since most BCG vaccines in use today were evaluated in clinical trials decades ago and are produced by outdated processes, the development of new BCG vaccines offers a number of advantages that include a modern well-defined manufacturing process along with state-of-the-art evaluation of safety and efficacy in target populations. We provide a description of the preclinical development of two novel rBCGs, VPM1002 that was constructed by adding a modified hly gene coding for the protein listeriolysin O (LLO) from Listeria monocytogenes and AERAS-422, which carries a modified pfoA gene coding for the protein perfringolysin O (PFO) from Clostridium perfringens, and three genes from Mycobacterium tuberculosis. Novel approaches like these should be helpful in generating stable and effective rBCG vaccine candidates that can be better characterized than traditional BCG vaccines.

Characterization of an intracellular ATP assay for evaluating the viability of live attenuated mycobacterial vaccine preparations.

The viability of BCG vaccine has traditionally been monitored using a colony-forming unit (CFU) assay. Despite its widespread use, results from the CFU assay can be highly variable because of the characteristic clumping of mycobacteria, their requirement for complex growth media, and the three week incubation period needed to cultivate slow-growing mycobacteria. In this study, we evaluated whether an ATP luminescence assay (which measures intracellular ATP content) could be used to rapidly estimate the viability of lyophilized and/or frozen preparations of six different BCG vaccine preparations - Danish, Tokyo, Russia, Brazil, Tice, and Pasteur - and two live attenuated mycobacterial vaccine candidates - a ΔlysAΔpanCD M. tuberculosis strain and a ΔmmaA4 BCG vaccine mutant. For every vaccine tested, a significant correlation was observed between intracellular ATP concentrations and the number of viable attenuated bacilli. However, the extractable intracellular ATP levels detected per cell among the different live vaccines varied suggesting that validated ATP luminescence assays with specific appropriate standards must be developed for each individual live attenuated vaccine preparation. Overall, these data indicate that the ATP luminescence assay is a rapid, sensitive, and reliable alternative method for quantifying the viability of varying live attenuated mycobacterial vaccine preparations.

Formulation of a mmaA4 gene deletion mutant of Mycobacterium bovis BCG in cationic liposomes significantly enhances protection against tuberculosis.

A new vaccination strategy is urgently needed for improved control of the global tuberculosis (TB) epidemic. Using a mouse aerosol Mycobacterium tuberculosis challenge model, we investigated the protective efficacy of a mmaA4 gene deletion mutant of Mycobacterium bovis BCG (ΔmmaA4BCG) formulated in dimethyl dioctadecyl ammonium bromide (DDA) - D(+) trehalose 6,6 dibenenate (TDB) (DDA/TDB) adjuvant. In previous studies, deletion of the mmaA4 gene was shown to reduce the suppression of IL-12 production often seen after mycobacterial infections. While the non-adjuvanted ΔmmaA4BCG strain did not protect mice substantially better than conventional BCG against a tuberculous challenge in four protection experiments, the protective responses induced by the ΔmmaA4BCG vaccine formulated in DDA/TDB adjuvant was consistently increased relative to nonadjuvanted BCG controls. Furthermore, the ΔmmaA4BCG-DDA/TDB vaccine induced significantly higher frequencies of multifunctional (MFT) CD4 T cells expressing both IFNγ and TNFα (double positive) or IFNγ, TNFα and IL-2 (triple positive) than CD4 T cells derived from mice vaccinated with BCG. These MFT cells were characterized by having higher IFNγ and TNFα median fluorescence intensity (MFI) values than monofunctional CD4 T cells. Interestingly, both BCG/adjuvant and ΔmmaA4BCG/adjuvant formulations induced significantly higher frequencies of CD4 T cells expressing TNFα and IL-2 than nonadjuvanted BCG or ΔmmaA4BCG vaccines indicating that BCG/adjuvant mixtures may be more effective at inducing central memory T cells. Importantly, when either conventional BCG or the mutant were formulated in adjuvant and administered to SCID mice or immunocompromised mice depleted of IFNγ, significantly lower vaccine-derived mycobacterial CFU were detected relative to immunodeficient mice injected with non-adjuvanted BCG. Overall, these data suggest that immunization with the ΔmmaA4BCG/adjuvant formulation may be an effective, safe, and relatively inexpensive alternative to vaccination with conventional BCG.

Malaria infections do not compromise vaccine-induced immunity against tuberculosis in mice.

BACKGROUND: Given the considerable geographic overlap in the endemic regions for malaria and tuberculosis, it is probable that co-infections with Mycobacterium tuberculosis and Plasmodium species are prevalent. Thus, it is quite likely that both malaria and TB vaccines may be used in the same populations in endemic areas. While novel vaccines are currently being developed and tested individually against each of these pathogens, the efficacy of these vaccines has not been evaluated in co-infection models. To further assess the effectiveness of these new immunization strategies, we investigated whether co-infection with malaria would impact the anti-tuberculosis protection induced by four different types of TB vaccines in a mouse model of pulmonary tuberculosis. PRINCIPAL FINDINGS: Here we show that the anti-tuberculosis protective immunity induced by four different tuberculosis vaccines was not impacted by a concurrent infection with Plasmodium yoelii NL, a nonlethal form of murine malaria. After an aerogenic challenge with virulent M. tuberculosis, the lung bacterial burdens of vaccinated animals were not statistically different in malaria infected and malaria naïve mice. Multi-parameter flow cytometric analysis showed that the frequency and the median fluorescence intensities (MFI) for specific multifunctional T (MFT) cells expressing IFN-γ, TNF-α, and/or IL-2 were suppressed by the presence of malaria parasites at 2 weeks following the malaria infection but was not affected after parasite clearance at 7 and 10 weeks post-challenge with P. yoelii NL. CONCLUSIONS: Our data indicate that the effectiveness of novel TB vaccines in protecting against tuberculosis was unaffected by a primary malaria co-infection in a mouse model of pulmonary tuberculosis. While the activities of specific MFT cell subsets were reduced at elevated levels of malaria parasitemia, the T cell suppression was short-lived. Our findings have important relevance in developing strategies for the deployment of new TB vaccines in malaria endemic areas.

Vaccine-induced anti-tuberculosis protective immunity in mice correlates with the magnitude and quality of multifunctional CD4 T cells.

The development of improved vaccines against Mycobacterium tuberculosis has been hindered by a limited understanding of the immune correlates of anti-tuberculosis protective immunity. In this study, we examined the relationship between long-term anti-tuberculosis protection and the mycobacterial-specific CD4 multifunctional T (MFT) cell responses induced by five different TB vaccines (live-attenuated, subunit, viral vectored, plasmid DNA, and combination vaccines) in a mouse model of pulmonary tuberculosis. In a 14-month experiment, we showed that TB vaccine-induced CD4 T cell responses were heterogenous. Antigen-specific monofunctional CD4 T cells expressing single cytokines and MFT CD4 T cells expressing multiple cytokines (IFN-γ and TNF-α, IFN-γ and IFN-γ, TNF-α, and IL-2, and all three cytokines) were identified after the immunizations. Interestingly, compared to the monofunctional cells, significantly higher median fluorescent intensities (MFIs) for IFN-γ and TNF-α were detected for triple-positive MFT CD4 T cells induced by the most protective vaccines while modest differences in relative MFI values were seen for the less protective preparations. Most importantly during the 14-month study, the levels of vaccine-induced pulmonary and splenic protective immunity correlated with the frequency and the integrated MFI (iMFI, frequency×MFI) values of triple-positive CD4 T cells that were induced by the same vaccines. These data support efforts to use MFT cell analyses as a measure of TB vaccine immunogenicity in human immunization studies.

Lysine auxotrophy combined with deletion of the SecA2 gene results in a safe and highly immunogenic candidate live attenuated vaccine for tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a major global health problem, despite the widespread use of the M. bovis Bacille Calmette-Guerin (BCG) vaccine and the availability of drug therapies. In recent years, the high incidence of coinfection of M. tuberculosis and HIV, as well as escalating problems associated with drug resistance, has raised ominous concerns with regard to TB control. Vaccination with BCG has not proven highly effective in controlling TB, and also has been associated with increasing concerns about the potential for the vaccine to cause disseminated mycobacterial infection in HIV infected hosts. Thus, the development of an efficacious and safe TB vaccine is generally viewed as a critical to achieving control of the ongoing global TB pandemic. In the current study, we have analyzed the vaccine efficacy of an attenuated M. tuberculosis strain that combines a mutation that enhances T cell priming (ΔsecA2) with a strongly attenuating lysine auxotrophy mutation (ΔlysA). The ΔsecA2 mutant was previously shown to be defective in the inhibition of apoptosis and markedly increased priming of antigen-specific CD8(+) T cells in vivo. Similarly, the ΔsecA2ΔlysA strain retained enhanced apoptosis and augmented CD8(+) T cell stimulatory effects, but with a noticeably improved safety profile in immunosuppressed mice. Thus, the M. tuberculosis ΔsecA2ΔlysA mutant represents a live attenuated TB vaccine strain with the potential to deliver increased protection and safety compared to standard BCG vaccination.