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.

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.

T-bet modulates the antibody response and immune protection during murine malaria.

CD4(+) T-cell subtypes govern the synthesis of different Ab isotypes and other immune functions. The influence of CD4(+) T-cell differentiation programs on isotype switching and other aspects of host immunological networks during malaria infection are currently poorly understood. Here, we used Tbx21(-/-) mice deficient for T-bet, a regulator of Th1 CD4(+) T-cell differentiation, to examine the effect of Th1 CD4(+) T cells on the immune protection to nonlethal murine malaria Plasmodium yoelii 17XNL. We found that Tbx21(-/-) mice exhibited significantly lower parasite burden that correlated with elevated levels of IgG1, indicating that T-bet-dependent Ab isotype switching may be responsible for lower parasite burden. Absence of T-bet was also associated with a transient but significant loss of T cells during the infection, suggesting that T-bet may suppress malaria-induced apoptosis or induce proliferation of T cells. However, Tbx21(-/-) mice produced greater numbers of Foxp3(+) CD25(+) regulatory CD4(+) T cells, which may contribute to the early contraction of T cells. Lastly, Tbx21(-/-) mice exhibited unimpaired production of IFN-γ by a diverse repertoire of immune cell subsets and a selective expansion of IFN-γ-producing T cells. These observations may have implications in malaria vaccine design.

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.

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.

Radiation-induced cellular and molecular alterations in asexual intraerythrocytic Plasmodium falciparum.

BACKGROUND: γ-irradiation is commonly used to create attenuation in Plasmodium parasites. However, there are no systematic studies on the survival, reversion of virulence, and molecular basis for γ-radiation-induced cell death in malaria parasites. METHODS: The effect of γ-irradiation on the growth of asexual Plasmodium falciparum was studied in erythrocyte cultures. Cellular and ultrastructural changes within the parasite were studied by fluorescence and electron microscopy, and genome-wide transcriptional profiling was performed to identify parasite biomarkers of attenuation and cell death. RESULTS: γ-radiation induced the death of P. falciparum in a dose-dependent manner. These parasites had defective mitosis, sparse cytoplasm, fewer ribosomes, disorganized and clumped organelles, and large vacuoles-observations consistent with "distressed" or dying parasites. A total of 185 parasite genes were transcriptionally altered in response to γ-irradiation (45.9% upregulated, 54.1% downregulated). Loss of parasite survival was correlated with the downregulation of genes encoding translation factors and with upregulation of genes associated with messenger RNA-sequestering stress granules. Genes pertaining to cell-surface interactions, host-cell remodeling, and secreted proteins were also altered. CONCLUSIONS: These studies provide a framework to assess the safety of γ-irradiation attenuation and promising targets for genetic deletion to produce whole parasite-based attenuated 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.

Induction of cell death after localization to the host cell mitochondria by the Mycobacterium tuberculosis PE_PGRS33 protein.

PE_PGRS33 is the most studied member of the unique PE family of mycobacterial proteins. These proteins are composed of a PE domain (Pro-Glu motif), a linker region and a PGRS domain (polymorphic GC-rich-repetitive sequence). Previous studies have shown that PE_PGRS33 is surface-exposed, constitutively expressed during growth and infection, involved in creating antigenic diversity, and able to induce death in transfected or infected eukaryotic cells. In this study, we showed that PE_PGRS33 co-localizes to the mitochondria of transfected cells, a phenomenon dependent on the linker region and the PGRS domain, but not the PE domain. Using different genetic fusions and chimeras, we also demonstrated a direct correlation between localization to the host mitochondria and the induction of cell death. Finally, although all constructs localizing to the mitochondria did induce apoptosis, only the wild-type PE_PGRS33 with its own PE domain also induced primary necrosis, indicating a potentially important role for the PE domain. Considering the importance of primary necrosis in Mycobacterium tuberculosis dissemination during natural infection, the PE_PGRS33 protein may play a crucial role in the pathogenesis of tuberculosis.

Highly persistent and effective prime/boost regimens against tuberculosis that use a multivalent modified vaccine virus Ankara-based tuberculosis vaccine with interleukin-15 as a molecular adjuvant.

Novel immunization strategies are needed to enhance the global control of tuberculosis (TB). In this study, we assessed the immunizing activity of a recombinant modified vaccinia Ankara (MVA) construct (MVA/IL-15/5Mtb) which overexpresses five Mycobacterium tuberculosis antigens (antigen 85A, antigen 85B, ESAT6, HSP60, and Mtb39), as well as the molecular adjuvant interleukin-15 (IL-15). Homologous prime/boost studies showed that the MVA/IL-15/5Mtb vaccine induced moderate but highly persistent protective immune responses for at least 16 months after the initial vaccination and that the interval between the prime and boost did not significantly alter vaccine-induced antituberculosis protective immunity. At 16 months, when the Mycobacterium bovis BCG and MVA/IL-15/5Mtb vaccine-induced protection was essentially equivalent, the protective responses after a tuberculous challenge were associated with elevated levels of gamma interferon (IFN-gamma), IL-17F, Cxcl9, and Cxcl10. To amplify the immunizing potential of the MVA/IL-15/5Mtb vaccine, a heterologous prime/boost regimen was tested using an ESAT6-antigen 85B (E6-85) fusion protein formulated in dimethyldiotacylammonium bromide/monophosphoryl lipid A (DDA/MPL) adjuvant as the priming vaccine and the MVA/IL-15/5Mtb recombinant virus as the boosting agent. When MVA/IL-15/5Mtb vaccine boosting was done at 2 or 6 months following the final fusion protein injections, the prime/boost regimen evoked protective responses against an aerogenic M. tuberculosis challenge which was equivalent to that induced by BCG immunization. Long-term memory after immunization with the E6-85-MVA/IL-15/5Mtb combination regimen was associated with the induction of monofunctional CD4 and CD8 IFN-gamma-producing T cells and multifunctional CD4 and CD8 T cells expressing IFN-gamma/tumor necrosis factor alpha (TNF-alpha), TNF-alpha/IL-2, and IFN-gamma/TNF-alpha/IL-2. In contrast, BCG-induced protection was characterized by fewer CD4 and CD8 monofunctional T cells expressing IFN-gamma and only IFN-gamma/TNF-alpha and IFN-gamma/TNF-alpha/IL-2 expressing multifunctional T (MFT) cells. Taken together, these results suggest that a heterologous prime/boost protocol using an MVA-based tuberculosis vaccines to boost after priming with TB protein/adjuvant preparations should be considered when designing long-lived TB immunization strategies.

A practical in vitro growth inhibition assay for the evaluation of TB vaccines.

New vaccines and novel immunization strategies are needed to improve the control of the global tuberculosis epidemic. To facilitate vaccine development, we have been creating in vitro mycobacterial intra-macrophage growth inhibition assays. Here we describe the development of an in vitro assay designed for BSL-2 laboratories which measures the capacity of vaccine-induced immune splenocytes to control the growth of isoniazid-resistant Mycobacterium bovis BCG (INH(r) BCG). The use of the INH(r) BCG as the infecting organism allows the discrimination of BCG bacilli used in murine vaccinations from BCG used in the in vitro assay. In this study, we showed that protective immune responses evoked by four different types of Mycobacterium tuberculosis vaccines [BCG, an ESAT6/Antigen 85B fusion protein formulated in DDA/MPL adjuvant, a DNA vaccine expressing the same fusion protein, and a TB Modified Vaccinia Ankara construct expressing four TB antigens (MVA-4TB)] were detected. Importantly, the levels of vaccine-induced protective immunity seen in the in vitro assay correlated with the results from in vivo protection studies in the mouse model of pulmonary tuberculosis. Furthermore, the growth inhibition data for the INH(r) BCG assay was similar to the previously reported results for a M. tuberculosis infection assay. The cytokine expression profiles at day 7 of the INH(r) BCG growth inhibition studies were also similar but not identical to the cytokine patterns detected in earlier M. tuberculosis co-culture assays. Overall, we have shown that a BSL-2 compatible in vitro growth inhibition assay using INH(r) BCG as the intra-macrophage target organism should be useful in developing and evaluating new TB immunization strategies.

Development of a murine mycobacterial growth inhibition assay for evaluating vaccines against Mycobacterium tuberculosis.

The development and characterization of new tuberculosis (TB) vaccines has been impeded by the lack of reproducible and reliable in vitro assays for measuring vaccine activity. In this study, we developed a murine in vitro mycobacterial growth inhibition assay for evaluating TB vaccines that directly assesses the capacity of immune splenocytes to control the growth of Mycobacterium tuberculosis within infected macrophages. Using this in vitro assay, protective immune responses induced by immunization with five different types of TB vaccine preparations (Mycobacterium bovis BCG, an attenuated M. tuberculosis mutant strain, a DNA vaccine, a modified vaccinia virus strain Ankara [MVA] construct expressing four TB antigens, and a TB fusion protein formulated in adjuvant) can be detected. Importantly, the levels of vaccine-induced mycobacterial growth-inhibitory responses seen in vitro after 1 week of coculture correlated with the protective immune responses detected in vivo at 28 days postchallenge in a mouse model of pulmonary tuberculosis. In addition, similar patterns of cytokine expression were evoked at day 7 of the in vitro culture by immune splenocytes taken from animals immunized with the different TB vaccines. Among the consistently upregulated cytokines detected in the immune cocultures are gamma interferon, growth differentiation factor 15, interleukin-21 (IL-21), IL-27, and tumor necrosis factor alpha. Overall, we have developed an in vitro functional assay that may be useful for screening and comparing new TB vaccine preparations, investigating vaccine-induced protective mechanisms, and assessing manufacturing issues, including product potency and stability.

A multi-valent vaccinia virus-based tuberculosis vaccine molecularly adjuvanted with interleukin-15 induces robust immune responses in mice.

Tuberculosis caused by Mycobacterium tuberculosis is responsible for nearly two million deaths every year globally. A single licensed vaccine derived from Mycobacterium bovis, bacille Calmette-Guerin (BCG) administered perinatally as a prophylactic vaccine has been in use for over 80 years and confers substantial protection against childhood tuberculous meningitis and miliary tuberculosis. However, the BCG vaccine is virtually ineffective against the adult pulmonary form of tuberculosis that is pivotal in the transmission of tuberculosis that has infected almost 33% of the global population. Thus, an effective vaccine to both prevent tuberculosis and reduce its transmission is urgently needed. We have generated a multi-valent, vectored vaccine candidate utilizing the modified virus Ankara (MVA) strain of vaccinia virus to tandemly express five antigens, ESAT6, Ag85A, Ag85B, HSP65 and Mtb39A of M. tuberculosis that have been reported to be protective individually in certain animal models together with an immunostimulatory cytokine interleukin-15 (MVA/IL-15/5Mtb). Although, immunological correlates of protection against tuberculosis in humans remain to be established, we demonstrate that our vaccine induced comparable CD4(+) T cell and greater CD8(+) T cell and antibody responses against M. tuberculosis in vaccinated mice in a direct comparison with the BCG vaccine and conferred protection against an aerogenic challenge of M. tuberculosis, thus warranting its further preclinical development.

Early pulmonary cytokine and chemokine responses in mice immunized with three different vaccines against Mycobacterium tuberculosis determined by PCR array.

In this study, the early pulmonary cytokine and chemokine responses in mice immunized with either BCG vaccine, a DeltasecA2 mutant of Mycobacterium tuberculosis, or a DNA vaccine expressing an ESAT6-antigen 85B fusion protein and then aerogenically challenged with a low dose of M. tuberculosis were evaluated by PCR array. The cellular immune responses at day 10 postchallenge were essentially equivalent in the lungs of mice immunized with either the highly immunogenic BCG vaccine or the DeltasecA2 M. tuberculosis mutant strain. Specifically, 12 immune biomolecules (including gamma interferon [IFN-gamma], interleukin-21 [IL-21], IL-27, IL-17f, CXCL9, CXCL10, and CXCL11) were differentially regulated, relative to the levels for naïve controls, in the lungs of vaccinated mice at this time point. Although the vaccine-related immune responses evoked in mice immunized with the DNA vaccine were relatively limited at 10 days postinfection, upregulation of IFN-gamma RNA synthesis as well as increased expression levels of CXCL9, CXCL10, and CXCL11 chemokines were detected.

The safety of post-exposure vaccination of mice infected with Mycobacterium tuberculosis.

New post-exposure tuberculosis vaccination strategies are being developed to prevent disease in individuals latently infected with Mycobacterium tuberculosis. However, concerns about the potential induction of deleterious Koch-like reactions after immunization of persons with latent tuberculosis has limited progress in assessing the effectiveness of post-exposure vaccination. To evaluate the safety of immunization after M. tuberculosis infection, two mouse models were established, a drug treatment low bacterial burden model and an active disease model. Twelve different M. tuberculosis antigen preparations and vaccines (including DNA, subunit, viral vectored, and live, attenuated vaccines) were evaluated using these mouse models. In the low bacterial burden model, post-exposure vaccination did not induce significant reactivational disease and only injection of BCG evoked increases in lung inflammatory responses at 1 month after the immunizations. Additionally, although significant increases in lung inflammation were seen for animals injected with the hps65 DNA vaccine or a M. tuberculosis culture supernatant preparation, no differences in the survival periods were detected between vaccinated and non-vaccinated mice at 10 months post-immunization using the low bacterial burden model. For the active disease model, significantly more lung inflammation was observed at 1 month after administration of the hsp65 DNA vaccine but none of the antigen preparations tested increased the lung bacterial burdens at this early time point. Furthermore, vaccination of diseased mice with BCG or TB DNA vaccines did not significantly affect mortality rates compared to non-vaccinated controls at 10 months post-immunization. Overall, these data suggest that while the potential risk of inducing Koch-like reactions is low after immunization of persons with latent tuberculosis, extreme caution is still needed as post-exposure vaccines progress from pre-clinical experiments into the initial phases of clinical testing.