Kristopher Kolibab

Mycobacterium bovis BCG Immunization Induces Protective Immunity against Nine Different Mycobacterium tuberculosis Strains in Mice

ABSTRACT Recent preclinical and epidemiologic studies have suggested that certain Mycobacterium tuberculosis genotypes (in particular, Beijing lineage strains) may be resistant to Mycobacterium bovis BCG vaccine-induced antituberculosis protective immunity. To investigate the strain specificity of BCG-induced protective responses in a murine model of pulmonary tuberculosis, C57BL/6 mice were vaccinated with BCG vaccine and then challenged 2 months later with one of nine M. tuberculosis isolates. Four of these strains were from the W-Beijing lineage (HN878, N4, NHN5, and ChS) while four were non-Beijing-type isolates (C913, CDC1551, NY669, and NY920). As a control, the WHO standard M. tuberculosis Erdman strain was evaluated in these vaccination/challenge experiments. To assess the protective responses evoked by BCG immunization, organ bacterial burdens and lung pathology were assessed in vaccinated and naïve mice at 4, 12, and 20 weeks postchallenge as well as during the day of infection. At 4 weeks after the aerosol challenge with each of these strains, significantly reduced bacterial growth in the lungs and spleens and significantly improved lung pathology were seen in all vaccinated animals compared to naïve controls. After 12 weeks, reduced organ bacterial burdens were detected in vaccinated animals infected with six of nine challenge strains. Although lung CFU values were lower in vaccinated mice for only three of nine groups at 20 weeks postchallenge, significantly decreased lung inflammation was seen in all immunized animals relative to controls at 20 weeks postchallenge. Taken together, these data demonstrate that BCG vaccination protects against infection with diverse M. tuberculosis strains in the mouse model of pulmonary tuberculosis and suggest that strain-specific resistance to BCG-induced protective immunity may be uncommon.

Development of a Murine Mycobacterial Growth Inhibition Assay for Evaluating Vaccines against Mycobacterium tuberculosis

ABSTRACT 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.

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

ABSTRACT 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-γ), 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-γ-producing T cells and multifunctional CD4 and CD8 T cells expressing IFN-γ/tumor necrosis factor alpha (TNF-α), TNF-α/IL-2, and IFN-γ/TNF-α/IL-2. In contrast, BCG-induced protection was characterized by fewer CD4 and CD8 monofunctional T cells expressing IFN-γ and only IFN-γ/TNF-α and IFN-γ/TNF-α/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 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.

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.

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.

Intranasal Administration of Mycobacterium bovis BCG Induces Superior Protection against Aerosol Infection with Mycobacterium tuberculosis in Mice

ABSTRACT 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.

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.

Sensitivity to Isoniazid of Mycobacterium bovis BCG Strains and BCG Disseminated Disease Isolates

Intravesical Mycobacterium bovis BCG instillation therapy is commonly used to prevent recurrences of carcinoma in situ (CIS) and/or Ta/T1 papillary tumors of the urinary bladder (8). A rare and serious complication associated with BCG treatment of bladder cancer is the development of disseminated BCG disease (1, 6). Current recommendations for bladder cancer patients (being treated with BCG) who develop persistent fever or experience an acute febrile illness consistent with a mycobacterial infection include administration of combination therapy with two or more antimycobacterial agents (3). Recently, U.S. federal public health authorities were notified of two cases of disseminated BCG disease resulting after bladder cancer therapy with BCG Connaught. Drug sensitivity testing (DST) of these strains suggested that both of these strains were resistant to low concentrations of isoniazid (0.2 μg/ml isoniazid [INH]), the primary drug used to treat Mycobacterium tuberculosis complex infections. These DST results raised concerns about acquired resistance in vivo or the generation of drug-resistant bacteria during manufacturing. Interpretation of the DST data was confounded because the levels of sensitivity to INH for many BCG strains were poorly defined. To address these concerns, the sensitivities to INH of 13 different BCG strains were determined using the standard proportion method (7). DST analysis showed that four of the BCG strains (Tokyo, Moreau, Russia, and Sweden) and two M. tuberculosis controls were extremely sensitive to INH at the 0.05-μg/ml level (Table 1). In contrast, the other 9 BCG strains tested (including BCG Connaught) consistently had 2- to 4-fold-higher MICs for INH. For these BCG strains, the MICs ranged from 0.1 to 0.2 μg/ml INH. Importantly, the MICs of INH for the patient isolates and the parental BCG Connaught strain were essentially identical. Table 1. Drug sensitivity testing of M. bovis BCG strains Interestingly, our DST analysis of the different BCG strains indicated that all BCG strains obtained by international public health officials from the Pasteur Institute after 1926 had moderately elevated levels of resistance to INH (Table 1). Following the original attenuation of BCG at the Pasteur Institute from 1908 to 1921, BCG strains were maintained by serial propagation in different laboratories (1921 to 1961) until frozen seed lot systems were developed. Behr et al. have reported that all BCG strains obtained from the Pasteur Institute after 1926 had a point mutation in the mma3 gene which resulted in impaired methoxy mycolic acid production (2). Since the target for INH antituberculosis activity is the mycolic acid biosynthetic pathway, it has been speculated that this mutation would cause decreased sensitivity to INH for BCG strains obtained after 1926. These data support this hypothesis since every strain obtained from the Pasteur Institute after 1926 has a modestly elevated MIC for INH. The increasing frequency of drug-resistant M. tuberculosis disease worldwide has enhanced public health concerns about the adequacy of antituberculosis therapeutic regimens and the overall effectiveness of tuberculosis control programs (4, 5). Within this environment, the identification of two BCG isolates with apparent increased resistance to INH from patients with disseminated mycobacterial disease was worrisome. However, these DST data suggest that the decreased sensitivity to INH seen in the patient isolates (relative to that seen in sensitive M. tuberculosis strains) was likely not caused by acquired resistance in vivo or a genotypic modification during the manufacturing process. The modestly decreased sensitivity to INH seen in these BCG strains likely resulted from a mutation in a gene involved in mycolic acid biosynthesis which occurred more than 8 decades ago.

Time to Detection of Mycobacterium tuberculosis Using the MGIT 320 System Correlates with Colony Counting in Preclinical Testing of New Vaccines

ABSTRACT Clinical studies have suggested that the enumeration of mycobacteria by using automated liquid systems is a faster and simpler alternative to quantitative cultures. Here, we show that the time to detection of M. tuberculosis growth as measured with the MGIT 320 liquid culture system inversely correlates with CFU determinations from culture on solid media and that mycobacterial quantification using the MGIT system is faster and easier to perform than CFU plating.