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

Transcription

1 CLINICAL AND VACCINE IMMUNOLOGY, July 2009, p Vol. 16, No /09/$ doi: /cvi Copyright 2009, American Society for Microbiology. All Rights Reserved. Development of a Murine Mycobacterial Growth Inhibition Assay for Evaluating Vaccines against Mycobacterium tuberculosis Marcela Parra, 1 Amy L. Yang, 1 JaeHyun Lim, 1 Kristopher Kolibab, 1 Steven Derrick, 1 Nathalie Cadieux, 1 Liyanage P. Perera, 2 William R. Jacobs, 3 Michael Brennan, 1,4 and Sheldon L. Morris 1 * Center for Biologics Evaluation and Research, United States Food and Drug Administration, Bethesda, Maryland 1 ; Metabolism Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 2 ; Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 3 ; and Aeras Global TB Vaccine Foundation, Rockville, Maryland 4 Received 6 February 2009/Returned for modification 7 March 2009/Accepted 11 May 2009 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 growthinhibitory 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. The tuberculosis (TB) epidemic is a global public health tragedy that is being fueled by the spread of human immunodeficiency virus/aids and the increasing incidence of multiple-drug-resistant Mycobacterium tuberculosis strains. Annually, about 2 million people worldwide die from tuberculosis and 8 to 9 million new cases of this disease are reported (34). Although the current TB vaccine, Mycobacterium bovis BCG, has been widely used for decades, its effectiveness has been shown to be highly variable in wellcontrolled clinical trials (5). While immunization with BCG is effective against severe childhood disease, BCG does not adequately protect against the most prevalent form of the disease, adult pulmonary tuberculosis (13). Vaccinated individuals who become infected with TB are susceptible to disease progression when the BCG-induced immune responses are suppressed or wane with time (32). Clearly, to curb the global TB epidemic, more effective immunization strategies must be generated and evaluated. The development of new vaccines against TB has been hindered by our limited understanding of the mechanisms of protective immunity against M. tuberculosis. While it is known that * Corresponding author. Mailing address: FDA/CBER, Building 29/ Room 502, 29 Lincoln Drive, Bethesda, MD Phone: (301) Fax: (301) sheldon.morris@fda.hhs.gov. Supplemental material for this article may be found at Published ahead of print on 20 May acquired cellular immune responses are critical for controlling tuberculosis infections, the cell subsets that confer antituberculosis protective immunity have not been adequately defined (14). In addition, the immune mechanisms that are responsible for inhibiting the intracellular growth of M. tuberculosis have not been fully delineated and the surrogate biomarkers of this growth inhibition remain unknown. Because it is difficult to study the multiple components of the immune system and their numerous interactions in vivo, the development of an in vitro system which models the in vivo immune responses should facilitate the identification of antituberculosis protective immune mechanisms. The availability of a relevant in vitro assay should allow a more direct study of the mediators of protective immunity against M. tuberculosis in a controlled system. Although in vitro mycobacterial growth inhibition assays for human cells have been developed and are being characterized for their capacity to detect vaccine-induced immunogenicity in human clinical trials, the development and assessment of preclinical assays to measure vaccineinduced activity against M. tuberculosis has thus far been limited (4, 6, 16, 18, 30, 35). To accelerate TB vaccine development and investigations of protective immune mechanisms, we initiated studies aimed at developing a murine in vitro functional assay for evaluating the protective activity of TB vaccines. For this assay, antituberculosis protection was evaluated by targeting an important end point, the control of M. tuberculosis growth within its primary host cell, the macrophage. By assessing the immune-mediated 1025

2 1026 PARRA ET AL. CLIN. VACCINE IMMUNOL. inhibition of mycobacterial growth, we hypothesized that our results would correlate more directly with in vivo protection than the measurement of other immune responses, including cytokine expression. In addition to assessing cellular immune mechanisms, a relevant in vivo assay could be useful for screening and comparing new TB vaccine candidates. From a manufacturing viewpoint, a standardized in vitro functional assay could also be adapted to measure vaccine potency, lot-to-lot production consistency, and vaccine stability. Here we describe our initial results from the characterization of a murine in vitro functional assay for assessing the activity of TB vaccines. We show that vaccine-induced protection seen in vitro for five different TB vaccines correlates with the antituberculosis protective immunity detected in a mouse model of pulmonary TB. Also, we establish an in vitro profile of cytokine expression which is associated with the activity of BCG vaccine and demonstrate that similar in vitro cytokine responses were detected for the four other types of TB vaccines that were tested in this study. MATERIALS AND METHODS Vaccines. The BCG Pasteur vaccine preparation was derived from the mycobacterial culture collection of the Trudeau Institute. Heat-killed BCG was prepared by autoclaving 10 8 CFU of BCG Pasteur at 121 C for 20 min. No mycobacterial growth was seen when aliquots of the heat-killed preparation were incubated for 3 to 4 weeks on Lowenstein-Jensen slants. The SD1 DNA vaccine was generated by cloning a fusion of the ESAT6 and antigen 85B genes into the pvax DNA vector (Invitrogen, San Diego, CA) as described previously (12). The E6-85B protein is an ESAT6-antigen 85B M. tuberculosis fusion protein which was purified by nickel affinity chromatography after cloning and expressing the ESAT6-antigen 85B fusion gene in the pet23b vector system (Novagen, San Diego CA). The protein-adjuvant formulation was prepared by mixing the fusion protein (50 g/ml) with dimethyldioctadecylammonium bromide (DDA; 150 g/ml; Kodak) and monophosphoryl lipid (MPL; 250 g/ml; Avanti Polar Lipids, Alabaster, AL). The seca2 gene deletion mutant was isolated by electroporating the pmb179 suicide vector containing a seca2 allele and a sacb marker into M. tuberculosis H37Rv and then counterselecting on Middlebrook 7H11 plates containing 38 mm (NH 4 ) 2 SO 4 and 3% sucrose (3). The MVA-4TB vaccine was generated by cloning four M. tuberculosis genes (antigen 85A, antigen 85B, ESAT6, and HSP65) as well as the interleukin-15 (IL-15) gene into a modified vaccinia virus Ankara (MVA) vector (27). Immunization schedules. In these in vivo studies, five C57BL/6 mice per group were utilized. For the live BCG and seca2 vaccines, 10 6 CFU were given once subcutaneously. A dose equivalent to 10 6 CFU of heat-killed BCG was also injected once by the subcutaneous route. Five micrograms of the E6-85B protein in the DDA (15 g) MPL (25 g) adjuvant was administered three times, 2 weeks apart, while an identical dose and schedule of the adjuvant was given as a control. For the DNA immunization, 200 g of the SD1 DNA vaccine or the pvax vector control was injected three times, 3 weeks apart, by the intramuscular route. Finally, two doses of PFU of the MVA-4TB construct or the MVA vector were given subcutaneously 1 month apart. In vitro coculture assay. The coculture assay for evaluating TB vaccines was based on procedures described earlier by Elkins and coworkers (1, 6, 7). The primary modification to the published methods included the preparation and use of the target cells for the assay, bone marrow macrophages (BMM ). These procedural changes included using 30% fewer and more purified (without red blood cells) macrophages, incubation after the M. tuberculosis macrophage infection without antibiotic, and infecting with the WHO standard M. tuberculosis Erdman strain. In our procedures, BMM were flushed through the femurs of C57BL/6 mice with Dulbecco s modified Eagle s medium (DMEM). The red blood cells were then lysed in ACK buffer solution for 3 min. After washing the cells and preparing a single-cell suspension, cells/ml were suspended in DMEM containing 10% fetal bovine serum, 10% L929a conditioned medium, and 1% of the following reagents: L-glutamine, modified Eagle s medium nonessential amino acids, HEPES buffer solution, and sodium pyruvate. The cells were then placed in each well of a 24-well plate and incubated for 7 days at 37 C in 5% CO 2. The medium was replaced every 2 to 3 days during the 7-day incubation. After the 7-day culture, the concentration of BMM was about 10 7 cells per well. For the mycobacterial infections, M. tuberculosis Erdman was added to each well at a multiplicity of infection of 1:100 (bacteria to BMM ) for 2 hours and then the wells were washed five times with phosphate-buffered saline (PBS). To determine the extent of bacterial uptake, a fraction of the macrophage cultures was immediately lysed with 0.1% saponin and the resulting cell lysates were diluted in PBS 0.04% Tween 80 and plated on Middlebrook 7H11 plates supplemented with 10% oleic acid, albumin, dextrose, catalase (OADC) enrichment medium (Becton Dickinson, Sparks MD). Typically, these plates were counted after 14 to 17 days of incubation at 37 C. Growth of the M. tuberculosis infection within BMMO was further monitored by lysing cultures at 4, 7, and 10 to 11 days after culture initiation and plating lysates on Middlebrook 7H11 plates with 10% OADC. The activity of the test vaccines was evaluated by coculturing splenocytes from immunized mice with the M. tuberculosis-infected BMM. For the live vaccines, the in vitro studies were initiated 6 weeks after the immunization. For the other vaccine preparations, the in vitro assays were begun 1 month following the final immunization. To harvest the splenocytes, the spleens were aseptically removed from three immunized and naïve C57BL/6 mice and disrupted to prepare a single-cell suspension. The erythrocytes were then lysed with ACK buffer for 4 min and the remaining spleen cells were washed with cold DMEM. To remove adherent splenic macrophages, spleen cells were added to culture flasks for 2 h at 37 C and nonadherent cells were recovered by gentle pipetting. Finally, of the nonadherent splenocytes were overlaid on 10 7 M. tuberculosis-infected macrophages and incubated at 37 C with 5% CO 2. At the specified time, the adherent cells were lysed with 0.1% saponin and diluted cell lysates were plated on Middlebrook 7H11 10% OADC plates for enumeration of mycobacterial CFU as described above. To test whether macrophage lysis contributed to the reduction in mycobacterial CFU during the course of the coculture assay, culture supernatants from selected samples at each time point were also plated on Middlebrook 7H11 plates. Since the number of mycobacteria detected in the supernatants at every time point from all vaccines and controls was at least 100-fold less than the bacteria present in adherent cells, intracellular killing (and not macrophage lysis) is likely the primary mechanism of bacterial reduction in the coculture system. Evaluation of vaccine-induced protective immunity in a mouse model of pulmonary tuberculosis. Six weeks after vaccination with the live attenuated vaccines and 4 weeks following the final vaccinations with subunit, viral-vectored, and DNA vaccines, the mice were aerogenically challenged with M. tuberculosis Erdman suspended in PBS with 0.04% Tween 80 at a concentration known to deliver about 200 CFU in the lungs over a 30-min exposure in a Middlebrook chamber (Glas Col, Terre Haute, IN). To determine the infection dose and the postinfection bacterial burden, mice were sacrificed at 4 h and 28 days postchallenge and then the lungs and spleens were removed aseptically and homogenized separately in PBS 0.04% Tween 80 using a Seward Stomacher 80 blender (Tekmar, Cincinnati OH). After serial dilutions in PBS-Tween 80, the lung and spleen homogenates were plated on Middlebrook 7H11 plates containing 10% OADC, 10 mg/ml ampicillin, 50 mg/ml cycloheximide, and 2 mg/ml 2-thiophenecarboxylic acid hydride (Sigma). The addition of 2-thiophenecarboxylic acid hydride to the growth medium inhibits BCG growth but not the growth of M. tuberculosis. Again, the plates were incubated for 14 to 17 days at 37 C before counting mycobacterial CFU. Evaluation of cytokine responses induced in the coculture assay. At the specified time period, nonadherent splenic cells were recovered from supernatants of the culture wells and stored in RNAlater (Qiagen, Valencia CA). Total RNA was isolated from these cellular suspensions using the RNAeasy minikit protocol (Qiagen). Equivalent amounts of RNA from these samples were reverse transcribed to cdna using the SuperScript first-strand synthesis kit (Invitrogen, San Diego CA). The effectiveness of the DNA synthesis for individual samples was assessed by analyzing the PCR products generated with primers for the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene. To quantitate the cytokine transcriptional responses in cells recovered from the in vitro system, the cdna was evaluated using RT2 profile cytokine PCR arrays (SAB Biosciences, Frederick, MD) and an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). For the cytokine PCR assay, the expression of 84 cytokine-like genes was evaluated (21). The mrna expression levels for each gene were then normalized to the expression of the GAPDH gene using the following equation: relative mrna expression 2 (Ct of cytokine Ct of GAPDH), where Ct is the threshold cycle. To determine whether the relative levels of gene expression were significantly different than the expression levels in naïve mice, the PCR array results were compared using the Wilcoxon matched pairs test (GraphPad Prism software, version 4; San Diego, CA). Outlier data points were formally removed using Grubb s test (GraphPad Prism, version 4). Finally, the relative gene expression values in

3 VOL. 16, 2009 IN VITRO ASSAY FOR EVALUATING TB VACCINES 1027 Vaccine TABLE 1. Tuberculosis vaccine preparations immune cell cultures were determined by dividing the gene expression levels in experimental samples by the expression values in naïve controls. Each reported value represents the mean increase (or decrease) of RNA expression relative to the naïve controls for 12 BCG vaccine experiments and three to five studies with the other vaccines. To assess cytokine protein concentrations by enzyme-linked immunosorbent assay (ELISA), culture supernatants were centrifuged to remove nonadherent cells. The levels of gamma interferon (IFN- ), tumor necrosis factor alpha (TNF- ), IL-27, and IL-10 were then evaluated using cytokine ELISA kits as described by the manufacturers. The levels of IFN-, TNF-, and IL-10 were measured using BD OptiEIA kits (Becton Dickinson), while IL-27 Quantikine ELISA kits were purchased from R&D Systems (Minneapolis, MN). Statistics. The data from these experiments were analyzed using the Graph- Pad Prism 4 program. The in vivo and in vitro protection results were evaluated by t tests and the Spearman correlation test (GraphPad Prism, version 4). The cytokine expression data were evaluated using t tests and the nonparametric Wilcoxon matched pairs test. RESULTS Description BCG...M. bovis BCG Pasteur seca2 mutant...m. tuberculosis proapoptotic attenuated strain with seca2 gene deletion SD1 DNA vaccine...dna vaccine expressing ESAT6 antigen 85B fusion protein E6 85B protein...esat6 antigen 85B fusion protein formulated in DDA-MPL adjuvant MVA-4 TB...Modified vaccinia virus Ankara strain expressing four TB antigens, ESAT6, antigen 85A, antigen 85B, and HSP65 Evaluation of five different vaccines against M. tuberculosis. To investigate whether vaccine-induced antituberculosis immunity could be detected in vitro for the various TB vaccine preparations, mice were immunized with either BCG vaccine, an M. tuberculosis attenuated mutant with a deletion in the seca2 gene, a TB DNA vaccine, a TB fusion protein, or a viral-vectored vaccine expressing four TB antigens (Table 1). These preparations included five types of TB vaccines which express different antigens and have distinct immunogenic capacities. The BCG vaccine preparation is derived from a BCG Pasteur stock culture. The seca2 strain is a highly protective proapoptotic deletion mutant of M. tuberculosis (17). The SD1 DNA vaccine construct expresses an ESAT6-antigen 85B fusion protein, which has been shown to boost BCG-induced immune responses and to protect against primary M. tuberculosis infections (12). The ESAT6-antigen 85B protein is a tuberculosis fusion antigen which induces substantial protective immunity when formulated in DDA/MPL adjuvant. Finally, the MVA-4 TB vaccine is a modified vaccinia Ankara construct that expresses four TB antigens, antigen 85A, antigen 85B, ESAT6, and Hsp 65 (27). As controls, mice were injected with either heat-killed BCG, the DNA vaccine vector, the MVA vector, or the DDA/MPL adjuvant. Characterization of in vitro mycobacterial growth inhibition for evaluating vaccines against tuberculosis. To assess whether vaccine activity could be evaluated using in vitro assays, the growth of M. tuberculosis in bone marrow macrophages that were cocultured with immune splenocytes was monitored over an 11-day period. Representative M. tuberculosis growth curves for a coculture assay which tested two vaccines (BCG and the SD1 protein-adjuvant formulation) are shown in Fig. 1. When M. tuberculosis-infected macrophages were incubated without splenocytes, logarithmic increases in the numbers of tubercle bacilli were observed. Interestingly, significant decreases in the in vitro mycobacterial growth were detected when naïve spleen cells were cultured with macrophages infected with M. tuberculosis. Typically, a 0.5-log 10 reduction in the proliferation of M. tuberculosis organisms (relative to the infected macrophages alone) was seen in naïve spleen cell cultures, presumably due to innate antimycobacterial mechanisms (29). However, greater decreases in the in vitro growth of M. tuberculosis were detected when splenocytes from mice immunized with the ESAT6-antigen 85B fusion protein-adjuvant formulation or BCG vaccine were cultured with M. tuberculosis-infected macrophages. For the experiment shown in Fig. 1, relative decreases in bacterial numbers of 0.8 and 0.9 log 10 (compared to naïve controls) were seen for the cultures containing the TB fusion protein and BCG immune splenocytes, respectively, at day 7 of the assay. In contrast, splenocytes from mice injected with an inactive control, the vector DNA, did not control the intramacrophage tubercular growth better than cells from naïve mice. To evaluate the reproducibility of this assay, four experiments were run using naïve and BCG immune splenocytes from four different groups of mice. In these studies, the temporal growth profiles and the mean protective responses for the BCG immune cells were consistent. The mean BCG-induced protection seen at day 7 (i.e., the difference between the naïve and experimental log 10 CFU) at day 7 of the coculture assays was (mean standard error of the mean [SEM]). Furthermore, similar mycobacterial growth patterns were seen when M. tuberculosis clinical isolates were substituted for the Erdman laboratory strain. For example, when naïve and BCG-immune splenocytes were cocultured with macrophages infected with the CDC1551 clinical isolate, the mycobacterial growth profile was essentially equivalent to the growth pattern seen in the M. tuberculosis Erdman infections (data not shown). To compare the in vitro mycobacterial growth inhibition responses induced by immunization with the different vaccine FIG. 1. Inhibition of intramacrophage growth of M. tuberculosis by splenocytes recovered from immunized mice. Murine bone marrow macrophages that were infected with M. tuberculosis were cocultured with splenocytes taken from mice immunized with BCG or the ESAT6- antigen 85B protein suspended in DDA-MPL adjuvant and the growth of M. tuberculosis was monitored over an 11-day culture period. As controls, M. tuberculosis-infected bone marrow macrophages were cultured with splenocytes from mice injected with vector DNA, naïve splenocytes, or in the absence of spleen cells (I-BMMO).

4 1028 PARRA ET AL. CLIN. VACCINE IMMUNOL. TABLE 2. Comparison of in vitro antituberculosis activity with in vivo protection results a Vaccine preparation In vitro activity b Protection c (lung) Protection c (spleen) BCG * * * seca2 mutant * * * E6-85B protein * * * Heat-killed BCG * * * MVA-4 TB * * * SD1 DNA vaccine * * * DNA vector MVA vector DDA-MPL adjuvant a *, significantly different than naïve controls (P 0.05). b In vitro activity is the difference between the naïve and experimental CFU (log 10 ) at day 7 of the in vitro coculture assay (mean SEM). c Protection is the difference between the naïve and experimental CFU (log 10 ) in mouse lungs (or spleens) at 28 days after an aerosol challenge with M. tuberculosis (mean SEM). preparations, at least three coculture assays were completed using splenocytes removed from mice vaccinated with each of the vaccines and the controls. Table 2 shows the mean in vitro growth inhibition responses for these vaccine preparations after 7 days of culture. A 7-day period was chosen because in most experiments the maximal differences in intramacrophage growth between naïve and immune cell cultures were usually seen at this time. Statistical analysis of these data indicated that immunization with each of the TB vaccines induced significantly elevated in vitro antituberculosis activity compared to the naïve controls (P 0.05). Moreover, the in vitro growthinhibitory responses evoked by each vaccine were significantly increased relative to the corresponding control (e.g., proteinadjuvant versus adjuvant alone). Overall, the inhibitory responses elicited by the vaccine preparations could be separated into two groups: high and moderate in vitro activity. The highly active vaccines (BCG, seca2, and the ESAT6-antigen 85B fusion protein-adjuvant formulation) induced substantial 0.87 to 0.93 log 10 CFU in vitro growth-inhibitory responses, while the moderately effective preparations, heat-killed BCG, MVA-4 TB, and the SD1 DNA vaccine, evoked less antituberculosis immunity (0.40 to 0.57 log 10 CFU). In fact, the in vitro activities detected in cocultures of BCG and seca2 immune splenocytes were significantly greater (P 0.05) than that induced in the moderately active group. Importantly, injection of the vector and adjuvant controls did not induce antituberculosis immunity that exceeded the growth-inhibitory responses seen in naïve controls. Comparison of in vivo protection with in vitro activity. The in vivo activities of these vaccine preparations were measured in standard vaccination/challenge experiments as described previously (11). Mice were vaccinated as described in Materials and Methods, challenged by the aerosol route with 200 CFU of M. tuberculosis Erdman, and sacrificed 28 days later to determine relative organ bacterial burdens. The in vivo activity was calculated by determining the mean protective responses (naïve control organ CFU vaccinated organ CFU) for two to four experiments (Table 2). Again, the vaccine-induced protection could be separated into the same two groups. The highly active vaccines, BCG, seca2 mutant, and the E6-85B fusion protein, induced substantial antituberculosis protective TABLE 3. Normalized cytokine mrna expression at days 5 and 7 of coculture with BCG-immune splenocytes Cytokine Mean ( SEM) normalized expression a on: Day 5 Day 7 GDF * IFN * * IL * IL * IL * * IL * * TNF * * Bmp # # IL # # Tnfsf # a Mean normalized expression values represent mean fold differences compared to expression by naïve controls, which were assigned a value of 1. *, upregulated cytokine expression in BCG-immune cell cultures or cytokine expression in cultures using naïve splenocytes; #, downregulated cytokine expression in BCG-immune cell cultures relative to expression in naïve control cultures. immunity. For these vaccines, greater than a 1-log 10 reduction in mycobacterial burden in the lung, relative to naïve controls, was seen at 28 days postchallenge. Although the moderately active vaccine preparations (heat-killed BCG, TB MVA, and the SD1 DNA vaccine) evoked modest protection in the lungs (0.57 to 0.79 log 10 CFU compared to naïve controls) at the 4-week postchallenge time point, these protective responses were significantly greater than the immune responses detected in naïve animals. Similar to the in vitro studies, injection of the vector and adjuvant controls did not evoke elevated in vivo antituberculosis responses relative to naïve mice. Finally, we compared the in vivo and in vitro data to assess the relevance of the vaccine activity seen in the coculture assay. Importantly, Spearman analysis showed that the correlation between in vitro vaccine-mediated activity and in vivo vaccine-induced protection in the lungs and spleens was highly significant (P 0.001) for these five different vaccines and the control preparations. Identification of immune biomarkers associated with vaccine-induced protective responses. To determine whether immune biomolecules were differentially regulated in cocultured naïve and BCG immune splenocytes, RNA expression of 84 cytokines in nonadherent cells was assessed using PCR arrays. The extent of expression was determined by normalizing the real-time PCR values to the expression of the GAPDH housekeeping gene and then by comparing the GAPDH-adjusted results to the level of expression in naïve controls. In these studies, vaccine-induced differential regulation was defined as significantly different levels of expression in naïve and immune cell cultures by the Wilcoxon matched pairs test and expression levels at least twofold higher (or lower) than the naïve controls. Table 3 shows the cytokine genes that were differentially regulated at days 5 and 7 of the coculture assay using BCGimmune cells. Cytokine expression was evaluated at days 5 and 7 of the coculture because significant inhibition of mycobacterial growth was detected on these days. At day 5, the expression levels of 5 of 84 cytokine-related genes were consistently upregulated and 2 were downregulated in the in vitro cultures. At day 7, the expression levels of nine cytokine-related genes were differentially regulated in BCG-immune cultures. At both time

5 VOL. 16, 2009 IN VITRO ASSAY FOR EVALUATING TB VACCINES 1029 FIG. 2. Protein expression levels of IFN-, TNF-, and IL-27 were higher in BCG coculture supernatants than controls. Bone marrow macrophages infected with M. tuberculosis were cocultured with BCG-immune splenocytes (BCG), naïve splenocytes, or in the absence of spleen cells (BMM). At day 7, the cytokine protein levels in culture supernatants were determined using cytokine ELISA protocols. *, P points, the expression levels of two cytokines known to be critical for conferring antituberculosis activity, IFN- and TNF-, were significantly upregulated. Furthermore, the expression levels of two other genes (IL-21 and IL-27) were upregulated and two were consistently downregulated (Bmp1 and IL-1) at 5 and 7 days after the initiation of a coculture of TB-infected macrophages with BCG-immune splenocytes. Importantly, the levels of mrna for many cytokines were not differentially regulated during these experiments. These cytokines included IFN-, IL-2, IL-3, Il-4, Il-7, Il-12, and IL-15 (see Table S1 in the supplemental material). To verify that the gene regulation values reflected cytokine protein expression levels for cytokines in which ELISA test kits were available, specific cytokine concentrations in supernatants from cocultures containing either BCG-immune or naïve splenocytes were assessed. For three cytokines which had elevated gene expression levels, IFN-, TNF-, and IL-27, cytokine ELISA responses were also significantly elevated in supernatants from BCG-immune cocultures compared to the naïve controls (Fig. 2). In contrast, the protein levels of IL-10, a molecule whose expression is not consistently differentially regulated in BCG-immune cultures, were increased less than 1.5-fold in the cocultures of BCG-immune cells compared to naïve controls (data not shown). For the validation of immune biomarkers against M. tuberculosis, it is important to define correlates of antituberculosis protective immunity which are applicable to all new TB vaccines as well as the BCG vaccine. To identify general antituberculosis biomarkers, cytokine gene expression analysis was extended at day 7 of cocultures to splenocytes recovered from mice immunized with the different TB vaccines. As shown in Table 4, the expression levels of five cytokine genes (GDF15, IFN-, IL-21, IL-27, and TNF- ) were often upregulated and three were frequently downregulated (Bmp1, IL-1, and Tnfsf14) in nonadherent cells recovered from coculture assays. It should be emphasized that elevated levels for IFN-, IL-21, and TNF- mrna and decreased levels of IL-1 mrna were seen in cocultures of all vaccines tested. Among the controls, only upregulation of IL-21 or downregulation of BMP1 was detected in the in vitro assays using splenocytes from mice injected with the DNA vector or the adjuvant, respectively. It is of interest that the only consistent differences in the cytokine profiles between the highly active vaccines (BCG, seca2 mutant, and ESAT6-antigen 85B fusion protein) and the moderately active preparations (heat-killed BCG and the SD1 DNA vaccine) were the higher IFN- levels (11.2, 17. 1, and 23.7 versus 3.8 and 7.1, respectively) detected in cocultures using spleen cells from mice immunized with the highly active vaccines. In addition, as seen in earlier in vivo studies, the cytokine responses in the cocultures containing BCG and seca2 immune cells were nearly identical (21). No substantive differences in the levels of cytokine regulation were detected in cocultures containing BCG- or seca2-immune cells. Surprisingly, despite the significantly higher levels of growth inhibition TABLE 4. Normalized cytokine mrna expression at day 7 of coculture for candidate TB vaccines Cytokine Normalized mrna expression BCG seca2 E6-85B kbcg a SD1DNA DNAVC b Adjuvant GDF IFN IL IL TNF Bmp IL Tnfsf a kbcg, heat-killed BCG. b DNAVC, vector DNA.

6 1030 PARRA ET AL. CLIN. VACCINE IMMUNOL. activity detected in BCG-immune cultures relative to heatkilled BCG controls, only modest differences were seen when the cytokine profiles of cultures using BCG-immune or heatkilled BCG-immune splenocytes were compared. Only elevated IFN- and IL-21 expression levels (2.4- and 3.2-fold increases, respectively) were detected in the BCG-immune cell cultures in comparison to cultures containing splenocytes recovered from mice injected with heat-killed BCG. DISCUSSION To facilitate the characterization of vaccines against tuberculosis, we have developed a murine in vitro functional assay for evaluating vaccine-induced antituberculosis activity. We reasoned that inhibition of intramacrophage growth of M. tuberculosis was a direct and relevant end point for assessing the potency of TB vaccines. With this functional assay, multiple protective mechanisms likely contribute to the in vitro reduction in mycobacterial proliferation, including both innate and adaptive immune responses. Importantly, we demonstrated that the vaccine-induced in vitro growth inhibition activity detected in our coculture assay significantly correlates with the protective immunity seen postvaccination in our mouse model of pulmonary tuberculosis and is relevant for estimating the in vivo potency of TB vaccines. In addition, we have shown that the protective responses evoked by different types of vaccines can be assessed using this assay. In the human in vitro functional studies reported thus far, only BCG-induced activity has been evaluated (4, 18, 35). Our results suggest that the antituberculosis protective immunity evoked by subunit, viral vector, or DNA vaccines should be detectable in similar assays designed to measure vaccine-induced immune responses in human clinical trials. An unexpected finding from our in vivo and in vitro protection studies was the moderate antituberculosis activity detected with the heat-killed BCG vaccine preparation. Although this result was surprising, it is consistent with earlier data reported by Opie and Freund, who showed that injection of heat-inactivated tubercle bacilli was nearly as effective as live BCG immunization in protecting rabbits against infection with M. tuberculosis (23). A major goal of tuberculosis vaccine research during the past 2 decades has been to identify the correlates of protective immunity against M. tuberculosis. The identification of protective correlates of immunity would clearly facilitate the evaluation and characterization of new TB vaccines both in preclinical studies and clinical trials. In vaccine studies, molecules that are upregulated (or downregulated) after the immunization are candidates as protective correlates. In our studies, using the in vitro coculture assay, cytokine profiles associated with antituberculosis protective activity were identified. Importantly, the patterns of cytokines that are up or downregulated after immunization with active TB vaccines were similar. For most of the vaccines, GDF15, IFN-, IL-21, IL-27, and TNF- expression levels were upregulated while BMP1, IL-1 and Tnfsf14 expression levels were downregulated (relative to naives) in cocultures of infected BMM and immune splenocytes. In contrast, the expression of these same cytokines was usually not differentially regulated for in vitro assays using control splenocytes. This result suggests that different types of TB vaccines evoke similar patterns of protective immune mediators in a mouse model of tuberculosis. Interestingly, the BCG-induced cytokine patterns detected with the in vitro assay resembled the cytokine responses seen in the lungs of BCGvaccinated mice at 10 days after an aerogenic challenge with M. tuberculosis (21). In both the in vitro and in vivo experiments, IFN-, IL-21, IL-27, and TNF- were upregulated (after exposure to a M. tuberculosis infection) in the cells of animals immunized with BCG. The IFN- and TNF- results are not surprising since these cytokines have been shown to be critical components of protective immunity against M. tuberculosis (14, 15, 22, 25). However, the roles of IL-21 and IL-27 during a mycobacterial infection are less certain. IL-21 is a type I cytokine which is produced largely by antigen-activated T cells. Although its major functions are to activate CD8 T cells and NK cells and to stimulate B-cell immunoglobulin production, IL-21 can also suppress dendritic cell activity (2, 20). Based on this immunosuppressive activity of IL-21, it has been suggested that this cytokine plays a pivotal role in the regulation of pathogen-induced immune responses. It has recently been shown that the combination of IL-21 and TGF- induces proinflammatory Th17 cells (36). It would be of interest to determine whether TH17 cell-promoting activity is also detected when IL-21 is combined with GDF15, another TGF- family member that was shown to be upregulated in this study. Similar to IL-21, IL-27 may have an important immune regulatory capacity since it has both proinflammatory and antiinflammatory properties. While IL-27 has been shown to promote inflammation, Th1 responses, and IFN- production, it can also inhibit inflammatory responses (19, 33). Surprisingly, animals deficient in the IL-27 receptor were able to limit M. tuberculosis infections more effectively than controls and neutralization of IL-27 in vitro led to enhanced antituberculosis activity in human monocytes (26, 28). Clearly, further studies will be needed to define the precise roles of IL-21 and IL-27 in mediating protective immunity against M. tuberculosis. Besides allowing the identification of contrasting immune responses to different vaccines, these data permit comparisons of the cytokine responses between highly active and less active vaccine formulations in the mouse model. It is surprising that the only consistent difference we observed in the cytokine profiles for the highly active and moderately effective vaccines was the increased IFN- levels seen for the most potent vaccines. We are currently investigating whether the elevated IFN- levels are associated with greater protection because of the increased expression of IFN- -inducible genes such as the CXCL9 and CXCL10 chemokines. In addition to identifying cytokine patterns that correlate with vaccine-induced protection, this murine in vitro system should be useful for defining cell subsets and cellular immune mechanisms which contribute to antituberculosis protective immunity. Using a similar murine coculture assay, Cowley and Elkins have demonstrated that double-negative T cells, membrane-bound TNF, and IFN- -independent processes partially mediate the anti-francisella protective immunity induced by the live vaccine strain of Francisella tularensis (7 10). In preliminary experiments, we found that purified splenic T cells from BCG-vaccinated mice inhibited mycobacterial growth in vitro and that the depletion of CD4 cells but not CD8 cells largely abrogated the protective effect of BCG vaccine (K. Kolibab and S. Derrick, unpublished results). These data are

7 VOL. 16, 2009 IN VITRO ASSAY FOR EVALUATING TB VACCINES 1031 consistent with earlier in vivo and in vitro results which indicated that the BCG vaccine induces a strong CD4 antituberculosis protective response (24, 31). It should be emphasized that cells from other relevant organs can be utilized in this assay. For example, we have shown that cells from the lymph nodes of BCG-vaccinated mice also inhibit the intramacrophage growth of M. tuberculosis in the coculture assay (M. Parra, unpublished results). Future experiments with the coculture assay will further examine the relative importance of T-cell subsets from the different relevant organs in mediating the protective immunity induced by the various types of TB vaccine preparations. In summary, we have described the development of a murine in vitro coculture assay to characterize the protective activity induced by TB vaccines. 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