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Immunogenicity and protective efficacy of DNA vaccines encoding secreted and non-secreted forms of Mycobacterium tuberculosis Ag85A
Tubercle and Lung Disease (1999) 79(4), 251–259 © 1999 Harcourt Publishers Ltd Article no. tuld.1998.0196
Immunogenicity and protective efficacy of DNA vaccines encoding secreted and non-secreted forms of Mycobacterium tuberculosis Ag85A S. L. Baldwin,† C. D. D’Souza,* I. M. Orme,* M. A. Liu,† K. Huygen,‡ O. Denis,‡ A. Tang,§ L. Zhu,§ D. Montgomery,§ J. B. Ulmer† *Mycobacterial Research Laboratories, Department of Microbiology, Colorado State University, Fort Collins Colorado, USA † Chiron Corporation, Emeryville, California, USA ‡ Department of Virology, Pasteur Institute, Brussels, Belgium § Department of Virus and Cell Biology, Merck Research Laboratories, West Point Pennsylvania, USA
Summary Objective: To determine the efficacy of Ag85A-DNA against challenge with a highly virulent human clinical isolate of Mycobacterium tuberculosis (CSU37) and to compare the potencies of two types of Ag85A-DNA vaccines; those expressing secreted and non-secreted forms of the protein. Design: Ag85A-DNA vaccinated mice were challenged with a highly virulent clinical isolate of M. tuberculosis (CSU37) in order to compare the efficacy of these vaccines. In vitro studies were also performed. Results: Enhanced humoral and cellular responses were induced in mice vaccinated with the secreted Ag85A-DNA compared to the non-secreted Ag85A-DNA. In addition, secreted Ag85A-DNA conferred protective immunity against infection with M. tuberculosis (CSU37). Conclusions: DNA vaccines encoding M. tuberculosis Ag85A have been shown to induce potent humoral and cellular immune responses leading to protection from M. tuberculosis (Erdman) challenge in mouse models.1 In this study we demonstrate that Ag85A can confer protection in a rigorous challenge model using a highly virulent human clinical isolate of M. tuberculosis (CSU37). This challenge model appears able to discriminate between DNA vaccines of differing potencies, as the more immunogenic DNA construct encoding a secreted form of Ag85A was protective, whereas the less immunogenic DNA construct encoding a non-secreted form of Ag85A was not. © 1999 Harcourt Publishers Ltd INTRODUCTION The only tuberculosis vaccine approved for human use is Mycobacterium bovis BCG, which is a live attenuated vaccine. The efficacy of the BCG vaccine has been the subject of controversy since several large human clinical trials revealed that the protection conferred by this vaccine varies greatly, and zero per cent efficacy was reported in some endemic areas such as Southern India.2 A significant limitation of the existing vaccine is that it elicits a delayed type hypersensitivity reaction to commercial purified protein derivative (PPD), the skin test reagent used in the diagnosis of tuberculosis. Furthermore, the live BCG
Correspondence to: Dr Susan L. Baldwin, Colorado State University, Department of Microbiology, Fort Collins, CO 80523, USA. Tel: +1 970 491 2694; Fax: +1 970 491 1815; E-mail address: [email protected]. Received: 16 April 1998; Revised: 7 July 1998; Accepted: 8 September 1998
vaccine represents a potential health risk, in itself, to immunocompromized individuals. Thus, many factors warrant the development of new vaccines against tuberculosis. The administration of DNA vaccines has been successful at generating both cell-mediated and humoral immune responses in many animal models of infectious disease, such as herpes simplex,3–7 hepatitis B,8–13 hepatitis C,14–18 HIV,19–23 Leishmania,24,25 Borrelia burgdorferi (lyme disease),26–28 M. tuberculosis,1,29–35 and others (for review see 36). Several mycobacterial antigens have been expressed using plasmid DNA vectors; 6 kDa,30 30–32 kDa [Ag85A, Ag85B, Ag85C],1,31 36 kDa,30,34 38 kDa,35 65 kDa29,30,32–34 and 70 kDa.30 Ag85A (mycolyl transferase)37 is an immunostimulatory protein which is an abundant component in the culture filtrate of M. tuberculosis.38 DNA vaccines encoding Ag85A have been shown to confer protection against an aerosol challenge with M. tuberculosis in mice.1 251
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To date, only laboratory strains of M. tuberculosis have been used in the low dose aerosol infected mouse model in order to study anti-tuberculosis DNA vaccine efficacy in vivo. In the present study, our aims were to determine the efficacy of Ag85A DNA against challenge with a highly virulent human clinical isolate of M. tuberculosis (CSU 37) and to compare the potency of two types of Ag85A DNA vaccines; those expressing secreted (tPA-Ag85) and nonsecreted (mature-Ag85) forms of the protein. Differences in the potency of the two DNA vaccines were determined based on the immunogenicity and protective efficacy in vivo against aerogenic challenge with a highly virulent clinical isolate of M. tuberculosis. We show that the DNA vaccine encoding the secreted form of Ag85A is superior to the mature form of the vaccine, as measured by the production of Ag85A specific antibodies, lymphoproliferation, and Ag85A specific CTL responses. We also show that the DNA vaccine encoding the secreted form of Ag85 was able to confer protection against a highly virulent human clinical isolate of tuberculosis.
MATERIALS AND METHODS Animals Specific pathogen-free, female C57BL/6 or BALB/c mice were purchased from Charles River Laboratories (North Wilmington, Mass, USA). C57BL/6 mice were held in barrier conditions in an ABL3 Biohazard Laboratory (Colorado State University, CO, USA). Mice were 4–8 weeks old at the beginning of the experiments and were housed four to a cage. Mice were allowed free access to water and standard mouse chow. Bacteria M. tuberculosis strains Erdman (TMCC 107), and CSU37 (kindly provided by Dr John Belisle, CSU, CO, USA) were grown to mid-log phase in Proskauer-Beck medium and stored in ampoules frozen at –70°C until use. DNA vaccine DNA encoding secreted or mature (non-secreted) Ag85A protein was cloned into the eukaryotic expression vector V1Jns as described previously.1 Briefly, the Ag85A gene was inserted into the V1Jns plasmid and was expressed under the control of the promoter and intron A of the immediate early protein-1 (IE1) from cytomegalovirus (CMV) followed by a polyadenylation site from the bovine growth hormone gene (BGH). Incorporation of the intron A and polyadenylation site into the plasmid allow for post-transcriptional processing within the eukaryotic host. In the secreted tPA-Ag85A DNA vaccine, the gene Tubercle and Lung Disease (1999) 79(4), 251–259
for the mature Ag85A (lacking the native mycobacterial signal sequence) is preceded by the eukaryotic signal sequence for human tissue plasminogen activator (tPA). The mature form of the DNA-Ag85A (mature-Ag85A) vaccine lacks any signal sequence. Vaccination protocol Mice (n=5/group) were immunized intramuscularly in both quadriceps, three times, at 3 week intervals or as noted in the figure legends. At each vaccination mice were given 50 µg/quadricep of either tPA-Ag85A DNA or matureAg85A DNA. Negative control animals were injected with saline or control plasmid vector DNA (which lacked a gene insert) in sterile saline. The positive control group was injected once, subcutaneously, with M. bovis BCG Pasteur at a concentration of 106 bacilli on the last day of immunization. IMMUNOGENICITY Determination of immune responses in mice included serum antibodies, as measured by ELISA; lymphoproliferation, as measured upon restimulation of spleen cells in vitro with antigen; and cytotoxic T lymphocytes, as measured upon in vitro restimulation with peptides (p7, corresponding to amino acids 71–78 (PVGGQSSF) and p15, corresponding to amino acids 145–152 (YAGAMSGL) of Ag85A). Details of these assays are given elsewhere.1,39 Briefly, lymphocytes harvested from pools of three spleens were restimulated for 7 days with peptide at 10 µg/ml then tested for cytotoxicity in a 3 hour 51Crrelease assay in round bottomed microwell plates on 104 51Cr-labeled P815 target cells. Results were similar whether individual spleens or pools of spleens were used in the restimulation. Effector cells were added to target cells at various E/T ratios in RPMI 1640 supplemented with 10% FCS in a total volume of 0.2 ml. Percent specific 51 Cr release was calculated as follows: 100X (release by CTLs-spontaneous release)/(total release–spontaneous release). Experiments were performed three times. Similar results were obtained for antibody responses and lymphoproliferation measured after one or multiple DNA immunizations, whereas differences in CTL responses were most readily seen after a single immunization. Experimental infections To determine bacterial growth curves, C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine, USA), 6–8 weeks of age, were aerogenically infected with either M. tuberculosis Erdman or M. tuberculosis CSU37 and the growth of bacilli in the lungs was determined over time. Mice were aerogenically exposed to either M. tuberculosis Erdman or © 1999 Harcourt Publishers Ltd
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M. tuberculosis CSU37, in a Middlebrook airborne infection apparatus (Glas-Col, Terre Haute, IN, USA) which was calibrated to deliver approximately 100 viable bacilli into the lung. Groups of four mice were euthanized either 7, 20 or 40 days post infection. Lungs were harvested, homogenized, serially diluted in sterile PBS and plated on 7H11 agar plates. Plates were incubated at 37°C in a humidified incubator and colonies were counted 3 weeks later. For the protection studies, mice were aerogenically exposed to approximately 100 viable M. tuberculosis CSU37 bacilli as described above. Animals were infected either 30 days or 90 days following administration of the last vaccination. Mice were euthanized 30 days following challenge with M. tuberculosis CSU37, lungs were harvested and the bacterial colonies were counted as described above.
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Fig. 1 Induction of anti-Ag85 antibodies by DNA vaccination. Female BALB/c mice (4–6 weeks) were injected with the indicated doses of Ag85 DNA three times at 3 week intervals and sera were analyzed by an ELISA at 3 weeks after the final immunization. Shown are geometric mean antibody titers ±SEM (n=5) for mice receiving DNA encoding secreted or mature forms of Ag85A.
Delayed type hypersensitivity Tuberculin purified protein derivative (PPD) lot CT68 was purchased from the Connaught Laboratories (Toronto, Canada). Concentrated PPD (2 mg/ml) was diluted 1:20 in sterile PBS and 50 µl containing 5 µg of PPD per mouse was injected in the left hind footpads using a 30-gauge needle. PBS was injected in the right footpads as negative controls. Footpad thickness/swelling was determined as the difference between the PPD injected footpads and the contralateral saline injected footpad. The DTH response was measured 48 h post-injection using calipers capable of measuring 0.05 mm increments in thickness.
RESULTS Immunogenicity of Ag85A DNA Mice were immunized with DNA encoding either secreted or mature Ag85A in order to compare the potency of each of these constructs to one another. Expression of Ag85A in vivo was assessed indirectly by the production of antiAg85 antibodies. DNA encoding the secreted form of Ag85A was more potent than that encoding the mature form, as seen by the overall higher titer of antibodies and by the induction of antibody responses at doses of DNA much lower than that required of the mature form (Fig. 1). Differences were statistically significant at all DNA doses tested (t-test; P=0.0016 to 0.049). Uninjected mice or those immunized with control plasmid DNA not encoding an antigen did not react to Ag85 in the ELISA (data not shown). Cellular responses were assessed in two ways. First, lymphoproliferation in response to antigen restimulation in vitro was measured. Both constructs induced lymphoproliferation in a dose-dependent fashion (Fig. 2). The © 1999 Harcourt Publishers Ltd
Fig. 2 Induction of lymphoproliferative responses by DNA vaccination. Female BALB/c mice (4–6 weeks) were injected with the indicated doses of Ag85 DNA three times at 3 week intervals and spleens were collected at 3 weeks after the final immunization. Pools of cells from three spleens per group were restimulated in vitro with M. bovis BCG culture filtrate proteins. Shown are data plotted as stimulation index versus DNA dose for mice receiving DNA encoding secreted or mature forms of Ag85A. Data are representative of three separate experiments.
tPA-Ag85 DNA, however, was more potent than matureAg85A DNA as seen by the elevated stimulation index at every dose of DNA tested (analysis of covariance; P=0.0008). Spleen cells from control mice did not proliferate upon restimulation (stimulation index < 2). Second, cytotoxic T lymphocyte (CTL) responses were measured in vitro. Two previously identified H-2d-specific CTL epitopes (amino acids 71–78, 145–152),39 were used to test for induction of CTL by Ag85A-DNA. After a single injection of high DNA dose (100 µg), Ag85-specific CTL were induced by both tPA-Ag85A DNA and mature-Ag85A Tubercle and Lung Disease (1999) 79(4), 251–259
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Fig. 3 Induction of cytotoxic T lymphocyte responses by DNA vaccination. Female BALB/c mice (4–6 weeks) were injected with either 5 or 100 µg of Ag85 DNA and spleens were collected at 4 weeks after a single immunization. Pools of cells from three spleens per group were restimulated with an H-2d-restricted CTL epitope (aa 71–78) and tested for lytic activity against P815 target cells that were untreated, pulsed with peptide p7 (corresponding to amino acids 71–78 (PVGGQSSF)) or stably transfected with Ag85A. Shown are data plotted as % specific lysis versus effector:target ratio for mice receiving DNA encoding secreted or mature forms of Ag85A. Data are representative of three separate experiments.
DNA using either the 71–78 and 145–152 epitopes (Figs 3 and 4). However, the magnitude of the CTL responses appeared to be slightly higher in mice immunized with tPA-Ag85A. This was true whether the P815 target cells were pulsed with the appropriate peptide or were stably expressing full-length Ag85A. Target cells untreated or pulsed with the inappropriate peptide were not lysed by specific CTL. After only a single injection of low DNA dose (5 µg), CTL were induced by tPA-Ag85A DNA only (Fig. 3). Therefore, by all measures tested, tPA-Ag85A DNA was more potent at inducing immune responses than was mature-Ag85A DNA. The differences in potency between DNA vaccines encoding secreted and non-secreted Ag85 were seen after one or multiple immunizations. For CTL, the biggest differences were seen after a single immunization. This may be due to the fact that previous experience in mice has suggested that maximum in vitro CTL responses (as measured by chromium release assays) are quickly achieved with multiple DNA immunizations (Ulmer et al., unpublished observations). Growth of M. tuberculosis CSU37 Growth of the widely used laboratory strain of M. tuberculosis (Erdman) was compared to the growth of a human clinical isolate of M. tuberculosis (CSU37). As seen in Figure 5, the clinical isolate (CSU 37) grew faster and to Tubercle and Lung Disease (1999) 79(4), 251–259
much higher titers within the lungs of mice than did the laboratory strain (Erdman). CSU37 was chosen for subsequent protection studies in light of greater virulence and its susceptibility to several antimycobacterial drugs (data not shown). Delayed type hypersensitivity The DTH response was examined in Ag85A DNA- and BCG-vaccinated mice. As seen in Figure 6, there was little or no DTH response to PPD in any of the mice which were vaccinated with Ag85A-DNA at either 30 or 90 days following vaccination. In contrast, BCG vaccinated animals mounted a significant DTH response 30 days following vaccination (> 0.4 mm); however, this response waned 90 days following vaccination, and was considered to be insignificant (Fig. 6). Protective efficacy The Ag85A-DNA vaccines were tested for their protective efficacy against infection with the highly virulent human clinical isolate M. tuberculosis CSU37. Two separate sets of mice were aerogenically infected with M. tuberculosis CSU37, either 30 or 90 days following vaccination. At 30 days following vaccination both Ag85A DNA vaccines showed slight protection (~0.7–0.8 log 10 reduction). © 1999 Harcourt Publishers Ltd
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Fig. 4 Induction of cytotoxic T lymphocyte responses by DNA vaccination. Female BALB/c mice (4–6 weeks) were injected with either 5 or 100 mg of Ag85 DNA and spleens were collected at 4 weeks after a single immunization. Pools of cells from three spleens per group were restimulated with an H-2d-restricted CTL epitope (aa 145–152) and tested for lytic activity against P815 target cells that were untreated, pulsed with peptide p15 (corresponding to amino acids 145–152 (YAGAMSGL)) or stably transfected with Ag85A. Shown are data plotted as % specific lysis versus effector:target ratio for mice receiving DNA encoding secreted or mature forms of Ag85A. Data are representative of three separate experiments.
good protection (~1.7 log 10 reduction in bacterial load within the lung, compared to saline injected animals). At 90 days following DNA vaccination, tPA-Ag85A DNA conferred significant protection (~0.8 log 10 reduction in bacterial load within the lung, compared to saline injected animals) against infection, whereas mature Ag85A DNA or control DNA did not (Fig. 7B). BCG also protected mice significantly against infection (similar to tPA-Ag85A DNA). However, BCG-mediated protection appeared to be waning compared to that seen at 30 days post vaccination. These results suggest that the antigen-specific protective immune response induced by Ag85A DNA required more than 30 days to mature or was masked by a short-lived non-specific response induced by the DNA itself. DISCUSSION Fig. 5 Growth of M. tuberculosis Erdman compared to M. tuberculosis CSU37 in the lungs of C57BL/6 mice following exposure to low-dose aerosol infections. Shown are data plotted as log 10 viable bacteria within the lung ±SEM (n=4).
However, this was likely due, at least in part, to a nonspecific effect of the DNA itself, as control DNAvaccinated mice also showed slightly reduced lung titers (Fig. 7A). In contrast, BCG-vaccinated animals exhibited © 1999 Harcourt Publishers Ltd
We have shown here that a DNA vaccine encoding the secreted form of M. tuberculosis Ag85A is more potent than a DNA vaccine encoding a mature form of the protein. tPA-Ag85A DNA induced higher levels of specific serum antibodies than the mature-Ag85A vaccine, in addition to enhanced cellular responses (including antigen specific lymphoproliferation and CTL mediated lytic activity). Furthermore, tPA-Ag85A DNA was able to confer immunity against a highly virulent human clinical Tubercle and Lung Disease (1999) 79(4), 251–259
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Fig. 6 Delayed type hypersensitivity to commercial PPD tuberculin in immunized C57BL/6 mice. Left footpads of the immunized mice (30 days or 90 days after vaccination) were injected with 5 µg of PPD in 50 µl sterile saline and contralateral footpads were injected with saline. Shown are data plotted as the mean increase in footpad thickness (mm) ±SD (n=5) over that seen in the contralateral saline injected footpads, 48 h after injection of PPD.
A B Fig. 7 Bacterial loads in the lungs of M. tuberculosis infected mice previously vaccinated with either vector control (control DNA), the secreted form of the vaccine (tPA-Ag85A), or the mature form of the vaccine (mature-Ag85A). Mice were infected aerogenically with approximately 102 M. tuberculosis CSU37 1 month or 3 months post-vaccination. Lungs were harvested 1 month following infection. BCG was the positive control and both saline and control DNA were the negative controls. Shown are data plotted as log10 viable bacteria within the lung (n=5) ±SEM. Groups showing significant protection [P < 0.05] within the lungs compared to saline controls are indicated with an asterisk. a) Challenge at Day 30 post vaccination, b) challenge at Day 90 post vaccination.
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isolate of M. tuberculosis, whereas neither the control DNA nor the mature-Ag85A vaccines was able to protect against this isolate for an extended period of time. Based on transient transfection in vitro, the Ag85A DNA constructs express proteins of expected molecular mass40,41 but differ qualitatively in that the secreted form is targeted to the endoplasmic reticulum for secretion, while the mature form is targeted to the cytoplasm. In addition, the secreted form is modified by the addition of an N-linked oligosaccharide (Montgomery et al., in preparation). The production of anti-Ag85 antibodies was used to assess the expression of Ag85A in vivo. An overall higher titer of antibodies and induction of antibody responses at lower doses of DNA suggest that the tPAAg85A DNA was at least 10-fold more potent than mature-Ag85A DNA. The higher antibody titers in mice vaccinated with tPA-Ag85A DNA are likely the result of more extracellular protein available for induction of B cell responses as a consequence of efficient secretion of Ag85A out of transfected cells in vivo. In contrast, the mature form of Ag85A is not efficiently secreted from transfected cells. In fact, multiple immunizations with mature-Ag85A DNA are required to induce significant levels of anti-Ag85 antibodies.40 It is interesting though, that after multiple immunizations of high doses of mature-Ag85A DNA the antibody titers which were measured approached the titers induced by tPA-Ag85A DNA, suggesting that significant protein is being made available to B cells. One possible explanation could be the release of protein from transfected cells, either as a consequence of toxicity of the protein or of immune-mediated responses directed toward antigen-expressing cells. The latter possibility would seem more likely, since no cellular toxicity was observed after transfection of cells in vitro (Montgomery et al., unpublished observations) and that immune-mediated clearance of transfected cells in vivo has been suggested by studies using reporter proteins.42 In support of this possibility, robust cellular immune responses induced by Ag85A DNA were observed. Cellular responses were measured using both lymphoproliferation and CTL assays. Lymphoproliferation was induced in a dose-dependent fashion with both the matureAg85A and the secreted-Ag85A DNA constructs. These results are consistent with previous observations that Ag85A DNA induced robust cellular immune responses.1 In the present study, DNA doses as low as 0.78 µg induced detectable proliferation with proliferation increasing in response to as much as 200 µg Ag85-DNA. CTL-responses were also measured to compare the cellular responses to the two DNA constructs, using two H-2d-specific CTL epitopes (amino-acids 71–78, 145–152). As shown here, the secreted form of the Ag85A-DNA vaccine was superior to the mature form of the vaccine in vivo as measured by antibody production, antigen specific lymphoprolifera© 1999 Harcourt Publishers Ltd
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tion responses, and CTL mediated lytic activity. To address whether these responses could contribute to increased protective efficacy in a mouse model of tuberculosis, the tPA-Ag85A DNA was compared to the matureAg85A DNA vaccine using a highly virulent clinical isolate of M. tuberculosis (CSU 37). We have previously shown that both the tPA-Ag85A and the mature-Ag85A DNA vaccines are able to confer immunity against the laboratory strain of M. tuberculosis (Erdman).1 Here we have shown that protective immunity against a highly virulent clinical isolate of M. tuberculosis can be attained, but only by the tPA-Ag85 DNA vaccine. Preliminary results in our laboratory suggest that tPA-Ag85 DNA induces a larger memory T cell population in infected mice, as suggested by increased levels of the putative ‘memory’ surface marker phenotype (CD4+/CD44hi/ CD45RBlo),43 compared to that seen in the mature-Ag85 DNA vaccinated mice. This could explain why one vaccine was more protective than the other, since the enhanced generation of memory T cells could lead to stronger anamnestic responses to subsequent infection with M. tuberculosis. This would be especially critical in cases involving infection with an extremely virulent M. tuberculosis isolate such as CSU37, which rapidly grow to very high levels within the lung. A heightened memory response in mice vaccinated with tPA-Ag85A DNA could lead to early containment of the infection within the lungs. Future studies should investigate the efficacy of this vaccine against multiple drug-resistant human clinical M. tuberculosis isolates. In addition to the antigen-specific protective immune response induced by tPA-Ag85A DNA observed at 90 days post-vaccination, a modest non-specific response was seen at 30 days post-vaccination in mice that received control DNA. A similar, but insignificant, reduction in lung titer was also seen in control DNA-vaccinated mice challenged with the Erdman strain (unpublished observations; 1). This effect could be due to the potential presence of immunostimulatory nucleotide sequences in the DNA vector. Such sequences, consisting of the basic motif purine-purine-C-G-pyrimidine, have potent effects on natural killer cells and lymphocytes in vitro, causing them to proliferate and secrete cytokines.44–46 Secretion of such cytokines, including interferon-γ, in vivo in response to the DNA vector could lead to activation of macrophages and the observed reduction in mycobacterial lung titers. This response, however, is short-lived and longterm immunity requires antigen-specific immunity. One goal of a new vaccine against tuberculosis is to formulate one which does not lead to a positive skin reaction to PPD, since this reactivity interferes with the ability to monitor individuals for prior exposure to M. tuberculosis. Regardless of the protection conferred, mice vaccinated with the Ag85A-DNA vaccines did not generate a signiTubercle and Lung Disease (1999) 79(4), 251–259
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ficant DTH response to commercial PPD. The fact that neither the tPA-Ag85A DNA vaccine (shown to be protective against M. tuberculosis CSU37) nor any of the other DNA-vaccines tested (not protective in this model) generated a DTH response suggests that there was no correlation between the ability to mount a DTH response to PPD, and the presence or absence of a protective response against infection with M. tuberculosis CSU37. Indeed, it was established several years ago that DTH and protective immunity to M. tuberculosis are dissociable phenomena.47 The protective immune response against tuberculosis is known to involve the generation of a Th1 type T cell response, which is predominantly characterized by the secretion of IFN-γ. Huygen et al. have shown increased levels of IFN-γ and IL-2 secreted from the splenocytes (pulsed with either PPD or Ag85A) of Ag85ADNA vaccinated donor mice, when compared to control DNA-vaccinated mice,1 and yet these mice do not elicit a DTH response to PPD. In addition, Cooper et al. have shown that IFN-γ knock out mice are in fact capable of mounting a DTH response, suggesting that IFN-γ (critical in a protective response) may not be essential to the DTH response.48 Animals vaccinated with these DNA vaccines do not elicit a DTH response to PPD, which may ultimately allow for the continued use of the tuberculosis diagnostic skin test. In summary, we have shown that Ag85A DNA can protect mice from challenge with a highly virulent human clinical isolate of M. tuberculosis. This rigorous challenge appears to be more able to discriminate between DNA vaccines of differing potencies, since DNA encoding a secreted form of Ag85A was protective but DNA encoding a non-secreted form was not, compared to challenge with a laboratory strain of M. tuberculosis (Erdman), where both DNA vaccines were equally protective. Hence, this model may be useful in the development of new vaccines against tuberculosis.
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ACKNOWLEDGMENTS This work was supported by NIH grant AI-75320, and by Merck Research Laboratories. We would like to thank the staff at the CSU laboratory of animal resources for their excellent care and monitoring of the mice.
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immune responses in mice receiving a DNA-based glycoprotein 120 vaccine. AIDS Res Hum Retroviruses 1994; 10: 1433–1441. Wang B, Boyer J, Srikantan V, et al. DNA inoculation induces neutralizing immune responses against human immunodeficiency virus type 1 in mice and nonhuman primates. DNA Cell Biol 1993; 12: 799–805. Wang B, Ugen K E, et al. Gene inoculation generates immune responses against human immunodeficiency virus type 1. Proc Natl Acad Sci USA 1993; 90: 4156–4160. Wang B, Boyer J, Srikantan V, et al. Induction of humoral and cellular immune responses to the human immunodeficiency Type 1 virus in nonhuman primates in vivo DNA inoculation. Virology 1995; 211: 102–112. Xu D, Liew F Y. Genetic vaccination against leishmaniasis. Vaccine 1994; 12: 1534–1536. Xu D, Liew F Y. Protection against leishmaniasis by injection of DNA encoding a major surface glycoprotein gp63 of L. major. Immunol 1995; 84: 173–176. Luke C J, Carner K, Liang X, Barbour A G. An OspA-based DNA vaccine protects mice against infection with Borrelia burgdorferi. J Infect Dis 1997; 175: 91–97. Simon M M, Gern L, Hauser P, et al. Protective immunization with plasmid DNA containing the outer surface lipoprotein A gene of Borrelia burgdorferi is independent of an eukaryotic promoter. Eur J Immunol 1996; 26: 2831–2840. Zhong W, Wiesmuller K H, Kramer M D, Wallich R, Simon M M. Plasmid DNA and protein vaccination of mice to the outer surface protein A of Borrelia burgdorferi leads to induction of T helper cells with specificity for a major epitope and augmentation of protective IgG antibodies in vivo. Eur J Immunol 1996; 26: 2749–2757. Lowrie D B, Tascon R E, Colston M J, Silva C L. Towards a DNA vaccine against tuberculosis. Vaccine 1994; 12: 1537–1539. Lowrie D B, Silva C L, Colston M J, Ragno S, Tascon R E. Protection against tuberculosis by a plasmid DNA vaccine. Vaccine 1997; 15: 834–838. Lozes E, Huygen K, Content J, et al. Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex. Vaccine 1997; 15: 830–833. Silva C L. New vaccines against tuberculosis. Braz J Med Biol Res 1995; 28: 843–851. Silva C L, Silva M F, Pietro R C, Lowrie D B. Characterization of T cells that confer a high degree of protective immunity against tuberculosis in mice after vaccination with tumor cells expressing hsp65. Infect Immun 1996; 64: 2400–2407. Tascon R E, Colston M J, Ragno S, Stavropoulos E, Gregory D, Lowrie D B. Vaccination against tuberculosis by DNA injection. Nat Med 1996; 2: 888–892
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35. Zhu X, Venkataprasad N, Thangaraj H S, et al. Functions and specificity of T Cells following nucleic acid vaccination of mice against Mycobacterium tuberculosis infection. J Immunol 1997; 158: 5921–5926. 36. Donnelly J J, Ulmer J B, Shiver J W, Liu M A. DNA vaccines [Review] Ann Rev Immunology 1997; 15: 617–648. 37. Belisle J T, Vissa V D, Sievert T, Takayama K, Brennan P J, Besra G S. Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis. Science 1997; 276: 1420–1422. 38. Wiker H G, Harboe M. The Antigen 85 complex: a major secretion product of Mycobacterium tuberculosis. Microb Reviews 1992; 56: 648–661. 39. Denis O, Tanghe A, Palfliet K, et al. Vaccination with plasmid DNA encoding antigen 85A stimulates a CD4+ and CD8+ T-Cell epitope repertoire broader than stimulated by M. tuberculosis H37Rv infection. Infect Immun 1998; 66: 1527–1533. 40. Montgomery D L, Huygen K, Yawman A M, et al. Induction of humoral and cellular immune responses by vaccination with M. tuberculosis antigen 85 DNA. Cell Mol Biol 1997; 43: 285–292. 41. Ulmer J B, Liu M A, Montgomery D L, et al. Expression and immunogenicity of M. tuberculosis antigen 85 by DNA vaccination. Vaccine 1997; 15: 792–794. 42. Davis H L, Millan C L, Watkins S C. Immune-mediated destruction of transfected muscle fibers after direct gene transfer with antigen-expressing plasmid DNA. Gene-Ther 1997; 4: 181–188. 43. Griffin J P, Orme I M. Evolution of CD4 T-cell subsets following infection of naive and memory immune mice with Mycobacterium tuberculosis. Infect Immun 62: 1683–1690. 44. Krieg A M, Yi A-K, Waldschmidt T J, et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 1995; 374: 546–549. 45. Messina J P, Gilkeson G S, Pisetsky D S. Stimulation of in vitro murine lymphocyte proliferation by bacterial DNA. J Immunol 1991; 147: 1759–1764. 46. Yamamoto S, Yamamoto T, Kataoka T, Kuramoto E, Yano O, Tokunaga T. Unique palindromic sequences in synthetic oligonucleotides are required to induce IFN and augment IFN-mediated natural killer activity. J Immunol 1992; 148: 4072–4076. 47. Orme I M, Collins F M. Adoptive protection of the Mycobacterium tuberculosis – infected lung: dissociation between cells that passively transfer protective immunity and those that transfer delayed-type hypersensitivity to tuberculin. Cellular Immunology 1984; 84: 113–120. 48. Cooper A M, Dalton D K, Stewart T A, Griffin J P, Russel D G, Orme I M. Disseminated tuberculosis in interferon γ genedisrupted mice. J Exp Med 1993; 178: 2243–2247.
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Immunogenicity and protective efficacy of DNA vaccines encoding secreted and non-secreted forms of Mycobacterium tuberculosis Ag85A S. L. Baldwin,† C. D. D’Souza,* I. M. Orme,* M. A. Liu,† K. Huygen,‡ O. Denis,‡ A. Tang,§ L. Zhu,§ D. Montgomery,§ J. B. Ulmer† *Mycobacterial Research Laboratories, Department of Microbiology, Colorado State University, Fort Collins Colorado, USA † Chiron Corporation, Emeryville, California, USA ‡ Department of Virology, Pasteur Institute, Brussels, Belgium § Department of Virus and Cell Biology, Merck Research Laboratories, West Point Pennsylvania, USA
Summary Objective: To determine the efficacy of Ag85A-DNA against challenge with a highly virulent human clinical isolate of Mycobacterium tuberculosis (CSU37) and to compare the potencies of two types of Ag85A-DNA vaccines; those expressing secreted and non-secreted forms of the protein. Design: Ag85A-DNA vaccinated mice were challenged with a highly virulent clinical isolate of M. tuberculosis (CSU37) in order to compare the efficacy of these vaccines. In vitro studies were also performed. Results: Enhanced humoral and cellular responses were induced in mice vaccinated with the secreted Ag85A-DNA compared to the non-secreted Ag85A-DNA. In addition, secreted Ag85A-DNA conferred protective immunity against infection with M. tuberculosis (CSU37). Conclusions: DNA vaccines encoding M. tuberculosis Ag85A have been shown to induce potent humoral and cellular immune responses leading to protection from M. tuberculosis (Erdman) challenge in mouse models.1 In this study we demonstrate that Ag85A can confer protection in a rigorous challenge model using a highly virulent human clinical isolate of M. tuberculosis (CSU37). This challenge model appears able to discriminate between DNA vaccines of differing potencies, as the more immunogenic DNA construct encoding a secreted form of Ag85A was protective, whereas the less immunogenic DNA construct encoding a non-secreted form of Ag85A was not. © 1999 Harcourt Publishers Ltd INTRODUCTION The only tuberculosis vaccine approved for human use is Mycobacterium bovis BCG, which is a live attenuated vaccine. The efficacy of the BCG vaccine has been the subject of controversy since several large human clinical trials revealed that the protection conferred by this vaccine varies greatly, and zero per cent efficacy was reported in some endemic areas such as Southern India.2 A significant limitation of the existing vaccine is that it elicits a delayed type hypersensitivity reaction to commercial purified protein derivative (PPD), the skin test reagent used in the diagnosis of tuberculosis. Furthermore, the live BCG
Correspondence to: Dr Susan L. Baldwin, Colorado State University, Department of Microbiology, Fort Collins, CO 80523, USA. Tel: +1 970 491 2694; Fax: +1 970 491 1815; E-mail address: [email protected]. Received: 16 April 1998; Revised: 7 July 1998; Accepted: 8 September 1998
vaccine represents a potential health risk, in itself, to immunocompromized individuals. Thus, many factors warrant the development of new vaccines against tuberculosis. The administration of DNA vaccines has been successful at generating both cell-mediated and humoral immune responses in many animal models of infectious disease, such as herpes simplex,3–7 hepatitis B,8–13 hepatitis C,14–18 HIV,19–23 Leishmania,24,25 Borrelia burgdorferi (lyme disease),26–28 M. tuberculosis,1,29–35 and others (for review see 36). Several mycobacterial antigens have been expressed using plasmid DNA vectors; 6 kDa,30 30–32 kDa [Ag85A, Ag85B, Ag85C],1,31 36 kDa,30,34 38 kDa,35 65 kDa29,30,32–34 and 70 kDa.30 Ag85A (mycolyl transferase)37 is an immunostimulatory protein which is an abundant component in the culture filtrate of M. tuberculosis.38 DNA vaccines encoding Ag85A have been shown to confer protection against an aerosol challenge with M. tuberculosis in mice.1 251
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To date, only laboratory strains of M. tuberculosis have been used in the low dose aerosol infected mouse model in order to study anti-tuberculosis DNA vaccine efficacy in vivo. In the present study, our aims were to determine the efficacy of Ag85A DNA against challenge with a highly virulent human clinical isolate of M. tuberculosis (CSU 37) and to compare the potency of two types of Ag85A DNA vaccines; those expressing secreted (tPA-Ag85) and nonsecreted (mature-Ag85) forms of the protein. Differences in the potency of the two DNA vaccines were determined based on the immunogenicity and protective efficacy in vivo against aerogenic challenge with a highly virulent clinical isolate of M. tuberculosis. We show that the DNA vaccine encoding the secreted form of Ag85A is superior to the mature form of the vaccine, as measured by the production of Ag85A specific antibodies, lymphoproliferation, and Ag85A specific CTL responses. We also show that the DNA vaccine encoding the secreted form of Ag85 was able to confer protection against a highly virulent human clinical isolate of tuberculosis.
MATERIALS AND METHODS Animals Specific pathogen-free, female C57BL/6 or BALB/c mice were purchased from Charles River Laboratories (North Wilmington, Mass, USA). C57BL/6 mice were held in barrier conditions in an ABL3 Biohazard Laboratory (Colorado State University, CO, USA). Mice were 4–8 weeks old at the beginning of the experiments and were housed four to a cage. Mice were allowed free access to water and standard mouse chow. Bacteria M. tuberculosis strains Erdman (TMCC 107), and CSU37 (kindly provided by Dr John Belisle, CSU, CO, USA) were grown to mid-log phase in Proskauer-Beck medium and stored in ampoules frozen at –70°C until use. DNA vaccine DNA encoding secreted or mature (non-secreted) Ag85A protein was cloned into the eukaryotic expression vector V1Jns as described previously.1 Briefly, the Ag85A gene was inserted into the V1Jns plasmid and was expressed under the control of the promoter and intron A of the immediate early protein-1 (IE1) from cytomegalovirus (CMV) followed by a polyadenylation site from the bovine growth hormone gene (BGH). Incorporation of the intron A and polyadenylation site into the plasmid allow for post-transcriptional processing within the eukaryotic host. In the secreted tPA-Ag85A DNA vaccine, the gene Tubercle and Lung Disease (1999) 79(4), 251–259
for the mature Ag85A (lacking the native mycobacterial signal sequence) is preceded by the eukaryotic signal sequence for human tissue plasminogen activator (tPA). The mature form of the DNA-Ag85A (mature-Ag85A) vaccine lacks any signal sequence. Vaccination protocol Mice (n=5/group) were immunized intramuscularly in both quadriceps, three times, at 3 week intervals or as noted in the figure legends. At each vaccination mice were given 50 µg/quadricep of either tPA-Ag85A DNA or matureAg85A DNA. Negative control animals were injected with saline or control plasmid vector DNA (which lacked a gene insert) in sterile saline. The positive control group was injected once, subcutaneously, with M. bovis BCG Pasteur at a concentration of 106 bacilli on the last day of immunization. IMMUNOGENICITY Determination of immune responses in mice included serum antibodies, as measured by ELISA; lymphoproliferation, as measured upon restimulation of spleen cells in vitro with antigen; and cytotoxic T lymphocytes, as measured upon in vitro restimulation with peptides (p7, corresponding to amino acids 71–78 (PVGGQSSF) and p15, corresponding to amino acids 145–152 (YAGAMSGL) of Ag85A). Details of these assays are given elsewhere.1,39 Briefly, lymphocytes harvested from pools of three spleens were restimulated for 7 days with peptide at 10 µg/ml then tested for cytotoxicity in a 3 hour 51Crrelease assay in round bottomed microwell plates on 104 51Cr-labeled P815 target cells. Results were similar whether individual spleens or pools of spleens were used in the restimulation. Effector cells were added to target cells at various E/T ratios in RPMI 1640 supplemented with 10% FCS in a total volume of 0.2 ml. Percent specific 51 Cr release was calculated as follows: 100X (release by CTLs-spontaneous release)/(total release–spontaneous release). Experiments were performed three times. Similar results were obtained for antibody responses and lymphoproliferation measured after one or multiple DNA immunizations, whereas differences in CTL responses were most readily seen after a single immunization. Experimental infections To determine bacterial growth curves, C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine, USA), 6–8 weeks of age, were aerogenically infected with either M. tuberculosis Erdman or M. tuberculosis CSU37 and the growth of bacilli in the lungs was determined over time. Mice were aerogenically exposed to either M. tuberculosis Erdman or © 1999 Harcourt Publishers Ltd
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M. tuberculosis CSU37, in a Middlebrook airborne infection apparatus (Glas-Col, Terre Haute, IN, USA) which was calibrated to deliver approximately 100 viable bacilli into the lung. Groups of four mice were euthanized either 7, 20 or 40 days post infection. Lungs were harvested, homogenized, serially diluted in sterile PBS and plated on 7H11 agar plates. Plates were incubated at 37°C in a humidified incubator and colonies were counted 3 weeks later. For the protection studies, mice were aerogenically exposed to approximately 100 viable M. tuberculosis CSU37 bacilli as described above. Animals were infected either 30 days or 90 days following administration of the last vaccination. Mice were euthanized 30 days following challenge with M. tuberculosis CSU37, lungs were harvested and the bacterial colonies were counted as described above.
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Fig. 1 Induction of anti-Ag85 antibodies by DNA vaccination. Female BALB/c mice (4–6 weeks) were injected with the indicated doses of Ag85 DNA three times at 3 week intervals and sera were analyzed by an ELISA at 3 weeks after the final immunization. Shown are geometric mean antibody titers ±SEM (n=5) for mice receiving DNA encoding secreted or mature forms of Ag85A.
Delayed type hypersensitivity Tuberculin purified protein derivative (PPD) lot CT68 was purchased from the Connaught Laboratories (Toronto, Canada). Concentrated PPD (2 mg/ml) was diluted 1:20 in sterile PBS and 50 µl containing 5 µg of PPD per mouse was injected in the left hind footpads using a 30-gauge needle. PBS was injected in the right footpads as negative controls. Footpad thickness/swelling was determined as the difference between the PPD injected footpads and the contralateral saline injected footpad. The DTH response was measured 48 h post-injection using calipers capable of measuring 0.05 mm increments in thickness.
RESULTS Immunogenicity of Ag85A DNA Mice were immunized with DNA encoding either secreted or mature Ag85A in order to compare the potency of each of these constructs to one another. Expression of Ag85A in vivo was assessed indirectly by the production of antiAg85 antibodies. DNA encoding the secreted form of Ag85A was more potent than that encoding the mature form, as seen by the overall higher titer of antibodies and by the induction of antibody responses at doses of DNA much lower than that required of the mature form (Fig. 1). Differences were statistically significant at all DNA doses tested (t-test; P=0.0016 to 0.049). Uninjected mice or those immunized with control plasmid DNA not encoding an antigen did not react to Ag85 in the ELISA (data not shown). Cellular responses were assessed in two ways. First, lymphoproliferation in response to antigen restimulation in vitro was measured. Both constructs induced lymphoproliferation in a dose-dependent fashion (Fig. 2). The © 1999 Harcourt Publishers Ltd
Fig. 2 Induction of lymphoproliferative responses by DNA vaccination. Female BALB/c mice (4–6 weeks) were injected with the indicated doses of Ag85 DNA three times at 3 week intervals and spleens were collected at 3 weeks after the final immunization. Pools of cells from three spleens per group were restimulated in vitro with M. bovis BCG culture filtrate proteins. Shown are data plotted as stimulation index versus DNA dose for mice receiving DNA encoding secreted or mature forms of Ag85A. Data are representative of three separate experiments.
tPA-Ag85 DNA, however, was more potent than matureAg85A DNA as seen by the elevated stimulation index at every dose of DNA tested (analysis of covariance; P=0.0008). Spleen cells from control mice did not proliferate upon restimulation (stimulation index < 2). Second, cytotoxic T lymphocyte (CTL) responses were measured in vitro. Two previously identified H-2d-specific CTL epitopes (amino acids 71–78, 145–152),39 were used to test for induction of CTL by Ag85A-DNA. After a single injection of high DNA dose (100 µg), Ag85-specific CTL were induced by both tPA-Ag85A DNA and mature-Ag85A Tubercle and Lung Disease (1999) 79(4), 251–259
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Fig. 3 Induction of cytotoxic T lymphocyte responses by DNA vaccination. Female BALB/c mice (4–6 weeks) were injected with either 5 or 100 µg of Ag85 DNA and spleens were collected at 4 weeks after a single immunization. Pools of cells from three spleens per group were restimulated with an H-2d-restricted CTL epitope (aa 71–78) and tested for lytic activity against P815 target cells that were untreated, pulsed with peptide p7 (corresponding to amino acids 71–78 (PVGGQSSF)) or stably transfected with Ag85A. Shown are data plotted as % specific lysis versus effector:target ratio for mice receiving DNA encoding secreted or mature forms of Ag85A. Data are representative of three separate experiments.
DNA using either the 71–78 and 145–152 epitopes (Figs 3 and 4). However, the magnitude of the CTL responses appeared to be slightly higher in mice immunized with tPA-Ag85A. This was true whether the P815 target cells were pulsed with the appropriate peptide or were stably expressing full-length Ag85A. Target cells untreated or pulsed with the inappropriate peptide were not lysed by specific CTL. After only a single injection of low DNA dose (5 µg), CTL were induced by tPA-Ag85A DNA only (Fig. 3). Therefore, by all measures tested, tPA-Ag85A DNA was more potent at inducing immune responses than was mature-Ag85A DNA. The differences in potency between DNA vaccines encoding secreted and non-secreted Ag85 were seen after one or multiple immunizations. For CTL, the biggest differences were seen after a single immunization. This may be due to the fact that previous experience in mice has suggested that maximum in vitro CTL responses (as measured by chromium release assays) are quickly achieved with multiple DNA immunizations (Ulmer et al., unpublished observations). Growth of M. tuberculosis CSU37 Growth of the widely used laboratory strain of M. tuberculosis (Erdman) was compared to the growth of a human clinical isolate of M. tuberculosis (CSU37). As seen in Figure 5, the clinical isolate (CSU 37) grew faster and to Tubercle and Lung Disease (1999) 79(4), 251–259
much higher titers within the lungs of mice than did the laboratory strain (Erdman). CSU37 was chosen for subsequent protection studies in light of greater virulence and its susceptibility to several antimycobacterial drugs (data not shown). Delayed type hypersensitivity The DTH response was examined in Ag85A DNA- and BCG-vaccinated mice. As seen in Figure 6, there was little or no DTH response to PPD in any of the mice which were vaccinated with Ag85A-DNA at either 30 or 90 days following vaccination. In contrast, BCG vaccinated animals mounted a significant DTH response 30 days following vaccination (> 0.4 mm); however, this response waned 90 days following vaccination, and was considered to be insignificant (Fig. 6). Protective efficacy The Ag85A-DNA vaccines were tested for their protective efficacy against infection with the highly virulent human clinical isolate M. tuberculosis CSU37. Two separate sets of mice were aerogenically infected with M. tuberculosis CSU37, either 30 or 90 days following vaccination. At 30 days following vaccination both Ag85A DNA vaccines showed slight protection (~0.7–0.8 log 10 reduction). © 1999 Harcourt Publishers Ltd
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Fig. 4 Induction of cytotoxic T lymphocyte responses by DNA vaccination. Female BALB/c mice (4–6 weeks) were injected with either 5 or 100 mg of Ag85 DNA and spleens were collected at 4 weeks after a single immunization. Pools of cells from three spleens per group were restimulated with an H-2d-restricted CTL epitope (aa 145–152) and tested for lytic activity against P815 target cells that were untreated, pulsed with peptide p15 (corresponding to amino acids 145–152 (YAGAMSGL)) or stably transfected with Ag85A. Shown are data plotted as % specific lysis versus effector:target ratio for mice receiving DNA encoding secreted or mature forms of Ag85A. Data are representative of three separate experiments.
good protection (~1.7 log 10 reduction in bacterial load within the lung, compared to saline injected animals). At 90 days following DNA vaccination, tPA-Ag85A DNA conferred significant protection (~0.8 log 10 reduction in bacterial load within the lung, compared to saline injected animals) against infection, whereas mature Ag85A DNA or control DNA did not (Fig. 7B). BCG also protected mice significantly against infection (similar to tPA-Ag85A DNA). However, BCG-mediated protection appeared to be waning compared to that seen at 30 days post vaccination. These results suggest that the antigen-specific protective immune response induced by Ag85A DNA required more than 30 days to mature or was masked by a short-lived non-specific response induced by the DNA itself. DISCUSSION Fig. 5 Growth of M. tuberculosis Erdman compared to M. tuberculosis CSU37 in the lungs of C57BL/6 mice following exposure to low-dose aerosol infections. Shown are data plotted as log 10 viable bacteria within the lung ±SEM (n=4).
However, this was likely due, at least in part, to a nonspecific effect of the DNA itself, as control DNAvaccinated mice also showed slightly reduced lung titers (Fig. 7A). In contrast, BCG-vaccinated animals exhibited © 1999 Harcourt Publishers Ltd
We have shown here that a DNA vaccine encoding the secreted form of M. tuberculosis Ag85A is more potent than a DNA vaccine encoding a mature form of the protein. tPA-Ag85A DNA induced higher levels of specific serum antibodies than the mature-Ag85A vaccine, in addition to enhanced cellular responses (including antigen specific lymphoproliferation and CTL mediated lytic activity). Furthermore, tPA-Ag85A DNA was able to confer immunity against a highly virulent human clinical Tubercle and Lung Disease (1999) 79(4), 251–259
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Fig. 6 Delayed type hypersensitivity to commercial PPD tuberculin in immunized C57BL/6 mice. Left footpads of the immunized mice (30 days or 90 days after vaccination) were injected with 5 µg of PPD in 50 µl sterile saline and contralateral footpads were injected with saline. Shown are data plotted as the mean increase in footpad thickness (mm) ±SD (n=5) over that seen in the contralateral saline injected footpads, 48 h after injection of PPD.
A B Fig. 7 Bacterial loads in the lungs of M. tuberculosis infected mice previously vaccinated with either vector control (control DNA), the secreted form of the vaccine (tPA-Ag85A), or the mature form of the vaccine (mature-Ag85A). Mice were infected aerogenically with approximately 102 M. tuberculosis CSU37 1 month or 3 months post-vaccination. Lungs were harvested 1 month following infection. BCG was the positive control and both saline and control DNA were the negative controls. Shown are data plotted as log10 viable bacteria within the lung (n=5) ±SEM. Groups showing significant protection [P < 0.05] within the lungs compared to saline controls are indicated with an asterisk. a) Challenge at Day 30 post vaccination, b) challenge at Day 90 post vaccination.
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isolate of M. tuberculosis, whereas neither the control DNA nor the mature-Ag85A vaccines was able to protect against this isolate for an extended period of time. Based on transient transfection in vitro, the Ag85A DNA constructs express proteins of expected molecular mass40,41 but differ qualitatively in that the secreted form is targeted to the endoplasmic reticulum for secretion, while the mature form is targeted to the cytoplasm. In addition, the secreted form is modified by the addition of an N-linked oligosaccharide (Montgomery et al., in preparation). The production of anti-Ag85 antibodies was used to assess the expression of Ag85A in vivo. An overall higher titer of antibodies and induction of antibody responses at lower doses of DNA suggest that the tPAAg85A DNA was at least 10-fold more potent than mature-Ag85A DNA. The higher antibody titers in mice vaccinated with tPA-Ag85A DNA are likely the result of more extracellular protein available for induction of B cell responses as a consequence of efficient secretion of Ag85A out of transfected cells in vivo. In contrast, the mature form of Ag85A is not efficiently secreted from transfected cells. In fact, multiple immunizations with mature-Ag85A DNA are required to induce significant levels of anti-Ag85 antibodies.40 It is interesting though, that after multiple immunizations of high doses of mature-Ag85A DNA the antibody titers which were measured approached the titers induced by tPA-Ag85A DNA, suggesting that significant protein is being made available to B cells. One possible explanation could be the release of protein from transfected cells, either as a consequence of toxicity of the protein or of immune-mediated responses directed toward antigen-expressing cells. The latter possibility would seem more likely, since no cellular toxicity was observed after transfection of cells in vitro (Montgomery et al., unpublished observations) and that immune-mediated clearance of transfected cells in vivo has been suggested by studies using reporter proteins.42 In support of this possibility, robust cellular immune responses induced by Ag85A DNA were observed. Cellular responses were measured using both lymphoproliferation and CTL assays. Lymphoproliferation was induced in a dose-dependent fashion with both the matureAg85A and the secreted-Ag85A DNA constructs. These results are consistent with previous observations that Ag85A DNA induced robust cellular immune responses.1 In the present study, DNA doses as low as 0.78 µg induced detectable proliferation with proliferation increasing in response to as much as 200 µg Ag85-DNA. CTL-responses were also measured to compare the cellular responses to the two DNA constructs, using two H-2d-specific CTL epitopes (amino-acids 71–78, 145–152). As shown here, the secreted form of the Ag85A-DNA vaccine was superior to the mature form of the vaccine in vivo as measured by antibody production, antigen specific lymphoprolifera© 1999 Harcourt Publishers Ltd
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tion responses, and CTL mediated lytic activity. To address whether these responses could contribute to increased protective efficacy in a mouse model of tuberculosis, the tPA-Ag85A DNA was compared to the matureAg85A DNA vaccine using a highly virulent clinical isolate of M. tuberculosis (CSU 37). We have previously shown that both the tPA-Ag85A and the mature-Ag85A DNA vaccines are able to confer immunity against the laboratory strain of M. tuberculosis (Erdman).1 Here we have shown that protective immunity against a highly virulent clinical isolate of M. tuberculosis can be attained, but only by the tPA-Ag85 DNA vaccine. Preliminary results in our laboratory suggest that tPA-Ag85 DNA induces a larger memory T cell population in infected mice, as suggested by increased levels of the putative ‘memory’ surface marker phenotype (CD4+/CD44hi/ CD45RBlo),43 compared to that seen in the mature-Ag85 DNA vaccinated mice. This could explain why one vaccine was more protective than the other, since the enhanced generation of memory T cells could lead to stronger anamnestic responses to subsequent infection with M. tuberculosis. This would be especially critical in cases involving infection with an extremely virulent M. tuberculosis isolate such as CSU37, which rapidly grow to very high levels within the lung. A heightened memory response in mice vaccinated with tPA-Ag85A DNA could lead to early containment of the infection within the lungs. Future studies should investigate the efficacy of this vaccine against multiple drug-resistant human clinical M. tuberculosis isolates. In addition to the antigen-specific protective immune response induced by tPA-Ag85A DNA observed at 90 days post-vaccination, a modest non-specific response was seen at 30 days post-vaccination in mice that received control DNA. A similar, but insignificant, reduction in lung titer was also seen in control DNA-vaccinated mice challenged with the Erdman strain (unpublished observations; 1). This effect could be due to the potential presence of immunostimulatory nucleotide sequences in the DNA vector. Such sequences, consisting of the basic motif purine-purine-C-G-pyrimidine, have potent effects on natural killer cells and lymphocytes in vitro, causing them to proliferate and secrete cytokines.44–46 Secretion of such cytokines, including interferon-γ, in vivo in response to the DNA vector could lead to activation of macrophages and the observed reduction in mycobacterial lung titers. This response, however, is short-lived and longterm immunity requires antigen-specific immunity. One goal of a new vaccine against tuberculosis is to formulate one which does not lead to a positive skin reaction to PPD, since this reactivity interferes with the ability to monitor individuals for prior exposure to M. tuberculosis. Regardless of the protection conferred, mice vaccinated with the Ag85A-DNA vaccines did not generate a signiTubercle and Lung Disease (1999) 79(4), 251–259
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ficant DTH response to commercial PPD. The fact that neither the tPA-Ag85A DNA vaccine (shown to be protective against M. tuberculosis CSU37) nor any of the other DNA-vaccines tested (not protective in this model) generated a DTH response suggests that there was no correlation between the ability to mount a DTH response to PPD, and the presence or absence of a protective response against infection with M. tuberculosis CSU37. Indeed, it was established several years ago that DTH and protective immunity to M. tuberculosis are dissociable phenomena.47 The protective immune response against tuberculosis is known to involve the generation of a Th1 type T cell response, which is predominantly characterized by the secretion of IFN-γ. Huygen et al. have shown increased levels of IFN-γ and IL-2 secreted from the splenocytes (pulsed with either PPD or Ag85A) of Ag85ADNA vaccinated donor mice, when compared to control DNA-vaccinated mice,1 and yet these mice do not elicit a DTH response to PPD. In addition, Cooper et al. have shown that IFN-γ knock out mice are in fact capable of mounting a DTH response, suggesting that IFN-γ (critical in a protective response) may not be essential to the DTH response.48 Animals vaccinated with these DNA vaccines do not elicit a DTH response to PPD, which may ultimately allow for the continued use of the tuberculosis diagnostic skin test. In summary, we have shown that Ag85A DNA can protect mice from challenge with a highly virulent human clinical isolate of M. tuberculosis. This rigorous challenge appears to be more able to discriminate between DNA vaccines of differing potencies, since DNA encoding a secreted form of Ag85A was protective but DNA encoding a non-secreted form was not, compared to challenge with a laboratory strain of M. tuberculosis (Erdman), where both DNA vaccines were equally protective. Hence, this model may be useful in the development of new vaccines against tuberculosis.
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ACKNOWLEDGMENTS This work was supported by NIH grant AI-75320, and by Merck Research Laboratories. We would like to thank the staff at the CSU laboratory of animal resources for their excellent care and monitoring of the mice.
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