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Improved protection in guinea pigs after vaccination with a recombinant BCG expressing MPT64 on its surface
Trials in Vaccinology 4 (2015) 29–32
Contents lists available at ScienceDirect
Trials in Vaccinology journal homepage: www.elsevier.com/locate/trivac
Improved protection in guinea pigs after vaccination with a recombinant BCG expressing MPT64 on its surface Simon O. Clark a, Giovanni Delogu b, Emma Rayner a, Michela Sali b, Ann Williams a, Riccardo Manganelli c,⇑ a b c
Public Health England, Microbiology Services, Porton Down, Salisbury, Wiltshire, United Kingdom Institute of Microbiology, University of the Sacred Heart, Rome, Italy Department of Molecular Medicine, University of Padova, Padova, Italy
a r t i c l e
i n f o
Article history: Received 6 March 2015 Accepted 26 March 2015 Available online 19 April 2015 Keywords: Tuberculosis Vaccines Animal model Guinea pigs
a b s t r a c t The lack of an efficient vaccine against tuberculosis is still one of the major problems threatening global human health. In previous work we showed that expression of the protective antigen MPT64 on the surface of Mycobacterium bovis BCG, the only approved vaccine against tuberculosis, strongly improved its immunogenicity and protective potential in mice. In this work we demonstrate that the same recombinant strain is able to induce better protection than wild type BCG also in guinea pigs preventing Mycobacterium tuberculosis dissemination and lung pathology, making this strain a strong candidate for further testing. Ó 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Tuberculosis (TB) is still one of the main scourges of human kind with about 9 million new cases of active disease and 1.5 million deaths each year [1]. Even though TB is a curable disease, treatment is long with patient compliance challenging, especially in developing countries where the infection is more diffused, boosting the emergence of strains resistant to the main antitubercular drugs [2]. The only approved vaccine against TB, Mycobacterium bovis BCG, protects against the most severe forms of extrapulmonary TB in infants, but its efficacy in preventing pulmonary disease in adults is poor [3]. For this reason, the development of a new vaccine against TB is one of the main challenges to stop the TB pandemic [4]. Given that it prevents severe disease in children, the use of BCG cannot be discontinued for ethical reasons, so any new vaccination strategy must incorporate BCG or an improved alternative e.g. recombinant BCG (rBCG) [5]. Since the mycobacterial surface is an excellent adjuvant, we hypothesized that expressing an antigen in its context could dramatically increase immunogenicity. Consequently, we developed a system to express proteins on the surface of BCG [6]. This is based on the PE domain of PE_PGRS33, recently identified as a functional ⇑ Corresponding author at: Department of Molecular Medicine, University of Padova, Via Gabelli 63, 35121 Padova, Italy. Tel.: +39 049 8272366; fax: +39 827 2355. E-mail address: [email protected] (R. Manganelli).
domain able to drive the localization of protein sequences fused at its N-terminus on the mycobacterial outer membrane [7]. We used this system to express MPT64, a protective antigen of Mycobacterium tuberculosis normally absent in BCG, on its surface and demonstrated increased protection and immunogenicity compared to parental strain following vaccination and challenge of immunized mice with virulent M. tuberculosis [8]. Moreover, homologous boosting resulted in a heightened degree of protection of mice immunized with this HPE-DMPT64-BCG strain [9]. In this paper we further characterize the protective potential of the HPE-DMPT64-BCG strain in guinea pigs, by demonstrating improved protection of vaccinated animals from infection through surface expression of MPT64. 2. Materials and methods 2.1. Bacteria The M. tuberculosis H37Rv (National Collection of Type Cultures (NCTC) 7416) challenge stock was generated from a chemostat grown to steady state in a defined medium which has been described previously [10]. 2.2. Vaccination Three groups of 8 Dunkin–Hartley guinea pigs, (specific pathogen-free, 250–300 g), obtained from a commercial supplier
http://dx.doi.org/10.1016/j.trivac.2015.03.003 1879-4378/Ó 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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(Harlan, UK), were inoculated subcutaneously with 5 104 CFU of HPE-DMPT64-BCG (rBCG expressing the PE_MPT64 chimera on its surface) [8] or BCG Danish 1331 (Statens Serum Institute, Copenhagen, Denmark), or were unvaccinated. Individual animals were identified using subcutaneously implanted microchips (PLEXX BV, The Netherlands). Guinea pig experimental work was conducted according to UK Home Office legislation for animal experimentation and was approved by the local ethical review process. 2.3. Aerosol challenge Animals were infected with a low aerosol dose (10–50 CFU retained in the lung) of M. tuberculosis H37Rv 12 weeks after vaccination using a Henderson apparatus in conjunction with the AeroMP (Biaera) control unit as previously described [11,12]. The aerosol was generated from a water suspension containing 5 106 CFU/ml. 2.4. Necropsy and bacteriological analysis At 4 weeks post challenge, guinea pigs were killed humanely by intraperitoneal injection of pentabarbitone (Euthatal) and a postmortem examination performed immediately. For each animal, the left middle and left and right cranial and the right caudal lobes were placed in one sterile container for bacteriology and the remaining lobes placed in 10% neutral-buffered formalin for histological examination. Tissues for determination of CFU were homogenized in 5 ml of sterile distilled water using a rotating blade macerator system (Ystral, UK). Viable counts were performed by plating 100 ll aliquots of serial dilutions on of the macerate onto Middlebrook 7H11 + OADC agar (BioMerieux, UK). Bacterial
load in lungs and spleen (CFU/tissue) of each group of animals was compared to identify statistically significant differences between the groups. 2.5. Histopathological examination Formalin-fixed samples were processed to paraffin wax, cut at 3–5 lm, and stained with haematoxylin and eosin. The nature and severity of the lesions in lungs and spleen was assessed blind by a veterinary pathologist using a subjective scoring system. Briefly, for the spleen, a score was assigned based on number and size of lesions, and presence of necrosis and calcification. For the lung, each lobe was assigned a score as follows: 0-normal; 1very few or small lesions, 0–10% consolidation; 2-few or small lesions, 10–20% consolidation; 3-medium sized, 20–33% consolidation; 4-moderate sized lesions, 33–50% consolidation; 5–50– 80% consolidation; extensive pneumonia; >80% consolidation; plus number of foci of necrosis. Scores from each lobe were combined. A mean score from lung lobe and from spleen was calculated for each group. Group mean differences were compared to subcutaneous BCG Danish and unvaccinated control groups. A reduction in consolidation, foci of necrosis, and foci of calcification (spleen) in the vaccinated animals when compared with the control groups was considered a protective effect of the vaccine. However, the scores are a product of a subjective scoring system and, therefore, are not regarded as numerical data suitable for statistical analysis. 2.6. Statistics Statistical analysis was performed with Minitab software version 15. The CFU data were analyzed by non-parametric Mann– Whitney test comparisons to compare median values of the
A
B
Fig. 1. (A) Determination of CFU in M. tuberculosis-infected guinea pigs. CFU were determined in lung (left) and spleen (right) homogenates derived from M. tuberculosisinfected guinea pigs that were unvaccinated (closed circle) or vaccinated with either BCG (open circle) or HPE-DMPT64-BCG (closed square) as indicated on the x-axis. Protective efficacies are expressed as total log10 bacterial counts/tissue. Groups included eight guinea pigs. Each symbol represents one guinea pig. (B) Table summarising the efficacy of HPE-DMPT64-BCG. CFU data were analyzed by non-parametric Mann–Whitney test comparisons to compare median values of the vaccine groups with either the unvaccinated or BCG control groups. A P value of <0.05 was considered significant.
S.O. Clark et al. / Trials in Vaccinology 4 (2015) 29–32
vaccine groups with either the unvaccinated or BCG control groups. A P value of <0.05 was considered significant.
3. Results and Discussion Fig. 1A shows the bacterial burden in lung and spleen of the three groups of guinea pigs. Both vaccinated groups showed a significant reduction of CFUs in lung and spleen compared to the unvaccinated group. While protection measured in the lung was equivalent for the two vaccines, the protective effect in the spleen
31
was significantly higher for the HPE-DMPT64-BCG strain ( 3.79 Log10 CFU/tissue) than that provided by wt BCG ( 2.95 Log10 CFU tissue) (Fig. 1B). A reduction in the severity of microscopic changes in both lung and spleen, was observed in both vaccinated groups. Interestingly, even if the bacterial burden in the lung of wt BCG and HPEDMPT64-BCG vaccinated animals was similar, animals vaccinated with HPE-DMPT64-BCG showed a reduced number of foci of necrosis in this organ. These results are in agreement with the reduced immunopathology observed in the lung of mice, both in the acute and chronic steps of M. tuberculosis infection, in HPE-DMPT64-BCG
Fig. 2. Guinea pig histopathology presented as a group mean score as stacked bars in lung (left) and spleen (right) following low dose, aerosol challenge with M. tuberculosis H37Rv. Histopathology was measured 4 weeks post-challenge. Black bar: consolidation score and white bar: number of foci of necrosis/caseation score. ⁄indicates that no lesions were observed in the tissue section.
Fig. 3. Photomicrographs of lung sections of infected animals stained with hematoxylin and eosin. (A) Unvaccinated animal. Large granulomas, three with central necrosis (arrows), and scattered, smaller granulomas. Calibration bar, 500 lm; (B) BCG vaccinated animal. Variably sized granulomas, one with central necrosis (arrow). Calibration bar, 500 lm; (C) the HPE-DMPT64-BCG vaccinated animal. A single, small, granuloma (arrow). Calibration bar, 500 lm. Inset, higher power of granuloma comprising mainly epithelioid macrophages with some lymphocytes. Calibration bar, 50 lm.
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vaccinated animal compared to BCG immunized mice [8], and suggest that immunization with HPE-DMPT64-BCG may result not only in an improved bacterial killing but also in a dampening of the host immune responses classically associated with disease and ultimately TB transmission [13]. The reduced pathology in HPE-DMPT64-BCG vaccinated animals was even more evident in spleen, where lesions were not observed (Figs. 2 and 3). Taken together these results support enhanced protective effect elicited by the HPE-DMPT64-BCG compared to BCG in the guinea pig model of TB. The reduced bacterial burden and absence of lesions in the spleen of HPE-DMPT64-BCG vaccinated animals compared to those immunized with wt BCG, suggests that the immune response elicited by the former was more effective in preventing bacterial dissemination. Similarly, homologous boosting of mice immunized with HPE-DMPT64-BCG resulted in a specific reduction in CFUs in the spleen [9]. In mice, the superior protection afforded by HPEDMPT64-BCG over BCG, or a recombinant BCG strain expressing MPT64 in the cytosol, was associated with the ability to elicit MPT64-specific IFN-c secreting cells carrying a specific T cell repertoire [8]. Hence, it is possible that also in guinea pigs these MPT64specific T lymphocytes may warrant enhanced anti-TB activity. Vaccines capable of eliciting an immune response that can more effectively control bacterial dissemination could be of relevance in humans, where dissemination from the site of primary infection is a key step in TB pathogenesis following primary infection [2,14] and during latent TB infection, when ‘‘scouting’’ bacilli arising from the dormant reservoir may actively replicate and promote active disease [15,16]. The reduced pathology observed in the lung of HPE-DMPT64BCG immunized animals compared to those vaccinated with BCG confirms previous observation in mice [8], further highlighting the enhanced protective activity afforded by this strain. Although many new live attenuated vaccines have been developed and tested in preclinical animal models, very few were able to consistently provide enhanced protection over BCG, which is used as the gold standard [17]. The results obtained in mice and guinea pigs for the HPE-DMPT64-BCG strain are very promising, making it a candidate for further testing. Moreover, it is worth noting that PE_MPT64 could be expressed on the surface of other rBCG strains, such as the BCG ureC hly+ strain [18], opening the possibility to further enhance the anti-TB immunity elicited by these recombinant BCG strains. Acknowledgements We gratefully acknowledge the support from the Biological Investigations Group at PHE, Porton Down. The views expressed
in this publication are those of the author(s) and not necessarily those of the Department of Health. This work was supported by the Bill and Melinda Gates Foundation and TuBerculosis Vaccine Initiative. References [1] WHO. Global tuberculosis report 2013. 2013. [2] C. Lienhardt, P. Glaziou, M. Uplekar, K. Lonnroth, H. Getahun, M. Raviglione, Global tuberculosis control: lessons learnt and future prospects, Nat. Rev. Microbiol. 10 (2012) 407–416. [3] P. Andersen, T.M. Doherty, The success and failure of BCG – implications for a novel tuberculosis vaccine, Nat. Rev. Microbiol. 3 (2005) 656–662. [4] G. Delogu, R. Manganelli, M.J. Brennan, Critical research concepts in tuberculosis vaccine development, Clin. Microbiol. Infect. 20 (Suppl. 5) (2014) 59–65. [5] P. Andersen, S.H. Kaufmann, Novel vaccination strategies against tuberculosis, Cold Spring Harb. Perspect. Med. 4 (2014) a018523. [6] A. Cascioferro, G. Delogu, M. Colone, M. Sali, A. Stringaro, G. Arancia, et al., PE is a functional domain responsible for protein translocation and localization on mycobacterial cell wall, Mol. Microbiol. 66 (2007) 1536–1547. [7] A. Cascioferro, M.H. Daleke, M. Ventura, V. Dona, G. Delogu, G. Palu, et al., Functional dissection of the PE domain responsible for translocation of PE_PGRS33 across the mycobacterial cell wall, 6 (2011) e27713. [8] M. Sali, G. Di Sante, A. Cascioferro, A. Zumbo, C. Nicolo, V. Dona, et al., Surface expression of MPT64 as a fusion with the PE domain of PE_PGRS33 enhances Mycobacterium bovis BCG protective activity against Mycobacterium tuberculosis in mice, Infect. Immun. 78 (2010) 5202–5213. [9] M. Sali, E. Dainese, M. Morandi, A. Zumbo, S. Rocca, S. Goussard, et al., Homologous prime boosting based on intranasal delivery of non-pathogenic invasive Escherichia coli expressing MPT64, decreases Mycobacterium tuberculosis dissemination, Vaccine 32 (2014) 4051–4058. [10] B.W. James, A. Williams, P.D. Marsh, The physiology and pathogenicity of Mycobacterium tuberculosis grown under controlled conditions in a defined medium, J. Appl. Microbiol. 88 (2000) 669–677. [11] S.O. Clark, Y. Hall, D.L. Kelly, G.J. Hatch, A. Williams, Survival of Mycobacterium tuberculosis during experimental aerosolization and implications for aerosol challenge models, J. Appl. Microbiol. 111 (2011) 350–359. [12] J.M. Hartings, C.J. Roy, The automated bioaerosol exposure system: preclinical platform development and a respiratory dosimetry application with nonhuman primates, J. Pharmacol. Toxicol. Methods 49 (2004) 39–55. [13] I.M. Orme, A.M. Cooper, Cytokine/chemokine cascades in immunity to tuberculosis, Immunol. Today 20 (1999) 307–312. [14] K. Pethe, S. Alonso, F. Biet, G. Delogu, M.J. Brennan, C. Locht, et al., The heparinbinding haemagglutinin of M. tuberculosis is required for extrapulmonary dissemination, Nature 412 (2001) 190–194. [15] M. Gengenbacher, S.H. Kaufmann, Mycobacterium tuberculosis: success through dormancy, FEMS Microbiol. Rev. 36 (2012) 514–532. [16] M.-C. Chao, E.J. Rubin, Letting sleeping dos lie: does dormancy play a role in tuberculosis?, Ann Rev. Microbiol. 64 (2010) 293–311. [17] H. McShane, W.R. Jacobs, P.E. Fine, S.G. Reed, D.N. McMurray, M. Behr, et al., BCG: myths, realities, and the need for alternative vaccine strategies, Tuberculosis 92 (2012) 283–288. [18] C. Desel, A. Dorhoi, S. Bandermann, L. Grode, B. Eisele, S.H. Kaufmann, Recombinant BCG DureC hly+ induces superior protection over parental BCG by stimulating a balanced combination of type 1 and type 17 cytokine responses, J. Infect. Dis. 204 (2011) 1573–1584.
Contents lists available at ScienceDirect
Trials in Vaccinology journal homepage: www.elsevier.com/locate/trivac
Improved protection in guinea pigs after vaccination with a recombinant BCG expressing MPT64 on its surface Simon O. Clark a, Giovanni Delogu b, Emma Rayner a, Michela Sali b, Ann Williams a, Riccardo Manganelli c,⇑ a b c
Public Health England, Microbiology Services, Porton Down, Salisbury, Wiltshire, United Kingdom Institute of Microbiology, University of the Sacred Heart, Rome, Italy Department of Molecular Medicine, University of Padova, Padova, Italy
a r t i c l e
i n f o
Article history: Received 6 March 2015 Accepted 26 March 2015 Available online 19 April 2015 Keywords: Tuberculosis Vaccines Animal model Guinea pigs
a b s t r a c t The lack of an efficient vaccine against tuberculosis is still one of the major problems threatening global human health. In previous work we showed that expression of the protective antigen MPT64 on the surface of Mycobacterium bovis BCG, the only approved vaccine against tuberculosis, strongly improved its immunogenicity and protective potential in mice. In this work we demonstrate that the same recombinant strain is able to induce better protection than wild type BCG also in guinea pigs preventing Mycobacterium tuberculosis dissemination and lung pathology, making this strain a strong candidate for further testing. Ó 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Tuberculosis (TB) is still one of the main scourges of human kind with about 9 million new cases of active disease and 1.5 million deaths each year [1]. Even though TB is a curable disease, treatment is long with patient compliance challenging, especially in developing countries where the infection is more diffused, boosting the emergence of strains resistant to the main antitubercular drugs [2]. The only approved vaccine against TB, Mycobacterium bovis BCG, protects against the most severe forms of extrapulmonary TB in infants, but its efficacy in preventing pulmonary disease in adults is poor [3]. For this reason, the development of a new vaccine against TB is one of the main challenges to stop the TB pandemic [4]. Given that it prevents severe disease in children, the use of BCG cannot be discontinued for ethical reasons, so any new vaccination strategy must incorporate BCG or an improved alternative e.g. recombinant BCG (rBCG) [5]. Since the mycobacterial surface is an excellent adjuvant, we hypothesized that expressing an antigen in its context could dramatically increase immunogenicity. Consequently, we developed a system to express proteins on the surface of BCG [6]. This is based on the PE domain of PE_PGRS33, recently identified as a functional ⇑ Corresponding author at: Department of Molecular Medicine, University of Padova, Via Gabelli 63, 35121 Padova, Italy. Tel.: +39 049 8272366; fax: +39 827 2355. E-mail address: [email protected] (R. Manganelli).
domain able to drive the localization of protein sequences fused at its N-terminus on the mycobacterial outer membrane [7]. We used this system to express MPT64, a protective antigen of Mycobacterium tuberculosis normally absent in BCG, on its surface and demonstrated increased protection and immunogenicity compared to parental strain following vaccination and challenge of immunized mice with virulent M. tuberculosis [8]. Moreover, homologous boosting resulted in a heightened degree of protection of mice immunized with this HPE-DMPT64-BCG strain [9]. In this paper we further characterize the protective potential of the HPE-DMPT64-BCG strain in guinea pigs, by demonstrating improved protection of vaccinated animals from infection through surface expression of MPT64. 2. Materials and methods 2.1. Bacteria The M. tuberculosis H37Rv (National Collection of Type Cultures (NCTC) 7416) challenge stock was generated from a chemostat grown to steady state in a defined medium which has been described previously [10]. 2.2. Vaccination Three groups of 8 Dunkin–Hartley guinea pigs, (specific pathogen-free, 250–300 g), obtained from a commercial supplier
http://dx.doi.org/10.1016/j.trivac.2015.03.003 1879-4378/Ó 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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S.O. Clark et al. / Trials in Vaccinology 4 (2015) 29–32
(Harlan, UK), were inoculated subcutaneously with 5 104 CFU of HPE-DMPT64-BCG (rBCG expressing the PE_MPT64 chimera on its surface) [8] or BCG Danish 1331 (Statens Serum Institute, Copenhagen, Denmark), or were unvaccinated. Individual animals were identified using subcutaneously implanted microchips (PLEXX BV, The Netherlands). Guinea pig experimental work was conducted according to UK Home Office legislation for animal experimentation and was approved by the local ethical review process. 2.3. Aerosol challenge Animals were infected with a low aerosol dose (10–50 CFU retained in the lung) of M. tuberculosis H37Rv 12 weeks after vaccination using a Henderson apparatus in conjunction with the AeroMP (Biaera) control unit as previously described [11,12]. The aerosol was generated from a water suspension containing 5 106 CFU/ml. 2.4. Necropsy and bacteriological analysis At 4 weeks post challenge, guinea pigs were killed humanely by intraperitoneal injection of pentabarbitone (Euthatal) and a postmortem examination performed immediately. For each animal, the left middle and left and right cranial and the right caudal lobes were placed in one sterile container for bacteriology and the remaining lobes placed in 10% neutral-buffered formalin for histological examination. Tissues for determination of CFU were homogenized in 5 ml of sterile distilled water using a rotating blade macerator system (Ystral, UK). Viable counts were performed by plating 100 ll aliquots of serial dilutions on of the macerate onto Middlebrook 7H11 + OADC agar (BioMerieux, UK). Bacterial
load in lungs and spleen (CFU/tissue) of each group of animals was compared to identify statistically significant differences between the groups. 2.5. Histopathological examination Formalin-fixed samples were processed to paraffin wax, cut at 3–5 lm, and stained with haematoxylin and eosin. The nature and severity of the lesions in lungs and spleen was assessed blind by a veterinary pathologist using a subjective scoring system. Briefly, for the spleen, a score was assigned based on number and size of lesions, and presence of necrosis and calcification. For the lung, each lobe was assigned a score as follows: 0-normal; 1very few or small lesions, 0–10% consolidation; 2-few or small lesions, 10–20% consolidation; 3-medium sized, 20–33% consolidation; 4-moderate sized lesions, 33–50% consolidation; 5–50– 80% consolidation; extensive pneumonia; >80% consolidation; plus number of foci of necrosis. Scores from each lobe were combined. A mean score from lung lobe and from spleen was calculated for each group. Group mean differences were compared to subcutaneous BCG Danish and unvaccinated control groups. A reduction in consolidation, foci of necrosis, and foci of calcification (spleen) in the vaccinated animals when compared with the control groups was considered a protective effect of the vaccine. However, the scores are a product of a subjective scoring system and, therefore, are not regarded as numerical data suitable for statistical analysis. 2.6. Statistics Statistical analysis was performed with Minitab software version 15. The CFU data were analyzed by non-parametric Mann– Whitney test comparisons to compare median values of the
A
B
Fig. 1. (A) Determination of CFU in M. tuberculosis-infected guinea pigs. CFU were determined in lung (left) and spleen (right) homogenates derived from M. tuberculosisinfected guinea pigs that were unvaccinated (closed circle) or vaccinated with either BCG (open circle) or HPE-DMPT64-BCG (closed square) as indicated on the x-axis. Protective efficacies are expressed as total log10 bacterial counts/tissue. Groups included eight guinea pigs. Each symbol represents one guinea pig. (B) Table summarising the efficacy of HPE-DMPT64-BCG. CFU data were analyzed by non-parametric Mann–Whitney test comparisons to compare median values of the vaccine groups with either the unvaccinated or BCG control groups. A P value of <0.05 was considered significant.
S.O. Clark et al. / Trials in Vaccinology 4 (2015) 29–32
vaccine groups with either the unvaccinated or BCG control groups. A P value of <0.05 was considered significant.
3. Results and Discussion Fig. 1A shows the bacterial burden in lung and spleen of the three groups of guinea pigs. Both vaccinated groups showed a significant reduction of CFUs in lung and spleen compared to the unvaccinated group. While protection measured in the lung was equivalent for the two vaccines, the protective effect in the spleen
31
was significantly higher for the HPE-DMPT64-BCG strain ( 3.79 Log10 CFU/tissue) than that provided by wt BCG ( 2.95 Log10 CFU tissue) (Fig. 1B). A reduction in the severity of microscopic changes in both lung and spleen, was observed in both vaccinated groups. Interestingly, even if the bacterial burden in the lung of wt BCG and HPEDMPT64-BCG vaccinated animals was similar, animals vaccinated with HPE-DMPT64-BCG showed a reduced number of foci of necrosis in this organ. These results are in agreement with the reduced immunopathology observed in the lung of mice, both in the acute and chronic steps of M. tuberculosis infection, in HPE-DMPT64-BCG
Fig. 2. Guinea pig histopathology presented as a group mean score as stacked bars in lung (left) and spleen (right) following low dose, aerosol challenge with M. tuberculosis H37Rv. Histopathology was measured 4 weeks post-challenge. Black bar: consolidation score and white bar: number of foci of necrosis/caseation score. ⁄indicates that no lesions were observed in the tissue section.
Fig. 3. Photomicrographs of lung sections of infected animals stained with hematoxylin and eosin. (A) Unvaccinated animal. Large granulomas, three with central necrosis (arrows), and scattered, smaller granulomas. Calibration bar, 500 lm; (B) BCG vaccinated animal. Variably sized granulomas, one with central necrosis (arrow). Calibration bar, 500 lm; (C) the HPE-DMPT64-BCG vaccinated animal. A single, small, granuloma (arrow). Calibration bar, 500 lm. Inset, higher power of granuloma comprising mainly epithelioid macrophages with some lymphocytes. Calibration bar, 50 lm.
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vaccinated animal compared to BCG immunized mice [8], and suggest that immunization with HPE-DMPT64-BCG may result not only in an improved bacterial killing but also in a dampening of the host immune responses classically associated with disease and ultimately TB transmission [13]. The reduced pathology in HPE-DMPT64-BCG vaccinated animals was even more evident in spleen, where lesions were not observed (Figs. 2 and 3). Taken together these results support enhanced protective effect elicited by the HPE-DMPT64-BCG compared to BCG in the guinea pig model of TB. The reduced bacterial burden and absence of lesions in the spleen of HPE-DMPT64-BCG vaccinated animals compared to those immunized with wt BCG, suggests that the immune response elicited by the former was more effective in preventing bacterial dissemination. Similarly, homologous boosting of mice immunized with HPE-DMPT64-BCG resulted in a specific reduction in CFUs in the spleen [9]. In mice, the superior protection afforded by HPEDMPT64-BCG over BCG, or a recombinant BCG strain expressing MPT64 in the cytosol, was associated with the ability to elicit MPT64-specific IFN-c secreting cells carrying a specific T cell repertoire [8]. Hence, it is possible that also in guinea pigs these MPT64specific T lymphocytes may warrant enhanced anti-TB activity. Vaccines capable of eliciting an immune response that can more effectively control bacterial dissemination could be of relevance in humans, where dissemination from the site of primary infection is a key step in TB pathogenesis following primary infection [2,14] and during latent TB infection, when ‘‘scouting’’ bacilli arising from the dormant reservoir may actively replicate and promote active disease [15,16]. The reduced pathology observed in the lung of HPE-DMPT64BCG immunized animals compared to those vaccinated with BCG confirms previous observation in mice [8], further highlighting the enhanced protective activity afforded by this strain. Although many new live attenuated vaccines have been developed and tested in preclinical animal models, very few were able to consistently provide enhanced protection over BCG, which is used as the gold standard [17]. The results obtained in mice and guinea pigs for the HPE-DMPT64-BCG strain are very promising, making it a candidate for further testing. Moreover, it is worth noting that PE_MPT64 could be expressed on the surface of other rBCG strains, such as the BCG ureC hly+ strain [18], opening the possibility to further enhance the anti-TB immunity elicited by these recombinant BCG strains. Acknowledgements We gratefully acknowledge the support from the Biological Investigations Group at PHE, Porton Down. The views expressed
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