Mycobacterium tuberculosis attenuated by multiple deletions of rpf genes effectively protects mice against TB infection

Tuberculosis 91 (2011) 219e223

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Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube

IMMUNOLOGICAL ASPECTS

Mycobacterium tuberculosis attenuated by multiple deletions of rpf genes effectively protects mice against TB infection Tatiana Kondratieva a, Elvira Rubakova a, Bavesh D. Kana b, Sergey Biketov c, Vasilii Potapov c, Arseny Kaprelyants d, Alexander Apt a, * a

Laboratory for Immunogenetics, Central Institute for Tuberculosis, Yauza alley, 2, Moscow 107564, Russia MRC/NHLS/WITS Molecular Mycobacteriology Research Unit, University of the Witwatersrand and The National Health Laboratory Service, Johannesburg, South Africa Scientific Research Center for Applied Microbiology and Biotechnology, Obolensk, Moscow Region, Russia d Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia b c

a r t i c l e i n f o

s u m m a r y

Article history: Received 12 December 2010 Received in revised form 18 January 2011 Accepted 26 January 2011

In this study, we investigated the residual virulence of mutants of Mycobacterium tuberculosis that are defective in 4 of the 5 rpf-like genes, their capacity to persist in the murine host and the utility present in these mutants to serve as novel vaccine candidates. Our data indicate that the two quadruple rpf deletion mutants, DACBD and DACDE, both display significant attenuation in the mouse lungs after aerosol infection, with no observable increase in bacillary loads upon aminoguanidine-induced immune suppression. However, after subcutaneous injection these strains were able to persist at the low level, similar to that of BCG, in the mouse lungs and lymphoid organs. Furthermore, both rpf quadruple mutants were able to enhance the numbers of IFN-g-producing T-cells in spleens to a level comparable to that of BCG, and conferred protection upon subsequent challenge with virulent M. tuberculosis in terms of mycobacterial multiplication in organs and survival time. The reduction in organ bacillary loads after vaccination with DACDE was comparable to that of BCG, while DACBD displayed a small but statistically significant enhancement in protection compared to BCG. Collectively, these data suggest that rpf deletion mutants show potential for further development as novel vaccine candidates for tuberculosis. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Resuscitation promoting factors Virulence Vaccine Protection

1. Introduction Currently, the only approved vaccine for tuberculosis (TB) is Bacille Calmette-Guérin (BCG) that has been used for prevention of tuberculosis for decades. Although there is ample evidence to suggest that it is highly successful in protecting against miliary TB, disseminated infection and TB meningitis in children,1,2 its efficacy against pulmonary infection in adults is questionable, and in certain clinical trials BCG failed to prevent pulmonary TB.3 Thus, more effective vaccines for successful TB prophylaxis and limitation of Mycobacterium tuberculosis transmission are urgently needed. New tools for genetic manipulation in mycobacteria substantially accelerated the development of vaccine candidates, and several designs for new vaccines have been proposed including protein subunit vaccines, DNA vaccines, virus vector-based vaccines and live vaccines (reviewed in Ref.4). In practical terms, each

* Corresponding author. Tel.: þ7 095 268 7810. E-mail address: [email protected] (A. Apt). 1472-9792/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tube.2011.01.005

approach has its intrinsic disadvantages, e.g., poor antigenic composition and strong adjuvant dependence of subunit vaccines, low immunogenicity of DNA vaccines, and potential danger of live, replicating vaccines for individuals with compromised immunity. However, there is growing consensus that the strategy of heterologous vaccination, combining priming injection of live vaccine (BCG, or modified BCG, or a novel live vaccine) with boosting injection of selected mycobacterial antigens or genetic constructs that encode such antigens would have maximal impact in endemic regions with a high TB incidence.5,6 There is no doubt that live, attenuated mycobacterial vaccines are beyond compare as a source of antigenic repertoire. BCG priming followed by boosting with particular antigens effectively induceds T-cell responses in mice, guinea pigs, cattle and humans,7 which explains persistent interest to live TB vaccines and repeating efforts of their improvement. Thus, the development of novel recombinant BCG vaccines has involved engineering of strains to express additional and/or to lose “undesirable” molecules to shift immune responses toward better TB protection. An example of this is the attempt to improve BCG by expressing mammalian cytokines in order to augment the immune

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response,8 but this approach did not resulted in an appreciable improvement in BCG performance. Conversely, the construction of a BCG strain expressing the listeriolysin gene (allowing for enhancement of antigen trafficking into cytoplasm for induction of the CD8þ T cell response), but deficient in the urease gene (aiming at maintenance of higher pH levels in the phagosome) resulted in elevation of immunogenicity in mice,9 and the strain has proceeded to clinical trials. Another approach to vaccine development has been the construction of live, attenuated strains of M. tuberculosis that have lost the capacity to cause disease but retain significant antigenic potency to induce a prolonged, protective immune response. The rationale for potential replacement of Mycobacterium bovis BCG with non-virulent M. tuberculosis is the closer antigenic similarity of the latter to the infectious agent, providing both a more adequate spectrum of immune response specificity and a possibility of boosting vaccination with important antigens absent in BCG but present in M. tuberculosis, e.g., RD1-encoded molecules. Several attenuated strains have been constructed by deleting genes to create auxotrophs that are dependent on metabolites scarcely present in host, e.g., auxotroph strains dependent on leucine, tryptophan, and pantothenate that provide protection comparable to that of BCG.10e12 However, there are two concerns which should be addressed during further development of auxotroph-based vaccines. Those are, firstly, low concentrations of substrates (aminoacids, vitamins, etc.) which could be present in the infected host tissues and promote the growth to inadmissible levels,13 and, secondly, assuring that the attenuated strain still retains the potential to persist in host tissue so as to induce full degree of protective immune responses. Therefore, it seems rational to attenuate live M. tuberculosis-based vaccines in such a way that substitution with host metabolites would be impossible and that the degree of attenuation could be varied. With this regard, strains bearing several mutations affecting mycobacterial metabolic pathways important for virulence and thus leading to attenuation14,15 may serve as interesting candidates for further vaccination studies. This may be equally applied to the strains lacking a few rpf proteins. The resuscitation promoting factor (Rpf) protein family contains secreted bacterial enzymes that are widely distributed in the actinobacteria and display the ability to enhance the culturability of dormant bacteria, possibly through muralytic cleavage of bacterial peptidoglycan (PG). Consequently, these proteins are thought to play an important role in controlling dormancy and resuscitation from non-culturable states, in many organisms including mycobacteria (reviewed in Ref.16). M. tuberculosis has five rpf-like genes, designated rpfAeE. Considering their structure, all seem to encode proteins with similar activity, likely serving as lytic transglycosylases. However, biological activity of individual rpf genes is different (reviewed in Ref.16). Thus, the rpfB gene is the only single gene whose deletion results in a delayed reactivation of chronic M. tuberculosis infection in mice. The rpfE is the only essential gene for the transition from slow to fast growth of M.bovis in a chemostate. The RpfA was shown to be regulated by cAMPdependent receptor, whereas the rpfC is regulated by the sigma factor SigD. In general, in vitro and in vivo studies of different multiple rpf KO-mutants revealed a functional hierarchy within the rpf genes, with rpfB and rpfE physiological importance ranking above that of rpfD and rpfC. Although these genes are individually and collectively dispensable for growth in vitro, deletion of multiple rpf-like genes results in a significant attenuation of virulence in the mouse model of TB infection and the inability to resume bacterial growth upon immune suppression.17,18 In addition, it has been shown that, with the exception of RpfC, all Rpfs of M. tuberculosis are immunogenic

and retain the ability to stimulate T-cell proliferation and IFN-g production.19 The previously reported in vitro attenuation of strains of M. tuberculosis defective in four rpf genes,20 prompted us to assess for the first time whether quadruple rpf deletion mutants could serve as novel live vaccine candidates. In particular, since the loss of rpfE or rpfB resulted in a more prominent in vivo attenuation compared to the loss of rpfD,20 in this study we used two quadruple mutants, DACBD and DACDE, which retained only rpfE and rpfB, respectively. Our data suggest that the two distinct quadruple rpf deletion mutants used in this study confer significant protection to disease comparable to BCG in the mouse model of TB infection.

2. Materials and methods 2.1. Bacterial strains The DACBD, DACDE quadruple mutants used in this study are those previously reported.20 M. bovis strain BCG-Pasteur and M. tuberculosis strain H37Rv (initially, a kind gift of G. Marchal, Institute Pasteur, Paris) were used for vaccination control and for challenge, respectively. Clump-free mid-log-phase suspensions of mycobacteria used for vaccination and challenge were prepared exactly as described elsewhere.21,22 2.2. Animal vaccination and infections C57BL/6JCit (B6) mice were bred under conventional, non-SPF conditions at the Animal Facilities of the Central Institute for Tuberculosis (Moscow, Russia), in accordance with guidelines from the Russian Ministry of Health # 755, US Office of Laboratory Animal Welfare (OLAW) Assurance #A5502-11. Water and food were provided ad labium. Female mice, 8e10 weeks of age were used to start all experiments. All experimental procedures were approved by the institutional animal care and use committee. Mice were vaccinated with 107 CFU in 0.3 ml of saline of either quadruple rpf M. tuberculosis mutant (DACBD or DACDE), or M. bovis BCG subcutaneously in the dorsum. Mice were infected with standard virulent M. tuberculosis strain H37RV via the respiratory tract either with 104 CFU intratracheally, or aerogenically with 3e6  102 CFU using an Inhalation Exposure System (Glas-Col, Terre Haute, IN), as previously described.22,23 Aminoguanidine carbonate (AG) (Sigma), 1 ml, 1% wt/vol, was administered 90 days post infection with stomach pump for a period of 10 days. Mortality was monitored daily starting 14 days post infection. To assess mycobacterial multiplication in spleens, lymph nodes and lungs, 0.1 ml of serial 10-fold dilutions of sterile whole-organ 2-ml homogenates were plated onto Dubos agar, and colonies were counted after 18e20 days of incubation at 37  C. 2.3. ELISPOT assays Sterile single cell suspensions were obtained from spleens as described.22 Sterile filter Millipore plates were coated with rat antibody against murine IFN-g (PharMingen, San Diego, CA), washed and blocked with RPMI-1640 containing 10% FCS (HiClone, Logan, UT). Cells from 3 individual animals per group were added to the wells with 4 doubling dilutions, starting 1  106 cells/well, and cultured for 48 h in medium alone or in the presence of 10 mg/ml mycobacterium sonicate. Following staining with biotin-labeled rat antibody against murine IFN-g (PharMingen), spots were counted using ELISPOT Bioreader 4000 Pro-X (BioSys, Karben, Germany), calculated and normalized for individual samples. The results are displayed as the mean  SD per organ.

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Figure 1. Persistence of rpf quadruple mutants and BCG in mice after subcutaneous injection. Both quadruple rpf quadruple mutants, ACDE and ACBD, and BCG persisted at low levels in lymphoid organs throughout the 3 month observation period. Results are presented as mean CFU  SEM, 4 mice per group, per time point.

3. Results and discussion 3.1. Persistence of quadruple rpf mutants in organs after subcutaneous administration and during immune suppression Since vaccination of mice with live anti-TB vaccines is performed almost exclusively via subcutaneous route, we first evaluated the level of persistence and the degree of dissemination to lymphoid organs and the lung of quadruple rpf mutants in comparison with BCG. As shown in Figure 1, following subcutaneous administration of 107 CFU of either mutant strain or BCG, mycobacteria were readily detected in low numbers in lymphoid organs throughout the 3 month period. In the lungs, mycobacteria were not present at early time points and were detected in low numbers 3 months post-injection. A 1-year monitoring of animal health following subcutaneous injection with quadruple rpf mutants revealed no visible signs of disease. No remarkable differences in persistence in

H37Rv H37Rv+AG ΔABCD ΔABCD+AG ΔACDE ΔACDE+AG

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lymph nodes and lungs were noted between BCG and the two rpf quadruple mutants. In spleens, the BCG population was smaller (P < 0.05) throughout the 3-month period of monitoring, but the total numbers remained very low for all three strains. The increasing prevalence of HIV-TB co-infection necessitates that any vaccine that is being developed for TB must be safe to use in immune compromised individuals.11 To assess the relative virulence of the rpf quadruple mutants upon immune suppression, we infected mice via aerosol route and administered aminonoguanidine (AG) during the chronic phase of infection. AG is a nitric oxide synthase inhibitor and has been shown to be an effective suppressor of protective immune response in murine TB models.24,25 As shown in Figure 2, at the chronic phase of the disease established within 90 days post aerosol challenge there was a dramatic difference between groups of mice infected with the wild type M. tuberculosis and its Drpf derivatives. While in the former group the 10-day AG treatment resulted in a 1.5-log increase in CFU counts in the lung, the growth of Drpf mutants started to decline at w100 days of the disease, and AG administration had no augmenting effect. Thus, genetic disruption

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Time post infection (days) Figure 2. Growth of rpf quadruple mutants after aerosol infection and immune suppression. Mice were infected by aerosol with 300e600 CFU H37Rv M. tuberculosis per animal and AG was administered 90 days post infection for a period of 10 days. At indicated time points, CFU counts in the lungs were estimated by plating serial dilutions of lung homogenates onto Dubos agar (4 mice per time point). Inhibition of iNOS by AG administration in vivo (arrow) augments multiplication of the wild type M. tuberculosis but not that of the quadruple mutants in the lungs of infected mice. The results are mean CFU  SEM, representative of two independent experiments.

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Vaccination Figure 3. Immunogenicity of rpf quadruple mutants compared to BCG. Vaccination with quadruple rpf mutants caused an increase in numbers of IFN-g-producing splenocytes equivalent to that caused by BCG vaccination. Mice in groups of 4 were vaccinated subcutaneously with 107 mid-log-phase single-cell (filtered through 4-mm filter) CFU of either attenuated strain, and after 5 weeks the numbers of IFN-gproducing cells in spleens in the presence or absence of M. tuberculosis sonicate were assessed individually in the ELISPOT format. Results are expressed as the mean number of positive cells per 1 million  SD.

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Figure 4. Protective efficacy of rpf quadruple mutants. Vaccination with quadruple rpf mutants and BCG confers protection against subsequent challenge with virulent mycobacteria in B6 mice. Mice were subcutaneously vaccinated with 107 CFU of either mutant strain or BCG. 3 months later, mice were infected intratracheally with 104 CFU of M. tuberculosis H37Rv. CFU counts in lungs (A) and spleens (B) were determined at weeks 5 and 10 post-challenge (4 mice per group). Significant (P < 0.05e0.01, ANOVA) differences between nonvaccinated controls and all vaccinated mice were observed at both time points. In addition, splenic CFU counts at week 10 post infection were significantly lower (P < 0.05, Student’s t-test) in DACBD-vaccinated mice compared to their DACDE- and BCG-vaccinated counterparts. PCR analysis of >100 individual agar colonies confirmed that cells recovered from organs in these experiments were the wild type H37Rv and not quadruple mutants (data not shown). (C) Vaccination with either attenuated strain substantially (P < 0.0001, Gohen’s criterion for survival curves) and equally prolonged the life span of animals infected 5 wk after vaccination.

of four out of five rpf genes diminished virulence of corresponding strains to appreciable levels. 3.2. Immunogenicity of rpf quadruple mutants compared to BCG It has been established that IFN-g-producing T-cells are crucial for the control of mycobacterial infections (reviewed in Ref.26,27). Hence, we sought to determine capacity of rpf quadruple mutants and BCG to induce IFN-g T cell response after subcutaneous vaccination. Injection of either BCG or DACDE/DACBD resulted in a significant and equal increase in the numbers of mycobacteriaspecific, IFN-g-producing cells in spleens of vaccinated compared to control mice (Figure 3). This suggested that attenuated multiple Drpf M. tuberculosis strains a priori would not yield BCG in the antiTB protective performance. 3.3. Protective efficacy of rpf quadruple mutants To directly compare capacities of BCG and rpf quadruple mutants to elicit protection against subsequent tuberculosis infection, mice were challenged via the intra-tracheal rout with 104 CFU of virulent M. tuberculosis H37Rv at 3 months post subcutaneous vaccination with 107 CFU of vaccine strain. Organ bacillary loads were assessed at 5 and 10 weeks post infection. Vaccination with either rpf quadruple mutant resulted in an approximately 1 log reduction in lung bacillary load at 5 weeks post infection which was similar to that observed for BCG vaccination (Figure 4A). However, vaccination with the DACBD mutant was significantly more effective than vaccination with BCG and DACDE strains in terms of early mycobacterial multiplication in the lungs at 5 weeks and post-dissemination growth in spleens at 10 weeks (P < 0.05, unpaired t-test, Figure 4B). Remarkably, comparison of CFU data obtained at weeks 5 and 10 support considerations of I. Orme and co-authors28 who pointed at a particular high discriminative and predictive value of CFU counts early after infection. In a good agreement with their hypothesis, at week 10 non-vaccinated animals expressed a small decline in CFU counts, probably due to some degree of immunity elicited by infection itself (Figure 4). In contrast, in vaccinated animals a mild increase in CFU counts between weeks 5 and 10 may be explained by partial arrest of mycobacterial growth at weeks 3e4 after challenge, resulting in

a lower level of antigenic stimulation prior to a full establishment of the chronic phase of infection. Protective efficacy of rpf mutant strains was also compared in terms of survival time. Mice were vaccinated subcutaneously with 107 CFU of either quadruple rpf mutant strain or BCG and subsequently challenged with virulence M. tuberculosis 5 weeks later. Since after the low-dose aerosol challenge even non-vaccinated B6 mice survive in excess of 200 days, we administered a higher dose of virulent mycobacteria (104 per mouse) via intratracheal route,22 to shorten the duration of the experiment. As shown in Figure 4C, animals vaccinated with either rpf quadruple mutant or BCG survived significantly longer than unvaccinated animals (P < 0.0001, Gohen’s criterion for survival curves). In this case, we found no significant differences in the protective efficacy of the rpf quadruple mutants and BCG. Overall, our results demonstrate that multiple depletion of rpflike genes attenuates M. tuberculosis to the extend making it a new live TB vaccine candidate. The protective efficacy of rpf mutant strains appeared to be very similar to that of BCG, and there is no obvious advantage in replacing BCG with these new strains per se. However, new attenuated strains were obtained on the M. tuberculosis genetic background and, unlike M. bovis BCG, retain all genomic segments that encode important immunogenic molecules, e.g., RD1 region. This opens opportunities to search for primeeboost combinations potentially more effective than those based upon BCG administration. This work is in progress in our laboratories.

Acknowledgments The authors thank Dr. Anna Goncharenko for PCR analysis. Funding: This study was financially supported by the Program “Molecular and Cellular Biology”, Russian Academy of Sciences. Competing interests: Ethical approval:

None declared. Not required.

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