New Approaches to TB Vaccination

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New Approaches to TB Vaccination Zhou Xing, MD, PhD; Mangalakumari Jeyanathan, PhD; and Fiona Smaill, MD

Pulmonary TB remains a leading global health issue, but the current Bacille Calmette-Guérin (BCG) vaccine fails to control it effectively. Much effort has gone into developing safe and effective boost vaccine candidates for use after the BCG prime vaccination. To date, almost all the lead candidates are being evaluated clinically via a parenteral route. Abundant experimental evidence suggests that parenteral boosting with a virus-based vaccine is much less effective than respiratory mucosal boosting, because the former fails to activate a type of T cell capable of rapidly transmigrating into the airway luminal space in the early phase of the Mycobacterium tuberculosis infection. The next few years will determine whether parenteral boosting with some of the lead vaccine candidates, particularly the protein-based vaccines, improves protection in humans over that by BCG. Much effort is needed to develop respiratory mucosal boost vaccines and to identify the reliable immune protective correlates CHEST 2014; 146(3):804-812

in humans.

ABBREVIATIONS: AdHu5 5 human type 5 adenovirus; ALT 5 airway luminal T cell ; APC 5 antigenpresenting cell; BCG 5 Bacille Calmette-Guérin; IFN 5 interferon; LIT 5 lung interstitial T cell; RM 5 respiratory mucosal; Th1 5 T helper cell type 1; WHO 5 World Health Organization

Pulmonary TB caused by Mycobacterium tuberculosis has haunted humankind for many centuries and still remains a top infectious killer. TB currently accounts for approximately 1.4 million deaths, second only to HIV/AIDS, and 9 million new cases each year. An estimated one-third of the world population is latently infected by M tuberculosis, and 5% to 10% of these people develop active TB at some point in their lives. Young children and HIV-positive hosts are much more susceptible to TB.1 Despite the availability of antibiotics, 20% of TB cases are multidrug resistant or extensively drug resistant. Although the overall incidence of TB in high-income countries

Manuscript received February 20, 2014; revision accepted March 11, 2014. AFFILIATIONS: From McMaster Immunology Research Centre and Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada. FUNDING/SUPPORT: The studies from the authors’ laboratory were supported by funds from the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, and the Canadian Foundation for Innovation.

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including Canada and the United States is much lower than in low/middle-income countries, TB continues to be a significant health issue in these countries because of greater TB rates in foreign-born immigrant populations, aboriginal communities, and HIV-positive and homeless people.2,3 The persisting global TB epidemic calls for the development of much improved TB vaccination strategies.

Global Bacille Calmette-Guérin Vaccination Program Bacille Calmette-Guérin (BCG) is the only anti-TB vaccine in use, to our knowledge, and it has been . 9 decades since its first

CORRESPONDENCE TO: Zhou Xing, MD, PhD, Room 4012-MDCL, Department of Pathology and Molecular Medicine, McMaster University, 1280 Main St W, Hamilton, ON L8S 4K1, Canada; e-mail: xingz@ mcmaster.ca © 2014 AMERICAN COLLEGE OF CHEST PHYSICIANS. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details. DOI: 10.1378/chest.14-0439

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use in humans. BCG represents the most widely administered vaccine in World Health Organization (WHO) immunization programs, being routinely used in . 180 countries.4 Canada and the United States are among a small number of high-income countries where BCG is not part of the national immunization program. In most countries, BCG, an attenuated Mycobacterium bovis-based vaccine, is administered once via the skin shortly after birth. The standard dose of BCG for infants , 12 months old is 1 to 4 3 105 colony-forming units. Nevertheless, because BCG is considered unsafe to HIV-positive hosts, it is not recommended by the WHO to be given to children who are HIV-positive.1,5,6 BCG effectively protects against disseminated childhood TB, but the current global TB epidemic speaks to its failure to effectively control adolescent and adult pulmonary TB.6 Among the major speculated reasons for the ineffectiveness of BCG vaccination are the use of genetically variant BCG strains, the interference from exposure to environmentally borne mycobacterial species, and the limited longevity of the protective immunity provided by BCG. Unfortunately, repeated BCG administrations are unable to improve its protective efficacy in humans or experimental animals.7,8 The current conviction by the scientific community and health policy makers is that the current BCG, or an improved version, will continue to be used as a childhood priming vaccine in the WHO immunization program, but there is an urgent need to develop novel TB vaccines that can be used as a booster following BCG immunization.

Global TB Vaccine Pipeline With the results reported recently of the first large-scale human trial of a TB vaccine since 1968, momentum is building in the field of TB vaccine research.9 The TB vaccine pipeline now has at least a dozen products in clinical trials (Fig 1) and a robust preclinical research program exploring novel approaches to vaccine development and delivery systems.1,5 A systems approach to designing TB vaccines based on a better understanding of the immunology of the host and the pathogenicity of the organism holds great promise for the next generation of candidate vaccines.10 The current slate of candidate TB vaccines is designed either to replace BCG with an improved organism-based vaccine or to boost BCG primed responses. With few exceptions, these new vaccines have been shown to have an excellent safety profile, including in HIV-positive and TB-infected people and in infants, with no evidence of

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immunopathology.9,11 MVAAg85A (MVA85A/AERAS485), a recombinant modified vaccinia virus expressing Ag85A, the immunodominant antigen of M tuberculosis, represents the most advanced candidate TB vaccine designed to boost prior vaccination with BCG. Disappointingly, despite being shown to be safe and to induce durable CD4 T-cell responses, intradermal vaccination with MVAAg85A in BCG-immunized infants failed to significantly improve protection over that by BCG against active TB disease or infection in a phase 2b, randomized, double-blind, placebo-controlled efficacy study involving 2,797 infants in TB-endemic South Africa.9,12 On reflection, experimental evidence suggests that this vaccine candidate did not significantly further enhance BCG-mediated protection in animal models.13 However, completing a large vaccine trial in a TB-endemic area is a success in itself.14-16 Another viral vector vaccine based on human type 35 adenovirus (Crucell Ad35/AERAS-402) that expresses multiple immunodominant TB antigens was shown to significantly enhance both CD4 and CD8 T-cell responses in healthy volunteers.17 Additional immunogenicity data are forthcoming from a number of recently completed clinical trials as well as an ongoing study in infants, but there is now an interest in developing Crucell Ad35 in a prime boost strategy with MVAAg85A. We have developed AdHu5Ag85A based on a recombinant replication-deficient human type 5 adenovirus (AdHu5). After demonstrating its protective efficacy in animal models, we have completed a phase 1 trial in healthy adults and demonstrated robust activation of multifunctional CD4 and CD8 cells in previously BCG-immunized individuals despite preexisting Ad5 antibodies, making it a promising vaccine candidate.18 Recombinant protein-based vaccines containing immunodominant antigens of M tuberculosis, delivered with a T helper cell type 1 (Th1)-activating adjuvant, have been shown to be safe and immunogenic in phase 1 studies. The M tuberculosis fusion protein M72 with the liposomalbased adjuvant AS01 (M721AS01E) induces multifunctional, long-lived CD4 T-cell responses and boosts T-cell populations in TB-infected individuals19,20; efficacy trials are currently being planned. Vaccines (H11IC31, H41IC31/AERAS-404, H561IC31/AERAS456) that include the antigens produced at different stages of TB infection (ie, during active disease [eg, Ag85B, ESAT6, and TB10.4] and latency [eg, Rv2660]) have the potential to be used as both prophylactic and therapeutic vaccines (Fig 1).21-23 The results of phase 1 and 2a safety and immunogenicity studies with different fusion

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Figure 1 – Global TB vaccine pipeline. The vaccine candidates (protein based, viral vectored, and mycobacterial organism based) currently undergoing various phases of clinical evaluation are shown.

proteins and new proprietary adjuvants (eg, IC31 and GLA-SE) will inform the decision as to which to advance to efficacy studies. New vaccines designed to replace BCG have had mixed results. Recombinant BCG has been engineered to overexpress immunodominant TB antigens or improve the presentation of antigens by changing the phagosomal membrane (VPM1002), and although earlier products were not shown convincingly to be safer or better, VPM1002 is entering a phase 2 safety and immunogenicity study in newborns in South Africa.24 Slow progress has been made in developing whole-cell or fragmented mycobacterial vaccine candidates. RUTI, a vaccine based on detoxified cellular fragments of M tuberculosis, is being considered as an adjunct to chemotherapy to treat both latent and active TB.25 There are plans to revisit earlier studies of an inactivated strain of Mycobacterium vaccae.26,27 In addition, an alternative approach of using attenuated strains of M tuberculosis with regulated or deleted virulence genes is entering phase 1 studies.28 There is no doubt that the ongoing clinical vaccine trials (Fig 1) will continue to provide new information. The ultimate success in the hunt for effective TB vaccination strategies rests on further improved knowledge in mucosal immune responses to TB and vaccine immunology and on our unrelenting effort to develop new vaccine formulations. 806 Translating Basic Research Into Clinical Practice

Current Progress and Challenges Natural Immunity Against Pulmonary M tuberculosis Infection

The first point of host-microbe interaction following pulmonary M tuberculosis exposure is the initial infection of antigen-presenting cells (APCs), alveolar macrophages,29 and dendritic cells30 by M tuberculosis, which leads to a cascade of host responses. Effective communication of M tuberculosis antigen-bearing APCs with naive T cells via antigen presentation and other cognate interaction in the draining lymph nodes initiates activation of predominantly Th1 cells.31,32 Mounting evidence suggests that CD8 T cells play a role in anti-TB immunity.33 Induction of Th1 immunity in the lung is essential to effectively control M tuberculosis growth within the APCs. It is widely believed that one of the most important roles of Th1 cells is to produce the interferon (IFN)-g required for activation of M tuberculosis-infected APCs. However, relative to many other types of respiratory intracellular infections, pulmonary mycobacterial infection significantly delays the induction of Th1 immunity in the lung of experimental animals.31,32 Indeed, anti-TB Th1 cells do not appear in significant numbers in the lung until 18 to 20 days after M tuberculosis exposure, resulting in much delayed protection.34-37 Although much still remains to be understood, delayed Th1 immunity has been linked to the innate immunosuppressive property of

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M tuberculosis on APCs, which causes delayed APC migration to draining lymph nodes and T-cell priming.36,38,39 Induction of regulatory T cells by a subtype of M tuberculosis-infected APCs has also been implicated in delayed Th1 immunity.40 A better understanding of the cellular and molecular mechanisms of delayed Th1 immunity is critical to understanding the immunologic reasons for ineffective lung protection by parenteral BCG vaccination in humans. In turn, such knowledge will help design effective boost vaccination strategies to improve BCG-induced immunity in the lung. BCG-Induced Immunity Against Pulmonary M tuberculosis Infection

Parenteral BCG vaccination has failed to effectively control pulmonary TB in humans, as evidenced by the current global TB epidemic. The reasons for its unsatisfactory effectiveness still remain largely speculative.6 Because M tuberculosis infection significantly delays the onset of Th1 immunity in a naive host, the question is whether Th1 immunity in the lung is also delayed upon M tuberculosis exposure even in parenterally BCGvaccinated humans. Although it is difficult to address this question in humans, murine studies have demonstrated the presence of effector memory Th1 cells in the lung interstitium, but not in the airway lumen,37 following parenteral BCG vaccination.41,42 Upon M tuberculosis exposure in such parenteral BCG-vaccinated animals, the expansion of this lung interstitial T-cell population and the appearance of airway luminal T cells (ALTs) were somewhat accelerated, compared with unvaccinated animals, but only by 4 to 5 days.37,43 In other words, the lungs of parenteral BCG-vaccinated animals are still left unprotected for at least 10 to 14 days.37 We have linked the lack of protection in the early phases of M tuberculosis infection in BCG-vaccinated animals to the lack of ALTs (Fig 2A).37 Thus, a significant delay in the appearance of Th1 immunity in the lung upon M tuberculosis exposure is a plausible mechanism for the ineffectiveness of parenteral BCG vaccination in humans. The drawback of these experimental studies is that the mice were vaccinated at or after 6 to 8 weeks of adult age. In comparison, BCG is given to humans shortly after birth.6 Thus, there is a need to investigate parenteral BCG-induced lung immunity in neonatal/infant animals. It is known that, in neonates/infants, the immune system is immature, with suboptimal APC functionality.44,45 Nonetheless, BCG vaccination induces Th1 cells in the peripheral blood of humans,46-48 which

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peak at 6 to 10 weeks and gradually wane over the first year of life.48 BCG also activates CD8 T cells but at a much lower rate than CD4 T cells.49 However, there is evidence from human studies that delaying BCG vaccination from birth to 10 weeks of age quantitatively and qualitatively enhanced BCG-specific T-cell responses in the circulation.50 Considering that most childhood vaccines are given after 2 months of age and that the immune system takes time to mature, the best time to administer BCG to infants in human immunization programs remains scientifically debatable. A similar question also applies to the timing of effective boost vaccination in humans (see subsequent discussion).

Route and Timing of Boost TB Vaccination: Parenteral or Respiratory Mucosal?

Both parenteral (IM or intradermal) and respiratory mucosal (RM) routes of boost vaccination have been investigated extensively in BCG-primed animal models. Whether parenteral boosting can significantly improve lung protection over that by BCG priming is determined by both the nature of the boost vaccine and the route of delivery.51 Repeated parenteral injections of selected protein-based boost vaccines are effective in this respect. In contrast, genetic, viral-based boost vaccines, when given once parenterally, are ineffective, particularly in murine models.52 There is evidence, however, that parenteral boosting with adenovirus-based TB vaccine can significantly improve protection over that by BCG priming in larger-size animals, including guinea pigs, goats and cattle.53-56 Experimental evidence suggests that whether a vaccination strategy can quickly mobilize systemically located Ag-specific T cells into the airway lumen holds the key to protective efficacy.52 The inability of parenteral virus-based boosting to improve protection is caused by its inability to generate ALTs (Fig 2A). Although it still remains to be verified experimentally, the ability of parenteral protein-based boosting to improve protection is speculated to result from the generation of a population of T cells that have acquired the homing molecules and, thus, can be mobilized quickly into the airway lumen upon M tuberculosis exposure (Fig 2B). Repeated injections of protein-based booster may have played a role in increasing such mucosal homing molecules on CD4 T cells.13,57 The potential drawbacks of protein-based boost vaccines are that they primarily activate CD4 T cells, but not CD8 T cells, and that repeated vaccinations are required. Furthermore, their application regarding RM boosting is limited, and further research is required to identify safe 807

Figure 2 – Geographical distribution of protective T cells determined by route of TB vaccination. A, T cells activated by parenteral vaccination with Bacille Calmette-Guérin or viral-based vaccines populate the lung interstitium via the pulmonary circulation, where they become long-lived memory T cells. Because of the lack of chemotactic signals in the lung and the lack of expression of mucosal-homing molecules, such antigen-specific T cells do not enter the airway lumen in naive state or in the early stage of Mycobacterium tuberculosis infection. B, T cells activated by respiratory mucosal vaccination populate both the lung interstitium and the airway lumen via the pulmonary circulation, where they become long-lived memory T cells. Such T cells express mucosal-homing receptors CCR1, CCR6, and CCR8 and aEb7 (CD103) and a4 b 1 (VLA4) integrins. aEb7 integrin binds to the E cadherin expressed on the basal-lateral surface of the airway epithelium. Circulating T cells that express the respiratory mucosal homing signatures following parenteral or respiratory mucosal vaccination are potential biomarkers used as a protective correlate. DC 5 dendritic cell; MF 5 macrophage.

and effective immune adjuvant. Nonetheless, the protein-based vaccine remains promising as an effective parenteral boosting platform. Of note, all candidate TB vaccines to date have been or are being evaluated in clinical trials via a parenteral route of boosting.1 Among these candidate vaccines is MVAAg85A, the most extensively clinically evaluated TB vaccine. However, parenteral boosting with this vaccine was ineffective in BCG-primed infants in the recently completed phase 2b efficacy trial in South Africa.9 This observation is in line with the results of preclinical models and highlights the importance of considering local lung immunity in boosting vaccination strategies (Figs 2A, 2B).13,51,52 RM boosting represents the most effective way to generate ALTs.52,58 In general, genetic, virus-based vaccines are amenable to effective RM vaccination. The chief mechanism by which RM boosting potently enhances protection is via the generation of long-lasting effector memory ALTs, critical to early control of mycobacterial infection within the airway (Fig 2B).59 RM boost vaccination also produces lung interstitial T cells (LITs), which help control infection when M tuberculosis spreads to the lung interstitium. Although parenteral vaccination with BCG or viralbased vaccine also generates LITs (Fig 2A),37,52 such LITs may differ from those induced by RM vaccination in that parenteral LITs lack certain RM homing receptors 808 Translating Basic Research Into Clinical Practice

probably including CCR1, CCR6, CCR8, and CD103 (Zhou Xing, MD, PhD, unpublished data, July 14, 2014). We have recently reported the importance of the choice of viral vector for RM boosting because different viral vectors may differentially imprint macrophages and dendritic cells in the lung, leading to different protective outcomes independent of T-cell immunity. In this regard, adenoviral vector is superior to vesicular stomatitis viral vector.60 A viral vector capable of robust, type 1 IFN induction should be avoided. Although extensively investigated in murine models, RM boost vaccination strategies still remain to be evaluated in clinical trials. MVAAg85A following aerosol delivery to human lungs is being evaluated. Regardless of the route of boost vaccination, whether there is preexisting immunity against the viral vaccine backbone needs to be considered. This is particularly relevant when using AdHu5 vectors.61 The efficacy of parenterally administered AdHu5 vectors was dampened in the animals that had preexisting AdHu5-neutralizing antibodies; this is also supported by clinical evidence. However, it is also accepted that the unrivaled potency of the AdHu5 vector system is able to overcome that concern. Indeed, we have recently observed potent boosting immunogenicity following intramuscular vaccination with a small dose of AdHu5Ag85A in human volunteers despite preexisting neutralizing

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antibody titers.18 Nevertheless, the dose of AdHu5-based vaccine could conceivably be further reduced should there be an absence of such preexisting antibodies. Furthermore, evidence suggests that preexisting anti-AdHu5 T-cell immunity may also mitigate the potency of AdHu5-based vaccines.62 Clinical investigation has provided the basis for using AdHu5-based vaccines via the respiratory tract, because few antiAdHu5 antibodies were found to be present on the surface of the respiratory mucosa as opposed to the peripheral blood.63 However, it remains to be investigated whether respiratory delivery of AdHu5-based vaccine may evade anti-AdHu5 T-cell immunity. On the other hand, rare serotypes of human adenoviruses such as AdHu35 have been used to develop TB vaccine; however, these vectors are known to be much less immunogenic than AdHu5.64 Along the same line of consideration, adenoviruses of nonhuman origin, such as chimpanzee-derived viruses, are being developed to circumvent the issue, because some chimpanzee adenoviruses are as immunogenic as AdHu5, and anti-AdHu5 antibodies do not cross-react with chimpanzee adenoviruses.64,65 Another relevant consideration is the timing of the boost vaccination following the BCG prime vaccination. Experimentally, boosting is often performed 4, 8, or 12 weeks after BCG priming. Scientifically, boosting ought to be at the point at which the T cells have largely entered into the memory phase. In animals, the selection of the timing is often limited by cost and other practical issues. In the later stage of clinical vaccine trials, the timing of boosting, when possible, ought to be aligned with the time when the booster is desired to be given to BCG-primed children in the real world.66 One time point could be 2 to 3 years of age, because in the first few years of life children are susceptible to developing TB after exposure because of immunologic immaturity.45,67 Another potential time point is 10 to 12 years of age, when BCG immunity begins to wane, exposure to M tuberculosis outside the household begins to increase, and adolescent/adult TB rates pick up. Of note, in the phase 2b MVAAg85A trial, boosting was performed within 4 to 6 months of neonatal BCG priming. 9 Much more is understood about primeboost TB vaccination strategies in fully developed adult animals and humans than in their immunologically underdeveloped counterparts, but there is still a need to better understand whether boost vaccination carried out at varying intervals after infant BCG priming may generate differential quantities and qualities of T cells. journal.publications.chestnet.org

TB Vaccine Trials and Immune Protective Correlates

A challenge facing TB vaccine scientists is the lack of immune protective correlates in humans. Experimental evidence has suggested qualitatively the importance of both CD4 and CD8 T cells and type 1 cytokines including IFN-g, IL-12, and tumor necrosis factor-a.68 Clinical reports on increased susceptibility to TB in HIV-infected humans and humans genetically deficient in some of these experimentally identified mechanisms also lend support.69 However, the bona fide vaccine-triggered protective correlates will not be known until a promising TB vaccine is identified successfully in efficacy trials. There are several potential approaches that may aid the prediction of such protective efficacy by the lead vaccine candidates. One is to use the parenteral BCG challenge in vaccinated human volunteers.70 The second approach is to use an in vitro mycobacterial or M tuberculosis growth-inhibition assay where the function of vaccineactivated immune cells can be studied by ex vivo infecting the vaccine trial-derived peripheral blood mononuclear cells.71 Recently, humanized murine models of TB have been established.72 Such models have the potential to allow rapid in vivo testing for vaccine efficacy in a surrogate human immunologic environment, identification of immune protective correlates, and examination of the circulating biomarkers indicative of the T cells with RM-homing properties. The current human TB vaccine trials are limited to analyzing the biologic samples obtained only from the peripheral blood following parenteral boost vaccination. Both clinical and experimental evidence suggest that the cytokine-producing T cells located in the peripheral tissue compartments (lung interstitium and/or peripheral blood) correlate poorly with immune protection, whereas the ALTs correlate well with protection in the lung (Figs 2A, 2B).52,73,74 Thus, in theory, the pulmonary, particularly the airway luminal, localization of T cells can be used as a protective correlate.41,52 In this regard, the attempt in human efficacy trials to correlate the cytokine- or multicytokine-producing T-cell responses in the peripheral blood with protective immunity can be misleading or unreliable (Fig 1A). However, in human vaccine trials, the lung interstitium is not readily accessible. The respiratory tract is accessible in practice via the BAL in the case of RM vaccine trials. One approach is to identify the circulating T cells that express the potential RM homing signatures including CCR1, CCR6, CCR8, aEb1 (CD103), and a4 b 1 (VLA-4)75 following parenteral or RM vaccination (Fig 2B). 809

Conclusions The world has seen a big leap forward in the development of promising TB vaccination strategies that can be used to boost protective immunity in the lung in BCG-primed hosts. About a dozen vaccines are currently in different stages of clinical evaluation, and more will be developed over the next few years. Some of these vaccine candidates were developed to replace the current BCG vaccine, whereas others are for boost vaccination. The next few years will also witness whether some of these vaccines are able to significantly improve protection over BCG in efficacy trials. However, a number of challenging issues remain: (1) It is unclear whether parenteral boost vaccination will prove effective in humans, (2) the effort and investment made into clinically developing RM boost vaccines lags far behind, (3) there is a lack of immune protective correlates in humans, (4) it remains unclear whether the immune responses detected in the peripheral blood are predictive of those in the lung or of protection in the lung, and (5) it remains poorly understood whether the vaccine candidates, which are currently largely designed for prophylactic application, will be effective in latently infected humans.

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Acknowledgments Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/ organizations whose products or services may be discussed in this article. Role of sponsors: The sponsors played no role in the design of authors’ own studies or in data analysis, or in the preparation of the manuscript.

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