Therapeutic Immunization against Mycobacterium tuberculosis Is an Effective Adjunct to Antibiotic Treatment

Rhea N. Coler 0 1 Sylvie Bertholet 0 1 2 Samuel O. Pine 0 1 Mark T. Orr 0 1 Valerie Reese 0 1 Hillarie Plessner Windish 0 1 Charles Davis 0 1 Maria Kahn 0 1 Susan L. Baldwin 0 1 Steven G. Reed () 0 1 0 Received 22 November 2011; accepted 13 March 2012; electronically published 13 August 2012 1 Infectious Disease Research Institute , Seattle, Washington 2 Present address: Novartis Vaccines & Diagnostics , via Fiorentina 1, Siena 53100, Italy. 1124 Columbia St, Suite 400, Seattle, WA (See the editorial commentary by McMurray on pages 1193-4.) Background. Recent advances in rational adjuvant design and antigen selection have enabled a new generation of vaccines with potential to treat and prevent infectious disease. The aim of this study was to assess whether therapeutic immunization could impact the course of Mycobacterium tuberculosis infection with use of a candidate tuberculosis vaccine antigen, ID93, formulated in a synthetic nanoemulsion adjuvant, GLA-SE, administered in combination with existing first-line chemotherapeutics rifampicin and isoniazid. Methods. We used a mouse model of fatal tuberculosis and the established cynomolgus monkey model to design an immuno-chemotherapeutic strategy to increase long-term survival and reduce bacterial burden, compared with standard antibiotic chemotherapy alone. Results. This combined approach induced robust and durable pluripotent antigen-specific T helper-1-type immune responses, decreased bacterial burden, reduced the duration of conventional chemotherapy required for survival, and decreased M. tuberculosis-induced lung pathology, compared with chemotherapy alone. Conclusions. These results demonstrate the ability of therapeutic immunization to significantly enhance the efficacy of chemotherapy against tuberculosis and other infectious diseases, with implications for treatment duration, patient compliance, and more optimal resource allocation. - Worldwide, the tuberculosis pandemic is associated with 1.72 million deaths annually, and the increase in multidrug-resistant tuberculosis (MDR-tuberculosis) further heightens this threat [1]. There is an urgent need for more effective therapeutic regimens to increase treatment compliance and decrease tuberculosis transmission [26]. The development of rationally designed molecular adjuvants that stimulate innate immune responses and shape the quality and strength of adaptive immunity, combined with select recombinant proteins, has enabled the development of a new generation of vaccines that can be used to treat and prevent infectious diseases. Although development of more effective prophylactic vaccines for tuberculosis is a high priority, therapeutic approaches, such as postexposure vaccines, which could be used in combination with antibiotics to shorten treatment regimens, clear bacilli [7], and limit the spread of MDR-tuberculosis [810], should be explored. Using a combination of drugs plus vaccine, we showed therapeutic efficacy among patients infected with another macrophage pathogen of the genus Leishmania [11], supporting the potential of an immune-therapeutic approach. In this regard, few tuberculosis vaccine candidates have been evaluated for therapeutic efficacy [1223]. We identified potent T cell antigens of Mycobacterium tuberculosis (M. tuberculosis), recognized by persons latently infected with M. tuberculosis, and used a synthetic nanoemulsion adjuvant, GLA-SE (a synthetic TLR-4 agonist [GLA], formulated in a stable oil-in-water emulsion [SE]), that adds an innate signal and potent Th1inducing properties [2426], to develop a vaccine candidate, ID93/GLA-SE [1214]. ID93 combines 4 antigens belonging to families of M. tuberculosis proteins associated with virulence (Rv2608, Rv3619, and Rv3620) or latency (Rv1813) [27]. To adequately evaluate therapeutic vaccines, it is essential that longterm models of protection against diseases be developed and used. To do this, we have used both mouse and nonhuman primate (NHP) models. Unlike C57BL/6 and BALB/c mice, which exhibit stabilized pulmonary bacterial growth and survive for >1 year [7, 2831], SWR/J mice exhibit extreme M. tuberculosis susceptibility, with progressive bacterial growth resulting in fatal disease, thus making them a good postexposure model to evaluate immunotherapeutic regimens [29]. The reasons for this increased susceptibility are unclear. Nevertheless, we have evidence that ID93/GLA-SEimmunized SWR/J mice mount effective immune responses and protection against M. tuberculosis (unpublished data). The cynomolgus monkey has been described as a good model for human tuberculosis, displaying a range of clinical and pathological changes [32], and may be efficiently protected against M. tuberculosis challenge [17, 33, 34]. To model synergy between chemo- and immune-therapy, we performed studies using ID93/GLA-SE, administered in combination with existing first-line antibiotics rifampicin (RIF) and isoniazid (INH) in SWR/J mice and cynomolgus monkeys. These data show that therapeutic immunization can be used to complement chemotherapy as an approach to treat tuberculosis. MATERIALS AND METHODS Mice, Treatment, and ID93/GLA-SE Immunization Female, age-matched (46 weeks) SWR/J and C57BL/6 mice were purchased from Jackson and Charles River Laboratories, respectively. All mice were maintained in the animal facility of The Infectious Disease Research Institute (IDRI) and were treated in accordance with the guidelines of the Animal Care and Use Committee. Mice were infected with a low dose (50100 bacteria) aerosol (LDA) of M. tuberculosis H37Rv (ATCC #27294) with use of a University of WisconsinMadison aerosol chamber. At 15 or 30 days after infection, a subset of mice was started on a drug regimen of INH (at 85 mg/L of drinking water) and RIF (at 50 mg/L of drinking water) administered for 30, 60, or 90 consecutive days. Female mice are estimated to drink 0.15 0.37 mL/g [35]. Assuming a mean intake of 0.26 mL/g per day, animals would receive approximately 22 mg/kg of INH and 13 mg/kg of RIF per day. The minimum inhibitory concentrations for M. tuberculosis H37Rv are 0.25 M for RIF and 1.0 M for INH. A subset of groups receiving the 6090day RIF-INH combination regimen was also injected with either GLA-SE alone (referred to as Rx + GLA-SE) or the ID93/GLA-SE vaccine (referred to as Rx + ID93/GLA-SE), which were produced as previously reported [14, 24, 25]. Mice were immunized 3 times, 3 weeks apart, with 8 g of protein plus 20 g of GLA-SE either during (DTT; days 15, 36, and 57) or after antibiotic treatment (PTT; days 107, 128, and 149). Therapeutic efficacy was determined by tracking survival over time and by plating lung homogenates as previously described [13]. Cytokine Profiling Assay Splenocytes (2 106/mL) were stimulated with ID93 (10 g), purified protein derivative (10 g; CSU), or phosphatebuffered saline. Cytokine concentrations (IFN-, IL-2, TNF, IL-5, IL-10, IL-13, and IL-17) were determined using a Pr (...truncated)