LVS ΔcapB-vectored multiantigenic melioidosis vaccines protect against lethal respiratory Burkholderia pseudomallei challenge in highly sensitive BALB/c mice.

Melioidosis, caused by the intracellular bacterial pathogen and Tier 1 select agent Burkholderia pseudomallei (Bp), is a highly fatal disease endemic in tropical areas. No licensed vaccine against melioidosis exists. In preclinical vaccine studies, demonstrating protection against respiratory infection in the highly sensitive BALB/c mouse has been especially challenging. To address this challenge, we have used a safe yet potent live attenuated platform vector, LVS ΔcapB, previously used successfully to develop vaccines against the Tier 1 select agents of tularemia, anthrax, and plague, to develop a melioidosis vaccine. We have engineered melioidosis vaccines (rLVS ΔcapB/Bp) expressing multiple immunoprotective Bp antigens among type VI secretion system proteins Hcp1, Hcp2, and Hcp6, and membrane protein LolC. Administered intradermally, rLVS ΔcapB/Bp vaccines strongly protect highly sensitive BALB/c mice against lethal respiratory Bp challenge, but protection is overwhelmed at very high challenge doses. In contrast, administered intranasally, rLVS ΔcapB/Bp vaccines remain strongly protective against even very high challenge doses. Under some conditions, the LVS ΔcapB vector itself provides significant protection against Bp challenge, and consistent with this, both the vector and vaccines induce humoral immune responses to Bp antigens. Three-antigen vaccines expressing Hcp6-Hcp1-Hcp2 or Hcp6-Hcp1-LolC are among the most potent and provide long-term protection and protection even with a single intranasal immunization. Protection via the intranasal route was either comparable to or statistically significantly better than the single-deletional Bp mutant Bp82, which served as a positive control. Thus, rLVS ΔcapB/Bp vaccines are exceptionally promising safe and potent melioidosis vaccines. IMPORTANCE: Melioidosis, a major neglected disease caused by the intracellular bacterial pathogen Burkholderia pseudomallei, is endemic in many tropical areas of the world and causes an estimated 165,000 cases and 89,000 deaths in humans annually. Moreover, B. pseudomallei is categorized as a Tier 1 select agent of bioterrorism, largely because inhalation of low doses can cause rapidly fatal pneumonia. No licensed vaccine is available to prevent melioidosis. Here, we describe a safe and potent melioidosis vaccine that protects against lethal respiratory challenge with B. pseudomallei in a highly sensitive small animal model-even a single immunization is highly protective, and the vaccine gives long-term protection. The vaccine utilizes a highly attenuated replicating intracellular bacterium as a vector to express multiple key proteins of B. pseudomallei; this vector platform has previously been used successfully to develop potent vaccines against other Tier 1 select agent diseases including tularemia, anthrax, and plague.

Listeria-vectored multi-antigenic tuberculosis vaccine protects C57BL/6 and BALB/c mice and guinea pigs against Mycobacterium tuberculosis challenge.

Mycobacterium tuberculosis (Mtb) infects one-third of the world's population and is a leading cause of death from a single infectious agent. New TB vaccines are urgently needed to augment immunity conferred by the current modestly protective BCG vaccine. We have developed live attenuated recombinant Listeria monocytogenes (rLm)-vectored TB vaccines expressing five [Mpt64/23.5-EsxH/TB10.4-EsxA/ESAT6-EsxB/CFP10-Ag85B/r30] (rLmMtb5Ag) or nine (additionally EsxN-PPE68-EspA-TB8.4) immunoprotective Mtb antigens (rLmMtb9Ag) and evaluated them for safety, immunogenicity and efficacy as standalone vaccines in two mouse models and an outbred guinea pig model. In immunogenicity studies, rLmMtb5Ag administered subcutaneously induces significantly enhanced antigen-specific CD4+ and CD8+ T-cell responses in C57BL/6 and BALB/c mice, and rLmMtb9Ag induces antigen-specific CD4+ and CD8+ T-cell proliferation in guinea pigs. In efficacy studies, both rLmMtb5Ag and rLmMtb9Ag are safe and protect C57BL/6 and BALB/c mice and guinea pigs against aerosol challenge with highly virulent Mtb. Hence, multi-antigenic rLm vaccines hold promise as new vaccines against TB.

Listeria-Vectored Multiantigenic Tuberculosis Vaccine Enhances Protective Immunity against Aerosol Challenge with Virulent Mycobacterium tuberculosis in BCG-Immunized C57BL/6 and BALB/c Mice.

Mycobacterium tuberculosis infects approximately one-third of the world's population, causing active tuberculosis (TB) in ~10 million people and death in ~1.5 million people annually. A potent vaccine is needed to boost the level of immunity conferred by the current Mycobacterium bovis BCG vaccine that provides moderate protection against childhood TB but variable protection against adult pulmonary TB. Previously, we developed a recombinant attenuated Listeria monocytogenes (rLm)-vectored M. tuberculosis vaccine expressing the M. tuberculosis 30-kDa major secretory protein (r30/Ag85B), recombinant attenuated L. monocytogenes ΔactA ΔinlB prfA*30 (rLm30), and showed that boosting BCG-primed mice and guinea pigs with rLm30 enhances immunoprotection against challenge with aerosolized M. tuberculosis Erdman strain. To broaden the antigen repertoire and robustness of rLm30, we constructed 16 recombinant attenuated L. monocytogenes vaccine candidates expressing 3, 4, or 5 among 15 selected M. tuberculosis antigens, verified their protein expression, genetic stability, and growth kinetics in macrophages, and evaluated them for capacity to boost protective efficacy in BCG-primed mice. We found that boosting BCG-primed C57BL/6 and BALB/c mice with recombinant attenuated L. monocytogenes multiantigenic M. tuberculosis vaccines, especially the rLm5Ag(30) vaccine expressing a fusion protein of 23.5/Mpt64, TB10.4/EsxH, ESAT6/EsxA, CFP10/EsxB, and r30, enhances BCG-induced protective immunity against M. tuberculosis aerosol challenge. In immunogenicity studies, rLm5Ag(30) strongly boosts M. tuberculosis antigen-specific CD4-positive (CD4+) and CD8+ T cell-mediated TH1-type immune responses in the spleens and lungs of BCG-primed C57BL/6 mice but does so only weakly in BCG-primed BALB/c mice. Hence, rLm5Ag(30) boosts BCG-primed immunoprotection against M. tuberculosis aerosol challenge in both C57BL/6 and BALB/c mice despite major differences in the magnitude of the vaccine-induced Th1 response in these mouse strains. Given the consistency with which recombinant attenuated L. monocytogenes vaccines expressing the 5 M. tuberculosis antigens in rLm5Ag(30) are able to boost the already high level of protection conferred by BCG alone in two rigorous mouse models of pulmonary TB and the broad CD4+ and CD8+ T cell immunity induced by rLm5Ag(30), this vaccine holds considerable promise as a new vaccine to combat the TB pandemic, especially for the majority of the world's population immunized with BCG in infancy. IMPORTANCE TB, one of the world's most important infectious diseases, afflicts approximately 10 million people and kills approximately 1.5 million people annually. The current vaccine, BCG, developed over a century ago, has been administered to about 5 billion people, mostly in infancy, but is only modestly protective. Hence, a vaccine is urgently needed to boost the level of protection afforded by BCG. Herein, we describe a safe potent live vaccine that utilizes as a vector an attenuated strain of Listeria monocytogenes, a bacterium that mimics the intracellular lifestyle of Mycobacterium tuberculosis, the causative agent of TB. The vaccine produces multiple immunologically protective proteins of M. tuberculosis. In two mouse models of pulmonary TB, the vaccine boosts the level of protection afforded by BCG. Thus, this vaccine holds considerable promise as a new vaccine to combat the TB pandemic, especially for the majority of the world's population immunized with BCG.

Artificial intelligence enabled parabolic response surface platform identifies ultra-rapid near-universal TB drug treatment regimens comprising approved drugs.

BACKGROUND: Shorter, more effective treatments for tuberculosis (TB) are urgently needed. While many TB drugs are available, identification of the best regimens is challenging because of the large number of possible drug-dose combinations. We have found consistently that responses of cells or whole animals to drug-dose stimulations fit a parabolic response surface (PRS), allowing us to identify and optimize the best drug combinations by testing only a small fraction of the total search space. Previously, we used PRS methodology to identify three regimens (PRS Regimens I-III) that in murine models are much more effective than the standard regimen used to treat TB. However, PRS Regimens I and II are unsuitable for treating drug-resistant TB and PRS Regimen III includes an experimental drug. Here, we use PRS methodology to identify from an expanded pool of drugs new highly effective near-universal drug regimens comprising only approved drugs. METHODS AND FINDINGS: We evaluated combinations of 15 different drugs in a human macrophage TB model and identified the most promising 4-drug combinations. We then tested 14 of these combinations in Mycobacterium tuberculosis-infected BALB/c mice and chose for PRS dose optimization and further study the two most potent regimens, designated PRS Regimens IV and V, consisting of clofazimine (CFZ), bedaquiline (BDQ), pyrazinamide (PZA), and either amoxicillin/clavulanate (AC) or delamanid (DLM), respectively. We then evaluated the efficacy in mice of optimized PRS Regimens IV and V, as well as a 3-drug regimen, PRS Regimen VI (CFZ, BDQ, and PZA), and compared their efficacy to PRS Regimen III (CFZ, BDQ, PZA, and SQ109), previously shown to reduce the time to achieve relapse-free cure in mice by 80% compared with the Standard Regimen (isoniazid, rifampicin, PZA, and ethambutol). Efficacy measurements included early bactericidal activity, time to lung sterilization, and time to relapse-free cure. PRS Regimens III-VI all rapidly sterilized the lungs and achieved relapse-free cure in 3 weeks (PRS Regimens III, V, and VI) or 5 weeks (PRS Regimen IV). In contrast, mice treated with the Standard Regimen still had high numbers of bacteria in their lungs after 6-weeks treatment and none achieved relapse-free cure in this time-period. CONCLUSIONS: We have identified three new regimens that rapidly sterilize the lungs of mice and dramatically shorten the time required to achieve relapse-free cure. All mouse drug doses in these regimens extrapolate to doses that are readily achievable in humans. Because PRS Regimens IV and V contain only one first line drug (PZA) and exclude fluoroquinolones and aminoglycosides, they should be effective against most TB cases that are multidrug resistant (MDR-TB) and many that are extensively drug-resistant (XDR-TB). Hence, these regimens have potential to shorten dramatically the time required for treatment of both drug-sensitive and drug-resistant TB. If clinical trials confirm that these regimens dramatically shorten the time required to achieve relapse-free cure in humans, then this radically shortened treatment has the potential to improve treatment compliance, decrease the emergence of drug resistance, and decrease the healthcare burden of treating both drug-sensitive and drug-resistant TB.

PPE37 Is Essential for Mycobacterium tuberculosis Heme-Iron Acquisition (HIA), and a Defective PPE37 in Mycobacterium bovis BCG Prevents HIA.

Mycobacterium tuberculosis, one of the world's leading causes of death, must acquire nutrients, such as iron, from the host to multiply and cause disease. Iron is an essential metal and M. tuberculosis possesses two different systems to acquire iron from its environment: siderophore-mediated iron acquisition (SMIA) and heme-iron acquisition (HIA), involving uptake and degradation of heme to release ferrous iron. We have discovered that Mycobacterium bovis BCG, the tuberculosis vaccine strain, is severely deficient in HIA, and we exploited this phenotypic difference between BCG and M. tuberculosis to identify genes involved in HIA by complementing BCG's defect with a fosmid library. We identified ppe37, an iron-regulated PPE family gene, as being essential for HIA. BCG complemented with M. tuberculosisppe37 exhibits HIA as efficient as that of M. tuberculosis, achieving robust growth with <0.2 µM hemin. Conversely, deletion of ppe37 from M. tuberculosis results in a strain severely attenuated in HIA, with a phenotype nearly identical to that of BCG, requiring a 200-fold higher concentration of hemin to achieve growth equivalent to that of its parental strain. A nine-amino-acid deletion near the N terminus of BCG PPE37 (amino acids 31 to 39 of the M. tuberculosis PPE37 protein) underlies BCG's profound defect in HIA. Significant genetic variability exists in ppe37 genes across different M. tuberculosis strains, with more than 60% of sequences from completely sequenced M. tuberculosis genomes having mutations that result in altered PPE37 proteins; furthermore, these altered PPE37 proteins are nonfunctional in HIA. Our findings should allow delineation of the relative roles of HIA and SMIA in M. tuberculosis pathogenesis.

Ultra-rapid near universal TB drug regimen identified via parabolic response surface platform cures mice of both conventional and high susceptibility.

As current treatment of tuberculosis is burdensomely long, provoking non-adherence and drug resistance, effective short-course treatments are needed. Using the output-driven parabolic response surface (PRS) platform, we have identified drug regimens that treat tuberculosis more rapidly in mice than the current Standard Regimen used in humans. We show that PRS Regimen III, comprising clofazimine, SQ109, bedaquiline and pyrazinamide, rapidly sterilizes the lung both in conventionally studied BALB/c mice and in C3HeB/FeJ mice, highly susceptible mice that develop massive necrotic granulomatous lung lesions akin to those in humans, achieving relapse-free cure in only 4 weeks (p<0.0001 versus Standard Regimen). In contrast, the Standard Regimen required 16 weeks to attain lung culture negative status and 20 weeks to achieve relapse-free cure. Thus, PRS Regimen III dramatically cuts by ~80% the time to relapse-free cure in mouse tuberculosis models. PRS Regimen III, with three nonstandard drugs, can potentially treat both drug-sensitive and most drug-resistant tuberculosis.

Drug regimens identified and optimized by output-driven platform markedly reduce tuberculosis treatment time.

The current drug regimens for treating tuberculosis are lengthy and onerous, and hence complicated by poor adherence leading to drug resistance and disease relapse. Previously, using an output-driven optimization platform and an in vitro macrophage model of Mycobacterium tuberculosis infection, we identified several experimental drug regimens among billions of possible drug-dose combinations that outperform the current standard regimen. Here we use this platform to optimize the in vivo drug doses of two of these regimens in a mouse model of pulmonary tuberculosis. The experimental regimens kill M. tuberculosis much more rapidly than the standard regimen and reduce treatment time to relapse-free cure by 75%. Thus, these regimens have the potential to provide a markedly shorter course of treatment for tuberculosis in humans. As these regimens omit isoniazid, rifampicin, fluoroquinolones and injectable aminoglycosides, they would be suitable for treating many cases of multidrug and extensively drug-resistant tuberculosis.