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Scholarly Works (21 results)

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UCLA Previously Published Works (2006)

Mycobacterium bovis infection of cattle and other domesticated animals exacts a significant economic toll in both economically developing and industrialized countries. Vaccination of herds and/or wild animals that share their grazing land and serve as reservoirs of infection has been proposed as a strategy to combat bovine tuberculosis. However, the only currently available vaccine, M. bovis Bacille Calmette-Guerin (BCG), is not highly efficacious. Here we show that a live recombinant vaccine, rBCG30, which expresses large amounts of the Mycobacterium tuberculosis 30kDa major secretory protein, is more efficacious against bovine tuberculosis than BCG in the highly demanding guinea pig model of pulmonary tuberculosis. Compared with the parental wild-type BCG strain, rBCG30 administered intradermally induced significantly greater cell-mediated and humoral immune responses against the 30kDa protein, as determined by measuring cutaneous delayed-type hypersensitivity and antibody titers. As for potency, in three independent experiments, rBCG30 induced greater protective immunity than BCG against aerosol challenge with a highly virulent strain of M. bovis, reducing the burden of M. bovis by 0.4 +/- 0.2 log colony-forming units (CFU) in the lung (P < 0.05) and by 1.1 +/- 0.4 log CFU in the spleen (P = 0.0005) below the level in BCG-immunized animals. A recombinant BCG vaccine overexpressing the identical M. bovis 30kDa protein, rBCG30Mb, also induced greater cell-mediated and humoral immunity against the 30 kDa protein than BCG and greater protective immunity against M. bovis challenge; however, its potency was not significantly different from rBCG30. As rBCG30 is significantly more potent than BCG against M. bovis challenge, it has potential as a vaccine against bovine tuberculosis in domesticated animals and in wild animal reservoirs. (c) 2005 Elsevier Ltd. All rights reserved.

    Cover page: A novel live recombinant mycobacterial vaccine against bovine tuberculosis more potent than BCG
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    UCLA Previously Published Works (2005)

    Tuberculosis continues to ravage humanity, killing 2 million people yearly. Most cases occur in areas of the world to which the disease is endemic, where almost everyone is vaccinated early in life with Mycobacterium bovis BCG, the currently available vaccine against tuberculosis. Thus, while more-potent vaccines are needed to replace BCG, new vaccines are also needed to boost the immune protection of the 4 billion people already vaccinated with BCG. Until now, no booster vaccine has been shown capable of significantly enhancing the level of protective immunity induced by BCG in the stringent guinea pig model of pulmonary tuberculosis, the "gold standard" for testing tuberculosis vaccines. In this paper, we describe a booster vaccine for BCG comprising the purified recombinant Mycobacterium tuberculosis 30-kDa protein, the major secreted protein of this pathogen. In the guinea pig model of pulmonary tuberculosis, boosting BCG-immunized animals once with the 30-kDa protein greatly increased cell-mediated and humoral immune responses to the protein in three consecutive experiments. Most importantly, boosting BCG-immunized animals once with the 30-kDa protein significantly enhanced protective immunity against aerosol challenge with highly virulent M. tuberculosis, as evidenced by a significantly reduced lung and spleen burden of M. tuberculosis compared with those for nonboosted BCG-immunized animals (mean additional reduction in CFU of 0.4 +/- 0.1 log in the lung [P = 0.03] and 0.6 +/- 0.1 log in the spleen [P = 0.002]). This study suggests that administering BCG-immunized people a booster vaccine comprising the 30-kDa protein may enhance their level of immunoprotection against tuberculosis.

      Cover page: Enhancing the protective efficacy of Mycobacterium bovis BCG vaccination against tuberculosis by boosting with the Mycobacterium tuberculosis major secretory protein
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      UCLA Previously Published Works (2006)

      In previous studies, we have described a live recombinant BCG vaccine (rBCG30) overexpressing the 30 kDa major secretory protein of Mycobacterium tuberculosis that induces greater protective immunity against tuberculosis than the current vaccine in the demanding guinea pig model of pulmonary tuberculosis. In this study, we have investigated the impact of vaccine dose on the development of cell-mediated and protective immunity in the guinea pig model. We found that the protective efficacy against M. tuberculosis aerosol challenge of both BCG and rBCG30 was essentially dose-independent over a dose range of 10(1)-10(6) live organisms. As previously observed, rBCG30 was more potent, reducing colony-forming units (CFU) below the level observed in animals immunized with the parental BCG vaccine by 0.7 logs in the lungs and 1.0 logs in the spleen (P < 0.0001). To gain abetter understanding of the influence of dose on bacterial clearance and immunity, we assessed animals immunized with 10(1), 10(3), or 10(6) CFU of rBCG30. The higher the dose, the higher the peak CFU level achieved in animal organs. However, whereas humoral immune responses to the 30kDa protein reflected the disparate CFU levels, cell-mediated immune responses did not; high and low doses of rBCG30 ultimately induced comparable peak lymphocyte proliferative responses and cutaneous delayed-type hypersensitivity responses to the 30kDa protein. We estimate that the amount of the 30 kDa protein required to induce a strong cell-mediated immune response when delivered via 10 rBCG30 organisms is about 9 orders of magnitude less than that required when the protein is delivered in a conventional protein/adjuvant vaccine. This study demonstrates that a very low inoculum of rBCG30 organisms has the capacity to induce strong protective immunity against tuberculosis and that rBCG30 is an extremely potent delivery system for mycobacterial antigens. (c) 2005 Elsevier Ltd. All rights reserved.

        Cover page: Extraordinarily few organisms of a live recombinant BCG vaccine against tuberculosis induce maximal cell-mediated and protective immunity
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        UCLA Previously Published Works (2000)
        Tuberculosis (TB) continues to ravage humanity, causing 2 million deaths per year. A vaccine against TB more potent than the current live vaccine, bacillus Calmette-Guérin (BCG), is desperately needed. Using two commercially available strains of BCG as host strains, BCG Connaught and Tice, we have constructed two recombinant BCG vaccines stably expressing and secreting the 30-kDa major secretory protein of Mycobacterium tuberculosis (M. tb.), the primary causative agent of TB. We have tested the efficacy of the two strains in the highly susceptible guinea pig model of pulmonary TB, a model noteworthy for its close resemblance to human TB. Animals immunized with the recombinant BCG vaccines and challenged by aerosol with a highly virulent strain of M. tb. had 0.5 logs fewer M. tb. bacilli in their lungs and 1 log fewer bacilli in their spleens on average than animals immunized with their parental conventional BCG vaccine counterparts. Statistically, these differences were highly significant. Paralleling these results, at necropsy, animals immunized with the recombinant BCG vaccines had fewer and smaller lesions in the lung, spleen, and liver and significantly less lung pathology than animals immunized with the parental BCG vaccines. The recombinant vaccines are the first vaccines against TB more potent than the current commercially available BCG vaccines, which were developed nearly a century ago.
          Creative Commons 'BY' version 4.0 license
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          UCLA Previously Published Works (2009)
          BCG has been administered to over 4 billion persons worldwide, but its efficacy in preventing tuberculosis in adults has been highly variable. One hypothesis for its variability is that different strains of BCG vary in protective efficacy, and moreover, that evolutionarily early strains are more efficacious than the more attenuated evolutionarily late strains, which lack region of deletion 2. To examine this hypothesis, we tested six widely used BCG strains--the evolutionarily early strain BCG Japanese, two evolutionarily late strains in DU2 Group III (BCG Danish and Glaxo), and three evolutionarily late strains in DU2 Group IV (BCG Connaught, Pasteur, and Tice)--in the guinea pig model of pulmonary tuberculosis. With the exception of BCG Glaxo, which had relatively poor efficacy, we found no substantial differences in efficacy between the early strain and the late strains, and only small differences in efficacy among late strains. BCG Tice was the most efficacious BCG vaccine, with significantly fewer Mycobacterium tuberculosis in the lung and spleen than BCG Danish and BCG Japanese, although absolute differences in the organ burden of M. tuberculosis among these three vaccines were small (< or =0.2 log). BCG Tice and Pasteur were not significantly different. rBCG30, a recombinant BCG Tice vaccine overexpressing the M. tuberculosis 30 kDa major secretory protein (Antigen 85B), was more potent than any BCG vaccine (P < 0.0001 for differences in organ burden). Our study shows that late strains are not less potent than an early strain and argues against strain differences as a major factor in the variability of outcomes in BCG vaccine trials.
            Creative Commons 'BY' version 4.0 license
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            UCLA Previously Published Works (2017)
            A potent vaccine against tuberculosis, one of the world's deadliest diseases, is needed to enhance the immunity of people worldwide, most of whom have been vaccinated with the partially effective Mycobacterium bovis BCG vaccine. Here we investigate novel live attenuated recombinant Listeria monocytogenes (rLm) vaccines expressing the Mycobacterium tuberculosis 30-kDa major secretory protein (r30/antigen 85B [Ag85B]) (rLm30) as heterologous booster vaccines in animals primed with BCG. Using three attenuated L. monocytogenes vectors, L. monocytogenes ΔactA (LmI), L. monocytogenes ΔactA ΔinlB (LmII), and L. monocytogenes ΔactA ΔinlB prfA* (LmIII), we constructed five rLm30 vaccine candidates expressing r30 linked in frame to the L. monocytogenes listeriolysin O signal sequence and driven by the hly promoter (h30) or linked in frame to the ActA N-terminal 100 amino acids and driven by the actA promoter (a30). All five rLm30 vaccines secreted r30 in broth and macrophages; while rLm30 expressing r30 via a constitutively active prfA* regulon (rLmIII/a30) expressed the largest amount of r30 in broth culture, all five rLm30 vaccines expressed equivalent amounts of r30 in infected macrophages. In comparative studies, boosting of BCG-immunized mice with rLmIII/a30 induced the strongest antigen-specific T-cell responses, including splenic and lung polyfunctional CD4+ T cells expressing the three cytokines interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-2 (IL-2) (P < 0.001) and splenic and lung CD8+ T cells expressing IFN-γ (P < 0.0001). In mice and guinea pigs, the rLmIII/a30 and rLmI/h30 vaccines were generally more potent booster vaccines than r30 with an adjuvant and a recombinant adenovirus vaccine expressing r30. In a setting in which BCG alone was highly immunoprotective, boosting of mice with rLmIII/a30, the most potent of the vaccines, significantly enhanced protection against aerosolized M. tuberculosis (P < 0.01).
              Creative Commons 'BY' version 4.0 license
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              UCLA Previously Published Works (2015)
              Tuberculosis is a major global health problem for which improved therapeutics are needed to shorten the course of treatment and combat emergence of drug resistance. Mycobacterium tuberculosis, the etiologic agent of tuberculosis, is an intracellular pathogen of mononuclear phagocytes. As such, it is an ideal pathogen for nanotherapeutics because macrophages avidly ingest nanoparticles even without specific targeting molecules. Hence, a nanoparticle drug delivery system has the potential to target and deliver high concentrations of drug directly into M. tuberculosis-infected cells-greatly enhancing efficacy while avoiding off-target toxicities. Stimulus-responsive mesoporous silica nanoparticles of two different sizes, 100 and 50 nm, are developed as carriers for the major anti-tuberculosis drug isoniazid in a prodrug configuration. The drug is captured by the aldehyde-functionalized nanoparticle via hydrazone bond formation and coated with poly(ethylene imine)-poly(ethylene glycol) (PEI-PEG). The drug is released from the nanoparticles in response to acidic pH at levels that naturally occur within acidified endolysosomes. It is demonstrated that isoniazid-loaded PEI-PEG-coated nanoparticles are avidly ingested by M. tuberculosis-infected human macrophages and kill the intracellular bacteria in a dose-dependent manner. It is further demonstrated in a mouse model of pulmonary tuberculosis that the nanoparticles are well tolerated and much more efficacious than an equivalent amount of free drug.
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                UCLA Previously Published Works (2010)
                Francisella tularensis, the causative agent of tularemia, is in the top category (category A) of potential agents of bioterrorism. The F. tularensis live vaccine strain (LVS) is the only vaccine currently available to protect against tularemia; however, this unlicensed vaccine is relatively toxic and provides incomplete protection against aerosolized F. tularensis, the most dangerous mode of transmission. Hence, a safer and more potent vaccine is needed. As a first step toward addressing this need, we have constructed and characterized an attenuated version of LVS, LVS ΔcapB, both as a safer vaccine and as a vector for the expression of recombinant F. tularensis proteins. LVS ΔcapB, with a targeted deletion in a putative capsule synthesis gene (capB), is antibiotic resistance marker free. LVS ΔcapB retains the immunoprotective O antigen, is serum resistant, and is outgrown by parental LVS in human macrophage-like THP-1 cells in a competition assay. LVS ΔcapB is significantly attenuated in mice; the 50% lethal dose (LD(50)) intranasally (i.n.) is >10,000-fold that of LVS. Providing CapB in trans to LVS ΔcapB partially restores its virulence in mice. Mice immunized with LVS ΔcapB i.n. or intradermally (i.d.) developed humoral and cellular immune responses comparable to those of mice immunized with LVS, and when challenged 4 or 8 weeks later with a lethal dose of LVS i.n., they were 100% protected from illness and death and had significantly lower levels (3 to 5 logs) of LVS in the lung, liver, and spleen than sham-immunized mice. Most importantly, mice immunized with LVS ΔcapB i.n. or i.d. and then challenged 6 weeks later by aerosol with 10× the LD(50) of the highly virulent type A F. tularensis strain SchuS4 were significantly protected (100% survival after i.n. immunization). These results show that LVS ΔcapB is significantly safer than LVS and yet provides potent protective immunity against virulent F. tularensis SchuS4 challenge.
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                  UCLA Previously Published Works (2016)
                  A safer and more effective vaccine than the unlicensed Francisella tularensis Live Vaccine Strain (LVS) is needed to protect against the biowarfare agent F. tularensis. Previously, we developed an LVS ΔcapB mutant that is significantly safer than LVS and provides potent protective immunity against F. tularensis respiratory challenge when administered intranasally but limited protection when administered intradermally unless as part of a prime-boost vaccination strategy. To improve the immunogenicity and efficacy of LVS ΔcapB, we developed recombinant LVS ΔcapB (rLVS ΔcapB) strains overexpressing various F. tularensis Francisella Pathogenicity Island (FPI) proteins - IglA, IglB and IglC, and a fusion protein (IglABC) comprising immunodominant epitopes of IglA, IglB, and IglC downstream of different Francisella promoters, including the bacterioferritin (bfr) promoter. We show that rLVS ΔcapB/bfr-iglA, iglB, iglC, and iglABC express more IglA, IglB, IglC or IglABC than parental LVS ΔcapB in broth and in human macrophages, and stably express FPI proteins in macrophages and mice absent antibiotic selection. In response to IglC and heat-inactivated LVS, spleen cells from mice immunized intradermally with rLVS ΔcapB/bfr-iglC or bfr-iglABC secrete greater amounts of interferon-gamma and/or interleukin-17 than those from mice immunized with LVS ΔcapB, comparable to those from LVS-immunized mice. Mice immunized with rLVS ΔcapB/bfr-iglA, iglB, iglC or iglABC produce serum antibodies at levels similar to LVS-immunized mice. Mice immunized intradermally with rLVS ΔcapB/bfr-iglABC and challenged intranasally with virulent F. tularensis Schu S4 survive longer than sham- and LVS ΔcapB-immunized mice. Mice immunized intranasally with rLVS ΔcapB/bfr-iglABC - but not with LVS - just before or after respiratory challenge with F. tularensis Schu S4 are partially protected; protection is correlated with induction of a strong innate immune response. Thus, rLVS ΔcapB/bfr-iglABC shows improved immunogenicity and protective efficacy compared with parental LVS ΔcapB and, in contrast to LVS, has partial efficacy as immediate pre- and post-exposure prophylaxis.
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                    UCLA Previously Published Works (2015)
                    We have optimized mesoporous silica nanoparticles (MSNs) functionalized with pH-sensitive nanovalves for the delivery of the broad spectrum fluoroquinolone moxifloxacin (MXF) and demonstrated its efficacy in treating Francisella tularensis infections both in vitro and in vivo. We compared two different nanovalve systems, positive and negative charge modifications of the mesopores, and different loading conditions-varying pH, cargo concentration, and duration of loading-and identified conditions that maximize both the uptake and release capacity of MXF by MSNs. We have demonstrated in macrophage cell culture that the MSN-MXF delivery platform is highly effective in killing F. tularensis in infected macrophages, and in a mouse model of lethal pneumonic tularemia, we have shown that the drug-loaded MSNs are much more effective in killing F. tularensis than an equivalent amount of free MXF.
                      Cover page: Mesoporous Silica Nanoparticles with pH-Sensitive Nanovalves for Delivery of Moxifloxacin Provide Improved Treatment of Lethal Pneumonic TularemiaCreative Commons 'BY' version 4.0 license
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