The facultative intracellular bacterium Listeria monocytogenes is being developed as a cancer vaccine platform because of its ability to induce potent innate and adaptive immunity. For successful clinical application, it is essential to develop a Listeria platform strain that is safe yet retains the potency of vaccines based on wild-type bacteria. Here, we report the development of a recombinant live-attenuated vaccine platform strain that retains the potency of the fully virulent pathogen, combined with a >1,000-fold reduction in toxicity, as compared with wild-type Listeria. By selectively deleting two virulence factors, ActA (⌬actA) and Internalin B (⌬inlB), the immunopotency of Listeria was maintained and its toxicity was diminished in vivo, largely by blocking the direct internalin B-mediated infection of nonphagocytic cells, such as hepatocytes, and the indirect ActA-mediated infection by cellto-cell spread from adjacent phagocytic cells. In contrast, infection of phagocytic cells was not affected, leaving intact the ability of Listeria to stimulate innate immunity and to induce antigenspecific cellular responses. Listeria ⌬actA͞⌬inlB-based vaccines were rapidly cleared from mice after immunization and induced potent and durable effector and memory T-cell responses with no measurable liver toxicity. Therapeutic vaccination of BALB͞c mice bearing murine CT26 colon tumor lung metastases or palpable s.c. tumors (>100 mm 3 ) with recombinant Listeria ⌬actA͞⌬inlB expressing an endogenous tumor antigen resulted in breaking of self-tolerance and long-term survival. We propose that recombinant Listeria ⌬actA͞⌬inlB expressing human tumor-associated antigens represents an attractive therapeutic strategy for further development and testing in human clinical trials.C ancer immunotherapy represents a promising treatment strategy that has produced some tantalizing clinical responses for a variety of malignant diseases. Although promising, there continues to be a strong need for a practical immunization strategy that can be routinely adopted to specific malignancies and that consistently yields durable and robust therapeutic antitumor responses.Progress in molecular and cellular immunology, combined with increasing understanding of pathogen physiology and hostpathogen interaction has facilitated the design and use of attenuated bacteria as conventional vaccine vectors. However, the practical utility of live attenuated vaccines relies on achieving a proper balance between the virulence͞toxicity and immunogenicity of the vaccine. The potency of a pathogen to elicit adaptive immunity is related in part to its ability to stimulate significant innate immunity through recognition of microbial pathogen-associated molecular patterns by Toll-like receptors. Microbial encounter with professional antigen-presenting cells (APC), such as dendritic cells, results in activation and maturation (1) as well as secretion of high levels of T helper-1-type cytokines (2). This interaction initiates adaptive immune responses and therefore link...

We developed a new class of vaccines, based on killed but metabolically active (KBMA) bacteria, that simultaneously takes advantage of the potency of live vaccines and the safety of killed vaccines. We removed genes required for nucleotide excision repair (uvrAB), rendering microbial-based vaccines exquisitely sensitive to photochemical inactivation with psoralen and long-wavelength ultraviolet light. Colony formation of the nucleotide excision repair mutants was blocked by infrequent, randomly distributed psoralen crosslinks, but the bacterial population was able to express its genes, synthesize and secrete proteins. Using the intracellular pathogen Listeria monocytogenes as a model platform, recombinant psoralen-inactivated Lm DeltauvrAB vaccines induced potent CD4(+) and CD8(+) T-cell responses and protected mice against virus challenge in an infectious disease model and provided therapeutic benefit in a mouse cancer model. Microbial KBMA vaccines used either as a recombinant vaccine platform or as a modified form of the pathogen itself may have broad use for the treatment of infectious disease and cancer.

Citrulline-malate (CM) has been proposed to provide an ergogenic effect during resistance exercise; however, there is a paucity of research investigating these claims. Therefore, we investigated the impact that CM supplementation would have on repeated bouts of resistance exercise. Fourteen resistance-trained males participated in a randomized, counterbalanced, double-blind study. Subjects were randomly assigned to placebo (PL) or CM (8 g) and performed three sets each of chin-ups, reverse chin-ups, and push-ups to failure. One week later, subjects ingested the other supplement and performed the same protocol. Blood lactate (BLa), heart rate (HR), and blood pressure (BP) were measured preexercise, with BLa measured a second time immediately following the last set, while HR and BP were measured 5 and 10 min postexercise. Citrulline-malate ingestion significantly increased the amount of repetitions performed for each exercise (chin-ups: PL = 28.4 ± 7.1, CM = 32.2 ± 5.6, p = .003; reverse chin-ups: PL = 26.6 ± 5.6, CM = 32.1 ± 7.1, p = .017; push-ups: PL = 89.1 ± 37.4, CM = 97.7 ± 36.1, p < .001). Blood lactate data indicated a time effect (p < .001), but no treatment differences (p = .935). Systolic BP data did not show differences for time (p = .078) or treatment (p = .119). Diastolic BP data did not show differences for time (p = .069), but indicated treatment differences (p = .014) for subjects ingesting CM. Collectively, these findings suggests that CM increased upper-body resistance performance in trained college-age males.

Recognition of the advantages of recombinant Listeria monocytogenes-based vaccines compared to those of other recombinant-vaccine platforms has facilitated the ongoing development and current evaluation of the former in early-phase clinical trials. These advantages include practical considerations, such as straightforward fermentation methods for manufacturing, and other desirable features, such as the ability to repeat administer even in the presence of protective L. monocytogenes-specific immunity (6,40,41). One compelling rationale for this vaccine platform is based on the well-known correlates of protection in the mouse listeriosis model: longlived functional CD4 ϩ and CD8 ϩ memory T cells induced in response to a single immunization with L. monocytogenes (19,28). There are now numerous publications that demonstrate the striking efficacy of recombinant L. monocytogenes vaccines in several animal models due to robust innate and adaptive cellular immunity (9,10,29). Recombinant L. monocytogenesbased vaccines represent an emerging approach to addressing an acute global need for effective vaccines that elicit functional cellular immunity to prevent or treat infections such as human immunodeficiency virus infection, hepatitis C virus infection, tuberculosis, and malaria as well as cancer.As L. monocytogenes is a food-borne pathogen having increased virulence among immunocompromised individuals, attenuated vaccine platforms are a prerequisite for advancement to evaluation with humans (23). We have previously described both live-attenuated and photochemically inactivated vaccine platforms derived from the wild-type (WT) strain 10403S (8, 9). The live-attenuated vaccine strain is deleted of both the actA and the inlB virulence genes (L. monocytogenes ⌬actA ⌬inlB vaccine strain), which in combination limit growth in the liver, a principal target organ of infection by the WT organism. Liver toxicity in mice, as measured by serum liver function tests for alanine transaminase and aspartate transaminase, is dramatically lower in mice injected intravenously (i.v.) with the L. monocytogenes ⌬actA ⌬inlB strain than in those injected i.v. with WT L. monocytogenes. Furthermore, liver toxicity was minimal and not dose limiting in two toxicology studies performed under good laboratory practice guidelines with cynomolgus monkeys given escalating doses of L. monocytogenes ⌬actA ⌬inlB-based strains (unpublished data). The L. monocytogenes ⌬actA ⌬inlB vaccine strain forms the basis for two ongoing FDA-approved phase 1 clinical trials being conducted with adult subjects with advanced cancers. The second vaccine platform, termed "killed but metabolically active" (KBMA), is derived from the L. monocytogenes ⌬actA ⌬inlB vaccine strain and is deleted of both uvrA and uvrB, genes encoding the DNA