Safety and Shedding of an Attenuated Strain ofListeria monocytogeneswith a Deletion ofactA/plcBin Adult Volunteers: a Dose Escalation Study of Oral Inoculation

Abstract

Listeria monocytogenes is an intracellular bacterial pathogen which causes bacteremia and has a tropism for the central nervous system and a propensity to cause maternofetal infection. L. monocytogenes has been shown to be an effective prophylactic and a therapeutic vaccine vector for viral and tumor antigens in animal models. L. monocytogenes mutants lacking the ActA protein, which is essential for intracellular movement, are attenuated but retain immunogenicity in mice. Given the pathogenic potential of L. monocytogenes, we created an attenuated mutant strain bearing double deletions in the actA and plcB virulence genes for an initial clinical safety study of a prototype L. monocytogenes vector in adults. Twenty healthy volunteers received single escalating oral doses (10 6 to 10 9 CFU, 4 volunteers per dose cohort) of this attenuated L. monocytogenes, designated LH1169. Volunteers were monitored in the hospital for 14 days with frequent clinical checks and daily blood and stool cultures, and they were monitored for six additional weeks as outpatients. There were no positive blood cultures and no fevers attributable to the investigational inoculation. Most volunteers shed vaccine bacteria for 4 days or less, without diarrhea. One volunteer had a late positive stool culture during outpatient follow-up. Three volunteers had abnormal liver function test results temporally associated with inoculation; one could be reasonably attributed to another cause. In the highest-dose cohort, humoral, mucosal, and cellular immune responses to the investigational organism were detected in individual volunteers. Attenuated L. monocytogenes can be studied in adult volunteers without serious long-term health sequelae.Listeria monocytogenes is a gram-positive bacterium which has long been studied in mice to elucidate mechanisms of cellular immune responses to intracellular pathogens (29, 35). Schafer and colleagues initially proposed that escape of L. monocytogenes from the phagocytic vacuole into the eukaryotic cytoplasm might make these organisms efficient vectors for delivery of antigens to the major histocompatibility complex class I-restricted antigen processing pathways (48). L. monocytogenes vectors have been used to deliver lymphocytic choriomeningitis virus (LCMV) nucleoprotein antigens to mice, with subsequent protection against fatal challenge with LCMV (21, 52, 53). Recombinant L. monocytogenes expressing cottontail rabbit papillomavirus E1 antigen has been successfully used as a therapeutic immunogen in animals bearing papillomavirus-induced cutaneous tumors (28). L. monocytogenes is being pursued as a vector for antigens derived from human papillomaviruses (23) and human immunodeficiency virus type 1 (HIV-1) (40,41). A murine study showed that oral inoculation of L. monocytogenes expressing HIV-1-gag induced mucosal and systemic immunity to this viral antigen (45). After careful review of available data on oral inoculation of wild-type L. monocytogenes in primates (17) and farm animals, (34,42,43) clinical data avail...

Live attenuated Listeria monocytogenes expressing HIV Gag: Immunogenicity in rhesus monkeys

“…Second, foreign antigens encoded by Lm are efficiently presented by both MHC class I and MHC class II molecules after being processed to peptides [23,24]; as Lm vectors deliver antigens directly to the DC cytosol, both CD8 + and CD4 + antigen-specific T cells can thus be activated [22]. Third, because the natural route of Lm infection involves oral exposure, Listeria-derived vaccine vectors may be given orally [25][26][27][28]. Fourth, evidence with a Listeria vector containing two genes of feline immunodeficiency virus (FIV) showed that pre-existing immunity against Lm does not preclude the generation of immunity to foreign antigens expressed by the Listeria vector [28].…”

“…As Lm can cause serious infections in neonates, pregnant women and immunocompromised hosts, different attenuation strategies have been considered: a) to delete the Lm actA gene (either alone or with the plcB or inlB genes) so that both intracellular movement and cell-tocell spreading of bacteria is stopped but immunogenicity is maintained [27,29,30]; b) to delete the uvrA and uvrB genes, which encode nucleotide excision repair genes [31]; and c) to delete essential genes in the D-alanine (D-ala) synthesis pathway. We have chosen the latter approach to develop a highly attenuated vaccine vector, called Lmdd.…”

Directed antigen delivery as a vaccine strategy for an intracellular bacterial pathogen

“…However, it remains questionable whether such strains are suitably attenuated for general use as a vaccine, especially in immunocompromised individuals. One attenuated Lm strain was found to be 1,000-fold less virulent than the WT parent Lm in mice, yet replication within primary infected host cells was identical to the parental strain, raising questions concerning the potential infectious risk to patients after administration (25).…”

“…The intracytosolic lifestyle of Lm has prompted the development of several attenuated strains as vaccine vectors (22)(23)(24)(25)(26). However, it remains questionable whether such strains are suitably attenuated for general use as a vaccine, especially in immunocompromised individuals.…”

“…Recently, vaccination with a metabolically active, yet replication-defective Lm strain (because of DNA crosslinking), has been shown to be similarly capable of priming CD8 ϩ T cells (34). Yet, cytoLLO Lm is the most attenuated Lm vaccine strain generated to date that can be administered as a viable organism, does not require any additional modifications to the bacteria or host, effectively stimulates protective antilisterial immunity, and is suitable for clinical safety testing in humans (25).…”