Ronald A. Barry

Listeria monocytogenes as a vaccine vector: virulence attenuation or existing antivector immunity does not diminish therapeutic efficacy.

The bacterium L. monocytogenes is a proposed vaccine carrier based upon the observation that this pathogen replicates within the intracytoplasmic environment facilitating delivery of Ag to the endogenous Ag processing and presentation pathway with subsequent stimulation of peptide specific MHC class I-restricted CD8+ effector cells. In this report, we evaluate virulence-attenuated strains of Listeria monocytogenes as vaccine vectors and examine whether existing antivector (antilisterial) immunity limits or alters its efficacy as a therapeutic cancer vaccine. Following immunization with virulence-attenuated mutants, we found that the effectiveness of L. monocytogenes as a recombinant cancer vaccine remains intact. In addition, we found that antibiotic treatment initiated 24 or 36 h following therapeutic immunization with recombinant L. monocytogenes allows full development of the antitumor response. We also demonstrate that the vaccine vector potential of L. monocytogenes is not limited in animals with existing antilisterial immunity. For these latter studies, mice previously immunized with wild-type L. monocytogenes were infused with melanoma cells and then 5 days later challenged with recombinant tumor Ag expressing L. monocytogenes. Collectively, these results add additional support for the use of L. monocytogenes as a vaccine vector and underscore its potential to be used repeatedly for stimulation of recall responses concomitant with primary cell-mediated responses to newly delivered heterologous tumor-associated epitopes.

Acquired immunity to an intracellular pathogen:immunologic recognition of L. monocytogenes‐infected cells

Summary: Listeria monocytogenes (L. monocytogenes) is a pathogenic bacterium, and subclinical infection in mice is utilized as a prototypic model to investigate the development and expression of acquired resistance to facultative intracellular organisms. A key virulence factor of L. monocytogenes is the hemolysin listeriolysin O (LLO), and BALB/c mice immunized with hemolysin‐secreting strains of L. monocytogenes develop specific acquired resistance, while mice immunized with hemolysin‐negative strains or non‐viable preparations of L. monocytogenes do not develop a protective immune response. Adoptive transfer studies show that L. monocytogenes‐Immune CD8+ T cells mediate acquired resistance. The L. monocytogenes‐immune CD8+ population is cytotoxic, and target cells infected with hemolysin‐secreting strains of L. monocytogenes are lysed, while target cells infected with hemolysin‐negative strains or non‐viable preparations of L. monocytogenes are not lysed. MHC dass la and Ib molecules present I. monocytogenes‐derived peptides, and we have identified Qa‐1b, a T‐region‐encoded MHC class Ib molecule, as a restriction element for L, monocytogenes‐specific CD8+ CTL. MHC class Ib‐restricted CTL are stimulated following infection with L. monocytogenes and are a significant component of the total MHC class I‐restricted CTL population. These findings support the observation that cytoplasmic L. monocytogenes‐derived antigens are endogenously processed and presented in association with MHC class Ia and Ib molecules to CD8+ effector cells, and that both populations of effector cells contribute to the immune response to this intracellular pathogen.

A vector-based minigene vaccine approach results in strong induction of T-cell responses specific of hepatitis C virus.

Multiepitope-based vaccines against hepatitis C virus (HCV) were designed in the form of three minigenes encompassing four domains of the NS3, NS4 and NS5B proteins that contain multiple class I/II restricted epitopes. The polyEp-WT minigene encodes all four domains in fusion, the polyEp-C minigene encodes the same fusion but optimised for mammalian translation and the polyEp-E3 minigene has an additional endoplasmic reticulum targeting sequence. Whereas the minigenes vectorised by DNA were poorly immunogenic, adenovirus vectorisation induced strong and broader IFNgamma-ELISpot and CTL responses in HLA-A2 transgenic mice. In addition, polyEp-WT and polyEp-E3 responses were found cross-reactive in a recombinant Listeria-NS3-based surrogate challenge. This study illustrates the potency of vectorised minigenes in the field of HCV vaccine development.

DNA Vaccination Protects Mice against Challenge with Listeria monocytogenes Expressing the Hepatitis C Virus NS3 Protein

ABSTRACT The goal of this study was to develop a new surrogate challenge model for use in evaluating protective cell-mediated immune responses against hepatitis C virus (HCV) antigens. The use of recombinant Listeria monocytogenes organisms which express HCV antigens provides novel tools with which to assay such in vivo protection, as expression of immunity against this hepatotropic bacterial pathogen is dependent on antigen-specific CD8+ T lymphocytes. A plasmid DNA vaccine encoding a ubiquitin-NS3 fusion protein was generated, and its efficacy was confirmed by in vivo induction of NS3-specific, gamma interferon-secreting T cells following vaccination of BALB/c mice. These immunized mice also exhibited specific in vivo protection against subsequent challenge with a recombinant L. monocytogenes strain (TC-LNS3) expressing the NS3 protein. Notably, sublethal infection of naive mice with strain TC-LNS3 induced similar NS3-specific T-cell responses. These findings suggest that recombinant strains of L. monocytogenes expressing HCV antigens should prove useful for evaluating, or even inducing, protective immune responses against HCV antigens.

Protection of interferon-γ knockout mice against Listeria monocytogenes challenge following intramuscular immunization with DNA vaccines encoding listeriolysin O

In this study we evaluated the efficacy of DNA vaccination of IFN-gamma knockout (GKO) mice against Listeria monocytogenes, as these immunodeficient mice are highly susceptible to infection with low numbers of this intracellular bacterial pathogen. Following intramuscular immunization of BALB/c GKO mice with plasmid DNA constructs encoding recombinant forms of the L. monocytogenes hemolysin, listeriolysin O (LLO), we detected the in vivo induction of a LLO(91-99) peptide-specific, protective immune CTL response equivalent to that observed following similar DNA vaccination of normal BALB/c mice. The observed protection represented greatly enhanced immunity for the GKO host, suggesting that DNA vaccination may provide a useful vaccine alternative for certain immunocompromised host populations.

Lack of Expansion of Major Histocompatibility Complex Class Ib- Restricted Effector Cells following Recovery from Secondary Infection with the Intracellular Pathogen Listeria monocytogenes

ABSTRACT Sublethal infection of BALB/c mice with the intracellular bacterial pathogen Listeria monocytogenes leads to the development of antilisterial immunity with concurrent stimulation of major histocompatibility complex (MHC) class Ia- and Ib-restricted CD8+ effector T cells. Secondary L. monocytogenes infection is followed by an accelerated increase in the number of Listeria-specific CD8+ cells and rapid clearance of the bacterium from the murine host. Recovery from secondary infection is associated with increased levels of effector cell function, as measured by gamma interferon secretion following coculture of immune cells with L. monocytogenes infected APCs in vitro, as well as antilisterial cytotoxicity, as measured by effector cell recognition of L. monocytogenes-infected target cells. We assessed the frequency ofL. monocytogenes-specific MHC class I-restricted cells following secondary infection by ELISPOT assays utilizing coculture of immune cells with L. monocytogenes-infected antigen-presenting cells that express MHC class Ia and/or Ib molecules. We found that the antilisterial Qa-1b (MHC class Ib)-restricted effector subset is not detected as an expanded population following secondary infection compared to the frequency of this effector population as measured following recovery from primary infection. This is in contrast to the frequency of antilisterial H2-Kd (MHC class Ia)-restricted effector cells, which following recovery from secondary infection are detected as an expanded population, and appears to undergo a substantial expansion event 3 to 4 days post-secondary infection. These results are consistent with the conclusion that althoughListeria-specific MHC class Ib-restricted effector cells are present following recovery from secondary infection, this subset does not appear to undergo the expansion phase that is detected for the MHC class Ia-restricted effector cell response.

Plasmid DNA Delivery by d‐Alanine‐Deficient Listeria monocytogenes

Optimal DNA vaccine efficacy requires circumventing several obstacles, including low immunogenicity, a need for adjuvant, and the costs of purifying injection grade plasmid DNA. Bacterial delivery of plasmid DNA may provide an efficient and low‐cost alternative to plasmid purification and injection. Also, the bacterial vector may exhibit potential as an immune adjuvant in vivo. Thus, we elected to examine the use of cell‐wall‐deficient Listeria monocytogenes as a DNA delivery vehicle in vitro. First, the d‐alanine‐deficient (Δdal‐dat) L. monocytogenes strain DP‐L3506, which undergoes autolysis inside eukaryotic host cells in the absence of d‐alanine, was transformed with a plasmid encoding green fluorescent protein (GFP) under control of the CMV promoter (pAM‐EGFP). Then COS‐7 and MC57G cell lines were infected with the transformed DP‐L3506 at various multiplicities of infection (MOI) in the presence or absence of d‐alanine. Subsequent GFP expression was observed in both cell lines by 24 h post‐infection with DP‐L3506(pAM‐EGFP). Notably, no GFP positive cells were observed when d‐alanine was omitted. Although transfection efficiency initially increased as a result of d‐alanine supplementation, high concentration or long‐term supplementation led to sustained bacterial growth that killed the infected host cells, resulting in fewer GFP‐expressing cells. Thus, efficient DNA delivery by transformed bacteria must balance bacterial invasion and survival with target cell health and survival.