Protection against murine listeriosis by oral vaccination with recombinant Salmonella expressing hybrid Yersinia type III proteins.

In the present study, we have investigated the possibility to engage the Yersinia outer protein E (YopE) as a carrier molecule for heterologous Ag delivery by the type III secretion system of Salmonella typhimurium. Defined secretion and translocation domains of YopE were fused to the immunodominant T cell Ags listeriolysin O and p60 of Listeria monocytogenes. In vitro experiments showed that S. typhimurium allows secretion and translocation of large hybrid YopE proteins in a type III-dependent fashion. Translocation and cytosolic delivery of these chimeric proteins into host cells, but not secretion into endosomal compartments, led to efficient MHC class I-restricted Ag presentation of listerial nonamer peptides. Mice orally vaccinated with a single dose of attenuated S. typhimurium expressing translocated hybrid YopE proteins revealed high numbers of IFN-gamma-producing cells reactive with listeriolysin O 91-99 or p60 217-225, respectively. This CD8 T cell response protected mice against a challenge with L. monocytogenes. In conclusion, these findings suggest that YopE is a versatile carrier molecule for type III-mediated foreign Ag delivery by Salmonella vaccine strains.     

The molecular mechanisms of listeriolysin O-induced lipid membrane damage

Listeria monocytogenes is an intracellular food-borne pathogen that causes listeriosis, a severe and potentially life-threatening disease. Listeria uses a number of virulence factors to proliferate and spread to various cells and tissues. In this process, three bacterial virulence factors, the pore-forming protein listeriolysin O and phospholipases PlcA and PlcB, play a crucial role. Listeriolysin O belongs to a family of cholesterol-dependent cytolysins that are mostly expressed by gram-positive bacteria. Its unique structural features in an otherwise conserved three-dimensional fold, such as the acidic triad and proline-glutamate-serine-threonine-like sequence, enable the regulation of its intracellular activity as well as distinct extracellular functions. The stability of listeriolysin O is pH- and temperature-dependent, and this provides another layer of control of its activity in cells. Moreover, many recent studies have demonstrated a unique mechanism of pore formation by listeriolysin O, i.e., the formation of arc-shaped oligomers that can subsequently fuse to form membrane defects of various shapes and sizes. During listerial invasion of host cells, these membrane defects can disrupt phagosome membranes, allowing bacteria to escape into the cytosol and rapidly multiply. The activity of listeriolysin O is profoundly dependent on the amount and accessibility of cholesterol in the lipid membrane, which can be modulated by the phospholipase PlcB. All these prominent features of listeriolysin O play a role during different stages of the L. monocytogenes life cycle by promoting the proliferation of the pathogen while mitigating excessive damage to its replicative niche in the cytosol of the host cell.     

Listeriolysin O: a genuine cytolysin optimized for an intracellular parasite

Cholesterol-dependent cytolysins (CDCs)* are produced by a large number of pathogenic gram-positive bacteria. A member of this family, listeriolysin O (LLO), is produced by the intracellular pathogen Listeria monocytogenes. A unique feature of LLO is its low optimal pH activity (approximately 6) which permits escape of the bacterium from the phagosome into the host cell cytosol without damaging the plasma membrane of the infected cell. In a recent study (Glomski et al., 2002, this issue), Portnoy's group has addressed the molecular mechanism underlying the pH sensitivity of LLO. Unexpectedly, a single amino acid substitution in LLO L461T results in a molecule more active at neutral pH and promoting premature permeabilization of the infected cells, leading to attenuated virulence. This finding highlights how subtle changes in proteins can be exploited by bacterial pathogens to establish and maintain the integrity of their specific niches.     

From evil to good: a cytolysin in vaccine development

Current vaccination strategies mainly target antigens into the phagosomal, major histocompatibility complex class II antigen-processing pathway and thus lead predominantly to humoral immune responses. The elicitation of cytotoxic T-cell responses instead requires introduction of antigens into the cytosol of professional antigen-presenting cells (APCs). The intracellular bacterium Listeria monocytogenes gains access to the host cell cytosol by means of a cytolysin, listeriolysin O. Vaccine researchers have successfully employed listeriolysin in novel vaccination approaches to provide access to the cytosol of professional APCs for purified protein antigens, attenuated bacterial vaccine strains, DNA vaccines and liposome contents.     

单增李斯特菌溶血素O的功能研究进展

单核细胞增生李斯特菌(Listeria monocytogenes, Lm,简称单增李斯特菌)是一种普遍存在的革兰阳性食源性病原体,可引起人类和一些动物的李斯特菌病。侵袭性李斯特菌病通常很严重,临床上表现为自然流产、败血症和脑膜脑炎,也可表现为发热性胃肠炎综合症。成孔蛋白单增李斯特菌溶血素O(Listeriolysin O,LLO,由hly基因编码)是一种重要的毒力因子,属于胆固醇依赖性细胞溶解素(cholesterol-dependent cytolysins,CDC)毒素,其通过膜穿孔机制介导Lm从吞噬体逃逸并引起李斯特菌病。最近的研究表明LLO除了主要的膜穿孔作用,还存在其他功能,在Lm感染过程中扮演了重要的角色。从LLO的功能和作用机制等方面综述了近些年对该毒素的研究进展,以便更好地理解单增李斯特菌的感染机制,为防治李斯特病的相关研究提供参考。     

Listeriolysin O: a key protein of Listeria monocytogenes with multiple functions

Cholesterol-dependent cytolysins (CDCs) are produced by a large number of pathogenic Gram-positive bacteria. Most of these single-chain proteins are secreted in the extracellular medium. Among the species producing CDCs, only two species belonging to the genus Listeria (Listeria monocytogenes and Listeria ivanovii) are able to multiply intracellularly and release their toxins in the phagosomal compartment of the infected host cell. This review provides an updated overview on the importance of listeriolysin O (LLO) in the pathogenicity of L. monocytogenes, focusing mainly on two aspects: (1) the structure-function relationship of LLO and (2) its role in intra- and extracellular signalling. We first examine the specific sequence determinants, or protein domains, that make this cytolysin so well adapted to the intracellular lifestyle of L. monocytogenes. The roles that LLO has in cellular signalling events in the context of relations to pathogenesis are also discussed.     

Alteration of epithelial cell lysosomal integrity induced by bacterial cholesterol‐dependent cytolysins

Bacterial pathogens can interfere during infection with host cell organelles, such as mitochondria, the endoplasmic reticulum‐Golgi system or nuclei. As important cellular functions are often compartmentalized in these organelles, their targeting allows pathogens to manipulate key host functions during infection. Here, we identify lysosomes as a new class of organelles targeted by the pathogenic bacterium Listeria monocytogenes. We demonstrate that extracellular Listeria, via secretion of the pore‐forming toxin listeriolysin O, alters lysosomal integrity in epithelial cells but not in macrophages. Listeriolysin O induces lysosomal membrane permeabilization and release of lysosomal content, such as cathepsins proteases, which remain transiently active in the host cytosol. We furthermore show that other bacterial pore‐forming toxins, such as perfringolysin O and pneumolysin, also induce lysosomes alteration. Together, our data unveil a novel activity of bacterial cholesterol‐dependent cytolysins.     

New routes of administration: epidermal, transcutaneous mucosal ways of vaccination

A successful vaccine triggers the interaction of various cells of the immune system as does a regular immune response. It is thus necessary to introduce the vaccine antigens into an anatomic site where they will contact immune cells. The route of administration is thus critical for the outcome of vaccination. Intramuscular or subcutaneous injections are the most popular. Antigens injected intramuscularly can form persistent precipitates that are dissolved and re-absorbed relatively slowly. If injecting antigens is a quick, easy and reproducible way to vaccination, it requires trained personnel. Alternatives exist, through non-invasive formulations which allow administration by the patient or a third party with no particular expertise. The skin, especially its epidermal layer, is an accessible and competent immune environment and an attractive target for vaccine delivery, through transcutaneous delivery or immunostimulant patches. Mucosal immunization is another strategy: its major rationale is that organisms invade the body via mucosal surfaces. Therefore, local protection at mucosal surface as well as systemic defense is beneficial. Various formulations of mucosal vaccines have been developed, such as the Sabin oral polio vaccine (OPV), rotavirus vaccines, cold-adapted influenza vaccines or vaccine against typhoid fever. Thus we are entering in an era where mucosal and transcutaneous immunisation will play an important role in disease management. However, it has not been so easy to obtain regulatory approval for mucosal or transcutaneous formulations and needle-based vaccines continue to dominate the market.     

Oral Somatic Transgene Vaccination Using Attenuated S. typhimurium

An attenuated strain of S. typhimurium has been used as a vehicle for oral genetic immunization. Eukaryotic expression vectors containing truncated genes of ActA and listeriolysin—two virulence factors of Listeria monocytogenes—have been used to transform S. typhimurium aroA. Multiple or even single oral immunizations with such transformants induced excellent cellular and humoral responses. In addition, protective immunity was induced with listeriolysin transformants. The quality of the responses suggested a transfer of plasmid DNA from the bacterial carrier to the host. Such transfer was unequivocally shown in vitro with primary peritoneal macrophages. We describe a highly versatile system for antigen delivery, identification of protective antigens for vaccination, and efficient generation of antibodies against the product of open reading frames present on virtually any DNA segment.     

Secretion of different listeriolysin cognates by recombinant attenuated Salmonella typhimurium: superior efficacy of haemolytic over non-haemolytic constructs after oral vaccination

Viable antigen (Ag) delivery systems expressing defined pathogen-derived proteins represent powerful candidates for future vaccination strategies. Here, recombinant (r)Salmonella typhimurium aroA strains secreting listeriolysin (Hly) of Listeria monocytogenes in haemolytic or non-haemolytic form were constructed to direct these carriers into cytosolic or phagosomal host cell compartments, respectively. Oral and intravenous (i.v.) vaccination of mice with either construct induced 'transporter associated with antigen processing'-dependent protection against the intracellular bacterial pathogen L. monocytogenes. Comparison of oral immunization with both rSalmonella constructs revealed superior vaccine efficacy of the haemolytic rS. typhimurium Hlys construct as compared to the non-haemolytic rSalmonella Hlys(492) strain. In contrast, efficacy of i.v. vaccination with either rSalmonella strain did not significantly differ. Therefore, rSalmonella strains secreting biologically active Hly represent valuable delivery systems for heterologous rAg or DNA which should be exploited for future mucosal vaccination strategies.     

Listeriolysin O suppresses phospholipase C-mediated activation of the microbicidal NADPH oxidase to promote Listeria monocytogenes infection

The intracellular bacterial pathogen Listeria monocytogenes produces phospholipases C (PI-PLC and PC-PLC) and the pore-forming cytolysin listeriolysin O (LLO) to escape the phagosome and replicate within the host cytosol. We found that PLCs can also activate the phagocyte NADPH oxidase during L.?monocytogenes infection, a response that would adversely affect pathogen survival. However, secretion of LLO inhibits the NADPH oxidase by preventing its localization to phagosomes. LLO-deficient bacteria can be complemented by perfringolysin O,?a related cytolysin, suggesting that other pathogens may also use pore-forming cytolysins to inhibit the NADPH oxidase. Our studies demonstrate that while the PLCs induce antimicrobial NADPH oxidase activity, this effect is alleviated by the pore-forming activity of LLO. Therefore, the combined activities of PLCs and LLO on membrane lysis and the inhibitory effects of LLO on NADPH oxidase activity allow L.?monocytogenes to efficiently escape the phagosome while avoiding the microbicidal respiratory burst.     

Escherichia coli expressing recombinant antigen and listeriolysin O stimulate class I-restricted CD8+ T cells following uptake by human APC

Vaccination against cancer or intracellular pathogens requires stimulation of class I-restricted CD8(+) T cells. It is therefore important to develop Ag delivery vectors that will promote cross-presentation by APCs and stimulate appropriate inflammatory responses. Toward this goal, we tested the potential of Escherichia coli as an Ag delivery vector in in vitro human culture. Bacteria expressing enhanced green fluorescent protein were internalized efficiently by dendritic cells, as shown by flow cytometry and fluorescence microscopy. Phenotypic changes in DC were observed, including up-regulation of costimulatory molecules and IL-12p40 production. We tested whether bacteria expressing recombinant Ags could stimulate human T cells using the influenza matrix protein as a model Ag. Specific responses against an immunodominant epitope were seen using IFN-gamma ELISPOT assays when the matrix protein was coexpressed with listeriolysin O, but not when expressed alone. THP-1 macrophages were also capable of stimulating T cells after uptake of bacteria, but showed slower kinetics and lower overall levels of T cell stimulation than dendritic cells. Increased phagocytosis of bacteria induced by differentiation of THP-1 increased their ability to stimulate T cells, as did opsonization. Presentation was blocked by proteasome inhibitors, but not by lysosomal protease inhibitors leupeptin and E64. These results demonstrate that recombinant E. coli can be engineered to direct Ags to the cytosol of human phagocytic APCs, and suggest possible vaccine strategies for generating CD8(+) T cell responses against pathogens or tumors.     

Oral delivery of DNA vaccines using attenuated Salmonella typhimurium as carrier

The efficacious delivery of eukaryotic expression plasmids to inductive cells of the immune system constitutes a key prerequisite for the generation of effective DNA vaccines. Here, we have explored the use of bacteria as vehicles to orally deliver expression plasmids. Attenuated Salmonella typhimurium aroA harbouring eukaryotic expression plasmids that encoded virulence factors of Listeria monocytogenes were administered orally to BALB/c mice. Strong cytotoxic and helper T cell responses as well as antibody production were elicited even after a single administration. Mice immunised four times with Salmonella that carried a eukaryotic expression plasmid encoding the secretory listerial protein listeriolysin were protected against a subsequent lethal challenge with this pathogen. A single dose was already partially protective. The efficiency of this vaccination procedure was due to transfer of the expression plasmid from the bacterial carrier to the mammalian host. Evidence for such an event could be obtained in vivo and in vitro. Expression of the desired antigen in various lymphoid tissues was already detectable 1 day after administration of the DNA vaccine and persisted for at least 1 month in spleen and mesenteric lymph nodes. Induction of cytotoxic and helper T cell responses was observed in all mouse strains tested including outbred strains whereas antibodies were mainly detected in BALB/c. Furthermore, we could show that immunogenicity could be improved by increasing the invasiveness of the bacterial carrier.     

Quantitation of CD8+ T cell expansion,memory, and protective immunity after immunization with peptide-coated dendritic cells

Dendritic cells (DCs) are potent APCs for naive CD8(+) T cells and are being investigated as vaccine delivery vehicles. In this study, we examine the CD8(+) T cell response to defined peptides from Listeria monocytogenes (LM), lymphocytic choriomeningitis virus, and murine CMV coated singly and in combination onto mature bone marrow-derived DCs (BMDCs). We show that immunization of mice with 2 x 10(5) mature BMDCs coated with multiple MHC class I peptides generates a significant Ag-specific CD8(+) T cell response in both the spleen and nonlymphoid organs. This immunization resulted in a peptide-specific hierarchy in the magnitude of CD8(+) T cell priming and noncoordinate kinetics in response to different peptide epitopes. Kinetics were not exclusively due to specific characteristics of the MHC class I molecule, and were not altered in an Ag-independent manner by concurrent LM infection. Mice immunized with listeriolysin O 91-99-coated BMDCs are protected against high dose challenge with virulent LM. This protection was enhanced by diversifying the memory CD8(+) T cell compartment, even in the absence of a large increase in Ag-specific CD8(+) memory T cells.     

Engineering of staphylococcal surfaces for biotechnological applications

Novel surface proteins can be introduced onto bacterial cell surfaces by recombinant means. Here, we describe various applications of two such display systems for the food-grade bacteria Staphylococcus carnosus and Staphylococcus xylosus, respectively. The achievements in the use of such staphylococci as live bacterial vaccine delivery vehicles will be described. Co-display of proteins and peptides with adhesive properties to enable targeting of the bacteria, have significantly improved the vaccine delivery potential. Recently, protective immunity to respiratory syncytial virus (RSV) could be evoked in mice by intranasal immunization using such 'second generation' vaccine delivery systems. Furthermore, antibody fragments and other 'affinity proteins' with capacity to specifically bind a certain protein, e.g. Staphylococcus aureus protein A-based affibodies, have been surface-displayed on staphylococci as initial efforts to create whole-cell diagnostic devices. Surface display of metal-binding peptides, or protein domains into which metal binding properties has been engineered by combinatorial protein engineering, have been exploited to create staphylococcal bioadsorbents for potential environmental or biosensor applications. The use of these staphylococcal surface display systems as alternatives for display of large protein libraries and subsequent affinity selection of relevant binding proteins by fluorescence-activated cell sorting (FACS) will be discussed.     

Cytolytic T-cell responses to human dendritic cells and macrophages infected with Mycobacterium bovis BCG and recombinant BCG secreting listeriolysin

Cytolytic T-cell responses from 63 normal blood donors were monitored in a Mycobacterium bovis BCG infection system in vitro. We wanted to know whether cultured dendritic cells were capable of potentiating the cytolytic T-cell responses to M. bovis BCG. Infected cultured dendritic cells were up to ten times more effective antigen-presenting cells than macrophages in proliferative assays, while cytolytic T-cell induction did not differ significantly between dendritic cells and macrophages. Separated CD4+ and CD8+ T-cell subsets contributed equally to lysis of infected targets. Experiments comparing wild-type M. bovis BCG strain with two new recombinant M. bovis BCG strains secreting listeriolysin revealed statistically significant higher maximal lysis values for recombinant M. bovis BCG. We conclude from our in vitro infection system with mycobacteria that dendritic cells are superior to macrophages in proliferative assays but equal to macrophages in their ability to induce cytolytic T-cell responses. Moreover, our data suggest that recombinant M. bovis BCG vaccine strains secreting listeriolysin improve cytolytic T-cell responses.     

The use of listeriolysin to identify in vivo induced genes in the gram-positive intracellular pathogen Listeria monocytogenes

Listeria monocytogenes is capable of growth within the cytoplasm of infected host cells. Escape from the host cell phagosome is mediated primarily through secretion of listeriolysin, a haemolytic factor which functions to actively lyse the phagosomal membrane. Listeriolysin negative mutants of L. monocytogenes are non-haemolytic on blood agar plates and demonstrate a significant reduction of virulence in the mouse model of infection. We have developed a system for the identification of in vivo induced genes in L. monocytogenes which utilizes the listeriolysin gene, hly, as both a reporter of gene expression and as a means of selection of promoter elements expressed in vivo. The system is analogous to in vivo expression technology (IVET) first reported for Salmonella, however, as listeriolysin functions in the environment of the host phagosome the loci identified in this study are most likely expressed during residence in the phagosome. The system was successfully tested using the promoter of the inducible virulence gene plcA. A bank was created by fusing a promoterless copy of hly to random promoter elements in a listeriolysin negative IVET host. Sequential inoculations of mice with this bank resulted in the isolation of clones with increased survival potential in the mouse model relative to a negative control, but which remained haemolysin negative on blood agar plates. Nine in vivo induced loci were identified including genes encoding a DNA topoisomerase III, a cellobiose transporter and a fumarase. Two isolates represented fusions to proteins of unknown function and three isolates contained no significant homologues in the database. A mutant in the fumarase gene demonstrated reduced virulence for mice and an inability to grow in cultured mouse phagocytes.     

Mucosal adjuvants and delivery systems for protein-, DNA- and RNA-based vaccines

Almost all vaccinations today are delivered through parenteral routes. Mucosal vaccination offers several benefits over parenteral routes of vaccination, including ease of administration, the possibility of self-administration, elimination of the chance of injection with infected needles, and induction of mucosal as well as systemic immunity. However, mucosal vaccines have to overcome several formidable barriers in the form of significant dilution and dispersion; competition with a myriad of various live replicating bacteria, viruses, inert food and dust particles; enzymatic degradation; and low pH before reaching the target immune cells. It has long been known that vaccination through mucosal membranes requires potent adjuvants to enhance immunogenicity, as well as delivery systems to decrease the rate of dilution and degradation and to target the vaccine to the site of immune function. This review is a summary of current approaches to mucosal vaccination, and it primarily focuses on adjuvants as immunopotentiators and vaccine delivery systems for mucosal vaccines based on protein, DNA or RNA. In this context, we define adjuvants as protein or oligonucleotides with immunopotentiating properties co-administered with pathogen-derived antigens, and vaccine delivery systems as chemical formulations that are more inert and have less immunomodulatory effects than adjuvants, and that protect and deliver the vaccine through the site of administration. Although vaccines can be quite diverse in their composition, including inactivated virus, virus-like particles and inactivated bacteria (which are inert), protein-like vaccines, and non-replicating viral vectors such as poxvirus and adenovirus (which can serve as DNA delivery systems), this review will focus primarily on recombinant protein antigens, plasmid DNA, and alphavirus-based replicon RNA vaccines and delivery systems. This review is not an exhaustive list of all available protein, DNA and RNA vaccines, with related adjuvants and delivery systems, but rather is an attempt to highlight many of the currently available approaches in immunopotentiation of mucosal vaccines.     

Listeriolysin O-deficient Listeria monocytogenes as a vaccine delivery vehicle: antigen-specific CD8 T cell priming and protective immunity

Strains of Listeria monocytogenes (LM) that are deficient in the virulence factor listeriolysin O (LLO) are highly attenuated and are thought not to elicit protective immunity. This failure has been attributed to the inability of the bacterium to enter the host cell cytosol and access MHC class I Ag processing machinery. We reexamined this issue using recombinant strains of LM that are deficient in LLO but express an additional CD8 T cell epitope derived from lymphocytic choriomeningitis virus. After infection with LLO-deficient strains, we find sizable priming of epitope-specific CD8 T cells and the development of a functional memory cell population. Mice primed with the LLO-deficient LM strain are equally resistant against high-dose challenge with virulent LM as mice primed with wild-type virulent bacteria and also resist heterologous challenge with lymphocytic choriomeningitis virus. Interestingly, priming with a low dose of LLO-deficient LM, which occurred in environment of reduced inflammation (IFN-gamma), allowed rapid amplification of Ag-specific CD8 T cells by booster immunization, despite an undetectable primary response. We conclude that the generation of protective immunity by LLO-deficient strains of LM does in fact occur and that this highly attenuated LM strain may be a useful platform for vaccine delivery.     

Progress in the development of malaria vaccines: context and constraints

Major technical advances in the field of vaccine development have culminated in an impressive array of prototype vaccines that may well provide 'proof of principle' that vaccines against all life-cycle stages may induce a degree of protection against malaria. As the mechanisms responsible for protection against this disease are not known, and vaccines for populations at greatest risk will be applied in the presence of ongoing infection and a degree of concomitant immunity, it is essential for us to learn from the 'experiments of nature' about acquired and ongoing immunity in order to determine when and how these vaccines may be applied. Successful interventions with chemoprophylaxis or vector control have provided obvious lessons and highlight the importance of recognising the lack of correlation between infection, clinical disease and mortality. Vaccines inducing sterile immunity raise concerns about rebound mortality in populations who will undoubtedly be re-challenged later in life, hence the need to review supplementary or alternative strategies for reducing disease through immune responses to toxins or molecules inducing pathology by adherence to host endothelium. Following antigen selection there are many challenges in choosing methods of antigen delivery and adjuvants, and measuring vaccine efficacy. A successful vaccine would need to be delivered through a national programme in the context of implementation of a wide range of components required for an effective control strategy.