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Lactic Acid Bacteria in Vaccine Development
PhD thesis by Jacob Glenting
Bioneer A/S
And
Biocentrum
Technical University o...
Preface
This thesis describes the results of a PhD study initiated in December 2003 and
finished in May 2007. The study wa...
Table of contents
Abstract(s)
UK and DK version ……………………………………………………………………………………….…………….1
Outline of thesis………………………………………...
Lactic Acid Bacteria in Vaccine Development
Abstract
This PhD study is focused on the use of lactic acid bacteria (LAB) in...
architecture of LAB and thereby affect their immune modulating activity. New
protein anchors were isolated from lactobacil...
The experiments presented in this thesis suggest new LAB based vaccine candidates:
(i) a live bacterial birch-pollen-aller...
Anvendelse af Mælkesyrebakterier til Vaccinefremstilling
Resume
Dette PhD studie fokuserer på brugen af mælkesyrebakterier...
Overflade display af antigener på bakterier er en attraktiv strategi til co-præsentation
af antigen og adjuvans fra bakter...
Eksperimenterne præsenteret i denne afhandling foreslår nye LAB baserede vaccine
kandidater: (i) en levende mælkesyrebakte...
Outline of thesis
The thesis begins with an introducing chapter, which is divided in two parts: (i) An
overview of lactic ...
Chapter 1
General Introduction
8
Introduction
Lactic acid bacteria (LAB) are a functionally related group of organisms known
primarily from their role in b...
The potential of LAB to survive through the gastro-intestinal tract, adhere to mucosal
surfaces, and activate the immune r...
allows for uptake through M-cells of the Peyer´s patches of the GI-tract and opens for
their subsequent distribution to th...
lipoteichoic acid of L. johnsonii have also been shown to participate in the adhesion to
intestinal cells [Granato et al.,...
The heterologous passenger protein can be targeted to three compartments:
intracellular accumulation, cell wall associated...
functional cell surface protein is unknown. But may simply be an increase in the rate
of turn over of the surface proteins...
heterologous protein, a plasmid replication unit, and a selectable marker for plasmid
maintenance during bacterial growth....
T7 promoter. Environmentally regulated promoters avoid the use of exogenous added
inducers. In L. lactis the P170 promoter...
with a requirement for either threonine or D-alanine has been constructed and
complemented with the relevant genes on plas...
References
Adlerberth I, Ahrne S, Johansson ML, Molin G, Hanson LA, Wold AE: A mannose-specific adherence mechanism in
Lac...
Gram GJ, Fomsgaard A, Thorn M, Madsen SM, Glenting J: Immunological analysis of a Lactococcus lactis-based DNA
vaccine exp...
Madsen SM, Arnau J, Vrang A, Givskov M, Israelsen H: Molecular characterization of the pH-inducible and growth
phase-depen...
Steidler L: In situ delivery of therapeutic cytokines by genetically engineered Lactococcus lactis. Meded Rijksuniv Gent
F...
BioMed Central
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Microbial Cell Factories
Open AccessReview
Live bacter...
Microbial Cell Factories 2006, 5:23 http://www.microbialcellfactories.com/content/5/1/23
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PhD thesis Glenting J

  1. 1. Lactic Acid Bacteria in Vaccine Development PhD thesis by Jacob Glenting Bioneer A/S And Biocentrum Technical University of Denmark June 2007
  2. 2. Preface This thesis describes the results of a PhD study initiated in December 2003 and finished in May 2007. The study was done mainly at Bioneer A/S, but with close interactions with Statens Serum Institute (SSI), Danish Toxicology Center (now DHI), ALK-Abello (ALK), and the Allergy Clinic, National University Hospital, DK. The PhD study was interrupted for 4 months because of a time consuming project work at Bioneer. Bioneer and the Danish Ministry of Science, Technology and Innovation have financially supported the work. I wish to thank the management of Bioneer for being supportive. Hanne Frøkiær has been my supervisor at DTU Biocentrum. I wish to thank Hanne for helpful discussions and teaching me how to work with dendritic cells. Hans Israelsen was my supervisor at Bioneer in the beginning of the study to whom I owe many thanks for teaching me how to do high quality science. However, as Hans left Bioneer, Søren Madsen has been taking over and contributed with ideas, assistance, and great discussions. Anders Fomsgaard (SSI) has been supervising the work on the gene vaccines and allowed me a peek into the world of virology and immunology. Thank you Anders for our collaboration. The work of this thesis overlaps with very different biological disciplines. Therefore I have relied on the expertise and help from several excellent researchers. Thanks to Mercedes Ferreras and Jens Brimnes (ALK), Ann Detmer and Stephen Wesssels (DHI), Gregers Gram (SSI), Lars K. Poulsen (Allergy Clinic), Bjørn Holst, Peter Ravn, Helle Wium and Simon Jensen (Bioneer). Outstanding technical assistance has been given by Ulla Poulsen, Pernille Smith, Annemette Brix, and Anne Cathrine Steenbjerg (Bioneer). Finally I wish to thanks Vera for being patient during the writing phase, cooking dinner, and her warm love! Jacob Glenting Hørsholm, June 2007
  3. 3. Table of contents Abstract(s) UK and DK version ……………………………………………………………………………………….…………….1 Outline of thesis…………………………………………………………………………………………………. 7 Chapter 1 General introduction…………………………………………………………………………………………………….8 Chapter 2 Immunological analysis of a Lactococcus lactis based DNA vaccine expressing HIV gp120…………………39 Chapter 3 Cell surface display of Bet v 1 on immunomodulatory lactobacilli: potential oral delivery vehicle for treatment of birch pollen allergy…………………………………………………………………………………..51 Chapter 4 DNA inversion Controls Expression of a Mannose Specific Adhesin from Lactobacillus plantarum …………70 Chapter 5 Recombinant Production of Immunological Active Peanut Allergen Ara h 2 using Lactococcus lactis……….94 Chapter 6 Conclusions and concluding remarks……………………………………………………………………………….114 Appendix A plasmid selection system in Lactococcus lactis and its use for gene expression in L. lactis and human kidney fibroblasts……………………………………………………………………………………….117
  4. 4. Lactic Acid Bacteria in Vaccine Development Abstract This PhD study is focused on the use of lactic acid bacteria (LAB) in development of vaccines and therapeutics. The applications of LAB in strategies to promote health and prevent diseases are several: (i) the bacterium can be used as a therapeutic itself, (ii) gene engineered LAB are suited for the delivery of medical components by the mucosal route, (iii) LAB are attractive microbial cell factories of heterologous proteins and pharmaceutical plasmid DNA. This thesis analysed these applications of LAB with the aim to develop novel vaccines and learn more about the interactions of LAB with the human mucosal surfaces and immune system. Three types of LAB based vaccines were developed and tested including a plasmid DNA vaccine, a live recombinant vaccine vehicle and a subunit protein vaccine. Because vaccines most often are given to healthy people, and therefore a minimum of risk is accepted, I felt compelled to analyse the safety aspects of vaccines and to give some suggestions for future development of safer vaccines. The result was two reviews focusing on either live bacterial vaccines or plasmid DNA vaccines. Both reviews include a discussion on the safety aspects of the vaccine technologies and give suggestions of aspects to consider in the early phases of vaccine development. For reasons of efficiency Escherichia coli is used today as the microbial factory for production of plasmid DNA vaccines. To avoid hazardous antibiotic resistance genes and endotoxins from plasmid systems used nowadays, we have developed a system based on the food-grade Lactococcus lactis and a plasmid without antibiotic resistance genes. The L. lactis system was compared to a traditional one in E. coli using identical vaccine constructs encoding the gp120 of HIV-1. Although the plasmid DNA vaccines encode similar antigens their immune effect differs. This provides information about the role of the “silent” plasmid backbone of DNA vaccines and the immune activating effect of DNA from human commensals. Antigen surface display on bacteria is an attractive strategy to co-present the antigen and the adjuvant effect of the bacterium. However, surface display of proteins can lower the access to vaccine epitopes by steric hindrance and change the surface 1
  5. 5. architecture of LAB and thereby affect their immune modulating activity. New protein anchors were isolated from lactobacilli and compared for their efficiency in protein display. By using a C-terminal anchor in combination with a long spacer the display of an active enzyme and a birch pollen allergen with preserved immunereactivity was obtained. Although surface molecules of LAB are key factors in the activation of the immune system, changing the cell wall by display of an allergen, did not alter their adjuvant properties. The developed allergen displaying LAB may represent a promising oral vaccine delivery vehicle for treatment of birch pollen allergy. An important feature of live LAB vaccines is their interaction with the mucosal surfaces. As mannose covers the mucosal surfaces we analysed the molecular factors mediating mannose adhesion in lactobacilli. A mannose specific adhesin was isolated and identified to be responsible for the binding to intestinal epithelial cells. Interestingly, the expression of the adhesion-gene was regulated by a flip-flop inversion of a DNA element present in the untranslated leader of the gene encoding the adhesin. The findings represent a new all-or-nothing transcriptional control in lactobacilli, which is also observed in other bacteria like Escherichia coli that reside in the human body. Lactococcus lactis is also an attractive microorganism for use in the production of protein therapeutics. L. lactis is considered food grade, free of endotoxins, and is able to secrete the heterologous product together with few other native proteins. Hypersensitivity to peanut represents a serious allergic problem. Some of the major allergens in peanut have been described. However, for therapeutic usage more information about the individual allergenic components is needed. In this thesis recombinant production of the Ara h 2 peanut allergen was tested using L. lactis. L. lactis could offer high yields of secreted, full length and immunologically active allergen. The L. lactis expression system can support recombinant allergen material for immunotherapy and component resolved allergen diagnostics. Furthermore, using the L. lactis expression system makes it relatively simple to engineer and screen allergen variants of Ara h 2 with reduced binding to IgE. 2
  6. 6. The experiments presented in this thesis suggest new LAB based vaccine candidates: (i) a live bacterial birch-pollen-allergen vaccine (ii) a plasmid DNA vaccine encoding a HIV-1 surface molecule (iii) a subunit peanut allergen vaccine. Furthermore, the developed LAB based vaccines are important tools to study the cross talk between commensals and the human body. 3
  7. 7. Anvendelse af Mælkesyrebakterier til Vaccinefremstilling Resume Dette PhD studie fokuserer på brugen af mælkesyrebakterier (LAB) til udvikling af vacciner og terapeutika. Anvendelserne af LAB til at promovere sundhed og forebygge sygdomme er flere: (i) bakterierne kan benyttes som terapeutika i sig selv, (ii) genmodificerede LAB er velegnet til aflevering af medicinske komponenter via den mukosale rute, (iii) LAB er attraktive mikrobielle cellefabrikker af heterologe proteiner og pharmaceutisk plasmid DNA. Denne afhandling analyserede disse applikationer af LAB med målet at udvikle nye vacciner og lære mere om interaktionerne mellem LAB og den humane mukosale overflade samt immunsystemet. Tre typer af LAB-baserede vacciner blev udviklet og testet. Disse inkluderer en plasmid DNA vaccine, en levende LAB-rekombinant vaccine og en subunit protein vaccine. Vacciner er ofte givet til raske personer. Derfor er den accepterede risiko ved vaccination meget lav. I denne afhandling er der derfor også fokuseret på sikkerhedsaspekterne af de udviklede vacciner. Resultatet var to reviews, der fokuserer på levende vacciner og DNA vacciner. Begge reviews inkluderer en diskussion af sikkerhedsaspekterne ved de to typer af vacciner og giver forslag til hvilke aspekter, der kan behandles i den tidlige udviklingsfase af vacciner. På grund af effektiviteten er Escherichia coli benyttet i dag som mikrobiel fabrik af plasmid DNA vacciner. For at undgå antibiotika resistens gener og endotoxin i disse anvendte produktionssystemer er her udviklet et system som er baseret på en sikker organisme, Lactococcus lactis, og som ikke anvender antibiotika. Dette system blev sammenlignet med et traditionelt E. coli baseret system ved brug af identiske vaccinekonstrukter, der koder for gp120 proteinet fra HIV-1. På trods af at plasmid vaccinerne koder for identiske antigener var det inducerede immunrespons forskelligt. Dette giver informationer om den ikke-kodende del af DNA vacciner og hvad DNA kompositionen betyder for adjuvanseffekten i DNA vacciner. Dette giver også information om den immunaktiverende effekt af DNA fra mælkesyrebakterier til stede i den humane bakterieflora. 4
  8. 8. Overflade display af antigener på bakterier er en attraktiv strategi til co-præsentation af antigen og adjuvans fra bakterien. Men overflade display af protein kan inhibere fremvisningen af vaccine epitoper til immunsystemet ved sterisk hindring og samtidigt ændre overflade arkitekturen af LAB, der derved ændrer den vigtige immunmodulerende aktivitet. Nye proteinankre blev isoleret fra laktobaciller og deres effektivitet mht. overflade display blev sammenlignet. Ved brug af et C-terminalt anker i kombination med en lang ”arm” kunne et aktivt enzym og et birkepollen allergen med konserveret immunreaktivitet immobiliseres til celle overfladen. På trods af at overfladekomponenter på LAB er nøglefaktorer i aktivering af immunsystemet ændrede display af birkepollen-allergenet ikke adjuvanseffekten af bakterien. Den udviklede allergen vaccine er kandidat til en ny oral behandling af birkepollen allergi. En vigtig evne af levende LAB vacciner er deres interaktion med den mukosale overflade. Mannose er en vigtig bestanddel af den mukosale overflade. Derfor analyserede vi de molekylære faktorer bag laktobacillers evne til at binde mannose. Et mannose specifikt adhesin blev identificeret som værende en central faktor i bindingen til epitel celler. Ekspression af adhesinet blev analyseret og var reguleret af en flip-flop mekanisme, hvor et DNA element opstrøms adhesinet inverteres. Denne opdagelse er en ny alt eller intet transskriptions reguleringsmekanisme i laktobaciller, som også er observeret i andre mave-tarm associerede bakterier som E. coli. Lactococcus lactis er også attraktiv til produktionen af heterologe proteiner. L. lactis er anerkendt som sikker og producerer ikke endotoxiner, samt sekreterer det heterologe produkt til det ekstracellulære miljø sammen med få andre native proteiner. Hypersensitivitet til peanuts er en alvorlig allergi. Nogle af allergenerne i peanuts er beskrevet. Men til terapeutisk brug er der brug for mere information om de enkelte allergener. I denne afhandling er rekombinant ekspression af Ara h 2 allergenet testet ved brug af L. lactis. Her opnåedes produktion af høje mængder af allergen med konserveret immunreaktivitet. L. lactis systemet kan benyttes til at producere Ara h 2 til immunterapi og til diagnostik af hypersensitivitet ved brug af isolerede allergen komponenter. Yderligere er det forholdsvist simpelt at udvikle Ara h 2 varianter med reduceret IgE binding. 5
  9. 9. Eksperimenterne præsenteret i denne afhandling foreslår nye LAB baserede vaccine kandidater: (i) en levende mælkesyrebakterie til terapeutisk behandling af birkepollen allergi (ii) en plasmid DNA vaccine udtrykkende gp120 fra HIV-1 (iii) en peanut allergen vaccine. Ud over at være lovende vaccineteknologier kan de anvendes som vigtige redskaber til analyse af interaktionen mellem den residerende flora og humane krop. 6
  10. 10. Outline of thesis The thesis begins with an introducing chapter, which is divided in two parts: (i) An overview of lactic acid bacteria (LAB) and their applications in vaccine development, (ii) two published reviews that give a more thorough description of the subject. The first review deals with the use of LAB as live microbial vehicles of vaccines and therapeutics. The other is a mini review and describes plasmid DNA vaccines and some aspects of their production. In both reviews the use of LAB in vaccine production is described and compared to alternative organisms. In addition to review the published literature the manuscripts discuss and give suggestion for the development of safer vaccines for the future. The second part of the thesis contains the experimental studies. Here a LAB and non- antibiotic based plasmid DNA vaccine was developed and compared to a routinely used Escherichia coli based gene vaccine. The use of Lactococcus lactis as new and antibiotic-free microbial factory of pharmaceutical plasmid DNA is discussed. The L. lactis host-vector plasmid selection system, which is the backbone of the suggested DNA vaccine, was developed before initiating this PhD study. However, as the plasmid and host-strain constructions indeed are relevant for this thesis I have attached my publication from 2002 in the appendix. In chapter 3, genetic elements were analysed to construct a live allergy vaccine with immunomodulatory activity. Chapter 4 represents a time consuming part of the thesis. Here the mucosal adhesive phenotype of lactobacilli was investigated. In chapter 5 L. lactis was used for recombinant production of a peanut allergen. This manuscript is submitted to Microbial Cell Factories. The summarising chapter 6 extracts the most important findings of the study and gives suggestions for future directions. 7
  11. 11. Chapter 1 General Introduction 8
  12. 12. Introduction Lactic acid bacteria (LAB) are a functionally related group of organisms known primarily from their role in bioprocessing of food products. LAB are gram-positive, anaerobic, with low G+C content, and acid tolerant. Acidification is important during food processing. However, LAB also contributes to the flavour, texture and nutritional level in the end product. The central role of LAB in industrial fermentation of food and beverages has driven the research on genetics and metabolisms of these bacteria. Today state of the art research on LAB in the industry develops and selects tailored strains with special metabolic characters. Although LAB are considered generally regarded as safe (GRAS) the manipulation of genes confer new challenges to this definition. To avoid labelling as genetically modified a mutagenesis strategy is usually employed. However, random mutagenesis of LAB, by use of chemicals or radiation, results in a relatively large strain library and isolation of the clone with proper gene modification can be a challenge. High throughput screening technology and the availability of genome sequences facilitate the selection and characterisation of the strain. Specific mutagenesis strategies using integration systems have been developed and optimized towards food grade status. These systems use the native LAB gene elements to obtain knock out or over expression mutants and in some cases alleviate the need for laborious screening activities. Although, gene modifications using site directed integration and gene manipulations ensure a fully characterised strain, the EU regulations demands GMO labelling, whereas a random mutagenisised strain escapes this process. Although a century has past by since the Noble prize awarded Elya Metchnikoff (1845-1916) proposed that LAB could promote health, the clinical and molecular data behind the acclaimed heath effects has been recently established. Today’s availability of genetic tools, appropriate in vitro and animal models, and clinical data allows for critical evaluation of this life-promoting effect of LAB. These “new” applications of LAB have fuelled the research activity and are by some researchers called the “LAB renaissance”. 9
  13. 13. The potential of LAB to survive through the gastro-intestinal tract, adhere to mucosal surfaces, and activate the immune responses make them attractive as transporters of vaccines and therapeutics. Today LAB has been used as delivery vehicles of cytokines [Steidler et al., 2001], therapeutic enzymes [Kiatpapen et al., 2001], antimicrobial peptides [Freitas et al., 2005], antigens [Pouwells et al., 1998], allergens [Daniel et al., 2006], hormones [Yao et al., 2006], antagonists [Ricci et al., 2003], and antibody fragments [Krüger et al., 2002]. Although obvious risks are associated with their recombinant status and non-controllable in situ antigen synthesis the scientific progress is promising. Especially after the positive outcome of the clinical trial with interleukin 10 secreting Lactococcus lactis [Braat et al., 2006]. Alongside the development of LAB as vaccine carriers several groups have focused on LAB as microbial cell factories of recombinant proteins or metabolic precursors. LAB as live mucosal vaccines Needle free and mucosal administration of vaccines is becoming increasingly relevant as the importance of mucosal immunity is acknowledged. In addition, non-parenteral administration avoids the risk of contaminated needles and need for a healthcare infrastructure. Live vaccines based on bacteria demands a less complicated down stream processing compared to subunit vaccines based on purified protein components. Some strains of LAB are attractive as live mucosal vaccines. Their GRAS status, ability to survive through the GI tract, adhesive properties, and immunomodulatory effect make them suitable for vaccine vehicles. However, their recombinant and live status adds certain issues that should be addressed (Table 1). A functional live vaccine based on LAB includes two basic elements: (i) the bacterial strain, and (ii) the recombinant expression unit that drives antigen synthesis. An overview of these is given below. Physical and immunological properties of LAB as vaccine vehicles Several physical properties make LAB interesting microbial vehicles of vaccine components. Especially antigens from pathogenic bacteria can be presented with close mimicry. Indeed, induction of protective immunity against Helicobacter pylori and Streptococcus pneumonia was obtained by immunization with L. lactis expressing the Cag12 membrane protein [Kim et al., 2006] and the PspA pneumococcal surface protein [Hannify et al., 2007], respectively. Although speculative, the size of LAB 10
  14. 14. allows for uptake through M-cells of the Peyer´s patches of the GI-tract and opens for their subsequent distribution to the mucosal associated lymphatic tissue. Indeed, L. plantarum expressing green fluorescent protein and given orally to mice was shown embedded in the mucus and in close contact with epithelial cells [Geoffroy et al., 2000]. This study also showed that L. plantarum was phagocytized by bronchoalveolar macrophages following nasal administration. LAB responds to the harsh milieu of the GI-tract by induction of genes that encode components to resist bile salts [Pfeiler et al., 2007], stress and metabolic changes [Bron et al., 2004]. Although concerns of prolonged persistency of the recombinant vaccine strain has been raised, the colonising capacity of some LAB may play a central role in their ability to induce an immune response. Bacterial colonisation requires adhesion of bacteria to the mucosal surfaces. Indeed, some strains of LAB express specific cell wall components or adhesins that mediate their adherence to the extracellular matrix (ECM) of the host. These molecular adhesion factors have been investigated using tissue samples, cell lines and components of the ECM [Miyoshi et al., 2006, Adlerberth et al., 1996, Granato et al., 1999, Greene et al., 1994, Henriksson et al., 1991, Henriksson et al., 1992, Henriksson et al., 1996, Hynonen et al., 2002, Rojas et al., 2002, Sillanpaa et al., 2000, Toba et al., 1995]. The ligands of these adhesins have been identified as sugar components [Adlerberth et al., 1996], and ECM proteins like fibronectin [Hynonen et al., 2002], mucin [Granato et al., 2004], and collagen [Sillanpaa et al., 2000]. The chemical identity of adhesion factors include both protein and non-protein components of the bacterial cell surface. Most often cell-surface-adhesins are proteins with signal sequences for their secretion and mechanisms for covalent anchoring to bacterial cell wall. One important mechanism anchors the carboxyl terminal via an LPXTG motif and a surface located sortase that catalyzes the covalent linkage of the adhesion [reviewed by Navarre & Schneewind, 1999]. However, adhesins without signal peptides and anchoring domains like the LPXTG motif have also been identified [Chhatwal et al., 2002]. The elongation factor EF-TU, normally involved in protein synthesis and without apparent signal peptide or cell wall anchoring motif, was identified as a cell surface protein mediating adhesion to intestinal cells [Granato et al., 2004]. Surprisingly was also the GroEL heat shock protein found on the surface of the same bacterial strain and identified as an adhesion factor [Bergonzelli et al., 2006]. Non-proteianous cell surface molecules like 11
  15. 15. lipoteichoic acid of L. johnsonii have also been shown to participate in the adhesion to intestinal cells [Granato et al., 1999]. The diversity of adhesins and their complex in vivo regulation illustrates the challenge associated with analysing interactions of LAB with the host. A central component of vaccines is adjuvant, which augments the induced response of both the innate and adaptive immune system. For most vaccines an exogenous added adjuvant is necessary. However, some strains of LAB have intrinsic adjuvant properties. Because the adjuvant effect of LAB differs from strain to strain and that both pro and anti-inflammatory strains have been isolated the term immunomodulatory is more appropriate. This effect has been evaluated in animal studies [Matsuzaki et al., 1998]. But more recently in vitro co-incubation with LAB and dendritic cells is used to analyse their immuneregulatory effect [Christensen et al., 2002, Mohamadzadeh et al., 2005, Zeuthen et al., 2006]. DNA, lipoteichoic acid, and bacterial surface proteins has been suggested as the molecular factors responsible for the immune activating effect of LAB [Pisetsky et al., 1999, Matsuguchi et al., 2003, Gram et al., 2007]. Although the DC model may provide new information on the communication between bacteria and the immune system the in vivo correlation may be questionable. Indeed, the metabolism and surface architecture of LAB changes considerable when bacteria are transferred from the laboratory to the environments in the GI-tract [Bron et al., 2004]. Genetic engineering of LAB for vaccine delivery The expression unit encoding the passenger protein can be episomal as plasmid DNA or integrated into the chromosome. Usually plasmid based gene expression support higher product yield due to the higher gene doses. However, plasmid systems adds to the associated with horizontal gene transfer to the indigenous flora. The risk of plasmid transfer can be lowered using narrow host range replicons or even replicons that are active only in a specific mutant strain. Gene units integrated on the chromosome are less promiscuous and were tested in L. lactis encoding IL10 [Steidler et al., 2003]. Here, the IL10 expression cassette was inserted into the thyA gene creating an auxotroph strain with a growth-requirement for external added thymine or thymidine. 12
  16. 16. The heterologous passenger protein can be targeted to three compartments: intracellular accumulation, cell wall associated, or secreted in a free form to the extra cellular milieu. Intracellular accumulation can protect the antigen during passage through the GI-tract. Protective immune reactions have been induced using intracellular accumulated tetanus toxin fragment C (TTFC) in L. lactis [Wells et al., 1993]. TTFC is a highly potent antigen and less immunogenic proteins may require a more efficient display. Antigen leaking mutants of LAB have therefore been developed. The alanine racemase mutants of L. lactis and L. plantarum contain a fragile cell wall when grown in absence of D-alanine and were more immunological potent using TTFC and the nasal route, than their wild type counterparts [Grangette et al., 2004]. Secretion of free form proteins has also induced immune responses using mucosal vaccination of LAB secreting and a variety of different proteins [Enouf et al., 2001, Chatel et al., 2001, Yao et al., 2006]. Bacterial surface display is often preferred to co-present adjuvant and antigen in close proximity to each other. Several display systems exist for association of a recombinant passenger protein to the surface of gram positive bacteria [Navarre & Schneewind, 1999]. Some involve interactions with the cytoplasmic membrane, residues of the lipotheicoic acid, whereas others are covalently linked to the cell wall. The sortase-mediated linkage is dictated by a sorting signal made of LPXTG followed by 20 hydrophobic aa residues and a tail of positively charged aa. Protein anchor signals using LPXTG from the Streptococcus pyogenes M6 protein was effective as surface display system in different lactobacilli but less so in L. lactis [Dieye et al., 2001]. However, other groups showed that the protein anchor of M6 could efficiently immobilize the L7/L12 Brucella abortus antigen to the surface of L. lactis [Ribeiro et al., 2002]. The lactococcal surface protease PrtP is also anchored by the LPXTG mechanism and was used as surface display system of chimeric malaria antigen Msa2 in L. lactis [Ramasamy et al., 2006]. Anchoring mechanisms that not relies on LPXTG has been identified in the autolysin AcmA of L. lactis, which is a non- covalently surface attached enzyme [Raha et al., 2005]. The C-terminal anchor domain of AcmA successfully targeted and immobilized the E. coli fimbrial F18 adhesin to the surface of L. lactis [Lindholm et al., 2004]. Although surface display ensures maximum exposure to the immune system it may lead to degradation by proteases present in the GI-tract. The mechanism of maintaining a non-degraded and 13
  17. 17. functional cell surface protein is unknown. But may simply be an increase in the rate of turn over of the surface proteins. Recently, non-recombinant but antigen displaying LAB has been developed. Here the protein anchor domain of AcmA is used to attach chimeric antigens to the peptidoglycan layer of wild type LAB [van Roosmalen et al.,2006]. Although the technology avoids the GMO issues the complexity of the technology may be problematic for large-scale vaccine manufacturing. LAB as microbial cell factories Besides applications in food processing, probiotics, and live vaccines, LAB are interesting microbial factories of industrial relevant metabolites and heterologous proteins. The scientific progress within metabolic engineering opens for bioproduction of specific chemical enantiomers like L-alanine [Hols et al., 1999] and L-lactate [Okano et al., 2007], which can be difficult to produce by chemical synthesis. Their GRAS status and lack of enodotoxins make LAB attractive producers of medical important components. In addition contain gram positive bacteria a cell wall mono layer and therefore absence of periplasmic space. This enables full secretion of the heterologous product to the culture medium simplifying the down stream purification steps. The genetic elements of heterologous expression systems For increased gene dosage plasmid based expression system are preferred. Several expression plasmids have been developed for various LAB and supports either a constitutive or regulated expression of proteins. The genetic elements of expression plasmids are similar and include an expression unit that drives the synthesis of the Table 1 Advantages and drawbacks of LAB as live vaccines Pros/Cons Description Pros Non-pathogenic status No risk of reversion to pathogenic status Mimicry of infection Bacterial antigens can be displayed in close resemble to native state Mucosal immunity Induction of mucosal immune response Mucosal administration Needle free vaccine administration Manufacturing process Established fermentation technology, simple down stream processing Cons GMO status Release of GMO in nature Dosage control In situ antigen synthesis may be difficult to control Undesired immune reactions Antigens may induce an immune reaction to the bacterium itself Prolonged persistency High stability of LAB in vivo is undesired Induction of tolerance Immunomodulatory effect of LAB in vivo is unclear 14
  18. 18. heterologous protein, a plasmid replication unit, and a selectable marker for plasmid maintenance during bacterial growth. A signal sequence is placed in translational fusion with the heterologous gene to allow secretion of the protein. Proteins that are targeted for secretion by the Sec-dependant pathway include a signal peptide of 25-35 aa in size, which is cleaved off by the signal peptidase during secretion. Several signal sequences have been identified in LAB using enzyme reporters of secretion like nuclease [Poquet et al., 1998] or ß- lactamase [Sibakov et al., 1991]. Furthermore, secretion efficiency can be enhanced using synthetic derivatives of signal sequences [Ravn et al., 2003], addition of a synthetic propeptide sequence (LEISSTCDA) to the N-terminal of the mature protein [Hazebrouck et al., 2007], and by co-expressing chaperones [Lindholm et al., 2006]. Highly active promoters are used for efficient transcription of the gene of interest. Promoters active in LAB usually contain a core region with a -10 region (TATAAT) and a -35 region (TTGACA) often spaced by 17 bp [Hawley & McCLure, 1982]. Regulatable promoters are preferred for high yield protein production. These are controlled by adding external components, by environmental conditions or the growth phase. The gene regulatory elements of the nisin gene cluster of L. lactis has been used for heterologous and regulatable expression in L. lactis [de Ruyter et al., 1996] and lactobacilli [Pavan et al., 2000]. Here expression is activated by addition of the peptide nisin to the growth medium. Genetic elements responsible for regulation and expression of the bacteriocin sakacin have been isolated from L. sakei [Axelsson et al., 1993] and used for development of an inducible expression system [Axelsson et al., 2003]. Here the SapA and SapI promoters are induced by addition of a peptide pheromone. By placing the genes encoding the response regulator and histidine kinase to the expression vector LAB without the sakacin operon can be used as hosts. A few studies describe the use of genetic components from the lac operon in heterologous production [deVos & Gasson, 1989]. Here the lacA promoter is repressed during growth on glucose but induced by a shift to medium with lactose as carbon source. Elements from the lac operon have been combined with elements from the E. coli bacteriophage T7 [wells et al., 1993]. Here, Wells et al. placed the T7 RNA polymerase under control of the lactose promoter. Growth on lactose induced T7 RNA polymerase expression, which in turn transcribes the heterologous gene via the 15
  19. 19. T7 promoter. Environmentally regulated promoters avoid the use of exogenous added inducers. In L. lactis the P170 promoter is induced in the transition to the stationary growth phase and also affected by the lactate concentration [Madsen et al., 1999]. Although most LAB are generally regarded as safe, their status can be compromised by the introduction of foreign DNA necessary for synthesis of recombinant proteins. Usually high copy number plasmids are used for high level expression of recombinant proteins. A simple way to prevent plasmid loss is to use plasmid-encoded antibiotic resistance markers and grow the bacteria in the presence of antibiotics. The chief drawbacks of this approach are the potential loss of selective pressure as a result of antibiotic degradation (as in the case of β-lactamase) and contamination of the biomass or purified protein by antibiotics and resistance genes, which is unacceptable from a medical point of view. Alternative genetic markers have been developed especially for L. lactis. Depending on the type of selection, they can be placed in two groups: resistance and complementation markers. Examples of resistance markers that confer immunity to an added agent such as nisin [Froseth et al., 1991] or the metal ions cadmium (Cd++ ) [Liu et al., 1996] and copper (Cu++ ) [Liu et al., 2002] have been designed for plasmid maintenance. Although some strains of LAB are naturally resistant to nisin and metal ions, the dominant nature of resistance markers make them versatile as they can be used in different lactococcal strains. The use of auxotrophic markers is based on complementation of a mutation or deletion in the host chromosome and is therefore strain-specific. In L. lactis, the first example was based on complementation of a lacF– strain deficient in lactose utilization [MacCormick et al., 1995]. In two other systems, auxotrophic markers complement purine and pyrimidine-auxotrophic strains using genes encoding nonsense tRNA suppressors [Dickely et al., 1995, Sørensen et al., 2000]. In these systems, expression of the plasmid-borne suppressor tRNA gene allows read-through of nonsense mutation(s) in the genes encoding purine or pyrimidine biosynthetic enzymes. Both systems permit selection in milk or other media that contain small or no amounts of purines or pyrimidines. Furthermore, amino acid-auxotrophic strains 16
  20. 20. with a requirement for either threonine or D-alanine has been constructed and complemented with the relevant genes on plasmid [Glenting et al., 2002] or on the chromosome [Bron et al., 2002]. Choice of a suitable cell factory Several key parameters must be addressed for the choice of particular protein production system. Posttranslational modifications like glycosylation or disulphide bridges may be essential for activity of the recombinant product. Here eukaryotes, rather than prokaryotes, should be used a cell factory. However, the fermentation costs and the production time are lowered using a bacterial production system. A major challenge facing biomanufacturing of proteins is down stream processing. Secretion of the recombinant product simplifies purification and can be achieved by eukaryotic and gram-positive bacterial systems. Furthermore, the relative expression level compared to the contaminants is important. Here, the complete lack of endotoxins in gram-positive organisms is an advantage as LPS often is co-purified with the target protein purified by ion exchange principles. LPS is a major challenge in production of pharmaceutical plasmid DNA as it is co-purified with the negatively charged DNA [Petsch & Anspach 2000]. The use of Gram-positive bacteria as plasmid DNA factories can avoid LPS-contamination, but may be problematic in terms of DNA yield [Gram et al., 2007]. Summary The new applications of LAB as gene engineered vehicles of mucosal vaccines and cell factories of pharmaceutical protein and plasmid DNA are promising. With the increasing knowledge on the interplay of LAB with the human body, specific strains with desired immune activity and adhesive properties can be selected. The biotechnological advantages of using LAB in vaccine development rely partly on the GRAS status and the good name of these bacteria. The challenge for the future vaccine development lies in harnessing the unique features of LAB, while maintaining their GRAS status. 17
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  25. 25. BioMed Central Page 1 of 12 (page number not for citation purposes) Microbial Cell Factories Open AccessReview Live bacterial vaccines – a review and identification of potential hazards Ann Detmer*1 and Jacob Glenting2 Address: 1Danish Toxicology Centre, Hørsholm, Denmark and 2Bioneer A/S, Hørsholm, Denmark Email: Ann Detmer* - ad@dhigroup.com; Jacob Glenting - jag@bioneer.dk * Corresponding author Abstract The use of live bacteria to induce an immune response to itself or to a carried vaccine component is an attractive vaccine strategy. Advantages of live bacterial vaccines include their mimicry of a natural infection, intrinsic adjuvant properties and their possibility to be administered orally. Derivatives of pathogenic and non-pathogenic food related bacteria are currently being evaluated as live vaccines. However, pathogenic bacteria demands for attenuation to weaken its virulence. The use of bacteria as vaccine delivery vehicles implies construction of recombinant strains that contain the gene cassette encoding the antigen. With the increased knowledge of mucosal immunity and the availability of genetic tools for heterologous gene expression the concept of live vaccine vehicles gains renewed interest. However, administration of live bacterial vaccines poses some risks. In addition, vaccination using recombinant bacteria results in the release of live recombinant organisms into nature. This places these vaccines in the debate on application of genetically modified organisms. In this review we give an overview of live bacterial vaccines on the market and describe the development of new live vaccines with a focus on attenuated bacteria and food-related lactic acid bacteria. Furthermore, we outline the safety concerns and identify the hazards associated with live bacterial vaccines and try to give some suggestions of what to consider during their development. Background Live vaccines have played a critical role from the begin- ning of vaccinology. Indeed, the very first vaccination experiment in the Western world was Jenner's inoculation of a boy with the milder cowpox virus to protect against the deadly smallpox. Although effective the technology has safety problems associated with the risk of reversion to a virulent organism and the possibility of causing dis- ease in immune compromised individuals. Within the last 20 years the concept of live vaccines gains renewed inter- est due to our increased immunological understanding and the availability of molecular techniques making the construction of safer live vaccines possible. This opens for the development of new live bacterial vaccines that can avoid the downsides of parenterally administered vaccine because it (i) mimics the route of entry of many patho- gens and stimulate the mucosal immune response (ii) can be administered orally or nasally avoiding the risk associ- ated with contaminated needles and need for a profes- sional healthcare infra structure (iii) has a simple down stream processing. Broadly, live bacterial vaccines can be classified as a self-limiting asymptomatic organism stimu- lating an immune response to one or more expressed anti- gens. Published: 23 June 2006 Microbial Cell Factories 2006, 5:23 doi:10.1186/1475-2859-5-23 Received: 25 April 2006 Accepted: 23 June 2006 This article is available from: http://www.microbialcellfactories.com/content/5/1/23 © 2006 Detmer and Glenting; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 22
  26. 26. Microbial Cell Factories 2006, 5:23 http://www.microbialcellfactories.com/content/5/1/23 Page 2 of 12 (page number not for citation purposes) Furthermore, live bacterial vaccines can be designed to induce an immune response to itself or to a carried heter- ologous antigen. A non-virulent or attenuated derivative of the pathogen is used to induce a response to the bacte- rium itself. When used as a vaccine vehicle the bacterium expresses an antigen from another species. Most com- monly, these vaccine vehicles are based on either attenu- ated pathogens or bacteria used in the food industry. Both classes of bacteria deliver the vaccine component to the immune system whereby immunization may benefit from the bacterium's intrinsic adjuvant. The vaccine compo- nent to be delivered can be either protein or DNA. In addi- tion, the vaccine component may be a classical antigen but may also be allergens or therapeutics. A recent devel- opment is the use of invasive bacteria for the delivery of plasmid DNA vaccines to mammalian cells obtaining in vivo synthesis of the plasmid-encoded antigen. As such, the applications of live bacterial vaccines are extensive and has lead to more than 2000 published papers. How- ever, only very few of the promising candidates have sur- vived the licensing process and become registered [1] illuminating the difficulty in developing a commercial live vaccine. One typhoid vaccine (Ty21a) contains live attenuated Salmonella typhi and is administered orally either as a liquid or as acid resistant capsules. Both formu- lations require three doses within one week to give immu- nity. The other registered vaccine based on live bacteria is against cholera and is given orally as a single dose of atten- uated Vibrio cholerae (CVD 103-HgR) in liquid formula- tion. This vaccine is used in a lower dose (5 × 108 live bacteria) for travellers from non-endemic regions and a one log higher dose for residents in endemic regions (5 × 109 live bacteria). The very few examples of live bacterial vaccines on the market may be due to lack of success in clinical trials. However, we believe that the safety of these vaccines is another issue. Indeed, prophylactic vaccines are given to healthy people and despite excellent safety record they remain targets of un-substantiated allegations by anti vaccine movements. Furthermore, future live vac- cines will most likely be either targeted mutagenised or equipped with foreign antigens and therefore considered recombinant. As such, they fall into the debate on releas- ing genetically modified organisms into nature. The feasi- bility of this new vaccine strategy will therefore in particular depend on considerations of safety issues. We believe that considering safety issues alongside the scien- tific consideration early in vaccine development will facil- itate its public acceptance and its entrance to the market. We therefore felt compelled to outline a review about live vaccines and their safety aspects. Attenuated pathogens as vaccines and vaccine vehicles Lindberg [2] has excellently reviewed the history of live bacterial vaccines. The first use of a live bacterial vaccine was in Spain in 1884 and consisted of a subcutaneous injection of weakened Vibrio cholerae. This study was fol- lowed a few years later by field trials in India with a more efficacious V. cholerae vaccine, however still parenteral. The first live oral V. cholerae vaccine candidate did not appear until the 1980s. Later the V. cholerae strain CVD 103 Hg-R has been found to be both safe and immuno- genic after a single oral dose. In 1996 a bivalent vaccine waspresented including two strains of V. cholerae called CVD 103 Hg-R and CVD 111 [3]. However, later on prob- lems with attenuation of strain CVD 111 appeared [4]. The development of the other registered live bacterial vac- cine began Hg-in the early 1970s using various live atten- uated S. typhi to vaccinate against typhoid fever. One proposed strain was made streptomycin-dependent, but failed to be efficacious in freeze-dried formulation [5]. Furthermore, the strain was genetically unstable and reverted to virulence. Another S. typhi strain (Ty21a) with a defect galE gene, as well as other not defined mutations, requires an external source of galactose. This strain was extensively evaluated in several field trials and has shown excellent safety record [6]. Later, other auxotrophic strains unable to synthesise essential compounds like aromatic amino acids were developed and tested on human volun- teers with variable safety and immunogenicity results [7- 10]. Attenuated live vaccines to prevent shigellosis have also been proposed. Both genetically engineered or selected mutants of Shigella have been tried but showed side effects in clinical trials and points to the need of addi- tional attenuation without hampering immunogenicity [11-13]. Kotloff et al attenuated the guanine auxotrophic Shigella flexneri 2a further by deleting two genes encoding enterotoxins [14]. In a phase 1 trial this strain with inacti- vated enterotoxin genes was better tolerated but still immunogenic compared to the guanine auxotrophic strain that contain active entoroxins. Recombinant Shigella has also been proposed as a vaccine vehicle [15]. Pathogenic Shigella has a virulence plasmid encoding proteins involved in thesecretion apparatus and proteins necessary for the entry process into human cells. This invasive capacity can be used to deliver plasmid DNA vaccines into mammalian cells [16]. Here, the delivered plasmid DNA encodes an antigen, which is expressed by the protein synthesis apparatus of the infected cells. Diaminopimelate Shigella auxotrophs undergo lysis unless diaminopimelate is present in the growth media [16]. Human cells contain low amounts of diami- nopimelate and upon entry the Shigella mutant lyse mak- ing the delivery of vaccine components more effective. Other attenuated bacteria have also been tested as vaccine vehicles of various proteins and plasmid DNA (Table 1). In conclusion, the mimicry of natural infection makes attenuated bacteria effective. The ability to deliver vaccine components of different origins like e.g., HIV [15,17,18] or piece of parasitic DNA [19] or gamete specific antigen 23
  27. 27. Microbial Cell Factories 2006, 5:23 http://www.microbialcellfactories.com/content/5/1/23 Page 3 of 12 (page number not for citation purposes) [20] make attenuated bacteria a versatile vaccinology tool. However, in spite of the efforts in constructing attenuated pathogens for use as bacterial vaccine vehicles none of them has reached the market yet. Lactic acid bacteria as vaccine vehicles The potential of using lactic acid bacteria (LAB) for the delivery of vaccine components is less exploited than attenuated pathogens. Due to their safe status and the availability of genetic tools for recombinant gene expres- sion LAB are attractive for use as vaccine vehicles. Further- more, their non-pathogenic status circumvents the need to construct attenuated mutants. However, LAB are non- invasive and the vaccine delivery to antigen presenting cells may be less effective than invasive bacteria. Still, anti- gen specific immune responses have been obtained with several LAB (Table 2). Geoffroy et al [21] used a green flu- orescent protein to visualize the phagocytosis of Lactoba- cillus plantarum by macrophages in vitro and in mice. Macrophages act as antigen presenting cells and this can explain a possible way to at least elicit a ClassII MHC receptor presentation of the antigen. Even though the transit time of Lactococcus lactis through the intestine is less than 24 h in mice [22], a potent immune response has been obtained with several antigens including tetanus toxin fragment C (TTFC). Surprisingly, a similar response was induced using dead or alive Lactococcus suggesting that in situ antigen synthesis is not essential [23]. A slightly better result was in the same study obtained with L. plantarum, but also here a similar response was induced from living or UV-light inactivated cells. Active vaccination using LAB The prospect of using live LAB as vaccine carriers has been reviewed [24,25]. The most frequently used model anti- gen is TTFC in which good results have been obtained both in intranasal and oral mice models using strains of L. plantarum and L. lactis [23,26]. Grangette et al [27] tested cytoplasmic expression of TTFC antigen in both L. plantarum and L. lactis showing protective effect in an oral mouse model. Shaw et al [28] tested both cytoplasmic and surface associated expression of same TTFC antigen and found that cytoplasmic expression was superior to surface exposed TTFC in L. lactis. In contrast, Bermúdez-Humarán et al [29] tested human papillomavirus type 16 E7 antigen sorted in different cellular compartments and found cell Table 1: Attenuated bacteria as vaccine vehicles Vaccine strain Attenuation Foreign insert Model Ref. Shigella flexneri Δasd pCMVβ Guinea pig, in vitro [80] Δasd CS3 and LTB/STm Mouse [81] ΔrfbF HIV-1 SF2Gag Mouse [17] ΔdapA ΔdapB β-gal, gene vaccine In vitro [16] ΔaroA ΔiscA gp120, gene vaccine Mouse [15] Salmonella enterica ΔaroA pCMVβ, pCMVactA and pCMVhly In vitro, mouse [82] ΔaroA ΔaroD C. tetani TTFC Mouse [83] ΔaroA ΔhtrA TTFC Mouse [83] ΔaroA+others GFP+cytokines Mouse [84] Δcya Δcrp Δasd SP10 cDNA Mouse [20] GalE + unspecified H. pylori, ureAB Human [85] Yersinia enterocolitica pYV- B. abortus, P39 Mouse [86] pYV- Ova Mouse [87] Listeria monocytogenes ΔactA Leichmania major Mouse [88] ΔactA LCM virus Mouse [89] Δdal Δdat HIV-1 gag gene vaccine Mouse [90] Δ2 M. bovis gene vaccine Mouse [91] Bordetella bronhiseptica ΔaroA TTFC Mouse [92] Erysipelotrix rhusiopatie Tn916- M. hyopneumonie Mouse, pig [93] Mycobacterium bovis unspecified P. falciparum, CSP Mouse [94] Brucella abortus Rough mutant (O-) lacZ or HSP65 Mouse [95] 24
  28. 28. Microbial Cell Factories 2006, 5:23 http://www.microbialcellfactories.com/content/5/1/23 Page 4 of 12 (page number not for citation purposes) wall-anchored antigen to induce the most potent immune response. The different outcome of these experiments may be explained by different stability of surface exposed TTFC and E7 antigen. Intracellular expression of a labile antigen can protect it from proteolytic degradation and environ- mental stress encountered at the mucosal surfaces. Genetic modification of the LAB cell wall rendering the strain more permeable increases the in vivo release of cyto- plasmic TTFC antigen and was tested by Grangette et al [27]. When administered orally these alanin racemase mutants were more immunogenic than their wild type counterparts. One explanation could be that oral immu- nization is very dependant on a sufficiently large dose of the antigen [27]. The use of live LAB as carriers of DNA vaccines has until now not been an option as they are non-invasive and therefore inefficiently deliver the plasmid DNA to the cytoplasma of antigen presenting cells. Recently Guima- rães et al [30] developed L. lactis expressing cell wall- anchored internalin from Listeria monocytogenes. This L. lactis inlA+ strain has been shown to enter eukaryotic cells in vitro, but also in vivo using an oral guinea pig model. To determine the tropism of recombinant invasive strains Critchley-Thorne el al used a perfusion bath with murine ileal tissue and tested an invasive E. coli vaccine candidate [31]. Although change of tropism of a bacterial carrier opens for targeted delivery it introduces new safety issues that should be addressed by persistence and distribution studies of the bacterial strain after vaccination. Active vaccination using recombinant L. johnsonii to treat allergy has been suggested [32]. IgE epitopes was fused to proteinase PrtB and cell wall-anchored. Subcutaneous and intranasal immunization of mice induced a systemic IgG response against human IgE. As such, allergy-induc- ing IgE may be cleared by IgG antibodies induced by the recombinant L. johnsonii. However, it remains to be proven if these antibodies are protective in human patients. In conclusion, LAB has been successfully used for active vaccination of animals like rodents (Table 2). Whether LAB will be effective as a mucosal vaccine in humans can only be answered by clinical trials. Furthermore, as the dose of recombinant LAB needed to elicit immune Table 2: LAB as vaccine vehicles Vaccine strain Foreign insert Model Ref. Lactococcus lactis C. tetani TTFC Mouse [23,96] TTFC+IL-2 or IL-6 Mouse [97] Human IL-10 Mouse [39] H. pylori ureB Mouse [98] B. abortus L7/L12 Mouse [99] S. pneumonie CPS Mouse [100] Rotavirus vp7 Mouse [101] B-lactoglobulin Mouse [102] HIV-1 gp120 Mouse [103] Malaria MSP-1 Mouse [104] SARS Nucleocapsid protein In vitro [105] E. rhusiopathiae SpaA Mouse [106] Lactobacillus plantarum TTFC Mouse [107] Allergen Der p1 Mouse [36] H. pylori (ureB) Mouse [108] Streptococcus gordonii Antibody Rat [34] Hornet venom Ag5.2 Mouse [109] TTFC Mouse [110] Lactobacillus casei B. anthracis (protective Ag) In vitro [111] SARS spike protein Mouse [112] Human papillomavirus L1 In vitro [113] Coronavirus S glycoprotein Mouse [114] S. pneumonie PsaA PspA In vitro [115] Lactobacillus zeae Antibody Rat [33] Lactobacillus johnsonii TTFC mimotope Mouse [116] 25
  29. 29. Microbial Cell Factories 2006, 5:23 http://www.microbialcellfactories.com/content/5/1/23 Page 5 of 12 (page number not for citation purposes) responses in animals is high it is unknown if the necessary dose for use in humans will be feasible and cost effective. Passive immunization using LAB Protection by preformed antibodies or antibody frag- ments is called passive vaccination. The pioneer experi- ments were based on injection of antisera produced by immunized animals like horse or sheep to combat for example rattlesnake venom. Recently, passive immunity was delivered using lactobacilli that secretes single-chain antibodies [33]. In a rat caries model, colonisation of the mouth with a L. zeae expressing a single-chain antibody fragment recognizing the adhesion molecule of Streptococ- cus mutans decreased the number of S. mutans and reduced the development of caries. Recombinant Streptococcus gor- donii displaying a microbiocidal single-chain antibody (H6) has been used to treat vaginal candidiasis in a rat model [34]. Although passive immunity has limits in its temporary nature, these results suggest that LAB elegantly can be used for the delivery of neutralising antibodies at mucosal sites. Allergy vaccines using LAB expressing allergens For a normal vaccination against an infectious disease, induction of tolerance to the infectious agent is consid- ered a side effect. This side effect is more prone to happen when vaccinating early in life [35]. However, induction of tolerance can have positive clinical implications when the purpose is to treat allergy. In a mouse model the use of a recombinant L. plantarum expressing the house dust mite allergen Der p1 as a fusion protein in the cytoplasm inhib- ited house dust mite-specific T-cell responses [36]. In this study mice were sensitized by immunization with the house dust mite peptide and then given either L. plantarum expressing Der p1 or L. plantarum without Der p1. Both strains inhibited IFN-γ production by T cells. But the decrease in production of-5 was only seen for the L. plantarum expressing the Der p1 peptide antigen. This indicates that the lactobacilli strain expressing Der p1 can suppress the cytokine milieu promoting the Th2 allergic response. Another example of the strain specific effect of LAB on induction and maintenance of oral tolerance has been shown using ί-lactoglobulin and gnotobiotic mice [37]. In this study L. paracasei (NCC 2461) was more effec- tive to induce and maintain oral tolerance in gnotobiotic mice than was L. johnsonii (NCC 533). The allergen can also be co-administered instead of recombinant expressed by the LAB. Mucosal co-application of L. plantarum or L. lactis together with birch pollen allergen Bet v1 shifted the immune response towards an anti-allergic Th1 response both in sensitized and un-sensitized animals [38]. Recom- binant strains expressing immune polarizing cytokines like IL-10 have also been developed and in vivo effects in both mice [39] and pigs [40] have been observed. More knowledge on the mechanisms behind skewing the immune response is however needed to select the proper strain with anti allergic immune polarization. Further- more, the immune regulatory effect of one strain of LAB may differ in allergic and non-allergic individuals. A down regulation in allergic persons and an immune stimulating effect in normal persons was observed when using same strain of LAB [41]. Immune stimulatory effects of LAB Among LAB's effect on the immune system there is a strain dependent induction of cytokines. Different LAB strains induce distinct mucosal cytokine profiles in BALB/c mice [42] pointing at the importance of using one strain for immune induction and another for induction of tolerance or a partial down regulation of the immune system. The same authors [43] also indicate growth phase dependent differences of orally administered LAB strains on the IgG1 (Th2)/IgG2a (Th1) antibody ratio in mice further compli- cating the process of choosing the proper strain for spe- cific modulation of the immune response. Adding to the complexity of these observations, a human study has shown that non-specific immune modulation by a given strain of L. rhamnosus (GG, ATCC 53103) differs in healthy and allergic subjects. In healthy persons the strain was immune stimulatory whereas in allergic persons it down-regulated an inflammatory response [44]. Interac- tions between different LAB strains can also interfere with the in vitro production of cytokines by dendritic cells [45]. As is shown in another study [46], two different lactoba- cilli with similar probiotic properties in vitro were shown to elicit divergent patterns of colonisation and immune response in germfree mice. Further evidence for an immune modulating effect is seen when either L. lactis or L. plantarum was used in a mouse model of birch pollen allergy [38]. In combination with birch pollen allergen Bet v1 both strains skewed the immune response from Th2 to Th1 in sensitised mice as indicated by the IFN-γ/IL- 5 ratio. The immune polarizing effect of LAB has also been observed in humans. A clinical trial showed a strain dependent immune modulation of two different LAB strains when administered together with an oral S. typhi vaccine (Ty21a) [47]. Here, thirty healthy volunteers were randomised into three groups receiving L. rhamnosus GG, L. lactis or placebo for 7 days. On days 1, 3 and 5 the Ty21a vaccine was given orally. Analysis showed a higher number of specific IgA-secreting cells in the group receiv- ing L. rhamnosus GG and a higher CR3 receptor expression on neutrophils in the group receiving L. lactis. A partial down regulation of the immune system has also been observed. Atopic children receiving 2 × 1010 L. rhamnosus GG daily for 30 days enhanced their IL-10 production in sera as well as in mitogen-induced peripheral blood mononuclear cells [41]. 26
  30. 30. Microbial Cell Factories 2006, 5:23 http://www.microbialcellfactories.com/content/5/1/23 Page 6 of 12 (page number not for citation purposes) It can be concluded that immune polarization towards either a Th2 or a Th1 response can be obtained using dif- ferent LAB. As such the intrinsic immune modulatory capacity of the LAB must be evaluated and selected to fit the purpose of vaccination. Safety concerns of the bacterial vaccine strain Several safety concerns of the bacterial vaccine strain have been raised (Table 3). Before using pathogenic bacteria for vaccination purposes, its pathogenicity must be weakened via attenuation. Attenuation usually involves deletion of essential virulence factors or mutation of genes encoding metabolic enzymes whose function is essential for sur- vival outside the laboratory. Inactivation of a metabolic gene has the advantage that the bacteria still express viru- lence determinants important to elicit a protective immune response. Appropriate stable auxotrophic strains are usually not able to replicate in the human body and can safely be used even in immune compromised individ- uals. Defined deletions of at least two metabolic essential genes are usually used [2] and decrease the probability of reversion to virulence. To reduce the risk of spreading for- eign genetic material to the environment the antigen encoding gene cassette can be inserted into the chromo- some replacing the metabolic essential gene. If the bacte- rium acquires the deleted gene it will automatically loose the antigen-encoding cassette. The use of antibiotic resist- ance genes as marker genes in vaccines is not encouraged as these genes can transfer to in the end humans and thus hamper the use of therapeutic antibiotics. Different alter- natives to antibiotic resistance marker genes have been published and should be used as soon as possible in the developmental process of a vaccine [48-50]. Another concern using live bacterial vaccines is the onset of autoimmune responses like arthritis especially in patients with the HLA-B27 tissue type [51]. However, the risk is certainly lower than after natural infection. The occurrence of such side effects can best be followed by post launch monitoring and must always be evaluated against the health risks associated to the disease itself. A theoretical side effect of vaccines is the possible induction of autoimmune reactions. However, there is no recom- mendation to avoid vaccination of people with an ongo- ing autoimmune disease like rheumatoid arthritis or systemic lupus erythematosus if vaccination otherwise is motivated [52]. In contrast, immune-compromised hosts can have difficulties in handling replicating live attenu- ated vaccines and should therefore not be vaccinated with such vaccines. However, new ways of further attenuating bacteria like combining auxotrophy with deletions of vir- ulence genes [14] may open for the use of live vaccines to immune-compromised hosts. In addition, immune-com- promised people close to hosts vaccinated with live atten- uated vaccines should be aware of the risk of cross contamination with the vaccine strain. Table 3: Safety concerns of the vaccine strain Systemic disturbance Systemic infection Conversion from avirulent to virulent bacterium Translocation to organs Disturbance of digestive processes Inhibition of bacterial production of nutrients Immune system Absorption of allergens through the intestinal epithelium Induction of tolerance to pathogen instead of immunity Induction or potentiation of autoimmunity Bacterial mimicry of self-antigen Metabolites Production of harmful/undesired metabolites including enzymatic activities Breakdown of chemicals to toxic metabolites Implications for natural flora in GI tract Permanent colonisation of cell substrate in the intestine Gene/plasmid transfer to host's indigenous flora "Competitive exclusion" of indigenous flora Unintentional transferral of cell substrate Unintentional transfer to other individuals Unintentional transfer to and viability/propagation in environments other than the intestines Contamination Extraneous or perceived adventitious DNA components should be removed (possibility of oncogenicity). 27
  31. 31. Microbial Cell Factories 2006, 5:23 http://www.microbialcellfactories.com/content/5/1/23 Page 7 of 12 (page number not for citation purposes) Adverse examples of human live vaccine strains causing death and illness among domesticated animals are rare but existent. In Mongolia in autumn 1979 the H1N1 influenza A vaccine virus may have caused a severe influ- enza epizootic among camels [53]. No examples of human bacterial vaccines causing problems among ani- mals have been found in the literature but the possibility exists and has to be both tested and evaluated before release of a live bacterial vaccine. In general the spread of live bacterial vaccines to the environment is a concern. However, attenuated human pathogens are usually not adapted to live outside its host. Therefore survival in the environment is usually short. Vaccines based on recom- binantLAB may result in the release of these bacteria in nature, as LAB are more suited to survive in the nature. Also here the use of auxotrophic mutants unable to repli- cate in the environment may be the answer. Releasing gene-modified organisms into the environment can cause debate and precautions to eliminate its spread are essen- tial. To avoid escape into the environment of the geneti- cally modified organism, Steidler et al. [40] replaced the thyA gene with the expressioncassette for human IL-10. As a consequence, the L. lactis mutant is dependant on thy- midine or thymine for growth, which is present in low amounts in nature and in the human body. Furthermore, acquirement of an intact thyA gene would recombine the transgene out of the genome, resulting in reversion to its wild type state. Safety concerns of the antigen encoding sequence In live bacterial vaccines the antigen-encoding gene is either plasmid located or integrated in to the chromo- some. In both cases several safety concerns can be raised (Table 4). For plasmid-encoded antigens the fate of the plasmid in the vaccinee must be evaluated. The use of a prokaryote plasmid replication unit of narrow host range can limit the horizontal plasmid transfer to other bacteria present in the vaccinated individual and prevent unde- sired persistence of the plasmid. In particular for plasmid DNA vaccines a study should identify which cells take up and/or express the DNA and what is the fate of the DNA within those cells as well as for how long the DNA persists in the cells [54]. Nasal administration of a naked DNA- vaccine in mice led to some accumulation of plasmid DNA in the brain [55] illustrating the diffusion of the plasmid after immunization. The amount of accumulat- ing plasmid that is acceptable outside the target cells needs to be further clarified. The recombinant plasmid harboured by bacterial vaccine vehicles may integrate in the genome of the recipient and potentially cause oncogenesesis. Concerns about the potential oncogenicity of biological products like contin- uous cell line products (CCL), DNA vaccines and gene therapy products have been raised [54]. In CCLs foreign DNA should be avoided in the final product and a limit has been defined as for maximal residual amount per human dose. In DNA vaccines DNA is obvious present but insertion of DNA should be avoided. Finally in the gene therapy product DNA is both present and inserted but insertional oncogenesis should be avoided. Integration of foreign DNA into the host genome is by definition inser- tional mutagenesis and can induce oncogenesis. There are three ways the extraneous DNA can lead to transforma- tion [54]: insertion of an active oncogene, insertional acti- vation of a host proto-oncogene, and by insertional deactivation of a host suppressor gene. The mechanism behind DNA integration into the chromosome is either by random integration, homologous recombination or retro- viral insertion [56]. The most probable cause of unwanted integration is by random integration which occurs at a fre- quency of approximately 10-4 [54]. Unwanted integration by homologous recombination and retroviral insertion can be avoided by omission of sequences necessary for insertion [57]. Analysing the antigen encoding unit car- ried by the bacteria for human homologous sequences and eliminating these can limit the integrative possibility. Although not similar to vaccination with bacteria the clin- ical trials using retroviral therapy can give some indica- tions of the hazards of DNA integration [58]. Indeed, activation of oncogenes is a risk associated with retroviral vaccination [59]. The report of adverse effects in a French gene therapy study, where 2 out of 10 patients developed leukaemia within 3 years of [60,61], illustrates occurrence of such a transformation event by activation of a proto- oncogene. Calculation of the probability of a harmful Table 4: Safety concerns of the antigen encoding sequence For protein and DNA vaccines Transfer of undesired genes via plasmid Transfer of vector to indigenous flora Open reading frames coding for injurious peptides (allergens) Imprecise transcription and translation Specifically for DNA-vaccines Persistence of DNA Permanent expression of the foreign antigen Formation of anti-DNA antibodies Transformation event Spread of antibiotic resistance genes 28
  32. 32. Microbial Cell Factories 2006, 5:23 http://www.microbialcellfactories.com/content/5/1/23 Page 8 of 12 (page number not for citation purposes) effect due to integration of foreign DNA into host genome has been performed and was found to be less than 10-16 to 10-19 per DNA molecule [62]. This frequency must be put in relation to the spontaneous mutation frequency which has been estimated in humans to occur at the rate of 1 in every 50 million nucleotides incorporated during DNA replication. This means that a human cell with 6 × 109 base pairs will contain 120 new mutations [63]. Possible insertions into the chromosome can be tested by PCR techniques [64,65]. However, insertion due to ran- dom integration can be difficult to detect this way [64]. Furthermore, insertion of foreign DNA can effect gene activity at sites remote from insertion [66]. Different ani- mal trial has foreseen possible adverse effects like in the following two examples. Foreign DNA ingested by mice has been shown to be covalently linked to mouse DNA [67]. Foreign DNA has also been shown in association with chromosomes in fetuses born by mice fed orally with bacteriophage M13 DNA [68]. There is however, no evi- dence for a germ line transmission of ingested foreign DNA [66]. The de novo methylation that frequently occurs with integrated foreign DNA has been suggested as being a natural defence mechanism [69]. In conclusion, integration of the plasmid harboured by bacterial vaccine vehicles is a potential hazard. Integration of gene therapy vectors has been observed, but omitting sequences driving the insertion may limit the possibility for integration of the plasmid carried by the bacterium. Plasmids for heterologous gene expression are usually preferred due to its multi copy nature and higher gene dosage. However, placing the antigen encoding genes on to the bacterial chromosome may limit the spread of the genes. The route of administration of the vaccine may also be important when evaluating hazards. As live bacterial vaccines is fit for mucosal administration one must remember that ingestion of foreign DNA does occur every day with our food and is as such not new. Peptides can be absorbed through the mucosa and some may induce an allergic reaction. The existence of genes in the bacterial vaccine coding for such potential allergens or other injurious peptides can be checked beforehand searching for homologies to known allergens, as the full sequence of the bacteria and plasmid should be known. Vaccination using live bacterial vaccines or exposure to the natural infections can lead to the formation of auto reactive antibodies, especially in people prone to autoim- mune diseases. However, the half life of the induced auto antibodies is usually short [70] and their specificity usu- ally polyclonal [71]. Several authors have tried to eluci- date the possibility of a link between autoimmunity and vaccination [70,72-77] and much controversy in this mat- ter is still existing. However, convincing data establishing a link between vaccination and autoimmunity in man are still not presented. In a mouse model a difference in clin- ical outcome was observed in two different mouse strains in relation with auto-antibodies induced by vaccination with dendritic cells loaded with apoptotic thymocytes [78]. In normal BALB/c mice the presence of post vaccina- tion autoantibodies was not associated with any clinical or histological sign of autoimmunity. However, in mice prone to autoimmunity (NZBxNZW) F1 a severe pathol- ogy attributed to autoimmunity was observed. This differ- ence in outcome attributed to the difference in genotype has also been observed in humans and it can be con- cluded that susceptibility to autoimmunity is determined more by genetic factors than by vaccine challenge despite the formation of post vaccination auto-antibodies [77]. A vaccination or treatment with adjuvant can also activate regulatory T cells and can thus be used as a method to pre- vent autoimmune disease if applied at the right time [79]. In the future tailor-made vaccines might be the solution for individuals with a genetic profile prone to autoimmu- nity. Conclusion Both attenuated bacteria like salmonella and food related lactic acid bacteria have been developed as live vaccines suitable for oral administration. Today, live vaccines based on attenuated S. typhi and V. cholerae are available. The development of bacterial vaccine vehicles carrying a heterologous gene or a DNA vaccine is more problematic and none has yet reached the market. Several bacteria have been suggested as vaccine vehicles and especially lac- tic acid bacteria are promising. Their safe status and immune modulating capacity have been tested using diverse vaccine components like antigens from infectious diseases, allergy promoting proteins and therapeutic anti- bodies. However, considerable safety issues against live vaccine vehicles can be raised. Their recombinant nature calls for a bio containment strategy and auxotroph mutants may be the answer. The bacterial host must be fully sequenced and evaluated using bioinformatics tools for the production of allergy inducing peptides. The anti- gen encoding gene cassette must be sequenced and homologies to self proteins or allergy inducing proteins should be addressed. Especially bacteria carrying recom- binant plasmids the probability of horizontal gene trans- fer to other bacteria present should be avoided by using host restricted replication units. Furthermore, the plas- mids should be evaluated for sequences facilitating inte- gration into the human genome. Authors' contributions The authors contributed equally to this work. Acknowledgements 29
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