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Struggling to get a universal meningococcal vaccineand novel uses for bacterial toxins in cancer treatment

Craig Daniels,1 Tino Krell,2 Carmen Michán3 andJuan L. Ramos2*1Structural Proteomics in Toronto, UHN and Universityof Toronto, Banting and Best Department of MedicalResearch; C.H. Best Institute 112 College Street, M5G1L6, Toronto, Ontario, Canada.2Consejo Superior de Investigaciones Científicas,Estación Experimental del Zaidín, Dept. ofEnvironmental Protection, C/ Prof. Albareda, 1, E-18008Granada, Spain.3Universidad de Córdoba, Campus de Rabanales, Dept.of Biochemistry and Molecular Biology, Edificio SeveroOchoa C-6, 2a Planta, 14071, Córdoba, Spain.

In the Early View section of Microbial Biotechnologyonline Engedal and colleagues (2010) provide an exten-sive review of the Shiga toxin and its use in targetedcancer therapy and imaging. Shiga toxins are producedby Shigella dysenteriae and some strains of Escherichiacoli. The toxins are composed of two non-covalentlylinked, modular parts: the A moiety (StxA) which containsthe enzymatically active A1 fragment, and the non-toxic,pentameric binding moiety (StxB). A unique property ofStx is that it binds specifically to the glycosphingolipidglobotriaosylceramide (Gb3) located on the surface oftarget cells and is then internalized by endocytosis. Con-sequently, in toxin-sensitive cells, the Stx/Gb3 complex istransported to the endoplasmic reticulum through theGolgi apparatus, the StxA is then translocated to thecytosol, where it irreversibly inhibits protein synthesis bymeans of modification of ribosomal 28S RNA. Interest-ingly, Gb3 shows a relatively restricted expression innormal human tissues, and has been reported to be highlyexpressed in many types of cancers.

Engedal and colleagues cover the introduction of Stxand its intracellular transport, a description of Gb3 and thecurrent methods being utilized to detect its cellularexpression, and a synopsis of previously publishedreports detailing Gb3 overexpression in human cancers.

Surprisingly, enhanced expression of Gb3 has beenreported in cancers of diverse tissues including but notlimited to colorectal carcinoma, primary human testicularcancer tissues and breast and ovarian cancers (Nudel-man et al., 1983; Ohyama et al., 1990; LaCasse et al.,1999). These findings along with the relatively restrictedexpression of Gb3 in normal human tissues raise theprospect that Stx derivatives, which specifically bind toGb3, could be used for targeted therapy and imaging oftumours. However, because the viability of such medicalapplications depends on several factors further researchis warranted into the potential application of Stx or StxBderivatives in cancer medicine, questions that arerequired to be addressed are: to what degree Gb3 isexpressed at the cell surface of normal cells, and to whatdegree and how Stx binds to and is taken up and trans-ported inside both normal and malignant cells. These andother aspects related to the use of Stx for imaging andtargeted cancer therapy of Gb3-expressing cancers areextensively discussed in this current review.

Neisseria meningitidis is a virulent human pathogenand continues to be a major cause of mortality throughoutthe world with children and adolescents forming theprimary risk group. The incidence of invasive meningo-coccal disease is low but infections can lead to deathwithin several hours if untreated. Neisseria meningitidiscan be classified into five serogroups, namely A, B, C,W-135 and Y. The capsular polysaccharides of serogroupA, C, W-135 and Y are highly immunogenic and conse-quently several polysaccharide-based vaccines coveringthese serotypes have been licensed. However, the cap-sular polysaccharide of serogroup B (MenB) resemblesstructures present on human cells which consequentlyresults in a poor immunogenicity of the bacterial polysac-charide and does not permit its use as vaccine antigen. Intheir review in Microbial Biotechnology titled ‘Vaccinedevelopment against Neisseria meningitidis’ Ulrich Vogeland Heike Claus summarize the challenges in the devel-opment and optimization of vaccines against this patho-gen (Vogel and Claus, 2010).

The major challenge consists in the development of auniversal N. meningitidis vaccine which includes MenB.The need for such a vaccine is underscored by data that

*For correspondence. E-mail juanluis.ramos@eez.csic.es; Tel. (+34)958 181608.

Microbial Biotechnology (2010) 3(4), 359–361 doi:10.1111/j.1751-7915.2010.00195.x

© 2010 The AuthorsJournal compilation © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd

show that MenB infections amount to around two-thirds ofall meningococcal infections in Germany. Since a vaccineapproach using unmodified polysaccharide is not feasiblefor MenB, the authors summarize alternative strategieswhich include the use of protein subunit antigens, mimo-tope antigens or the use chemically modified polysaccha-ride antigens. Due to the resemblance of the capsularpolysaccharides with structures present on human cellsthe induction of autoantibodies using modified polysac-charide as antigens cannot be ruled out. In addition, thelack of success in generating a MenB vaccine using tra-ditional approaches has led to the exploration of wayswhich are novel in vaccinology, such as the use of inac-tivated lipopolysaccharide (LPS) as antigen. LPS is ahighly toxic compound and its detoxification is a delicateissue and future studies are required to reveal the effi-ciency of this approach.

On of the main challenges in vaccine development isthe evaluation of the efficiency of new vaccine candidatesthe testing is seriously hampered by the low incidence ofthe meningococcal disease and implies that vaccine effi-ciency cannot be determined in clinical trials. Therefore,other surrogates of protection need to be used, such asbactericidal activity of the induced antibodies, to estimatethe efficiency of a vaccine candidate.

Another major difficulty is related to the fact that thehuman nasopharynx is the natural habitat of N. meningiti-dis and immune selection drives an extensive evolution ofsurface antigens. As a result the meningococcal popula-tion is diverse and dynamic. As a consequence the issueof strain coverage of any vaccine prototype needs to beaddressed, i.e. the vaccine efficiency towards a panel ofstrains has to be monitored. The extent of strain coverageis likely to be an important criterion for vaccine licensing.Humans are also host to non-pathogenic meningococci,which were shown to contribute to natural immunityagainst disease. Therefore, care needs to be taken indefining the broadness of the vaccine. The vaccine shouldstrongly impact on a wide range of different pathogenicmeningococci but should preferentially not react with thebeneficial, non-pathogenic strains.

A number of MenB vaccine prototypes are currentlyunder investigation. Many questions concerning thesevaccines will only be answered following licensure, suchas their efficiency, impact on non-pathogenic strains andthe frequency of vaccine-induced immune escape of thebacteria. Therefore, a first generation MenB vaccine willonly be a starting point for the continuous developmentand optimization of such a vaccine. The speed by whichinvestigation advances will determine whether childrenand adolescents of the generations to come will be pro-tected from this dreadful disease.

Continuing on the vaccine development topic is thereview written by Kroll and colleagues; plasmids serve as

important tools for the construction of recombinant micro-organisms with multiple biotechnological potential. Theauthors present an extensive review dealing with differentways to create stable plasmids while avoiding antibioticresistances (Kroll et al., 2010). The article includes a cleardescription of the diverse plasmid addiction systems(PASs) based on toxin/antitoxin (TA), metabolic require-ments or operator repressor titration. Following a briefdescription of the latter two mechanisms of plasmid sta-bility, the authors provide a substantial narrative of multipleapplications which utilize the TA system. All of the appli-cations combine a stable toxin with an unstable (or lessstable) antitoxin, either of a proteinaceous or of a nucleo-tidic nature. Since the initial description of the first TAsystem based on the hok gene for programmed cell death(PCD) by S. Molin and colleagues (1987) these systemshave been widely used in bioremediation. Of particularinterest are the suggestions of PAS applications in thedesign of live vaccine therapies capable of responding toenvironmental signals and on the construction ofantibiotic-free genetic tool/vectors for the study of highlypathogenic organisms. The authors anticipate that the useof new PASs, both natural and artificial, for the construc-tion of heterologous expression systems will reduce notonly their environmental risk but also their productioncosts. Clearly further research in this exciting field willprovide us with many potential biotechnologically usefultools.

Pathogens pose a significant threat to human andanimal health and are an important facet of robust agri-cultural practices; naturally, dealing with these pathogensis of utmost importance. This subject has been dealt withbefore in Microbial Biotechnology by Di Stefano and col-leagues (2009) who reported that biofilms formed by Sta-phylococcus aureus and S. epidermidis (two referencestrains and two ocular isolates) were very relevant to theoverall virulence of the bacteria. Based on this article andothers Daniels and colleagues (2010) proposed that reli-able detection tools for rapid and sensitive analysis andtyping of infectious pathogens are needed, a topic that ispresently undertaken by Kostic and colleagues (2010) inMicrobial Biotechnology. The authors present a diagnosticmicroarray for the detection of relevant bacterial food- andwater-borne pathogens. Microarray technology offersseveral advantages in comparison with conventionalmicrobiological culture-based techniques and othermolecular methods, including the possibility of parallel,specific and rapid detection of many different organismsin one single assay. The current microarray platform isbased on sequence-specific end labelling of oligonucle-otides. The extended probe set consists of 63 oligonucle-otides targeting the gyrB gene of the 24 most commonfood- and water-borne pathogens. The gene gyrB is arobust phylogenetic marker (Yamamoto and Harayama,

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© 2010 The AuthorsJournal compilation © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Microbial Biotechnology, 3, 359–361

1996). Another relevant feature of this detection method issensitivity. The sensitivity of microbial diagnostic microar-rays is defined as the lowest amount of nucleic acid (cells)needed for successful detection (absolute sensitivity). Themicroarray presented by Kostic and colleagues alloweddetection of as few as 104 cfu. Another key issue in theuse of array technology is validation; Kostic and col-leagues (2010) approached this by using a set of refer-ence strains and a set of spiked environmental samples.The reliability of the obtained data was additionallyverified by independent analysis of the samples usingfluorescence in situ hybridization and conventional micro-biological reference methods. Last but not least themicroarray-based pathogen detection microarray wascompared with the current microbiological referencemethods and provided comparable results. Thus, theadvantage of microarray technology for the detection ofmultiple pathogenic strains is clear; hopefully this technol-ogy will be functionally implemented in the near future.

References

Daniels, C., Espinosa-Urgel, M., Niqui-Arroyo, J.L., Michán,C., and Ramos, J.L. (2010) Metabolic engineering, newantibiotics and biofilm viscoelasticity. Microb Biotechnol 3:10–14.

Di Stefano, A., D’Aurizio, E., Trubiani, O., Grande, R., DiCampli, E., Di Giulio M., et al. (2009) Viscoelastic proper-ties of Staphylococcus aureus and Staphylococcus epider-midis mono-microbial biofilms. Microb Biotechnol 2: 634–641.

Engedal, N., Skotland, T., Torgersen, M.L., and Sandvig, K.(2010) Shiga toxin and its use in targeted cancer therapy

and imaging. Microb Biotechnol (in press): doi:10.1111/j.1751-7915.2010.00180.x.

Kostic, T., Stessl, B., Wagner, M., Sessitsch, A., andBodrossy, L. (2010) Microbial diagnostic microarray forfood- and water-borne pathogens. Microb Biotechnol 3:444–454.

Kroll, J., Klinter, S., Schneider, C., Voß, I., and Steinbüchel,A. (2010) Plasmid addiction systems: perspectivesand applications in biotechnology. Microb Biotechnol (inpress): doi:10.1111/j.1751-7915.2010.00170.x.

LaCasse, E.C., Bray, M.R., Patterson, B., Lim, W.M., Peram-palam, S., Radvanyi, L.G., et al. (1999) Shiga-like toxin-1receptor on human breast cancer, lymphoma, andmyeloma and absence from CD34(+) hematopoietic stemcells: implications for ex vivo tumor purging and autologousstem cell transplantation. Blood 94: 2901–2910.

Molin, S., Klemm, P., Poulsen, K., Biehl, H., Gerdes, K., andAndersson, P. (1987) Conditional suicide system for con-tainment of bacteria and plasmids. Nat Biotechnol 5: 1315–1318.

Nudelman, E., Kannagi, R., Hakomori, S., Parsons, M., Lip-inski, M., Wiels, J., et al. (1983) A glycolipid antigen asso-ciated with Burkitt lymphoma defined by a monoclonalantibody. Science 220: 509–511.

Ohyama, C., Fukushi, Y., Satoh, M., Saitoh, S., Orikasa, S.,Nudelman, E., et al. (1990) Changes in glycolipid expres-sion in human testicular tumor. Int J Cancer 45: 1040–1044.

Vogel, U., and Claus, H. (2010) Vaccine developmentagainst Neisseria meningitidis. Microb Biotechnol (in press):doi:10.1111/j.1751-7915.2010.00178.x.

Yamamoto, S., and Harayama, S. (1996) Phylogenetic analy-sis of Acinetobacter strains based on the nucleotidesequences of gyrB genes and on the amino acidsequences of their products. Int J Syst Bacteriol 46: 506–511.

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© 2010 The AuthorsJournal compilation © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Microbial Biotechnology, 3, 359–361