Commensal bacteria armed against biological warfare
1
SIGA Pharmaceuticals Inc. (New York, NY,USA) has been awarded a research grant of over $800 000 by the US Defense Advanced Research Projects Agency (DARPA) to develop vaccines and antitoxins to counteract the effects of biological warfare agents. SIGA’s technology, which Dennis Hruby (vice-president of research at SIGA) describes as ‘potentially one of the biggest breakthroughs in vaccine technology’, uses commensal bacteria to deliver any protein of interest continuously to an appropriate mucosal surface. ‘Because most infections begin at mucosal sites, an attractive strategy to prevent such infections is to induce an immune response geared to surface molecules of pathogens when they contact mucosal surfaces’, explains Vincent Fischetti of Rockefeller University (New York, NY, USA), one of SIGA’s founders. The trick is to engineer harmless bacteria that normally inhabit mucosa, such as the oral commensal Streptococcus gordonii, to express a foreign protein on its cell surface. This is done by exploiting the highly conserved anchor motif, which is present on most surface proteins of Gram-positive bacteria and helps to attach them to the cell surface. One protein that contains the anchor motif is the M protein. By genetically deleting much of its surface-exposed segment and replacing it with the foreign protein, the bacterium can be made to express any antigen on its surface. SIGA use an Escherichia coli plasmid that integrates into the host bacterium’s chromosome (Fig. 1), rather than remaining episomal. This ensures that expression of the gene remains stable. The recombinant commensal can then be allowed to colonize a mucosal site, where it will continue to express the foreign protein over many generations. If the foreign protein is antigenic, it will then stimulate a potent mucosal immune response. ‘We don’t know exactly how this works, but in experiments in mice with a vector-borne vaccine against group A streptococci, we found that animals colonized with the recombinant bacteria produced secretory IgA, serum IgG and a cellular response to the recombinant protein’, says Fischetti. ‘We believe the recombinant organisms are taken into the M cells (specialized epithelial cells that transcytose pathogens and antigens across the mucosal surface to the lymphoid tissue beneath) as part of the normal surveillance of the mucosal surface, and, in doing so, an immune response is induced to the recombinant protein.’ The same technique can be used to provide short-term protection against agents of biological warfare, by engineering the bacteria to express neutralizing agents instead of antigens. To combat agents of biological warfare, SIGA plans to express single-chain antibodies or bacterial antitoxins on the surface of the commensal bacteria. Once established on the relevant mucosal surface, the organisms should then neutralize any incoming toxins. ‘By combining a specific antigen with a specific commensal’, says Fischetti, ‘vaccines or neutralizing agents could be tailored to specific antigens.’Passive protection with immunoglobulins, as with any non-humanized antibodies, would generate a host immune response that would reduce their long-term effectiveness. ‘If short-term protection is successful, single-chain humanized antibodies could be engineered for longer protective immunity,’says Fischetti. ‘The promise of this technology is to produce a vaccine or neutralizing agent in a very short time’, Fischetti adds. ‘Our ability to mobilize countermeasure agents quickly could save many lives in the event of an attack.’This should allow SIGA to meet the US military’s need for rapidly deployable defences against agents of biological warfare. ‘If possible, the organism that is producing, say, an antitoxin would be administered to the population before exposure, so that, when they are exposed, the toxin would be neutralized’, says Fischetti. Oral or nasal delivery probably offers the best protection against biological warfare agents, he adds, because most of them would attack the respiratory mucosa. Injection would be less effective because it bypasses the mucosal response. The development of mucosal vaccines against agents of biological warfare is a departure for SIGA’s research team. So far, the company has been concentrating on developing a vaccine to prevent throat infections caused by group A streptococci. Fischetti expects US Food and Drug Administration approval to begin clinical trials with the vaccine, which has a group A streptococcal antigen expressed on the surface of S. gordonii, later this year. About 20 million cases of group A streptococcal infections occur each year in the USA and, at present, there is no vaccine for them. Untreated, streptococcal sore throat can progress to rheumatic fever. As the anchor motif for surface proteins is highly conserved in Gram-positive bacteria, applications of this technology need not stop at protecting the oral and respiratory mucosa from attack: a wide variety of commensals could be engineered for surface expression and used to deliver antigens to intestinal and vaginal sites too. Moreover, says Fischetti, ‘the prolonged exposure of the immune system to a recombinant antigen achieved by stable colonization of a recombinant commensal after a single immunizing dose could be a safe and effective way of overcoming the need for repeated doses of antigen’. Dorothy Bonn 369 N e w s MOLECULAR MEDICINE TODAY, SEPTEMBER 1998 Copyright ©1998 Elsevier Science Ltd. All rights reserved. 1357 - 4310/98/$19.00 Figure 1. Chromosomal integration of the fusion protein gene into Streptococcus gordonii. The plasmid (a) contains a construct encoding the signal sequence (S) and the extreme N terminus (N) of the S. gordonii M protein, the foreign protein, the M protein anchor motif and ermC, which confers erythromycin resistance. This is used to transform (b) S. gordonii cells that have been engineered to contain a cat gene (which confers chloramphenicol resistance) flanked by sequences that are homologous to each end of the plasmid construct. The cat gene construct is placed in front of a strong promoter. The plasmid becomes linearized during transfor- mation and recombines with the sequences flanking the cat gene. This results in (c) the fusion gene becoming integrated into the S. gordonii chromosome. Recombinants can then be selected for erythromycin resistance and the loss of chloramphenicol resistance. Modified from Fischetti, V.A. (1996) ASM News 62, 405–410. Linearized plasmid Chromosome of S. gordonii Chromosome of S. gordonii S S N N Foreign protein Foreign protein Anchor Anchor Recombination ermc ermc cat P P a b c Commensal bacteria armed against biological warfare
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Transcript of Commensal bacteria armed against biological warfare
SIGA Pharmaceuticals Inc. (New York, NY, USA)has been awarded a research grant of over $800000 by the US Defense Advanced ResearchProjects Agency (DARPA) to develop vaccinesand antitoxins to counteract the effects ofbiological warfare agents. SIGA’s technology,which Dennis Hruby (vice-president of researchat SIGA) describes as ‘potentially one of thebiggest breakthroughs in vaccine technology’,uses commensal bacteria to deliver any protein ofinterest continuously to an appropriate mucosalsurface. ‘Because most infections begin atmucosal sites, an attractive strategy to preventsuch infections is to induce an immune responsegeared to surface molecules of pathogens whenthey contact mucosal surfaces’, explains VincentFischetti of Rockefeller University (New York,NY, USA), one of SIGA’s founders.
The trick is to engineer harmless bacteria thatnormally inhabit mucosa, such as the oralcommensal Streptococcus gordonii, to express aforeign protein on its cell surface. This is done byexploiting the highly conserved anchor motif,which is present on most surface proteins ofGram-positive bacteria and helps to attach themto the cell surface.
One protein that contains the anchor motif isthe M protein. By genetically deleting much of itssurface-exposed segment and replacing it with theforeign protein, the bacterium can be made toexpress any antigen on its surface. SIGA use anEscherichia coli plasmid that integrates into thehost bacterium’s chromosome (Fig. 1), rather thanremaining episomal. This ensures that expressionof the gene remains stable.
The recombinant commensal can then beallowed to colonize a mucosal site, where it willcontinue to express the foreign protein over manygenerations. If the foreign protein is antigenic, itwill then stimulate a potent mucosal immuneresponse. ‘We don’t know exactly how this works,but in experiments in mice with a vector-bornevaccine against group A streptococci, we foundthat animals colonized with the recombinantbacteria produced secretory IgA, serum IgG and acellular response to the recombinant protein’, saysFischetti. ‘We believe the recombinant organismsare taken into the M cells (specialized epithelialcells that transcytose pathogens and antigensacross the mucosal surface to the lymphoid tissuebeneath) as part of the normal surveillance of themucosal surface, and, in doing so, an immuneresponse is induced to the recombinant protein.’
The same technique can be used to provideshort-term protection against agents of biologicalwarfare, by engineering the bacteria to express
neutralizing agents instead of antigens. To combatagents of biological warfare, SIGA plans to expresssingle-chain antibodies or bacterial antitoxins onthe surface of the commensal bacteria. Onceestablished on the relevant mucosal surface, theorganisms should then neutralize any incomingtoxins. ‘By combining a specific antigen with aspecific commensal’, says Fischetti, ‘vaccines orneutralizing agents could be tailored to specificantigens.’ Passive protection with immunoglobulins,as with any non-humanized antibodies, wouldgenerate a host immune response that would reducetheir long-term effectiveness. ‘If short-termprotection is successful, single-chain humanizedantibodies could be engineered for longer protectiveimmunity,’ says Fischetti.
‘The promise of this technology is to producea vaccine or neutralizing agent in a very shorttime’, Fischetti adds. ‘Our ability to mobilizecountermeasure agents quickly could save manylives in the event of an attack.’ This should allowSIGA to meet the US military’s need for rapidlydeployable defences against agents of biologicalwarfare. ‘If possible, the organism that isproducing, say, an antitoxin would beadministered to the population before exposure,so that, when they are exposed, the toxin would
be neutralized’, says Fischetti. Oral or nasaldelivery probably offers the best protectionagainst biological warfare agents, he adds,because most of them would attack the respiratorymucosa. Injection would be less effective becauseit bypasses the mucosal response.
The development of mucosal vaccines againstagents of biological warfare is a departure forSIGA’s research team. So far, the company hasbeen concentrating on developing a vaccine toprevent throat infections caused by group Astreptococci. Fischetti expects US Food and DrugAdministration approval to begin clinical trialswith the vaccine, which has a group Astreptococcal antigen expressed on the surface ofS. gordonii, later this year. About 20 million casesof group A streptococcal infections occur eachyear in the USA and, at present, there is novaccine for them. Untreated, streptococcal sorethroat can progress to rheumatic fever.
As the anchor motif for surface proteins ishighly conserved in Gram-positive bacteria,applications of this technology need not stop atprotecting the oral and respiratory mucosa fromattack: a wide variety of commensals could beengineered for surface expression and used todeliver antigens to intestinal and vaginal sites too.Moreover, says Fischetti, ‘the prolonged exposureof the immune system to a recombinant antigenachieved by stable colonization of a recombinantcommensal after a single immunizing dose couldbe a safe and effective way of overcoming theneed for repeated doses of antigen’.
Dorothy Bonn
369
N e w sMOLECULAR MEDICINE TODAY, SEPTEMBER 1998
Copyright ©1998 Elsevier Science Ltd. All rights reserved. 1357 - 4310/98/$19.00
Figure 1. Chromosomal integration of the fusion protein gene into Streptococcus gordonii. The plasmid (a)contains a construct encoding the signal sequence (S) and the extreme N terminus (N) of the S. gordoniiM protein, the foreign protein, the M protein anchor motif and ermC, which confers erythromycin resistance.This is used to transform (b) S. gordonii cells that have been engineered to contain a cat gene (which conferschloramphenicol resistance) flanked by sequences that are homologous to each end of the plasmid construct.The cat gene construct is placed in front of a strong promoter. The plasmid becomes linearized during transfor-mation and recombines with the sequences flanking the cat gene. This results in (c) the fusion gene becomingintegrated into the S. gordonii chromosome. Recombinants can then be selected for erythromycin resistanceand the loss of chloramphenicol resistance. Modified from Fischetti, V.A. (1996) ASM News 62, 405–410.
Linearizedplasmid
Chromosome
of S. gordonii
Chromosome
of S. gordonii
S
S
N
N
Foreign protein
Foreign protein
Anchor
Anchor
Recombination
ermc
ermc
catP
P
a
b
c
Commensal bacteria armedagainst biological warfare
