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Antimicrob Agents Chemother. Dec 2011; 55(12): 5469–5474.
PMCID: PMC3232761
An Escherichia coli O157-Specific Engineered Pyocin Prevents and Ameliorates Infection by E. coli O157:H7 in an Animal Model of Diarrheal Disease[down-pointing small open triangle]
Jennifer M. Ritchie,1 Jennifer L. Greenwich,1 Brigid M. Davis,1 Roderick T. Bronson,2 Dana Gebhart,3 Steven R. Williams,3 David Martin,3 Dean Scholl,3* and Matthew K. Waldor1*
1Channing Laboratory, Brigham and Women's Hospital/Harvard Medical School and HHMI, 181 Longwood Avenue, Boston, Massachusetts 02115
2Department of Pathology, Harvard Medical School, 77 Ave. Louis Pasteur, Boston, Massachusetts 02115
3AvidBiotics Corporation, 300 Utah Ave., Suite 150, S. San Francisco, California 94080
*Corresponding author. Mailing address for Dean Scholl: AvidBiotics Corporation, 300 Utah Ave., Suite 150, S. San Francisco, CA 94080. Phone: (650) 873-1117. Fax: (650) 873-1010. E-mail: dean/at/avidbiotics.com. Mailing address for Matthew Waldor: Channing Laboratory, Brigham and Women's Hospital/Harvard Medical School and HHMI, 181 Longwood Avenue, Boston, MA 02115. Phone: (617) 525-4646. Fax: (617) 525-4660. E-mail: mwaldor/at/rics.bwh.harvard.edu.
Received June 8, 2011; Revisions requested May 21, 2011; Accepted September 15, 2011.
AvR2-V10.3 is an engineered R-type pyocin that specifically kills Escherichia coli O157, an enteric pathogen that is a major cause of food-borne diarrheal disease. New therapeutics to counteract E. coli O157 are needed, as currently available antibiotics can exacerbate the consequences of infection. We show here that orogastric administration of AvR2-V10.3 can prevent or ameliorate E. coli O157:H7-induced diarrhea and intestinal inflammation in an infant rabbit model of infection when the compound is administered either in a postexposure prophylactic regimen or after the onset of symptoms. Notably, administration of AvR2-V10.3 also reduces bacterial carriage and fecal shedding of this pathogen. Our findings support the further development of pathogen-specific R-type pyocins as a way to treat enteric infections.
Enterohemorrhagic Escherichia coli (EHEC) is a major cause of food-borne colitis and diarrhea. E. coli O157:H7 is the most commonly isolated EHEC serotype that has been associated with human disease (10). Since its initial isolation during a multistate outbreak due to contaminated ground beef in 1982 (12), E. coli O157:H7 has been linked to outbreaks and/or sporadic disease in over 30 countries on six continents (4). In the United States, E. coli O157:H7 is estimated to be responsible for approximately 97,000 reported illnesses per year (8). Although most patients recover within 10 days, infections can be life-threatening, as development of hemolytic-uremic syndrome (HUS) occurs in ~10 to 15% of cases (10).
The severe consequences of infection with E. coli O157:H7 or other EHEC serotypes are due largely to the bacterium's production of Shiga toxins (Stx1 and/or Stx2), which are potent AB5-type exotoxins (9). Stx has been shown to contribute to both intestinal and systemic manifestations of disease in several animal models of infection (1, 11, 13, 14, 1921). Notably, Stx production and its release from bacterial cells can be induced by antibiotics; consequently, administration of antibiotics is contraindicated for patients infected with EHEC (26, 27). Antimotility agents are also not deemed beneficial, as prolonged intestinal retention of bacteria can augment toxin exposure (22). Hence, clinical management of individuals with EHEC infection is usually limited to supportive interventions such as hydration. Novel means for preventing infection and limiting disease duration and/or pathogen dissemination could therefore be beneficial for both individual patients and affected communities.
R-type pyocins are high-molecular-weight bacteriocins produced by some Pseudomonas aeruginosa strains that specifically kill competing P. aeruginosa strains (for reviews, see references 6 and 18). These protein complexes, which resemble the tail structures of bacteriophages of the Myoviridae family, first bind to specific cell surface receptors and then contract and insert their core into the cell envelope. The protein forms a channel through the inner membrane that causes membrane depolarization and, ultimately, cell death (23). Replacing the pyocin tail fiber or fusing it to receptor-recognizing tail proteins of bacteriophages enables the complex to recognize new targets (25). To date, R-type pyocins have been engineered to have an altered killing spectrum for a subset of Pseudomonas strains as well as to recognize other organisms, including E. coli and Yersinia pestis (16, 25). In theory, R-type pyocins could serve as a platform for production of numerous highly specific antimicrobial agents and thus might enable pathogens to be targeted without disruption of the normal host flora.
Scholl et al. demonstrated that E. coli O157:H7 can be killed by an engineered R-type pyocin, AvR2-V10, in which the tail spike from bacteriophage phiV10 is fused to the R2 pyocin tail fiber (16). The phiV10 bacteriophage recognizes the O157 antigen, which is also a virulence factor (3); consequently, emergence of pathogenic O157:H7 strains that are resistant to AvR2-V10 (e.g., due to changes in expression or structure of the O antigen) seems unlikely. Notably, unlike the case with many antibiotics, AvR2-V10-mediated cell killing does not induce the release of Stx and thus should not exacerbate the symptoms of EHEC infection (16). Here we demonstrate that a variant of AvR2-V10, termed AvR2-V10.3, is active against E. coli O157:H7 in vivo, using the infant rabbit model of E. coli O157:H7 infection. Early administration of AvR2-V10.3 prevented diarrhea induced by E. coli O157:H7 and dramatically reduced bacterial carriage and shedding. Administration of AvR2-V10.3 after the onset of diarrhea was also effective, reducing bacterial carriage and the severity of disease symptoms, suggesting that the engineered pyocin has potential as both a prophylactic and therapeutic agent.
Construction of the AvR2-V10.3 expression vector pDG389.
pDG389 (Fig. 1), which is derived from the expression vector pETcoco (Novagen), was used to construct AvR2-V10.3. Details of the construction of pDG389 and the sequence of this plasmid can be found in Fig. S1 in the supplemental material. The copy number of this plasmid can be increased from 1 to ~40 copies per cell by the addition of arabinose to the culture medium, due to the induction of the replication regulator TrfA, which is under the control of the PBAD promoter. pDG389 carries the R2 pyocin gene cluster (prf3 to prf24) (7), from which the tail fiber gene, prf15, has been deleted (16). Additionally, pDG389 encodes an R2-phiV10.3 tail fiber spike fusion protein under the control of the arabinose-inducible promoter PBAD. This protien consists of amino acids 1 to 164 of Prf15 fused to amino acids 217 to 875 of the tail spike protein from the O157-specific phage phiV10.3, a derivative of phiV10 that was selected for its efficient plaque formation on a derivative of strain EDL933 that produces reduced levels of O157 antigen (described below). The mutant tail spike protein differs from the wild-type version at a single amino acid (Asp417Gly).
Fig. 1.
Fig. 1.
Expression construct for AvR2-V10.3. pDG389, which is a derivative of pETcoco, contains the entire R2 pyocin gene cluster (prf3 to prf24), with the exception of the tail fiber gene (prf15). The pyocin structural genes are transcribed to the right and (more ...)
Selection of EDL933 R2-V10r0.3 and phiV10.3.
EDL933 R2-V10r0.3 was identified as a spontaneous mutant derivative of EDL933 that was resistant to killing by AvR2-V10 and subsequently found to be resistant to phiV10 as well. On lipopolysaccharide (LPS) gels, EDL933 R2-V10r0.3 appears to produce a reduced amount of O antigen. A spontaneously arising derivative of phiV10 that could infect EDL933 R2-V10r0.3 (termed phiV10.3) was sequenced and found to contain a single mutation within prf15, resulting in the amino acid replacement Asp417Gly. Pyocin containing the mutant tail spike fusion (AvR2-V10.3), unlike AvR2-V10, displayed killing activity against EDL933 R2-V10r0.3 (see Fig. S2 in the supplemental material).
Production and purification of wild-type and recombinant pyocins.
AvR2-V10.3 was purified from E. coli BL21 containing the expression vector pDG389 as follows. BL21/pDG389 was grown in 200 ml of tryptic soy broth with no dextrose (BD)-1% KNO3-12.5 μg ml−1 chloramphenicol at 37°C with shaking at 250 rpm. At an optical density at 600 nm (OD600) of 1.0, arabinose was added to a final concentration of 0.05%. After ~3 h, the cell culture lysed and was centrifuged at 6,000 × g for 20 min to remove heavy cellular debris. The cleared supernatant was centrifuged again at 24,000 × g for 1 h to remove finer debris, before the pyocin was pelleted by high-speed centrifugation at 61,000 × g (1 h). The pyocin pellet was resuspended in 5 ml of TN50 buffer (10 mM Tris, pH 7.5, 50 mM NaCl) containing 3% mannitol. Total pyocin activity recovered in the pellet was typically >90% of the total starting activity, and typical yields were about 1013 killing units (KU; see below) per liter of culture. Pyocin material was estimated to be ~90% pure by SDS gel electrophoresis. Wild-type R2 pyocin was purified from P. aeruginosa strain PAO1 by a similar centrifugation method, with the exception that bacteria were grown in Trypticase soy broth (BD) and pyocins were induced with mitomycin C at an OD600 of 0.5 as described previously (17).
AvR2-V10.3 killing activity was measured by the titration survival method as described previously, using strain EDL933 as an indicator (16). The survival assay indirectly measures individual bactericidal killing events, expressed in KU. R2 activity was measured similarly, using P. aeruginosa strain 13s as the target (17). The killing activity of AvR2-V10.3 on EDL933 did not differ from that of previously described AvR2-V10 (16), as both yielded activities in the range of 2 × 1012 to 4 × 1012 KU per mg of protein.
Infection and analysis of infant rabbits.
All animal studies performed in this study complied with local, institutional, and federal guidelines. Three-day-old New Zealand White infant rabbits were inoculated orogastrically with 5 × 108 CFU of E. coli O157:H7 strain EDL933 as previously described (13). Purified pyocin (1011 to 1012 KU/dose) or pyocin buffer (TN50) was administered orogastrically to the rabbits either starting shortly after EDL933 administration (at 3, 26, and 52 h) or after the onset of diarrhea (once daily, starting at day 3 post-EDL933 administration). Three hours prior to each pyocin/buffer administration, rabbits were treated with cimetidine (intraperitoneal injection at 50 mg kg of body weight−1; Hospira, IL). Each litter of infant rabbits (typically 6 to 8 rabbits) was divided so that half received pyocin treatment and half received buffer. The efficacy of the highest pyocin dose (1012 KU) was assessed in two independent litters for both prophylactic and therapeutic protocols. The rabbits were examined for the development of diarrhea daily. Evaluations were based on signs of fecal staining on the hind legs and tail as well as the physical appearance of stool released from the rabbits during handling. Stool was considered normal if it consisted of hard, formed, dark-colored pellets and abnormal if it consisted of soft or liquid yellow-green fecal material. Intestinal tissue and stool samples were collected at necropsy at 72 h (prophylaxis study) or 9 days (therapeutic study) postinfection, and EHEC CFU g−1 were determined as described previously (13). Intestinal inflammation (heterophil score) was scored on a scale of 0 (none) to 4 (severe), as described previously (13).
Pyocin activity in the intestinal tract was assessed using uninfected rabbits. AvR2-V10.3 was administered to infant rabbits as described above, and at various times posttreatment, the rabbits were euthanized and their intestines removed for further processing. Pyocin activity was tested in both tissue homogenates, with the entire small intestine, cecum, or large intestine homogenized in 4 ml of TN50 buffer, and luminal contents, with 3 ml TN50 buffer used to flush out the contents of each section. Pyocin activity was assayed in a semiquantitative manner by using the spot method as previously described (16). In this assay, bactericidal activity is detected by a zone of clearing on a lawn of bacteria on which 5-fold serial dilutions of intestinal contents or homogenates are spotted as 5-μl aliquots.
Expression of AvR2-V10.3 in E. coli.
In this study, we used a variant of AvR2-V10 in which the R2 pyocin tail fiber is fused to the tail spike of a mutant version of phage phiV10, called phiV10.3. The tail fiber of phiV10.3 differs from that of phiV10 by a single amino acid substitution (Asp417Gly). No difference was observed in the killing ability of AvR2-V10 versus AvR2-V10.3 against EDL933 or any other wild-type E. coli O157:H7 isolate that was tested. However, AvR2-V10.3 was found to form plaques more efficiently than AvR2-V10 on some E. coli O157:H7 strains that produced low levels of O antigen (see Fig. S2 in the supplemental material), which could provide therapeutic advantages. It is not known why AvR2-V10.3 exhibits enhanced binding to these mutant strains. Like AvR2-V10, AvR2-V10.3 did not induce Shiga toxin production from EDL933 (data not shown).
For ease of production, AvR2-V10.3 was synthesized in E. coli rather than in P. aeruginosa as was done previously (16). Both the tail fiber fusion protein and the full-length R2 pyocin gene cluster (except for the wild-type tail fiber gene, prf15) were expressed from a copy number-controlled single plasmid, pDG389 (Fig. 1). Production of AvR2-V10.3 from pDG389 was induced by addition of 0.05% arabinose; no pyocin production was observed in the absence of arabinose. Bacterial lysis (presumably caused by the pyocin's lysis genes, prf9 and prf24) initiated ~2.5 h after induction and became maximal after ~3 h. Induction with 0.05% arabinose led to release of ~1010 KU of pyocin particles per ml into the medium; higher levels of arabinose did not increase pyocin yields. The yield of AvR2-V10.3 from E. coli containing pDG389 was 3- to 5-fold higher than that of AvR2-V10 from P. aeruginosa.
AvR2-V10.3 is active in the intestine.
For this study, since we wished to assess the efficacy of orogastrically administered AvR2-V10.3 against E. coli O157:H7 within a mammalian host, we first assessed whether AvR2-V10.3 could maintain activity within the infant rabbit intestinal tract. Because AvR2-V10.3 particles are inactivated by low pH (16), rabbits were pretreated with the histamine H2 receptor antagonist cimetidine, which transiently prevents stomach acidification (15). Bactericidal activities in luminal contents and tissue homogenates from AvR2-V10.3-treated animals at various times after AvR2-V10.3 administration were compared to those for samples from unexposed control rabbits. When samples were spotted on lawns of EDL933, both tissue homogenates and luminal contents from treated rabbits caused clearance at dilutions as high as 1:75. In contrast, no bactericidal activity was apparent in samples obtained from a non-pyocin-treated (control) rabbit. Low levels of bactericidal activity (present in undiluted or 1:5-diluted samples) remained in cecal contents at 24 h postadministration; consequently, AvR2-V10.3 was administered once daily in all subsequent experiments.
AvR2-V10.3 ameliorates E. coli O157:H7-induced disease and reduces E. coli O157:H7 colonization.
Typically, infant rabbits develop severe diarrhea approximately 2 to 3 days after orogastric inoculation of 5 × 108 CFU E. coli O157:H7 (13). At this time, intestinal inflammation, particularly infiltration by heterophils (the rabbit equivalent of neutrophils), is evident in colonic tissue sections, and abundant EHEC can be isolated from the intestinal tract (13). To assess the efficacy of AvR2-V10.3 in counteracting E. coli O157:H7 infection, we first determined whether AvR2-V10.3 given shortly after infant rabbits were inoculated with E. coli O157:H7 could inhibit bacterial colonization and/or the symptoms of infection. In the studies described below, infant rabbits were treated with AvR2-V10.3 3, 26, and 52 h after inoculation with E. coli O157:H7 (Fig. 2A). Prior to each AvR2-V10.3 administration, rabbits were treated with cimetidine. Mock-treated (control) rabbits infected with E. coli O157:H7 were also given cimetidine prior to administration of pyocin vehicle (TN50 buffer).
Fig. 2.
Fig. 2.
Prophylactic administration of AvR2-V10.3 prevents or mitigates symptoms of E. coli O157:H7 infection. (A) Schematic of the onset of diarrhea and timing of E. coli O157:H7 infection coupled with prophylactic treatment protocols. Low (L; 1011 KU), medium (more ...)
The effects of various doses of AvR2-V10.3 (1011 to 1012 KU) upon E. coli O157:H7-infected rabbits were assessed. Notably, rabbits that received the highest dose (1012 AvR2-V10.3 KU/dose) did not develop diarrhea at any time during the 3-day experiment (Fig. 2A). In contrast, rabbits given buffer alone exhibited smears of fecal material on their hind legs and tails within approximately 1 to 2 days postinfection. By the start of the third day postinfection, the area of fecal contamination was larger, and liquid feces were detectable at the rabbits' anuses. Fecal contamination and/or diarrhea was also observed on animals that received lower doses of pyocins (1011 or 2 × 1011), although the onset and severity of symptoms were delayed by the higher of these doses (Fig. 2A). At necropsy (day 3), the colonic contents from rabbits treated with 1012 AvR2-V10.3 KU appeared markedly different from those of untreated rabbits. The colonic contents of treated rabbits consisted of discrete, formed pellets rather than the largely liquid material obtained from untreated animals (Fig. 2B). Histological analyses of colonic intestinal tissue revealed that administration of 1012 AvR2-V10.3 KU also reduced the severity of E. coli O157:H7-induced intestinal inflammation. In general, the mid- and distal colons of most rabbits given 1012 AvR2-V10.3 KU contained fewer heterophils than did the intestines of untreated rabbits (Fig. 2C), although these differences reached statistical significance only for the midcolon (P ≤ 0.05). Nevertheless, these findings provide further evidence that the treatment regimen reduces the ability of E. coli O157:H7 to cause intestinal disease.
Administration of 1012 AvR2-V10.3 KU also greatly reduced the number of E. coli O157:H7 cells recovered from the intestine and present in the stools of treated rabbits. At 72 h postinfection, there was a >5-log reduction in the number of E. coli O157:H7 CFU present in the stools of rabbits treated with this dose of AvR2-V10.3 compared to the untreated rabbits (mean, 1 × 104 versus 2 × 109 CFU g−1) (Fig. 3). Significant (3- to 4-log) reductions were also observed in the numbers of organisms colonizing the small and large intestines of these animals (Fig. 3). Lower doses of AvR2-V10.3 were not as effective at reducing the number of E. coli O157:H7 cells recovered in the intestine (Fig. 3), consistent with the observed diarrhea in these animals (Fig. 2A).
Fig. 3.
Fig. 3.
Recovery of E. coli O157:H7 from infected rabbits following treatment with various doses of AvR2-V10.3. For each litter of infected rabbits, half were treated with buffer alone (closed symbols) and half were treated with AvR2-V10.3 (open symbols) (L, (more ...)
Overall, our results indicate that high doses of AvR2-V10.3 markedly reduce the ability of E. coli O157:H7 to replicate and persist in the infant rabbit intestine and to cause disease when given shortly after ingestion of the pathogen. To confirm that this is a specific effect of AvR2-V10.3, we treated E. coli O157:H7-infected rabbits with an equivalent regimen of R2 (3 doses at 1012 KU/dose), the wild-type Pseudomonas-targeting pyocin from which AvR2-V10.3 was derived, which does not kill EDL933 in vitro. These rabbits developed diarrhea similar to that seen in untreated rabbits (Fig. 2A). Furthermore, colonic homogenates from infected rabbits given R2 pyocin contained similar numbers of E. coli O157:H7 cells to those in homogenates from untreated rabbits (geometric mean [95% confidence interval], 4 × 108 [1 × 107 to 2 × 1010] and 1 × 109 [8 × 107 to 4 × 1010] CFU g−1, respectively). These experiments suggest that the effects of AvR2-V10.3 on E. coli O157:H7 colonization and perturbation of the infant rabbit intestine reflect the O157-specific bactericidal activity of this engineered pyocin.
AvR2-V10.3 reduces the severity of E. coli O157:H7-induced diarrhea when given after the onset of disease.
To assess the utility of AvR2-V10.3 as a therapeutic and/or antishedding agent, we also investigated its effects on E. coli O157:H7-infected infant rabbits that already displayed signs of disease. In these studies, treatment of infant rabbits did not commence until 3 days after their infection with E. coli O157:H7, a point at which all animals had developed diarrhea. Infected animals were given daily doses of either AvR2-V10.3 (1012 KU) or the buffer solution, in both cases preceded by the administration of cimetidine. Within 2 days of treatment initiation, rabbits receiving AvR2-V10.3 began to resolve the diarrhea, as evidenced by a reduction in the amount of fecal material present on their fur relative to that of mock-treated animals. This relative reduction in fecal accumulation persisted until necropsy, which was performed at 9 days postinfection. At this point, in addition to the reduced amount of fecal material on AvR2-V10.3-treated rabbits, the material also appeared drier than on rabbits in the control group, suggesting that the release of liquid stool (diarrhea) had largely ceased. In contrast, liquid stool was observed at the anuses of untreated animals, and the fur of these animals was contaminated extensively with wet fecal material (Fig. 4A). Thus, even after the onset of diarrhea in E. coli O157:H7-infected animals, administration of AvR2-V10.3 could still mitigate the signs of infection.
Fig. 4.
Fig. 4.
AvR2-V10.3 reduces diarrhea severity and intestinal bacterial load when administered after the onset of diarrhea. (A) Fecal contamination at necropsy (9 days postinfection) on E. coli O157:H7-infected rabbits treated with AvR2-10.3 or buffer daily after (more ...)
Histological analyses also suggested that treatment of established diarrheal disease with AvR2-V10.3 is efficacious. Colonic tissue from untreated rabbits appeared to contain more heterophils than did tissue sections from AvR2-V10.3-treated rabbits, although this finding did not reach statistical significance (P = 0.16). Additionally, colonic sections from untreated rabbits were hyperemic and edematous and contained many mononuclear cells (Fig. 4B), similar to previously reported findings for rabbits at day 7 postinfection (13) and consistent with a more severe disease state in these animals.
Consistent with its diminution of diarrheal symptoms, administration of AvR2-V10.3 after the onset of diarrhea reduced the abundance of E. coli O157:H7 cells in the intestines of infected rabbits. The numbers of E. coli O157:H7 cells recovered from ileal and colonic homogenates from AvR2-V10.3-treated rabbits were reduced significantly (1 to 2 logs) compared to those of the untreated group (Fig. 4C). Also, significantly fewer E. coli O157:H7 cells were detected in the stools of AvR2-V10.3-treated rabbits (P ≤ 0.05). It is likely that comparisons of the numbers of EHEC cells recovered in stools underestimate the net effect of AvR2-V10.3 on EHEC shedding, as treated animals appear to produce less stool overall; however, we have been unable to precisely quantify diarrheal output. Regardless, it is clear that administration of AvR2-V10.3 after the onset of EHEC-induced symptoms can reduce bacterial carriage within the intestine. Notably, the E. coli O157:H7 cells isolated from the intestines and stools of AvR2-V10.3-treated animals maintained susceptibility to killing by AvR2-V10.3 in in vitro assays, suggesting that their persistence in vivo likely reflects an insufficient pyocin dose or limitations in tissue accessibility rather than selection for pyocin-resistant bacteria. Thus, it is possible that increased pyocin doses, alternative formulations, or altered treatment regimens might enable even greater mitigation of disease symptoms, colonization, and bacterial shedding.
Our findings that the engineered pyocin AvR2-V10.3 can reduce intestinal colonization by E. coli O157:H7 and can mitigate or prevent symptoms of infection clearly demonstrate its promise as a prophylactic and therapeutic counter to infection by this important enteric pathogen. Use of AvR2-V10.3 rather than conventional antibiotics with antimicrobial activity against E. coli O157:H7 provides several significant advantages, including a lack of drug-induced toxin production (16) and a probable maintenance of the normal intestinal flora. Additionally, since mutation of genes enabling O157 antigen synthesis dramatically curtails colonization of the intestine (3), it is unlikely that pathogenic mutants of E. coli O157:H7 that are resistant to AvR2-V10.3 would arise during the course of treatment. Although O157:H7 is not the only EHEC serotype that causes disease in humans, it does account for the majority of cases in many countries (10). With that said, we are currently constructing specific pyocins to target other EHEC serotypes as well as other enteric pathogens.
Intraperitoneal and intravenous administration of natural R-type pyocins has previously been demonstrated to promote survival of mice with peritoneal Pseudomonas aeruginosa infections (2, 5, 17); however, the efficacy of orogastrically administered pyocins against either EHEC or other bacterial pathogens has not been reported previously. Notably, although AvR2-V10.3 is inactivated by acids (16), the pyocin maintained some bactericidal activity in the intestinal tracts of animals pretreated with cimetidine, which transiently elevates gastric pH. Although natural R-type pyocins are generally protease resistant (6), it could not be presumed that the O157 targeting domain that was introduced via genetic engineering would also have this property. Future research is planned to develop methods to quantitatively study the pharmacokinetics and pharmacodynamics of orally administered AvR2-V10.3 through the gastrointestinal tract and to optimize formulation and delivery methods. Recent advances in the formulation of orally delivered protein therapeutics and vaccines (24) will provide a basis for improving oral formulations. Given that AvR2-V10.3 efficacy was observed with what is likely a suboptimal delivery method, we anticipate that even better results can be achieved by enhancing this agent's pharmacologic properties.
Supplementary Material
Supplemental material
ACKNOWLEDGMENTS
This work was funded by NIH grant 1R43A1084295-01 to J.M.R., D.S., and M.K.W. and by grant AI-R37 042347 and the HHMI (M.K.W.).
Dana Gebhart, Steve Williams, Dean Scholl, and David Martin are employees and shareholders of AvidBiotics. Matthew K. Waldor is also a shareholder of AvidBiotics.
Footnotes
Supplemental material for this article may be found at http://aac.asm.org/.
[down-pointing small open triangle]Published ahead of print on 26 September 2011.
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