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Surface-expressed colonization factors and their subunits are promising candidates for inclusion into a multivalent vaccine targeting enterotoxigenic Escherichia coli (ETEC), a leading cause of acute bacterial diarrhea in developing regions. However, soluble antigens are often poorly immunogenic in the absence of an adjuvant. We show here that the serum immune response to CfaE, the adhesin of the ETEC colonization factor CFA/I, can be enhanced in BALB/c mice by immunization with a chimeric antigen containing CfaE and pentameric cholera toxin B subunit (CTB) of cholera toxin from Vibrio cholerae. We constructed this antigen by replacing the coding sequence for the A1 domain of the cholera toxin A subunit (CTA) with the sequence of donor strand complemented CfaE (dscCfaE) within the cholera toxin operon, resulting in a dscCfaE-CTA2 fusion. After expression, via non-covalent interactions between CTA2 and CTB, the fusion and CTB polypeptides assemble into a complex containing a single dscCfaE-CTA2 protein bound to pentameric CTB (dscCfaE-CTA2/CTB). This holotoxin-like chimera retained the GM1 ganglioside binding activity of CTB, as well as the ability of CfaE to mediate the agglutination of bovine red blood cells when adsorbed to polystyrene beads. When administered intranasally to mice, the presence of CTB in the chimera significantly increased the serum immune response to CfaE compared to dscCfaE alone, stimulating a response similar to that obtained with a matched admixture of dscCfaE and CTB. However, by the orogastric route, immunization with the chimera elicited a superior functional immune response compared to an equivalent admixture of dscCfaE and CTB, supporting further investigation of the chimera as an ETEC vaccine candidate.

Background. Tip-localized adhesive proteins of bacterial fimbriae from diverse pathogens confer protection in animal models, but efficacy in humans has not been reported. Enterotoxigenic Escherichia coli (ETEC) commonly elaborate colonization factors comprising a minor tip adhesin and major stalk-forming subunit. We assessed the efficacy of antiadhesin bovine colostral IgG (bIgG) antibodies against ETEC challenge in volunteers. Methods. Adults were randomly assigned (1:1:1) to take oral hyperimmune bIgG raised against CFA/I minor pilin subunit (CfaE) tip adhesin or colonization factor I (CFA/I) fimbraie (positive control) or placebo. Two days before challenge, volunteers began a thrice-daily, 7-day course of investigational product administered in sodium bicarbonate 15 minutes after each meal. On day 3, subjects drank 1 × 109 colony-forming units of colonization factor I (CFA/I)-ETEC strain H10407 with buffer. The primary efficacy endpoint was diarrhea within 120 hours of challenge. Results. After enrollment and randomization, 31 volunteers received product, underwent ETEC challenge, and were included in the per protocol efficacy analysis. Nine of 11 placebos developed diarrhea, 7 experiencing moderate to severe disease. Protective efficacy of 63% (P = .03) and 88% (P = .002) was observed in the antiadhesin bIgG and positive control groups, respectively. Conclusions. Oral administration of anti-CFA/I minor pilin subunit (CfaE) antibodies conferred significant protection against ETEC, providing the first clinical evidence that fimbrial tip adhesins function as protective antigens.

Enterotoxigenic Escherichia coli (ETEC) commonly cause diarrhea in children living in developing countries and in travelers to those regions. ETEC are characterized by colonization factors (CFs) that mediate intestinal adherence. We assessed if bovine colostral IgG (bIgG) antibodies against a CF, CS17, or antibodies against CsbD, the minor tip subunit of CS17, would protect subjects against diarrhea following challenge with a CS17-expressing ETEC strain. Adult subjects were randomized (1:1:1) to receive oral bIgG against CS17, CsbD, or placebo. Two days prior to challenge, subjects began dosing 3 times daily with the bIgG products (or placebo). On day 3, subjects ingested 5 × 109 cfu ETEC strain LSN03-016011/A in buffer. Subjects were assessed for diarrhea for 120 hours postchallenge. A total of 36 subjects began oral prophylaxis and 35 were challenged with ETEC. While 50.0% of the placebo recipients had watery diarrhea, none of the subjects receiving anti-CS17 had diarrhea (P = .01). In contrast, diarrhea rates between placebo and anti-CsbD recipients (41.7%) were comparable (P = 1.0). This is the first study to demonstrate anti-CS17 antibodies provide significant protection against ETEC expressing CS17. More research is needed to better understand why anti-CsbD was not comparably efficacious. Clinical Trials Registration. NCT00524004.

Enterotoxigenic Escherichia coli (ETEC) are the most common cause of bacterial diarrhea in young children in developing countries and in travelers. Efforts to develop an ETEC vaccine have intensified in the past decade, and intestinal colonization factors (CFs) are somatic components of most investigational vaccines. CFA/I and related Class 5 fimbrial CFs feature a major stalk-forming subunit and a minor, antigenically conserved tip adhesin. We hypothesized that the tip adhesin is critical for stimulating antibodies that specifically inhibit ETEC attachment to the small intestine. To address this, we compared the capacity of donor strand complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, and CFA/I fimbriae to elicit anti-adhesive antibodies in mice, using hemagglutination inhibition (HAI) as proxy for neutralization of intestinal adhesion. When given with genetically attenuated heat-labile enterotoxin LTR192G as adjuvant by intranasal (IN) or orogastric (OG) vaccination, dscCfaE exceeded CFA/I fimbriae in eliciting serum HAI titers and anti-CfaE antibody titers. Based on these findings, we vaccinated Aotus nancymaae nonhuman primates (NHP) with dscCfaE alone or admixed with one of two adjuvants, LTR192G and cholera toxin B-subunit, by IN and OG administration. Only IN vaccination with dscCfaE with either adjuvant elicited substantial serum HAI titers and IgA and IgG anti-adhesin responses, with the latter detectable a year after vaccination. In conclusion, we have shown that dscCfaE elicits robust HAI and anti-adhesin antibody responses in both mice and NHPs when given with adjuvant by IN vaccination, encouraging further evaluation of an ETEC adhesin-based vaccine approach.

Human challenges with enterotoxigenic Escherichia coli (ETEC) have broadened our understanding of this important enteropathogen. We report findings from the first challenge studies using ETEC-expressing colonization factor fimbria CS17 and CS19. LSN03-016011/A (LT, CS17) elicited a dosedependent effect, with the upper dose (6 X 10⁹ organisms) causing diarrhea in 88% of recipients. WS0115A (LTSTp, CS19) also showed a dose response, with a 44% diarrhea rate at 9 X 10⁹ organisms. Both strains elicited homologous antifimbrial and anti-LT antibody seroconversion. These studies establish the relative pathogenicity of ETEC expressing newer class 5 fimbriae and suggest suitability of the LT|CS17-ETEC challenge model for interventional trials.

This study describes the efforts and outcomes associated with the establishment of a clinical sample repository during the 2016 Zika virus epidemic. To overcome the challenge of limited access to clinical samples to support diagnostic test development, multiple US Department of Health and Human Services (HHS) agencies formed a partnership to create the HHS Zika Specimen Repository. In 2016-2017, the Biomedical Advanced Research and Development Authority and the Centers for Disease Control and Prevention collected patient specimens (4420 convalescent sera aliquots from 100 donors and 7171 plasma aliquots from 239 donors), confirmed Zika virus test results, assembled 1 panel for molecular testing (n = 25 sets) and 7 panels for serologic testing (n = 92), and distributed the panels to test developers. We manufactured 8 test panels and distributed 74 sets of panels to 32 commercial companies, public health partners, and research institutions. Manufacturers used these panels to generate data that supported 14 US Food and Drug Administration (FDA) emergency use authorizations and 1 FDA approval. To develop a repository that can respond immediately to future disease outbreaks, we recommend that organizations pre-position procedures, resources, and partnerships to optimize each partner’s contribution.

The microbiology of war wounds has changed as medicine and warfare have evolved. This study was designed to determine the microbial flora and bacterial quantification of present-day war wounds in US troops from Iraq and Afghanistan upon arrival at the National Naval Medical Center (NNMC). Patients with extremity combat wounds treated with a vacuum-assisted wound closure device were enrolled in study. Wounds were biopsied every 48 to 72 hours with quantitative microbiology performed on all biopsies. Two hundred forty-two wound biopsies from 34 patients; 167 (69%) showed no growth, and 75 (31%) showed positive growth. The incidence of any bacterial isolation from biopsies weekly from the time of injury was 28% (first), 31% (second), and 37% (≥third). Acinetobacter baumannii was the most prevalent isolate. Most soft-tissue wounds from Iraq and Afghanistan do not have significant bacterial burden upon arrival to and during initial treatment at NNMC. Improved evaluation of combat wound microbiology at all levels of care is warranted to determine shifts in microbiology and to impact care practices.

Biofilms in medical devices such as catheters are a source of recurring infections in chronically ill patients. Eradication of microorganisms within biofilms is very difficult using standard antimicrobial treatments. Examination of bacteria growing as biofilms revealed that these matrix-enclosed populations can survive exposure to antimicrobial agents at higher concentrations than can their planktonic counterparts. In order to study this process, hydrogel-biofilm constructs were employed. Using a thermoreversible hydrogel, we have shown that it is possible to recover individual biofilm cells and visualize the spatial and physiological heterogeneity within biofilm communities. Klebsiella pneumoniae and Escherichia coli cells seeded within the hydrogel formed dense biofilms characterized by an exopolysaccharide matrix, complex structural features (i.e., microcolonies, channels), and decreased antibiotic susceptibility. Quantitative comparison of biofilm structures revealed little variation between bacterial strains. Over time (24 to 48 hours), mean biomass and thickness of biofilms increased significantly; however, the mean roughness, an indicator of biofilm structural heterogeneity, deceased significantly. Multiple equivalent biofilms were prepared and exposed to imipenem, ceftazidime, or gentamicin. Plate-count data showed that exposure to these antibiotics had little effect on established biofilms, whereas planktonic control cultures were eradicated. Transport limitation was not a significant factor in reduced biofilm susceptibility. Imipenem and ceftazidime easily penetrated through the hydrogel-biofilm constructs and retained sufficient activity to inhibit growth of susceptible organisms; gentamicin penetration was slowed but not halted. None of the antibiotics studied was capable of completely eradicating biofilms, but all caused significant damage at high concentrations. At the single-cell level, subpopulations of viable, injured, and dead cells were identified and enumerated based on membrane permeability and differential uptake of nucleic-acid dyes. Results showed a dose-dependent decrease in viable cells and an increase in injured and dead cells. At the community level, in situ viability staining in conjunction with confocal microscopy was used to visualize and quantify the physiological responses within intact, hydrated biofilms. Biofilms treated with oxygen-dependent or growth-rate dependent antibiotics demonstrated that cell location plays a major role in biofilm resistance to killing. Patterns of antibiotic-induced damage within microcolonies reflected the physiological heterogeneity of biofilms.