Robert H. Reid

Enteral immunization and challenge of volunteers given enterotoxigenic E. coli CFA/II encapsulated in biodegradable microspheres

The development of a safe and effective vaccine against enterotoxigenic Escherichia coli (ETEC) would be useful for travellers and for young children in endemic areas. A feasibility study of an enteral ETEC vaccine prototype consisting of colonization factor antigen II (CFA/II), containing two component antigens CS1 and CS3, encapsulated in biodegradable polymer microspheres (BPM) was conducted in healthy volunteers. Ten adult volunteers swallowed intestinal tubes on days 0, 7, 14 and 28; after collection of jejunal fluid samples, 1 mg of CFA/II in BPM was administered via the tube. Volunteers kept a diary of symptoms after each dose. Secretory IgA in jejunal fluids, serum responses and circulating antibody-secreting cells (ASC) were measured before and after vaccination. The vaccine was well tolerated. Five of ten volunteers developed IgA anti-CFA/II ASC by 7 days after the last dose of vaccine; these same five vaccinees had IgA anti-CS3 ASC, and three of these five vaccinees had IgA anti-CS1 ASC. Five of ten vaccinees developed rises in jejunal fluid sIgA anti-CFA/II with peak GMT of 1:42. About 8 weeks after the first dose of vaccine, ten vaccinees and ten unvaccinated control volunteers underwent challenge with 10(9) c.f.u. ETEC E24377A (O139:H28 LT+ST+CS1+CS3+). Ten of ten controls and seven of ten vaccinees developed diarrhoea (p = 0.11, 30% vaccine efficacy). Two of the three protected vaccinees had the highest numbers of ASC and highest sIgA titres during the course of immunization, suggesting that these responses were protective and that this vaccine development strategy has merit. Future studies with higher dosages and a different dosing schedule are planned.

Preclinical evaluation of microencapsulated CFA/II oral vaccine against enterotoxigenic E. coli

Colonization Factor Antigen (CFA/II) from enterotoxigenic Escherichia coli (ETEC) prepared under good manufacturing practices (GMP) was successfully incorporated into biodegradable poly(D,L-lactide-co-glycolide) (PLGA) polymer microspheres (BPM) under GMP and found to be safe and immunogenic when administered intraduodenally to rabbits. Following vaccination, Peyers patch cells responded by lymphocyte proliferation to in vitro challenge with CFA/II. Also, B cells secreting specific anti-CFA/II antibodies were found in spleens following vaccination. No pathological changes were found following total necropsies of ten rabbits vaccinated with CFA/II BPM. Sixty-three per cent of the CFA/II BPM were between 5 and 10 microns diameter by volume particle size distribution; 1.17% protein content; 2.15% moisture; < 0.01% acetonitrile; 1.6% heptane; 22 non-pathogenic bacteria and three fungi per 1 mg protein dose; and passed the general safety test. We conclude that the CFA/II BPM oral vaccine is immunogenic and safe to begin a Phase I clinical safety study following Investigational New Drug approval.

Pili in microspheres protect rabbits from diarrhoea induced by E. coli strain RDEC-1

We tested whether pilus proteins of rabbit diarrhoeagenic Escherichia coli (RDEC-1), incorporated into biodegradable microspheres, could function as safe and effective oral immunogens in the rabbit diarrhoea model. The RDEC-1 adhesin, AF/R1, incorporated into poly(D,L-lactide-co-glycolide) microspheres, was administered intraduodenally. Vaccinated and unvaccinated rabbits were challenged with RDEC-1 and killed 1 week later. Vaccination with AF/R1 in microspheres did not cause diarrhoea or weight loss. After challenge, rabbits given AF/R1 in microspheres, in contrast to unvaccinated animals, remained in good health. RDEC-1 attachment to caecal epithelium of vaccinated rabbits was reduced (p = 0.02), whereas numbers of RDEC-1 in intestinal fluids were little affected. Also, in vaccinated animals, biliary anti-AF/R1 IgA levels were increased, and AF/R1-induced blast-cell transformation was vigorous in spleen cell cultures. We conclude that vaccination with AF/R1 in microspheres was safe and protected rabbits against RDEC-1 disease, probably by interfering with adherence of the bacteria to the intestinal mucosa. The interference might have been due to the presence of specific antibodies secreted in bile.

Linear epitopes of colonization factor antigen I and peptide vaccine approach to enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) cause diarrhea in infants and in travelers to developing countries. The bacteria utilize colonization factors (CF) for adherence to intestinal epithelia, then release toxins causing diarrhea. CF are strong immunogens as well as protective antigens. While 20 ETEC CF have been described in the literature, 11 CF are prominent enough to be considered for vaccine targeting. Of this group, six of the members fall into the CFA/I family of CF. Geysen pin (peptide) linear epitope analysis demonstrated that three regions containing linear epitopes exist in CFA/I, and that both B- and T-cell linear epitopes of CFA/I were concentrated at the N-terminus of the protein. We have determined N-terminal sequence of the CFA/I family members not previously sequenced. Comparison of the protein sequence of the six members of the family showed a strong homology up to residue 36. A peptide of 36 amino acids representing a consensus of the six sequences was synthesized and used to immunize animals. The antibody induced to the peptide was reactive to the peptide as well as cross-reactive to each member of the CFA/I family in Western blots. In addition, this antibody agglutinated three of the six members of the CFA/I family when added to whole cells expressing the native CF. We are currently evaluating different carriers and conjugation methods to maximize production of high titer, agglutinating antibody. It is hoped that this and related research will result in an effective and inexpensive cross-reactive and cross-protective ETEC vaccine.

Development and Validation of Murabutide Assay by High Performance Capillary Electrophoresis

Abstract The objective of this study was to develop an analytical technique for the assay of murabutide, a synthetic peptide, by high performance capillary electrophoresis. The analyte concentration was monitored by UV detection. Murabutide was eluted by using pH 8.0 borate buffer as the running buffer. The effect of the running buffer concentration on peak sharpness was investigated. Increasing concentration of the running buffer increased peak migration time and also improved the peak sharpness significantly. The assay technique was validated by plotting the standard curve and performing the inter-and intra-day variability studies.

Primary in vitro immunisation of rabbit Peyer’s patch B-cells with keyhole limpet haemocyanin

The major protective barrier of the gut against ingested pathogens is the gut-associated immune system. Immune response to foreign substances or organisms is modulated by several interactive cellular components of the gut-associated lymphoid tissue (GALT) by direct cell contact and by soluable mediators (lymphokines). If the bulk of the immune system resides in the GALT, then the intestinal lymphocyte populations and mechanisms of cell-mediated immunity become extremely important in carrying out specialized effector functions. Peyer’s patch, which appears crucial for antigen recognition, is one such lymphoid compartment. Hence, the purpose of this study is to determine if KLH would be immunogenic in vitro for rabbit Peyer’s patch cells. The present study describes primary in vitro immunization, examines the in vitro antibody response by the Enzyme-Linked Immunosorbent Assay (ELISA), and explores the kinetics of the immune response of the Peyer’s patch cells.

Rabbit ileal lamina propria mononuclear cells suppress the primary in vitro antibody response of spleen cells to KLH

Intestinal lamina propria (LP) mononuclear cells have been found to suppress the peripheral T-cell response (human) and the spleen T-cell response (rabbit) to PHA. Rabbit spleen mononuclear cells have been shown to respond to primary in vitro immunization with KLH giving an IgM antibody response. The purpose of this study was to determine what effect rabbit ileal LP mononuclear cells would have on this primary in vitro antibody response to KLH.

Lymphocyte proliferation in response to the antigens of colonisation factor antigen/I following primary in vitro immunisation

CFA/I is a pilus composed of repeating pilin protein subunits found on many serogroups of enterotoxigenic Escherichia coli which promotes attachment. Primary in vitro immunization of rabbit spleen cells with a synthetic peptide representing the n-terminal 1–13 amino acid fragment of the CFA/I subunit (CFA/I 1–13) produced anti-peptide antibodies which also recognized native CFA/I. We have developed a unique system of in vitro immunization followed by lymphocyte proliferation to determine that CFA/I 1–13 contains a T cell epitope.