Functional characterization and evaluation of protective efficacy of EA752-862 monoclonal antibody against B. anthracis vegetative cell and spores.

The most promising means of controlling anthrax, a lethal zoonotic disease during the early infection stages, entail restricting the resilient infectious form, i.e., the spores from proliferating to replicating bacilli in the host. The extractible antigen (EA1), a major S-layer protein present on the vegetative cells and spores of Bacillus anthracis, is highly immunogenic and protects mice against lethal challenge upon immunization. In the present study, mice were immunized with r-EA1C, the C terminal crystallization domain of EA1, to generate a neutralizing monoclonal antibody EA752-862, that was evaluated for its anti-spore and anti-bacterial properties. The monoclonal antibody EA752-862 had a minimum inhibitory concentration of 0.08 mg/ml, was bactericidal at a concentration of 0.1 mg/ml and resulted in 100% survival of mice against challenge with B. anthracis vegetative cells. Bacterial cell lysis as observed by scanning electron microscopy and nucleic acid leakage assay could be attributed as a possible mechanism for the bactericidal property. The association of mAb EA752-862 with spores inhibits their subsequent germination to vegetative cells in vitro, enhances phagocytosis of the spores and killing of the vegetative cells within the macrophage, and subsequently resulted in 90% survival of mice upon B. anthracis Ames spore challenge. Therefore, owing to its anti-spore and bactericidal properties, the present study demonstrates mAb EA752-862 as an efficient neutralizing antibody that hinders the establishment of early infection before massive multiplication and toxin release takes place.

Induction of immune response and prevention of alveolar bone loss with recombinant Porphyromonas gingivalis peptidylarginine deiminase.

OBJECTIVE: Treatments for periodontitis are not absolutely perfect, and a vaccine against Porphyromonas gingivalis (P. gingivalis) could become a valuable adjunct therapy for periodontitis. DESIGN: In this study, a vaccine of peptidylarginine deiminase (PAD) from P. gingivalis was evaluated in P. gingivalis-induced murine lesion and periodontitis models. The prevention of alveolar bone loss analysis determined by micro-computed X-ray tomography (micro-CT), and histological assays. Furthermore, the induction of immune response of mouse anti-PAD done with ELISA and Western Blot analysis. RESULTS: Compared with animal immunization with incomplete Freund's adjuvant (IFA) alone, PAD group significantly inhibited (P<0.05) bone resorption. ELISA and Western Blot showed that PAD induced response involving immunoglobulin G1 (Ig G1) predominantly. CONCLUSIONS: These results suggest that PAD could be a candidate antigen for a vaccine against P. gingivalis infection.

Comparison of efficacies of bovine immune colostral antibody and each immunoglobulin class against verotoxin 2, flagellum and somatic cells of Escherichia coli O157:H7 in mice.

PURPOSE: The efficacy of bovine immune colostral (colostral) antibodies against verotoxin (VT) 2, flagellum and somatic cells of Escherichia coli (E. coli) O157:H7 in mice was determined. METHODS: Three major immunoglobulin (Ig) classes were isolated from the colostral antibody against VT2 by affinity chromatography and were used for estimation. Mice inoculated with VT2 were administered each Ig class from the colostral antibody, colostral antibody (colostral whey containing antibody) or serum antibody against VT2 at 1 hour after VT2 inoculation. RESULTS: All control mice (20/20) died after administration of sterilized saline instead of the colostral antibody. The survival rate was 93.3% (14/15) after administration of S-IgA or IgM antibody, or colostral antibody. Survival rates for IgG antibody and serum antibody administration were 80% (12/15) and 60% (9/15), respectively. Serum concentrations of VT2, which was absorbed from the small intestine in mice after administration of VT2 and colostral antibody, were measured by fluorescence enzyme immunoassay (FEIA). Serum concentrations of VT2 after administration of colostral antibody were lower than those after administration of sterilized saline. Mice inoculated with VT2-producing E. coli 157:H7 were administered anti-flagellum or anti-somatic colostral antibodies. Survival rates for E. coli O157:H7-infected mice administered the anti-flagellum and anti-somatic colostral antibodies were 52.4% (11/21) and 22.2% (4/18), respectively. Furthermore, survival rates increased to 89.5% (17/19) with combined administration of anti-flagellum and anti-VT2 colostral antibodies. CONCLUSION: These results suggest that colostral antibodies against VT2, flagellum and somatic cells are effective against E. coli O157:H7 infection.

Phenotype-information-phenotype cycle for deconvolution of combinatorial antibody libraries selected against complex systems.

Use of large combinatorial antibody libraries and next-generation sequencing of nucleic acids are two of the most powerful methods in modern molecular biology. The libraries are screened using the principles of evolutionary selection, albeit in real time, to enrich for members with a particular phenotype. This selective process necessarily results in the loss of information about less-fit molecules. On the other hand, sequencing of the library, by itself, gives information that is mostly unrelated to phenotype. If the two methods could be combined, the full potential of very large molecular libraries could be realized. Here we report the implementation of a phenotype-information-phenotype cycle that integrates information and gene recovery. After selection for phage-encoded antibodies that bind to targets expressed on the surface of Escherichia coli, the information content of the selected pool is obtained by pyrosequencing. Sequences that encode specific antibodies are identified by a bioinformatic analysis and recovered by a stringent affinity method that is uniquely suited for gene isolation from a highly degenerate collection of nucleic acids. This approach can be generalized for selection of antibodies against targets that are present as minor components of complex systems.

Antibodies enhance interaction of Vibrio cholerae with intestinal M-like cells.

Intestinal M cells bear a receptor for secretory immunoglobulin A (IgA) (sIgA) facing the lumen of the epithelial surfaces. Cells bearing this receptor are also found throughout an experimental monolayer consisting of polarized Caco-2 cells, a colon adenocarcinoma cell line. The presence of antibodies (mainly sIgA) in the lumen of the small intestine led us to explore the participation of the sIgA receptor and antibodies in the interaction of Caco-2-associated M-like cells with the mucosal pathogen Vibrio cholerae. Here, we demonstrate that sIgA antibodies isolated from pooled healthy human colostrums, as well as IgG from pooled healthy human serum, can recognize V. cholerae. Furthermore, opsonization enhances M-like-cell transcytosis of V. cholerae strains. We also show that the cholera toxin (CT) receptor ganglioside GM(1) colocalizes with the sIgA receptor in cells of the epithelial monolayer. Both sIgA and IgG antibodies compete for the attachment of soluble CT subunit B to immobilized GM(1). Our results indicate that in this in vitro model system of intestinal epithelia, human sIgA and IgG contribute to the uptake of V. cholerae by M-like cells, probably through an interaction with GM(1). Our results support previous findings of others showing that sIgA can act as an endogenous adjuvant and that sIgA is important for the antigen-sampling function of M cells.

Specific immune response of mares and their newborn foals to Actinobacillus spp. present in the oral cavity.

Oral swab samples, serum and colostrum was taken from 15 mares and 14 of their foals, within 24 h of birth. The presence of antibody against Actinobacillus spp. isolated from the oral cavity was investigated using agar gel immunodiffusion. Antibodies against 48 out of the 77 Actinobacillus isolates from all horses in the study were present in the respective sera of 13 mares and 9 foals. In 11 mother-foal pairs, the antibody content of the foal serum was similar to that of the mare, and in 9 cases this was reflected in the antibody content of colostrum from the mare. The results indicate that an immune response to Actinobacillus spp. colonising the oral cavity is present in many adult horses and that this immune response can be transferred from mother to foal via colostrum.

A whirlpool associated outbreak of Pontiac fever at a hotel in Northern Sweden.

In April 1999, an outbreak of Pontiac fever occurred at a hotel in Northern Sweden. A retrospective cohort study to find the source and define the extent of the outbreak was carried out among 530 Swedish and Norwegian guests. Twenty-nine epidemiological cases (8% of 378 responders) aged 21-57 years were identified. Antibodies against Legionella micdadei were detected in 17 of 27 tested cases and 3 other symptomatic persons. Visiting the whirlpool area was identified as the sole risk factor (RR 86; 95% CI 21-352) and infected cases were confined to visitors to this area over three successive days. The attack rate was 71% (27/38) and 24 cases (83%) used the whirlpool. Environmental sampling was negative for Legionella sp. But epidemiological investigation strongly suggests that the whirlpool was the source of the outbreak. The possibility of serious legionella infections underlines the importance of strict maintenance practices to maintain hygiene of whirlpools.

Immunologic response to tetanus toxoid in geriatric patients.

STUDY OBJECTIVE: Tetanus antibody levels have been shown to be inadequate in 50% of patients older than 65 years. Although immunization recommendations have been made for this age group, the efficacy of this intervention has not been well documented. We sought to determine the difference in tetanus antibody levels after the administration of one tetanus toxoid immunization to geriatric patients without adequate titers. METHODS: Thirty-five patients older than 65 years at a large urban comprehensive care geriatric center who were documented to have inadequate tetanus antibody titers were each given one tetanus toxoid immunization. Repeat titers were obtained at least 2 months after the immunization with the use of enzyme-linked immunosorbent assay (Bindazyme kit; the Binding Site Corporation, Birmingham, England). We considered tetanus antibody levels greater than .17 IU/mL protective. RESULTS: The mean age was 79.4 years; 30 of 35 (86%) were female. Repeat tetanus antibody titers were obtained an average of 123 days (range, 63 to 204 days) after immunization with tetanus toxoid. The mean preimmunization antibody titer was .1 IU/mL (range, .04 to .16 IU/mL). After immunization, antibody titers increased a mean of .61 IU/mL (range, -.01 to 2.23 IU/mL; 95% confidence interval, .35 to .87 IU/mL). Thirty of the 35 patients who received a single injection of tetanus toxoid (86%) developed protective titers. We found no relationship between seroconversion and age, sex, or medical history; nor did we find a relationship between antibody level and time elapsed since immunization when repeat titers were obtained. CONCLUSION: Administration of one tetanus toxoid injection affords protective immunity in many geriatric patients.

Oral immunization of rabbits against Pasteurella multocida with an alginate microsphere delivery system.

Oral delivery of microencapsulated antigens is a potential means to vaccinate rabbits against Pasteurella multocida, a common bacterial pathogen. Groups of five rabbits were dosed orally on days 0, 7, and 14 with alginate microspheres prepared to contain no added protein, 5 mg of a potassium thiocyanate extract of P. multocida (PTE), or 5 mg of PTE with 200 micrograms of cholera toxin (CT). In addition, groups were dosed orally with 5 mg of soluble PTE with or without 200 micrograms CT, intranasally (IN) with 1 mg of soluble PTE, or with saline. Serum and nasal lavage samples collected prior to initial immunization and 10, 16, and 21 days later were assayed by ELISA for anti-PTE IgG and IgA. Strong nasal lavage IgA and serum IgG activities were found in samples from rabbits immunized with PTE IN or orally when incorporated into microspheres. Addition of CT did not significantly enhance either response. To examine the development of protective immunity, groups were similarly immunized and challenge-exposed IN on day 16 with 10(6) CFU of P. multocida. One week later, rabbits were euthanized, and specimens from the lungs, nasopharynx, liver, and inner ear were cultured for P. multocida. Less severe infections of the lung and nasopharynx developed in rabbits immunized with PTE IN or orally in microspheres, with or without added CT. In addition, culture of liver and tympanic bullae samples from these rabbits yielded growth of P. multocida less frequently compared to other P. multocida-challenged rabbits. Coadministration of CT and PTE did not significantly improve protective immunity to challenge.

Relationship between Legionella spp. and antibody titres at a therapeutic thermal spa in Portugal.

The presence of Legionella spp. in the water of a Portuguese spa was ascertained during the spa season, between May and November. Simultaneously the prevalence of anti-legionella antibodies in people attending the spa was also investigated. The antibody titres of 172 randomly selected patients and 42 therapists were determined, and compared with a control group of 503 blood donors. Legionellae were present in the spa water at low concentrations, generally lower than 10(3) c.f.u./l. A total of 92 strains representing eight different species or serogroups were isolated; the predominant isolates belonged to Legionella pneumophila serogroup 6 and to L. londiniensis. During the study, no clinical cases of Legionnaires' disease were observed, and the antibody titres were generally low in the groups studied. However, the antibody titres of the patients increased slightly during their stay at the spa, approaching the values for the therapists. Mean antibody titres in the groups related with the spa were significantly higher than those in the blood donors against five of the seven legionella antigens tested. The largest number of elevated antibody titres in the exposed groups were to the L. pneumophila sg 5 and sg 6 antigens.

Detection of antibodies against Actinobacillus pleuropneumoniae, serotype 2 in porcine colostrum using a blocking enzyme-linked immunosorbent assay specific for serotype 2.

A blocking enzyme-linked immunosorbent assay (ELISA) developed for detection of antibodies to Actinobacillus pleuropneumoniae serotype 2 in sera from pigs (Nielsen et al., 1991) was evaluated for its suitability to detect antibodies in colostrum to this serotype. Using colostrum from sows experimentally infected with serotype 2 and from herds known to be infected with this serotype, the sensitivity of the test was 100%. Antibodies to A. pleuropneumoniae serotype 2 could be detected in colostrum of experimentally infected sows until at least 5 days after farrowing. Positive results were not observed with colostrum samples from herds known to be free from A. pleuropneumoniae. The high diagnostic sensitivity and specificity of the assay indicated that screening of herds for A. pleuropneumoniae serotype 2 infection by testing colostrum would be a reliable and simple method for herd monitoring.

[A field experiment using E. coli-K99 vaccine]. / Een praktijkproef met een E. coli-K99 vaccin.

In a field experiment in which vaccine containing E. coli-K99 antigen were administered to approximately thirty per cent of the pregnant dairy cows in herds in which the calves were affected with scours, during the first five days of life (the other animals were treated with a placebo), following results were obtained: The colostrum of vaccinated cows contained significantly higher concentrations of immunoglobulins to E. coli-K99 than did that of controls (Table 1). From the start of the experiment, severe scouring disappeared in the calves of vaccinated cows, and from those of the controls. E. coli-K99 could no longer be isolated. Zoötechnical measures such as adequate supplementation colostrum, hygienic procedures, etc., probably played an important role in the disappearance of the problems of scouring in these herds.

Colostral transfer of immunity to Haemophilus parahaemolyticus in pigs.

Complement fixating antibodies for Haemophilus parahaemolyticus were shown to be transferred from immune sows to their offspring. Colostrum-fed 4-day-old piglets from immune sows resisted intranasal inoculation whereas their littermates, fed on cows' milk, were fully susceptible to the infection. Piglets inoculated later in the suckling period (3 to 8 weeks after birth) when their serumtiters had declined to very low levels, showed some degree of resistance, but the infection was not eliminated from the mucous membranes of the respiratory tract. In chronically infected breeding herds piglets are usually affected during the later part of the suckling period and clinical symptoms are often vague. Though a positive titer is indicative of resistance the results presented above show that protection is usually not complete. Further studies are required to ascertain whether the results obtained here are applicable in a rational control.