Low Levels Of Specific Antibodies Research Articles

New approach to generating of human monoclonal antibodies specific to the proteolytic domain of botulinum neurotoxin A.

Botulinum neurotoxins (BoNTs) cause botulism and are the most potent natural toxins known. Immunotherapy with neutralizing monoclonal antibodies (MAbs) is considered to be the most effective immediate response to BoNT exposure. Hybridoma technology remains the preferred method for producing MAbs with naturally paired immunoglobulin genes and with preserved innate functions of immune cells. The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against BoNTs. In an effort to develop novel BoNT type A (BoNT/A) immunotherapeutics, sorted by flow cytometry plasmablasts and activated memory B cells from a donor repeatedly injected with BoNT/A for aesthetic botulinum therapy could be used due to obtain hybridomas producing native antibodies. Plasmablasts and activated memory B-cells were isolated from whole blood collected 7 days after BoNT/A injection and sorted by flow cytometry. The sorted cells were then electrofused with the K6H6/B5 cell line, resulting in a producer of native human monoclonal antibodies (huMAbs). The 3 antibodies obtained were then purified by affinity chromatography, analyzed for binding by Western blot assay and neutralization by FRET assay. We have succeeded in creating 3 hybridomas that secrete huMAbs specific to native BoNT/A and the proteolytic domain (LC) of BoNT/A. The 1B9 antibody also directly inhibited BoNT/A catalytic activity in vitro. The use activated plasmablasts and memory B-cells isolated at the peak of the immune response (at day 7 of immunogenesis) that have not yet completed the terminal stage of differentiation but have undergone somatic hypermutation for hybridization allows us to obtain specific huMAbs even when the immune response of the donor is weak (with low levels of specific antibodies and specific B-cells in blood). A BoNT/A LC-specific antibody is capable of effectively inhibiting BoNT/A by mechanisms not previously associated with antibodies that neutralize BoNT. Antibodies specific to BoNT LC can be valuable components of a mixture of antibodies against BoNT exposure.

Identification and characterization of two salmon louse heme peroxidases and their potential as vaccine antigens

Salmon louse, Lepeophtheirus salmonis, represents major challenge for salmon farming. Current treatments impose welfare issues and are costly, whereas prophylactic measures are unavailable. Two salmon louse heme peroxidases (LsPxtl-1 and LsPxtl-2) were tested for their importance for parasite development and as potential vaccine candidates. LsPxtl-1 possesses two heme peroxidase domains and is expressed in ovaries and gut, whereas LsPxtl-2 encodes one domain and contains N-terminal signal peptide and an Eph receptor ligand-binding domain. LsPxtl-1, but not LsPxtl-2, knockdown in nauplius II stage caused poor swimming and death, indicating its importance for parasite development. Immunizations using single DNA plasmid injection encoding the peroxidases or heterologous prime (DNA) and boost (recombinant LsPxtl-2 protein) gave non-significant reduction in lice numbers. Single injection gave low specific antibody levels compared with the prime-boost. The findings suggest LsPxtl-1 is important for parasite development but formulations and vaccination modalities used did not significantly reduce lice infestation.

Secondary antibody deficiency is associated with development of infection in kidney transplantation: Results of a multicenter study.

We performed a multicenter study to assess the association between secondary antibody deficiency (immunoglobulin G [IgG] hypogammaglobulinemia combined with low levels of specific antibodies) and development of infection in kidney transplantation. We prospectively analyzed 250 adult kidney recipients at four centers. The assessment points were before transplantation and 7 and 30days after transplantation. The immune parameters were as follows: IgG, IgA, and IgM and complement factors C3 and C4 tested by nephelometry; specific IgG antibodies to cytomegalovirus (CMV) and IgG and IgG2 antibodies to pneumococcal polysaccharide (anti-PPS) determined using enzyme-linked immunosorbent assay. The clinical follow-up period lasted 6months. The clinical outcomes were CMV disease and recurrent bacterial infections requiring antimicrobial therapy. Multivariate logistic regression. At day 7, IgG hypogammaglobulinemia (IgG levels<700mg/dL) combined with low IgG anti-CMV antibody titers (defined as levels<10000 units) was present in 12% of kidney recipients. IgG hypogammaglobulinemia combined with low IgG anti-PPS antibody titers (defined as levels<10mg/dL) at 1month after kidney transplantation were recorded in 16% of patients. At day 7 the combination of IgG hypogammaglobulinemia and low anti-CMV titers was independently associated with the development of CMV disease (odds ratio [OR], 6.95; 95% confidence interval [CI], 1.17-41.31; P=.033). At day 30 after transplantation, the combination of IgG<700mg/dL and IgG anti-PPS<10mg/dL, was independently associated with recurrent bacterial infection (OR, 5.942; 95% CI, 1.943-18.172; P=.002). In a prospective multicenter study, early immunologic monitoring of secondary antibody deficiency proved useful for the identification of kidney recipients who developed severe infection.

Comel-Netherton syndrome: A local skin barrier defect in the absence of an underlying systemic immunodeficiency.

BackgroundComel‐Netherton syndrome (NS) is a rare autosomal disease, characterized by severe skin disease, hair shaft defects, atopic diathesis, and increased susceptibility for skin infections. Since patients with NS suffer from recurrent infections, it has been hypothesized that an underlying immunodeficiency attributes to this. Here, we studied clinical and immunological characteristics of the cohort of NS patients in the Netherlands in order to identify whether potential immunodeficiencies result in the increased risk of infectious complications.MethodsPhenotypes were scored for severity of skin condition, specific hair shaft defects, atopy, and recurrent infections. Patients’ blood samples were collected for quantification of serum immunoglobulin (Ig) levels, specific antibodies against Streptococcus pneumoniae, and allergen‐specific IgE, as well as detailed immunophenotyping of blood leukocyte and lymphocyte subsets by flow cytometry.Results A total of 14 patients were included with age range 3‐46 years and varying degrees of skin involvement. All patients presented with atopic symptoms (food allergy, n = 13; hay fever, n = 10; asthma, n = 7). Recurrent skin infections were common, particularly in childhood (n = 12). Low levels of specific antibodies against S pneumoniae were found in 10 of 11 evaluated patients. Detailed immunological analysis was performed on 9 adult patients. Absolute numbers of lymphocyte subsets and serum immunoglobulin levels were all within normal ranges.ConclusionMultidisciplinary evaluation of our national cohort showed no evidence for a severe, clinically relevant systemic immunodeficiency. Therefore, we conclude that in Dutch NS patients the increased risk of infections most likely results from the skin barrier disruption and that increased allergen penetration predisposes to allergic sensitization.

Cancer Vaccines Co-Targeting HER2/Neu and IGF1R.

(1) Background: Human epidermal growth factor receptor 2 (HER2)/neu-driven carcinogenesis is delayed by preventive vaccines able to elicit autochthonous antibodies against HER2/neu. Since cooperation between different receptor tyrosine kinases (RTKs) can occur in human as well as in experimental tumors, we investigated the set-up of DNA and cell vaccines to elicit an antibody response co-targeting two RTKs: HER2/neu and the Insulin-like Growth Factor Receptor-1 (IGF1R). (2) Methods: Plasmid vectors carrying the murine optimized IGF1R sequence or the human IGF1R isoform were used as electroporated DNA vaccines. IGF1R plasmids were transfected in allogeneic HER2/neu-positive IL12-producing murine cancer cells to obtain adjuvanted cell vaccines co-expressing HER2/neu and IGF1R. Vaccination was administered in the preneoplastic stage to mice prone to develop HER2/neu-driven, IGF1R-dependent rhabdomyosarcoma. (3) Results: Electroporated DNA vaccines for murine IGF1R did not elicit anti-mIGF1R antibodies, even when combined with Treg-depletion and/or IL12, while DNA vaccines carrying the human IGF1R elicited antibodies recognizing only the human IGF1R isoform. Cell vaccines co-expressing HER2/neu and murine or human IGF1R succeeded in eliciting antibodies recognizing the murine IGF1R isoform. Cell vaccines co-targeting HER2/neu and murine IGF1R induced the highest level of anti-IGF1R antibodies and nearly significantly delayed the onset of spontaneous rhabdomyosarcomas. (4) Conclusions: Multi-engineered adjuvanted cancer cell vaccines can break the tolerance towards a highly tolerized RTK, such as IGF1R. Cell vaccines co-targeting HER2/neu and IGF1R elicited low levels of specific antibodies that slightly delayed onset of HER2/neu-driven, IGF1R-dependent tumors.

Immune responses result in misdiagnosis of Schistosoma japonicum by immunodiagnosis kits in egg-positive patients living in a low schistosomiasis transmission area of China.

BackgroundIn recent field surveys, we failed to detect the presence of specific antibody against Schistosoma japonicum in some egg-positive patients by commonly used immunodiagnostic kits. To find out whether low levels of specific antibody truly exist among egg-positive individuals and elucidate the underlying immune mechanisms, we carried out a cross-sectional epidemiologic study in a S. japonicum low transmission endemic area of Poyang Lake region, China and compared the humoral and cellular immune characteristics between S. japonicum high and low antibody responders.MethodsKato–Katz thick smear assay was used to determine the schistosomiasis status of 3,384 participants residing in two Poyang Lake region villages, Jiangxi, China. Among the 142 stool egg-positive participants, we identified low and high S. japonicum antibody responders with soluble egg antigen (SEA) and adult worm antigen (AWA) specific IgG levels by adopting ROC curve analysis. To compare the humoral and cellular immune responses between high and low S. japonicum antibody responders, serum specific antibody levels as well as the percentage of T lymphocyte subpopulation in PMBC, and cell stimulated cytokines (IFN- gamma and interlukin-10) were detected.ResultsEight S. japonicum egg-positive participants were defined as low antibody responders. Although the percentage of CD3+T cells in low responders was slightly higher and the percentage of CD4+ T cells, CD8+ T cells, the ratio of CD4+/CD8+ and CD4+ CD25+ Treg cells were lower than those in high responders, the differences between the two groups were not significant (P > 0.05). AWA -stimulated interlukin-10 level was significantly higher in high responders, while other cytokines did not show differences between two groups. For antibody profiles, except AWA specific IgA, significant differences of each antibody isotype between low and high responders were detected (P < 0.05).ConclusionsOur study confirmed that there are S. japonicum antibody low responders among schistosome egg-positive residents in S. japonicum low-transmission areas in China. Thus, mis-diagnosis using immune-diagnosis kits do exist. Significant differences of responding antibody levels between low and high responders were detected, while no major cellular response changes were observed.

The effects of viral vaccination of dairy heifer calves on the incidence of respiratory disease, mortality, and growth

Bovine respiratory disease (BRD) is one of the most common infectious causes of morbidity and mortality in young dairy cattle. The objective of this randomized clinical trial was to determine the effectiveness of 1 or 2 doses of a 5-way, modified-live viral vaccine, administered to heifer calves before weaning to aid in the prevention of BRD. The hypotheses were that vaccination would reduce the incidence of BRD and mortality, and that 2 doses would be more effective than 1. A total of 2,874 heifer calves from 19 commercial dairy farms in Minnesota and Ontario were enrolled at 1 to 7d of age and were followed until 3mo of age. Calves were randomly assigned to receive a commercial, intramuscular, modified-live vaccine against bovine viral diarrhea virus types 1 and 2, bovine respiratory syncytial virus, bovine herpesvirus type 1, and parainfluenza virus type 3 at 15 to 21d of age (2wk only), 35 to 42d (5wk only), both 2 and 5wk, or sterile saline at both times (unvaccinated controls). The incidence of failure of passive transfer was 11 or 32%, using cut-points of serum total protein of 5.2 and 5.7g/dL, respectively. Overall, 22% of calves were treated at least once for BRD. The incidence risk of naturally occurring BRD was 7.7% before 2wk of age, 8.0% between 2 and 5wk, and 9.5% between 5wk and 3mo of age, and was not different between vaccination groups. Overall mortality throughout the 3-mo study period was 3.5%. Mortality was 1.6% before 2wk of age, 0.5% between 2 and 5wk, and 1.2% between 5wk and 3mo of age. The risk of mortality was not affected by vaccination. Mean average daily gain of 1.07kg/d from 5wk to 3mo of age was not different between vaccine groups. In this population of commercial, home-raised calves, with an overall low incidence of failure of passive transfer, intramuscular vaccination with a multivalent, modified live viral vaccine at 2 or 5wk of age or both was not associated with a decreased risk of BRD or mortality, or with growth until 3mo of age. Reasons for these findings may include interference by maternal antibodies, unresponsiveness of the neonatal immune system, timing of immunity relative to pathogen exposure, disease caused by pathogens other than the viruses in the vaccine, or herd immunity. However, in populations with higher incidence of failure of passive transfer or risk of BRD, calves with low levels of specific antibodies may respond differently to vaccination.

Prevalence of Low Titers of Pneumococcal Antibodies

Objective: 1) Determine the prevalence of low titers of pneumococcal antibodies (PAB) in patients with recurrent infections. 2) Assess whether there is a difference in PAB titers between patients unimmunized and patients vaccinated against pneumococcus. Method: Cross-sectional study between September 2006 and May 2008 in patients with recurrent infections. PAB levels against 10 serotypes were measured in all patients, and it was established if they had been vaccinated against pneumococcus. We compared the frequency of PAB low titers between both groups using the chi-square test. Results: A total of 257 patients were included in the study, aged between 0 and 68 years (median: 5 years) and with 54% being male. Sixty-one patients (24%) were vaccinated against pneumococcus. The prevalence of low titers of PAB (more than 50% of the serotypes with levels lower than 1.3 ug/mL) was 43% overall, with 8.2% and 53.6% in vaccinated and unvaccinated patients, respectively ( P &lt; .0001). Conclusion: Low levels of specific antibodies to pneumococcus is a common condition in patients with recurrent infections. It appears that the pneumococcal vaccine plays an important role in reducing the prevalence of this condition, but prospective studies are needed to prove it and show the clinical usefulness of the vaccine.

Complement‐mediated killing of Philasterides dicentrarchi (Ciliophora) by turbot serum: relative importance of alternative and classical pathways

The present study was carried out to elucidate the in vitro killing activity of turbot complement and specific antibodies against the ciliate parasite Philasterides dicentrarchi. Fresh serum from nonimmunized fish showed a moderate ability to kill the parasite, which indicates that P. dicentrarchi is able to activate the alternative complement pathway (ACP). Fresh serum from immunized fish, which contained high levels of specific antibodies, showed greater killing activity. Heat-inactivated serum, with or without antibodies, and antibodies alone did not have any effect on parasite viability, which indicates that serum mainly kills P. dicentrarchi through the antibody-mediated classical complement pathway (CCP). Ascitic fluid from infected fish, but containing low levels of specific antibodies, showed a low ability to kill the parasite, and fresh serum from nonimmunized infected fish did not kill the parasite. The latter serum contained some specific antibodies but lower levels of complement than serum from control and vaccinated fish, and the lack of ability of this serum to kill the parasite was probably related to low levels of complement. In addition, serum and ascitic fluid from infected turbot showed high proteolytic activity which degraded fish Igs. The proteolytic activity generated may favour survival of the parasite during infection.

Utilization of Complement-Dependent Cytotoxicity To Measure Low Levels of Antibodies: Application to Nonstructural Protein 1 in a Model of Japanese Encephalitis Virus

Enzyme-linked immunosorbent assay (ELISA) and related assays are representative of methods currently used for antibody tests. However, they occasionally produce nonspecific reactions, thus making it difficult to reliably measure low levels of specific antibodies. To find a test method that minimizes nonspecific reactions, we introduced the principle of antibody-mediated complement-dependent cytotoxicity (CDC) into an antibody assay. The procedure has three steps: (i) the mixing of test samples with a suspension of cells expressing the antigen of interest on their surfaces, (ii) the addition of rabbit complement, and (iii) the measurement of lactose dehydrogenase (LDH) activities by adding a chromogenic substrate to the reaction mixture. When the specific antibodies exist in the sample, complement activation triggered by antibody binding on the surface of the antigen-expressing cells may lyse the cells, releasing LDH into the medium. Mouse and rabbit sera hyperimmune to nonstructural protein 1 (NS1) of Japanese encephalitis virus (JEV) lysed NS1-expressing cells in a dose-dependent manner. Evaluations using sera from horses naturally infected with JEV showed that the CDC assay had quantitative correlation and qualitative agreement with previously established NS1 antibody-detecting immunostaining and ELISA methods. The assay method also detected NS1 antibodies in sera of mice 2 days after experimental infection with JEV; specific, but not natural, immunoglobulin M antibodies were detected. Since almost all sera examined in this study showed no nonspecific reactions, the CDC assay was shown to be a reliable method for measuring low levels of specific antibodies.

Evaluation of a commercial assay for the detection of mumps specific IgM antibodies in oral fluid and serum specimens

Background An IgM antibody capture radioimmunoassay (MACRIA) has been used in the UK Reference Laboratory for the detection of mumps specific IgM antibodies in oral fluid and serum samples. The method has proved both sensitive and specific and has been used for the surveillance of mumps since 1994. The use of radioactive labels has restricted the use of MACRIA to specialised laboratories and alternative, non-isotopic, sensitive assays capable of detecting the low levels of specific antibodies in oral fluid have not been available. Recently, a novel mumps specific IgM capture enzyme immunoassay (MACEIA) utilising recombinant mumps nucleoprotein (rMuVN) and monoclonal antibodies to the nucleoprotein, produced by Microimmune Limited, has been developed for use with both serum and oral fluid samples. Objectives In this study, we have evaluated the performance of the Microimmune MACEIA for both serum and oral fluid against specimens tested by MACRIA. Study design The panel consisted of matched serum and oral fluid specimens from 137 cases of suspected mumps received in the Virus Reference Department for routine investigation from March 2003 to October 2004. Results The sensitivity, specificity, positive and negative predictive value of the Microimmune MACEIA on serum samples compared to MACRIA were 98.8%, 100.0%, 100.0% and 98.5%, respectively. The sensitivity, specificity, positive and negative predictive value of the Microimmune MACEIA on oral fluid samples compared to Microimmune MACEIA and MACRIA consensus results on the paired serum samples were 90.3%, 97.6%, 98.3% and 87.1%, respectively. Conclusions The Microimmune MACEIA was found to be a rapid, sensitive and specific alternative to MACRIA for the detection of mumps specific IgM in sera and oral fluids.

Clinical development and serological antibody responses in sheep and rabbits experimentally infested with Psoroptes ovis and Psoroptes cuniculi

Psoroptes ovis of sheep origin, and Psoroptes cuniculi of rabbit origin were used in experimental infestations. In experiment I, groups of four rabbits and four sheep were infested with 50–100 mites of each isolate on the skin of the back (skin infestation, SI) or in the external auditory canal (aural infestation, AI). In rabbits, SI and AI with P. cuniculi and AI with P. ovis induced in all animals typical ear lesions and pronounced antibody reactions to P. cuniculi antigens in ELISA. After SI of rabbits with P. ovis no clinical signs were detected, no mites could be reisolated and no specific antibodies were detected. In sheep, P. ovis SI induced mange whereas AI did not induce typical clinical signs and mites could not be reisolated. In both these animal groups, ELISA revealed pronounced and comparable specific antibody reactions. After SI and AI with P. cuniculi no clinical symptoms were observed and no mites could be reisolated. Nevertheless, low levels of specific antibody were detected. In experiment II, clinical progression and antibody reactions to P. ovis SI in naive sheep were compared with sheep previously exposed to P. ovis or P. cuniculi. In both pre-exposed groups of animals, clinical signs appeared within 2 days after challenge infestation and three days earlier than in primarily infested sheep. Subsequently, no obvious difference in the clinical progression was observed between the three groups of animals. The results of this study document antigenetic crossreactivity of the two morphologically and genetically distinguishable Psoroptes species but differences in their biological behaviour and virulence which both are of epidemiological and taxonomic relevance.

Leucocyte populations and responses to immunization and photoperiod manipulation in Atlantic salmon ( Salmo salar L.) 0+ smolt

The immune status of season-independent Atlantic salmon smolt may be affected by changes in photoperiod and high rearing temperatures. Variations in different leucocyte subpopulations were measured in photo-manipulated out-of-season (0+) Atlantic salmon smolt by use of flow cytometry and specific monoclonal antibodies. 0+ smolts were produced by exposing parr to continuous light (LD 24:0) until June 10 followed by a “winter” photoperiod (LD 12:12) for 6 weeks, and then continuous light. Three different groups were studied; untreated smolt (UT), smolt injected with a commercial multivalent oil-adjuvanted vaccine (VA), and smolt injected with the saline/oil-adjuvant used in the vaccine (AD). The fish were injected in the “winter” photoperiod. From introduction of “winter” photoperiod until 2 weeks after seawater transfer, fractions of B-cells (identified by G2H3), neutrophiles (identified by E3D9) as well as polymorphonuclear and B-cells (identified by C4B6) were monitored. From introducing continuous photoperiod and until seawater transfer, there were variations for all groups within the leucocyte subpopulations identified by the monoclonal antibodies (MAb) used. Neutrophiles had a minor increase, while the number of B-cells decreased. C4B6, identifying both types of cells recognised by E3D9 and G2H3 remained stable, indicating that the leucocyte fraction identified by C4B6 was not changed, but there was redistribution with respect to the numbers of B-cells and neutrophiles. After seawater transfer, another redistribution of leucocytes took place. A minor reduction for the neutrophiles was observed, while at the same time, both B-cells and the polymorphonuclear cells increased. It was not possible to identify any effect of vaccine and/or adjuvant on the leucocytes studied. The specific serum antibody levels measured by enzyme-linked immunosorbent assay (ELISA) were low during the experimental period. A low immune response was observed measured as antibody-producing cells (APC) by ELISPOT. The low levels of specific antibodies supported the findings of low APC numbers. There were indications that UT had lower numbers of APC compared to AD and VA. The results show that changes with respect to fractions of leucocyte populations take place during parr–smolt transformation, and these changes may affect the cellular and humoral immune response to vaccination. Different photoperiod regimes may have different effects on leucocyte populations and general conclusions should not be made from the single photoperiod studied.

DNA vaccination against tuberculosis: expression of a ubiquitin-conjugated tuberculosis protein enhances antimycobacterial immunity.

Genetic immunization is a promising new technology for developing vaccines against tuberculosis that are more effective. In the present study, we evaluated the effects of intracellular turnover of antigens expressed by DNA vaccines on the immune response induced by these vaccines in a mouse model of pulmonary tuberculosis. The mycobacterial culture filtrate protein MPT64 was expressed as a chimeric protein fused to one of three variants of the ubiquitin protein (UbG, UbA, and UbGR) known to differentially affect the intracellular processing of the coexpressed antigens. Immunoblot analysis of cell lysates of in vitro-transfected cells showed substantial differences in the degradation rate of ubiquinated MPT64 (i.e., UbG64 < UbA64 < UbGR64). The specific immune response generated in mice correlated with the stability of the ubiquitin-conjugated antigen. The UbA64 DNA vaccine induced a weak humoral response compared to UbG64, and a mixed population of interleukin-4 (IL-4)- and gamma interferon (IFN-gamma)-secreting cells. Vaccination with the UbGR64 plasmid generated a strong Th1 cell response (high IFN-gamma, low IL-4) in the absence of a detectable humoral response. Aerogenic challenge of vaccinated mice with Mycobacterium tuberculosis indicated that immunization with both the UbA64- and UbGR64-expressing plasmids evoked an enhanced protective response compared to the vector control. The expression of mycobacterial antigens from DNA vaccines as fusion proteins with a destabilizing ubiquitin molecule (UbA or UbGR) shifted the host response toward a stronger Th1-type immunity which was characterized by low specific antibody levels, high numbers of IFN-gamma-secreting cells, and significant resistance to a tuberculous challenge.

Proteus syndrome and immunodeficiency

A 10 year old boy with Proteus syndrome presented with a pericardial effusion of unknown aetiology. Immunological investigation revealed low serum IgG and IgA, accompanied by low levels of specific...

The stability and immunogenicity of a protein antigen encapsulated in biodegradable microparticles based on blends of lactide polymers and polyethylene glycol

Protein-loaded microparticles were produced from blends of poly(ethylene glycol) (PEG) with poly( l-lactide) (PLA) homopolymer or poly( dl-lactide co-glycolide) copolymers (PLG) using a water-in oil-in oil method. The stability of ovalbumin (OVA) associated with microparticles prepared using PEG and 50:50 PLG, 75:25 PLG and PLA, respectively, was analysed by SDS-PAGE and quantified by scanning densitometry following incubation in PBS at 37°C for up to 1 month. Fragmentation and aggregation of OVA was detected with all 3 formulations. The extent of both processes correlated with the degradation rate of the lactide polymer used and decreased in the order PLA<75:25 PLG<50:50 PLG. Extensive degradation of the PLG/PEG microparticles also occurred over 4 weeks whereas the use of PLA/PEG blends resulted in a stable microparticle morphology and much reduced fragmentation and aggregation of the associated protein. Following a single sub-cutaneous immunisation, high levels of specific serum IgG antibody were elicited by OVA associated with the PLA/PEG particles. Injection of OVA associated with the 75:25 PLG/PEG microparticles resulted in very low levels of specific antibody. A higher response was induced by the 50:50 PLG/PEG formulation but there was very large inter-animal variation in this group. Antibody levels elicited by all 3 formulations were significantly higher than those elicited by a single injection of soluble OVA. Analysis of antigen specific IgG1 and IgG2a antibody subtype levels also revealed the greater efficacy of the PLA/PEG microparticles as an adjuvant system. The use of PLA/PEG microparticles shows improved protein loading and delivery capacity while maintaining a high level of stability of the associated protein. These results indicate a strong correlation between the stability of microencapsulated antigen and the magnitude of the immune response following sub-cutaneous immunisation.

Antibody and Th1/Th2-type responses in BALB/c mice inoculated with live or dead Echinococcus granulosus protoscoleces.

The aims of this study were to investigate whether a Th1- or a Th2-type response is stimulated in the first stages of experimental infection with Echinococcus granulosus, and to determine whether live or dead protoscoleces equally contribute to such Th1/Th2-type polarization. Live parasites stimulated the production of IL-10, IL-4 and IL-5 as early as week 1 postinoculation. The levels of IL-10 and IL-4 decreased towards week 4 p.i. and that of IFN gamma increased. The production of specific antibodies was characterized by high levels of systemic IgG1 and local IgM and IgG3 (measured in peritoneal lavages). In contrast, dead parasites induced elevated levels of IL-4, IFN gamma, IL-10 and IL-5 on week 1 postinoculation followed by a decrease of IFN gamma and an increase of IL-4. Low levels of specific antibodies were stimulated by dead parasites both systemically and in the peritoneal cavity. These results show that E. granulosus infection induced an early Th2-type response and that live parasites stimulated stronger antibody responses than dead parasites. In addition, they strongly suggest that both phenomena were modulated by live protoscoleces.

Passive transfer of specific maternal immunity does not protect Atlantic salmon ( Salmo salarL.) fry against yersiniosis

In order to elucidate whether specific protective immunity can be transferred from mother to offspring in Atlantic salmon, brood fish were vaccinated repeatedly by injection against yersiniosis during the period of egg development. An increase in specific antibodies measured by ELISA against Yersinia ruckeriserovar O2, and of bacterial agglutinating titres against both serovar O1 and O2 of Y. ruckeriwas demonstrated in the serum of vaccinated brood fish. Agglutination was also detected in homogenates of freshly stripped eggs and of fry, but at equal levels in groups from vaccinated and nonvaccinated mothers. Vaccination of the brood females resulted in low levels of specific antibodies against Y. ruckeriserovar O2 in their eggs and in fry sampled one week after hatching. However, mortalities after challenge were not reduced in fry descending from vaccinated mothers. These results indicate that specific maternal antibodies are transferred from Atlantic salmon mothers to eggs and fry, but at very low levels, insufficient to protect the offspring against yersiniosis.

Serologic responses to Rhodococcus equi in individuals with and without human immunodeficiency virus infection.

Thirty healthy blood donors, 15 workers from horse-breeding farms, 69 human immunodeficiency virus (HIV)-negative persons at risk for HIV infection, 125 HIV-infected subjects without Rhodococcus equi infection, and nine HIV-infected patients with Rhodococcus equi pneumonia were evaluated in order to detect serum antibodies to Rhodococcus equi precipitate-soluble antigen by an enzyme immunoassay (EIA). Whereas EIA values for healthy donors, horse farm workers, individuals at risk for HIV infection, and HIV-positive subjects without Rhodococcus equi infection were comparable, HIV-infected patients with rhodococcal disease had significantly higher Rhodococcus equi antibody levels (p < 0.0001). The clinical outcome of Rhodococcus equi pneumonia was more severe in subjects who had low levels of specific antibodies, whereas patients who recovered had elevated Rhodococcus equi antibody levels over time. Immunoblot studies showed that both Rhodococcus equi-infected patients and foals recognized a protein band of approximately 60 kDa in the Rhodococcus equi precipitate-soluble antigen. On the other hand, the Rhodococcus equi-infected patients did not react with the diffuse 15 to 17 kDa virulence-associated proteins that represent important virulence factors both in mice and horses.

Helicobacter pylori infection, nutrition and growth of West African infants

Helicobacter pylori is probably the commonest bacterial infection of humankind. In adults, colonization of the stomach is associated with chronic gastritis and duodenal ulcer disease. However, children in the developing world acquire H. pylori soon after birth, and there is evidence that it plays a part, through suppression of the gastric acid barrier, in the pathogenesis of the syndrome of diarrhoea, malnutrition and growth failure. Infants born of mothers who secrete milk with high levels of anti- H. pylori immunoglobulin A (IgA) antibody acquire the infection later than those born of mothers with low specific antibody levels. Enhancement of maternal breast milk anti- H. pylori IgA levels may protect infants from H. pylori infection during the vulnerable weaning period when many are susceptible to enteric infections, leading to recurrent diarrhoea and adverse consequences on nutrition and growth.