Immune response of horses to vaccination with the recombinant Hc domain of botulinum neurotoxin types C and D

Botulinum neurotoxins, predominantly serotypes C and D, cause equine botulism through forage poisoning. The C-terminal part of the heavy chain of botulinum neurotoxin types C and D (HcBoNT/C and D) was expressed in Escherichia coli and evaluated as a recombinant mono- and bivalent vaccine in twelve horses in comparison to a commercially available toxoid vaccine. A three-dose subcutaneous immunization of adult horses elicited robust serum antibody response in an ELISA using the immunogen as a capture antigen. Immune sera showed dose-dependent high potency in neutralizing specifically the active BoNT/C and D in the mouse protection assay. The aluminium hydroxide based mono- and bivalent recombinant HcBoNT/C and D vaccines were characterized by good compatibility and the ability to elicit protective antibody titers similar or superior to the commercially available toxoid vaccine.

Vaccine efficacy of recombinant cathepsin D aspartic protease from Schistosoma japonicum

Mice were vaccinated with recombinant Schistosoma japonicum cathepsin D aspartic protease, expressed in both insect cells and bacteria, in order to evaluate the vaccine efficacy of the schistosome protease. Mean total worm burdens were significantly reduced in vaccinated mice by 21-38%, and significant reductions in female worm burdens were also recorded (22-40%). Vaccination did not reduce fecundity; rather, we recorded increased egg output per female worm in vaccinated animals, suggesting a crowding effect. Vaccinated mice developed high levels of antibodies (predominantly IgG1, IgG2a and IgG2b isotypes), but there was no correlation between antibody levels and protective efficacy. Immune sera from vaccinated mice did not inhibit the in vitro degradation of human haemoglobin by the recombinant protease, and passive transfer of serum or antibodies from vaccinated animals, before and after parasite challenge, did not significantly reduce worm or egg burdens in recipient animals. These results suggest that antibodies may not play a key role in the protective effect elicited, and that protection may be due to a combination of humoral and cell-mediated responses.

Entamoeba histolytica: antigenic characterization of axenic strains from Brazil

Trophozoites from cultures of Entamoeba histolytica strains isolated and grown axenically in Brazil (ICB-CSP, ICB-462 and ICB-32) were used for immune sera production and for characterization of their antigens by using electrophoretic and glycoproteic profiles, in parallel with a standard strain isolated and kept under axenic conditions in USA (HK-9). Hyperimmune sera, presenting high antibody titers with homologous and heterologous antigens, were obtained. The four strains in study revealed similar and complex electrophoretic and glycoproteic profiles showing polypep-tides with molecular weights ranging from 200 to less than 29 kDa. No significant differences were detected between the pathogenic and non-pathogenic strains.

Production of monoclonal antibodies anti-Taenia crassiceps cysticerci with cross-reactivity with Taenia solium antigens

We describe the production of the potential monoclonal antibodies (MoAbs) using BALB/c mice immunized with vesicular fluid (VF)-Tcra (T. crassiceps) antigen. Immune sera presented anti-VF-Tcra (<20kD) IgG and IgM antibodies with cross-reactivity with T. solium (Tso) antigen (8-12, 14, and 18 kD). After cell fusion, we selected 33 anti-Tcra and anti-Tso reactive IgM-clones and 53 anti-Tcra specific IgG-clones, 5 of them also recognizing Tso antigens. Two clones identified the 8-14 and 18kD peptides of VF-Tcra.

Sharing of antigens between Plasmodium falciparum and Anopheles albimanus

The presence of common antigens between Plasmodium falciparum and Anopheles albimanus was demonstrated. Different groups of rabbits were immunized with: crude extract from female An. albimanus (EAaF), red blood cells infected with Plasmodium falciparum (EPfs), and the SPf66 synthetic malaria vaccine. The rabbit's polyclonal antibodies were evaluated by ELISA, Multiple Antigen Blot Assay (MABA), and immunoblotting. All extracts were immunogenic in rabbits according to these three techniques, when they were evaluated against the homologous antigens. Ten molecules were identified in female mosquitoes and also in P. falciparum antigens by the autologous sera. The electrophoretic pattern by SDS-PAGE was different for the three antigens evaluated. Cross-reactions between An. albimanus and P. falciparum were found by ELISA, MABA, and immunoblotting. Anti-P. falciparum and anti-SPf66 antibodies recognized ten and five components in the EAaF crude extract, respectively. Likewise, immune sera against female An. albimanus identified four molecules in the P. falciparum extract antigen. As far as we know, this is the first work that demonstrates shared antigens between anophelines and malaria parasites. This finding could be useful for diagnosis, vaccines, and the study of physiology of the immune response to malaria.

Trypanosoma cruzi: antigen-receptor mediated endocytosis of antibody

Trypanomastigote forms of Trypanosoma cruzi were derived from tissue culture and incubated with immune and non-immune human sera. All immune sera showed high titers of specific humoral antibodies of the IgM or the IgG type. Agglutination and swelling of parasites were observed after incubation at 37��C, but many trypomastigotes remained free-swimming in the sera for two to three days. The quantitiy of immune serum capable of lysing a maximum of 10 x 10 [raised to the power of 6] sensitized red cells was not capable of lysing 4 x 10 [raised to the power of 3] tripomastigotes. Typically, the parasites underwent cyclical changes with the formation of clumps of amastigotes and the appearance of epimastigote forms. Multiplication of the parasites was observed in immune sera. Further, the infectivity of the parasites to susceptible mice was not lost. All sera used produced similar general effects on the growth of the parasite. The antibody bound to T. cruzi appeard to enter cells by antigen-receptor mediated endocytosis. The ferritin-conjugated antibody was internalized and delivered to phagolysosomes where they might be completely degraded to amino-acids. This seemed to be a coupled process by which the immunoglobulin is first bound to specific parasite surface receptor and then rapidly endocytosed by the cell.

Identification of immunogenic proteins of Waddlia chondrophila.

Evidence is growing for a role of Waddlia chondrophila as an agent of adverse pregnancy outcomes in both humans and ruminants. This emerging pathogen, member of the order Chlamydiales, is also implicated in bronchiolitis and lower respiratory tract infections. Until now, the serological diagnosis of W. chondrophila infection has mainly relied on manually intensive tests including micro-immunofluorescence and Western blotting. Thus, there is an urgent need to establish reliable high throughput serological assays. Using a combined genomic and proteomic approach, we detected 57 immunogenic proteins of W. chondrophila, of which 17 were analysed by mass spectrometry. Two novel hypothetical proteins, Wim3 and Wim4, were expressed as recombinant proteins in Escherichia coli, purified and used as antigens in an ELISA test. Both proteins were recognized by sera of rabbits immunized with W. chondrophila as well as by human W. chondrophila positive sera but not by rabbit pre-immune sera nor human W. chondrophila negative sera. These results demonstrated that the approach chosen is suitable to identify immunogenic proteins that can be used to develop a serological test. This latter will be a valuable tool to further clarify the pathogenic potential of W. chondrophila.

The use of ferromagnetic dacron as solid-phase in enzyme immunoassays

Ferromagnetic dacron is proposed as an alternative solid-phase for magnetic enzyme immunoassays. Human serum albumin (HSA) was covalentlyimmobilized onto ferromagnetic dacron and as enzyme immunoassay was developed using anti-HSA rabbit sera. Peroxidase, o-phenylenediamine (OPD) and hydrogen peroxide were used anti-HSA rabbit sera. Peroxidase, o-phenylenediamine (OPD) and hydrogen peroxide were used as the enzymatic label and substrates, respectively. Best results were observed when particles of 63-100 ��m (diameter) and 10 ��g of immobilized antigen were used. Positive reactions were detected until dilutions of1:51200 of immune sera. Its reproducibility was similar to standard ELISA. Disruption of the immunocomplexes formed and recuperation of the immobilized antigen in other immunoassays also proved to be reliable.

Plasmodium falciparum: nitric oxide modulates heme speciation in isolated food vacuoles.

Nitric oxide (NO) and NO-derived reactive nitrogen species (RNS) are present in the food vacuole (FV) of Plasmodium falciparum trophozoites. The product of PFL1555w, a putative cytochrome b(5), localizes in the FV membrane, similar to what was previously observed for the product of PF13_0353, a putative cytochrome b(5) reductase. These two gene products may contribute to NO generation by denitrification chemistry from nitrate and/or nitrite present in the erythrocyte cytosol. The possible coordination of NO to heme species present in the food vacuole was probed by resonance Raman spectroscopy. The spectroscopic data revealed that in situ generated NO interacts with heme inside the intact FVs to form ferrous heme nitrosyl complexes that influence intra-vacuolar heme solubility. The formation of heme nitrosyl complexes within the FV is a previously unrecognized factor that could affect the equilibrium between soluble and crystallized heme within the FV in vivo.

Immunological and protective properties of novel malaria blood stage peptide antigens

ABSTRACT Malaria is a major worldwide public health problem, with transmission occurring throughout Africa, Asia, Oceania and Latin America. Over two billion people live in malarious areas of the world and it is estimated that 300-500 million cases and 1.5-2.7 million deaths occur annually. The increase in multi-drug resistant parasites and insecticide-resistant vectors has made the development of malaria vaccine a public health priority. The published genome offers tremendous opportunity for the identification of new antigens that can befast-tracked for vaccine development. We identified potential protein antigens present on the surface of asexual malaria blood stages through bioinformatics and published transcriptome and proteorn�� analysis. Amongst the proteins identified, we selected those that contain predicted a-helical coiled-coil regions, which are generally short and structurally stable as isolated fragments. Peptides were synthesized and used to immunize mice. Most peptides tested were immunogenic as demonstrated in ELISA assays, and induced antibodies of varying titres. In immunofluorescence assays, anti-sera from immunized mice reacted with native proteins expressed at different intraerythrocytic developmental stages of the parasite's cycle. In parallel in vitro ADCI functional studies, human antibodies affinity purified on some of these peptides inhibited parasite growth in association with monocytes in magnitudes similar to that seen in semiimmune African adults. Siudies using human immune sera taken from different malaria endemic regions, demonstrated that majority of peptides were recognized at high prevalence. 73 peptides were next tested in longitudinal studies in two cohorts separated in space and time in coastal Kenya. In these longitudinal analyses, antibody responses to peptides were sequentially examined in two cohorts of children at risk of clinical malaria in order to characterize the level of peptide recognition by age, and the role of anti-peptide antibodies in protection from clinical malaria. Ten peptides were associated ?with a significantly reduced odds ratio for an episode of clinical malaria in the first cohort of children and two of these peptides (LR146 and ��S202.11) were associated with a significantly reduced odds ratio in both cohorts. This study has identified proteins PFB0145c and MAL6P1.37 among others as likely targets of protective antibodies. Our findings support further studies to systematically assess immunogenicity of peptides of interest in order to establish clear criteria for optimal design of potential vaccine constructs to be tested in clinical trials. RESUME La malaria est un probl��me de sant�� publique mondial principalement en Afrique, en Asie, en Oc��anie et en Am��rique latine. Plus de 2 milliards de personnes vivent dans des r��gions end��miques et le nombre de cas par ann��e est estim�� entre 300 et 500 millions. 1.5 �� 2.7 millions de d��c��s surviennent annuellement dans ces zones. L'augmentation de la r��sistance aux m��dicaments et aux insecticides fait du d��veloppement d'un vaccin une priorit��. Le s��quen��age complet du g��nome du parasite offre l'opportunit�� d'identifier de nouveaux antig��nes qui peuvent rapidement mener au d��veloppement d'un vaccin. Des prot��ines antig��niques potentielles pr��sentes �� la surface des globules rouges infect��s ont ��t�� identifi��es par bioinformatique et par l'analyse du prot��ome et du transcriptome. Nous avons s��lectionn��, parmi ces prot��ines, celles contenant des motifs dits "a helical coiled-coil" qui sont g��n��ralement courts et structurellement stables. Ces r��gions ont ��t�� obtenues par synth��se peptidique et utilis��es pour immuniser des souris. La plupart des peptides test��s sont immunog��niques et induisent un titre variable d'anticorps d��termin�� par ELISA. Les r��sultats de tests d'immunofluorescence indiquent que les sera produits chez la souris reconnaissent les prot��ines natives exprim��es aux diff��rents stades de d��veloppement du parasite. En parall��le, des ��tudes d'ADCI in vitro montrent qu�� des anticorps humains purifi��s �� partir de ces peptides associ��s �� des monocytes inhibent la croissance du parasite aussi bien que celle observ��e chez des adultes africains prot��g��s. Des ��tudes d'antig��nicit�� utilisant des sera de personnes prot��g��es de diff��rents ��ges vivant dans des r��gions end��miques montrent que la majorit�� des peptides sont reconnus avec une haute pr��valence. 73 peptides ont ��t�� test��s dans une ��tude longitudinale avec 2 cohortes de la c��te du Kenya. Ces 2 groupes viennent de zones bien distinctes et les pr��l��vements n'ont pas ��t�� effectu��s pendant la m��me p��riode. Dans cette ��tude, la r��ponse anticorps contre les peptides synth��tiques a ��t�� test��e dans les 2 cohortes d'enfants �� risque de d��velopper un ��pisode de malaria afin de caract��riser le niveau de reconnaissance des peptides en fonction de l'��ge et de d��terminer le r��le des anticorps anti-peptides dans la protection contre la malaria. Parmi ces peptides, 10 sont associ��s �� une r��duction significative des risques de d��velopper un ��pisode de malaria dans la premi��re cohorte alors qu'un seul (LR146 et AS202.11) l'est dans les 2 cohortes. Cette ��tude a identifi��, parmi d'autres, les prot��ines PFB0145c et MAL6P1.37 comme pouvant ��tre la cible d'anticorps. Ces r��sultats sont en faveur de futures ��tudes qui ��valueraient syst��matiquement l'immunog��nicit�� des peptides d'int��r��t dans le but d'��tablir des crit��res de s��lection clairs pour le d��veloppement d'un vaccin. R��sum�� pour un large public La malaria est un probl��me de sant�� publique mondial principalement en Afrique, en Asie, en Oc��anie et en Am��rique latine. Plus de 2 milliards de personnes vivent dans des r��gions end��miques et le nombre de cas par ann��e est estim�� entre 300 et 500 millions. 1.5 �� 2.7 millions de d��c��s surviennent annuellement dans ces zones. La r��sistance aux m��dicaments et aux insecticides augmente de plus en plus d'o�� la n��cessit�� de d��velopper un vaccin. Le s��quen��age complet du g��nome (ensemble des g��nes) de P. falciparum a conduit au d��veloppement de nouvelles .��tudes �� large ��chelle dans le domaine des prot��ines du parasite (prot��ome) ; dans l'utilisation d'algorithmes, de techniques informatiques et statistiques pour l'analyse de donn��es biologiques (bioinformatique) et dans les technologies de transcription et de profiles d'expression (transcriptome). Nous avons identifi��, en utilisant les outils ci-dessus, des nouvelles prot��ines antig��niques qui sont pr��sentes au stade sanguin de la malaria. Nous avons s��lectionn��, parmi ces prot��ines, celles contenant un motif dit "a-helical coiled-coil" qui sont des domaines impliqu��s dans un large ��ventail de fonctions biologiques. Des peptides repr��sentant ces r��gions structurellement stables ont ��t�� synth��tis��s et utilis��s pour immuniser des souris. La plupart des peptides test��s sont immunog��niques et induisent un titre variable d'anticorps d��termin�� par ELISA. Les r��sultats de tests d'immunofluorescence indiquent que plusieurs sera de souris immunis��es avec ces peptides reconnaissent les prot��ines natives exprim��es �� la surface des globules rouges infect��s. En parall��le, des ��tudes d'ADCI in vitro montrent que des anticorps humains purifi��s �� partir de ces peptides en pr��sence de monocytes inhibent la croissance du parasite de mani��re similaire �� celle observ��e chez des adultes africains prot��g��s. Des ��tudes d'antig��nicit�� utilisant des sera de personnes immunes de diff��rents ��ges (adultes et enfants) vivant dans des r��gions end��miques montrent que la majorit�� des peptides sont reconnus avec une haute pr��valence. 73 peptides ont ��t�� test��s dans des ��tudes ��pid��miologiques dans 2 villages c��tiers du Kenya Ces 2 groupes vivent dans des zones bien distinctes et les pr��l��vements n'ont pas ��t�� effectu��s pendant la m��me p��riode. Dans ces ��tudes, la r��ponse anticorps dirig��e contre les peptides synth��tiques a ��t�� test��e en utilisant 467 ��chantillons sanguins d'enfants �� risque de d��velopper un ��pisode de malaria afin de caract��riser le niveau de reconnaissance des peptides en fonction de l'��ge et de d��terminer le r��le des anticorps anti-peptides dans la protection contre la malaria c��r��brale. Parmi ces peptides, 10 sont associ��s �� une protection contre un ��pisode de malaria dans le premier village alors qu'un seul l'est dans les 2 villages. Ces r��sultats sont en faveur de futures ��tudes qui ��valueraient syst��matiquement l'immunog��nicit�� des peptides int��ressants dans le but d'��tablir des crit��res de s��lection clairs pour le d��veloppement d'un vaccin.

Serological cross-reactivity between different Chlamydia-like organisms.

The serological cross-reactivity between different recently described Chlamydia-related organisms was determined. Mouse sera exhibited a strong reactivity against autologous antigen and closely related heterologous antigen but no cross-reactivity with distantly related species. These results are important to better interpret serological studies and assess the pathogenic role of these obligate intracellular bacteria.

Equine secretory IgA and secretory component.

A complete secretory immunologie system has been identified in the equine species. It is characterised by the presence of a secretory component either bound to secretory IgA (SigA) or remaining in the free form (FSC). The mean molecular weights of SigA, serum lgA and FSC have been estimated. The homology of the equine and human IgA classes have been demonstrated by cross-reaction with anti-human lgA antisera. A quantit ative study of equine immunoglobulins in various fluids have shown that SlgA is predominant in saliva, mature milk, nasal and lacrimal secretions, but not in colostrum. In vitro binding of human and bovine FSC is found to occur mostly with the polymerie form of equine serum lgA.

Immunostimulatory property of a synthetic peptide belonging to the soluble ATP diphosphohydro-lase isoform (SmATPDase 2) and immunolocalisation of this protein in the Schistosoma mansoni egg

A peptide (SmB2LJ; r175-194) that belongs to a conserved domain from Schistosoma mansoni SmATPDase 2 and is shared with potato apyrase, as predicted by in silico analysis as antigenic, was synthesised and its immunostimulatory property was analysed. When inoculated in BALB/c mice, this peptide induced high levels of SmB2LJ-specific IgG1 and IgG2a subtypes, as detected by enzyme linked immunosorbent assay. In addition, dot blots were found to be positive for immune sera against potato apyrase and SmB2LJ. These results suggest that the conserved domain r175-194 from the S. mansoni SmATPDase 2 is antigenic. Western blots were performed and the anti-SmB2LJ antibody recognised in adult worm (soluble worm antigen preparation) or soluble egg antigen antigenic preparations two bands of approximately 63 and 55 kDa, molecular masses similar to those predicted for adult worm SmATPDase 2. This finding strongly suggests the expression of this same isoform in S. mansoni eggs. To assess localisation of SmATPDase 2, confocal fluorescence microscopy was performed using cryostat sections of infected mouse liver and polyclonal antiserum against SmB2LJ. Positive reactions were identified on the external surface from the miracidium in von Lichtenberg's envelope and, in the outer side of the egg-shell, showing that this soluble isoform is secreted from the S. mansoni eggs.

Tissue microarray and immunohistochemistry as tools for evaluation of antibodies against Chlamydia-like bacteria.

Tissue microarray technology was used to establish immunohistochemistry protocols and to determine the specificity of new antisera against various Chlamydia-like bacteria for future use on formalin-fixed and paraffin-embedded tissues. The antisera exhibited strong reactivity against autologous antigen and closely related heterologous antigen, but no cross-reactivity with distantly related species.

Time of injection determines the effect of alpha-MSH antiserum on DA neurons in psychological stress

Male rats were subjected to "psychological stress" which consisted in 10 sec footshock on the first day followed 24 hr later by a 10 sec stay in the experimental chamber without shock. Intravenous antiserum against alpha-MSH markedly changed the functional state of mesencephalic and hypothalamic DA neurons (assessed by histochemical microfluorimetry) when administered before the second session but not when given before the first session. These observations reveal an interesting parallelism in the temporal characteristics of the effects of alpha-MSH on avoidance behavior and central DA systems.