Immunological and Metabolomic Impacts of Administration of Cry1Ab Protein and MON 810 Maize in Mouse

We have investigated the immunological and metabolomic impacts of Cry1Ab administration to mice, either as a purified protein or as the Cry1Ab-expressing genetically modified (GM) MON810 maize. Humoral and cellular specific immune responses induced in BALB/cJ mice after intra-gastric (i.g.) or intra-peritoneal (i.p.) administration of purified Cry1Ab were analyzed and compared with those induced by proteins of various immunogenic and allergic potencies. Possible unintended effects of the genetic modification on the pattern of expression of maize natural allergens were studied using IgE-immunoblot and sera from maize-allergic patients. Mice were experimentally sensitized (i.g. or i.p. route) with protein extracts from GM or non-GM maize, and then anti-maize proteins and anti-Cry1Ab–induced immune responses were analyzed. In parallel, longitudinal metabolomic studies were performed on the urine of mice treated via the i.g. route. Weak immune responses were observed after i.g. administration of the different proteins. Using the i.p. route, a clear Th2 response was observed with the known allergenic proteins, whereas a mixed Th1/Th2 immune response was observed with immunogenic protein not known to be allergenic and with Cry1Ab. This then reflects protein immunogenicity in the BALB/c Th2-biased mouse strain rather than allergenicity. No difference in natural maize allergen profiles was evidenced between MON810 and its non-GM comparator. Immune responses against maize proteins were quantitatively equivalent in mice treated with MON810 vs the non-GM counterpart and no anti-Cry1Ab–specific immune response was detected in mice that received MON810. Metabolomic studies showed a slight “cultivar” effect, which represented less than 1% of the initial metabolic information. Our results confirm the immunogenicity of purified Cry1Ab without evidence of allergenic potential. Immunological and metabolomic studies revealed slight differences in mouse metabolic profiles after i.g. administration of MON810 vs its non-GM counterpart, but no significant unintended effect of the genetic modification on immune responses was seen

A MAP6-Related Protein Is Present in Protozoa and Is Involved in Flagellum Motility

In vertebrates the microtubule-associated proteins MAP6 and MAP6d1 stabilize cold-resistant microtubules. Cilia and flagella have cold-stable microtubules but MAP6 proteins have not been identified in these organelles. Here, we describe TbSAXO as the first MAP6-related protein to be identified in a protozoan, Trypanosoma brucei. Using a heterologous expression system, we show that TbSAXO is a microtubule stabilizing protein. Furthermore we identify the domains of the protein responsible for microtubule binding and stabilizing and show that they share homologies with the microtubule-stabilizing Mn domains of the MAP6 proteins. We demonstrate, in the flagellated parasite, that TbSAXO is an axonemal protein that plays a role in flagellum motility. Lastly we provide evidence that TbSAXO belongs to a group of MAP6-related proteins (SAXO proteins) present only in ciliated or flagellated organisms ranging from protozoa to mammals. We discuss the potential roles of the SAXO proteins in cilia and flagella function

Phosphorylcholine is favorable for antibody production from hybridoma cells

Growth of antibody-secreting hybridomas requires special conditions such as serum-free defined media containing growth factors and vitamins. However, the surface on which these cells can proliferate has been shown to play an important role. Phosphorylcholine (PC)-based polymers are zwitterionic compounds with nonbiofouling properties. These polymers are characterized by having reduced protein absorption properties. Our aim was to determine whether well-established hybridoma cell lines were able to proliferate and produce measurable amounts of monoclonal antibodies when grown on PC-polymer-coated surfaces. Comparative experiments using four well-known hybridoma cell lines (PAb421, PAb246, PAb1801 which recognize p53, and PAb280 which recognizes SV40 small t antigen) grown on PC-polymer-coated, uncoated, and two commercially available tissue culture plates showed that PC-polymer-coated plates were more efficient than uncoated plates in sustaining cell growth and monoclonal antibody production/secretion as defined by growth assays and ELISA. Also, results demonstrated that PC-polymer-coated plates were able to perform better than commercially available plates. These observations suggest that PC polymers could be used as an alternative, efficient surface coating to grow hybridoma cell lines and allow detectable antibody secretion. © 2005 Wiley Periodicals, Inc.Peer reviewe

An ELISA for sensitive and specific detection of circulating antigen of Angiostrongylus vasorum in serum samples of naturally and experimentally infected dogs

Canine angiostrongylosis is an emerging cardiopulmonary disease in Europe which can be fatal if left untreated. We developed a sandwich-ELISA based on a monoclonal antibody (mAb Av 56/1/2) and on polyclonal rabbit antibodies directed against Angiostrongylus vasorum adult excretory/secretory – antigen for the detection of circulating serum antigen of A. vasorum. The sensitivity of the test was 95.7% (78.1–99.9, 95% CI) as determined with sera of 23 dogs naturally infected with A. vasorum. The specificity was 94.0% (83.5–98.7, 95% CI) using 50 dog sera (control group) submitted for reasons other than parasitic infections. Potential cross-reactions were investigated with sera of a group of totally 61 dogs with proven infections with Dirofilaria immitis (n = 23), Crenosoma vulpis (n = 14), Ancylostoma caninum (n=4) or Toxocara canis (n = 20). No significant difference was observed concerning the proportion of positive reactions between the control group and the group with proven helminth infections other than A. vasorum. In experimentally inoculated dogs with proven worm burdens of A. vasorum, the proportion of seropositive dogs increased over the first 3 months of infection, starting from 35 days post inoculation (dpi) which was before the onset of larval excretion. Ten weeks post inoculation, 98.6% of the dogs were seropositive, and circulating antigen persisted in two dogs with long-term follow-up over 286 and 356 days, respectively. In contrast, in dogs with a single treatment with imidacloprid/ moxidectin at four or 32 dpi, no circulating antigen was observed, while in dogs treated at 88–92 dpi, OD values decreased within 13–34 days. The specific detection of circulating A. vasorum antigen by ELISA represents a valid alternative for reliable diagnosis and for follow-up investigations after anthelmintic treatment. Moreover, the test can be used for mass screening in large epidemiological investigations

Identification of Linear Epitopes in Bacillus anthracis Protective Antigen Bound by Neutralizing Antibodies*

Protective antigen (PA), the binding subunit of anthrax toxin, is the major component in the current anthrax vaccine, but the fine antigenic structure of PA is not well defined. To identify linear neutralizing epitopes of PA, 145 overlapping peptides covering the entire sequence of the protein were synthesized. Six monoclonal antibodies (mAbs) and antisera from mice specific for PA were tested for their reactivity to the peptides by enzyme-linked immunosorbent assays. Three major linear immunodominant B-cell epitopes were mapped to residues Leu156 to Ser170, Val196 to Ile210, and Ser312 to Asn326 of the PA protein. Two mAbs with toxin-neutralizing activity recognized two different epitopes in close proximity to the furin cleavage site in domain 1. The three-dimensional complex structure of PA and its neutralizing mAbs 7.5G and 19D9 were modeled using the molecular docking method providing models for the interacting epitope and paratope residues. For both mAbs, LeTx neutralization was associated with interference with furin cleavage, but they differed in effectiveness depending on whether they bound on the N- or C-terminal aspect of the cleaved products. The two peptides containing these epitopes that include amino acids Leu156–Ser170 and Val196–Ile210 were immunogenic and elicited neutralizing antibody responses to PA. These results identify the first linear neutralizing epitopes of PA and show that peptides containing epitope sequences can elicit neutralizing antibody responses, a finding that could be exploited for vaccine design

Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis▿

The control of intracellular pathogens such as Mycobacterium tuberculosis is dependent on the activation and maintenance of pathogen-reactive T cells. Dendritic cells (DCs) are the major antigen-presenting cells initiating antimycobacterial T-cell responses in vivo. To investigate if immunization strategies that aim to optimize DC function can improve protective immunity against virulent mycobacterial infection, we exploited the ability of the hematopoietic growth factor Fms-like tyrosine kinase 3 ligand (Flt3L) to expand the number of DCs in vivo. A DNA fusion of the genes encoding murine Flt3L and M. tuberculosis antigen 85B stimulated enhanced gamma interferon (IFN-γ) release by T cells and provided better protection against virulent M. tuberculosis than DNA encoding the single components. Vaccination of mice with a recombinant Mycobacterium bovis BCG strain secreting Flt3L (BCG:Flt3L) led to early expansion of DCs compared to immunization with BCG alone, and this effect was associated with increased stimulation of BCG-reactive IFN-γ-secreting T cells. BCG and BCG:Flt3L provided similar protective efficacies against low-dose aerosol M. tuberculosis; however, immunization of immunodeficient mice revealed that BCG:Flt3L was markedly less virulent than conventional BCG. These results demonstrate the potential of in vivo targeting of DCs to improve antimycobacterial vaccine efficacy

Detection of specific antibodies in dogs infected with Angiostrongylus vasorum

Canine angiostrongylosis, caused by the nematode Angiostrongylus vasorum, is an emerging cardiopulmonary disease in Europe which can be fatal if left untreated. We determined the diagnostic value of the specific detection of antibodies against A. vasorum adult somatic antigen, adult excretory/secretory (E/S) antigen and first stage larvae (L1) somatic antigen in ELISAs. Also, A. vasorum adult somatic antigen purified by monoclonal antibodies (mAb) was evaluated in a sandwich-ELISA. Among the crude antigens, the best sensitivities when testing 21 naturally infected dogs were obtained using adult E/S and somatic antigen (85.7% and 76.2%, respectively), which were comparable with the results of the sandwich-ELISA based on mAb-purified antigens (81%). The ELISA performed with L1 antigen had the lowest sensitivity (42.9%). In experimentally inoculated dogs, the sensitivities ranged from 97.7% to 100% with all test settings. The specificity was 98.8% (92.5-99.9%, 95% CI) with all ELISAs using sera of 82 randomly selected dogs. Cross-reactions using adult somatic, adult E/S and L1 somatic antigen were observed in sera of dogs infected with Crenosoma vulpis, Dirofilaria immitis, Dirofilaria repens, and Eucoleus aerophilus. In contrast, using the mAb-purified antigens, the cross-reactions were minimal. Depending on the antigens used, specific antibodies were detected starting between 13 and 21 days post experimental inoculation (dpi), and at latest between 35 and 48 dpi, thus before or around the onset of patency. The serological follow-up of four A. vasorum-infected dogs after anthelmintic treatment at 88 dpi showed a decrease of antibody levels after drug administration, and the animals became seronegative 2-9 weeks later. Two untreated dogs remained seropositive. In four dogs treated 4 dpi, virtually no antibody-reaction was detectable, with the exception of the ELISA performed with L1 antigen. The early detection of specific antibodies against A. vasorum by ELISA represents a valid alternative for a reliable diagnosis and for follow-up investigations after anthelmintic treatment