In vitro and in vivo evaluation of chlorhexidine salts as potential alternatives to potassium dichromate for Eimeria maxima M6 oocyst preservation.

Introduction: Coccidiosis caused by the Eimeria spp., an Apicomplexan protozoon, is a major intestinal disease that affects the poultry industry. Although most cases of coccidiosis are subclinical, Eimeria infections impair bird health and decrease overall performance, which can result in compromised welfare and major economic losses. Viable sporulated Eimeria oocysts are required for challenge studies and live coccidiosis vaccines. Potassium dichromate (PDC) is typically used as a preservative for these stocks during storage. Although effective and inexpensive, PDC is also toxic and carcinogenic. Chlorhexidine (CHX) salts may be a possible alternative, as this is a widely used disinfectant with less toxicity and no known carcinogenic associations. Methods: In vitro testing of CHX gluconate and CHX digluconate exhibited comparable oocyst integrity and viability maintenance with equivalent bacteriostatic and bactericidal activity to PDC. Subsequent use of CHX gluconate or digluconate-preserved Eimeria oocysts, cold-stored at 4°C for 5 months, as the inoculum also resulted in similar oocyst shedding and recovery rates when compared to PDC-preserved oocysts. Results and discussion: These data show that using 0.20% CHX gluconate could be a suitable replacement for PDC. Additionally, autofluorescence was used as a method to evaluate oocyst viability. Administration of artificially aged oocysts exhibiting >99% autofluorescence from each preserved treatment resulted in no oocyst output for CHX salt groups.

DNA aptamer-based rolling circle amplification product as a novel immunological adjuvant.

Several agonists to CD40 have shown to induce acquired immune responses. Here, we developed and evaluated the rolling circle amplification (RCA) products that are based on anti-CD40 DNA aptamers as a novel vaccine adjuvant. First, we developed DNA aptamers with specific binding affinity to chicken CD40 extra domain (chCD40ED). Next, we prepared the RCA products that consist of these aptamers to increase the spanning space and overall binding affinity to chCD40ED. Using 8 DNA aptamer candidates, 4 aptamer-based RCA products (aptamer RCAs) were generated, each consisting of two distinct aptamers. We demonstrated that all 4 aptamer RCAs significantly induced the signal transduction in chicken HD11 macrophage cell line (p < 0.05). Finally, we conjugated one of the aptamer RCAs (Aptamer RCA II) to M2e epitope peptide of influenza virus as a model hapten, and the immune complex was injected to chickens. Aptamer RCA II stimulated anti-M2e IgG antibody production to the level significantly higher as compared to the control (M2e epitope alone; p < 0.05). The results of our work suggest that aptamer RCA is a novel platform to boost the efficacy of vaccines, which might find broad applications to other antigens beyond M2e epitope evaluated in this study using chicken infection model.

Golden Gate assembly with a bi-directional promoter (GBid): A simple, scalable method for phage display Fab library creation.

Fabs offer an attractive platform for monoclonal antibody discovery/engineering, but library construction can be cumbersome. We report a simple method - Golden Gate assembly with a bi-directional promoter (GBid) - for constructing phage display Fab libraries. In GBid, the constant domains of the Fabs are located in the backbone of the phagemid vector and the library insert comprises only the variable regions of the antibodies and a central bi-directional promoter. This vector design reduces the process of Fab library construction to "scFv-like" simplicity and the double promoter ensures robust expression of both constituent chains. To maximize the library size, the 3 fragments comprising the insert - two variable chains and one bi-directional promoter - are assembled via a 3-fragment overlap extension PCR and the insert is incorporated into the vector via a high-efficiency one-fragment, one-pot Golden Gate assembly. The reaction setup requires minimal preparatory work and enzyme quantities, making GBid highly scalable. Using GBid, we constructed a chimeric chicken-human Fab phage display library comprising 1010 variants targeting the multi-transmembrane protein human CD20 (hCD20). Selection/counter-selection on transfected whole cells yielded hCD20-specific antibodies in four rounds of panning. The simplicity and scalability of GBid makes it a powerful tool for the discovery/engineering of Fabs and IgGs.

Crude Inactivated Influenza A Virus Adjuvated with a Bispecific Antibody Complex Targeting Chicken CD40 and AIV M2e Confers Protection Against Lethal HPAI Challenge in Chickens.

In vivo targeting an immunogen to the CD40 receptor expressed on professional antigen-presenting cells (APCs) dramatically enhances speed, magnitude, and quality of the immune response. Our previous evaluation of this strategy in poultry was limited to immunogenicity studies using CD40-targeted synthetic peptides, which demonstrated significant antigen-specific serum IgG and tracheal IgA levels <1 week after primary administration. In this study, this antibody-guided immunization strategy was modified to permit incorporation of inactivated highly pathogenic avian influenza virions (in lieu of short synthetic peptides) as the immunogen by simply mixing a bispecific antibody complex (anti-CD40/M2e) with crude inactivated virus before injection. Adjuvated avian influenza virus (AIV) induced significant hemagglutination inhibition titers up to 6 weeks postimmunization. In efficacy studies, administration of a single vaccine dose yielded 56%-64% survival against challenge with highly pathogenic H5N1, and 100% protection was achieved upon boosting. These results represent a feasible strategy to effectively target whole inactivated influenza A virus to chicken APCs, regardless of AIV clade and without phenotyping or purifying the virus from crude allantoic fluid. The data represent proof of principle for the unique prophylactic efficacy and versatility of a CD40-targeting adjuvation strategy that can in principle also be harnessed in other poultry vaccines.

Significant mucosal sIgA production after a single oral or parenteral administration using in vivo CD40 targeting in the chicken.

Many pathogens enter the host through mucosal surfaces and spread rapidly via the circulation. The most effective way to prevent disease is to establish mucosal and systemic immunity against the pathogen. However, current vaccination programs in poultry industry require repeated administrations of live-attenuated virus or large amounts (10 to 100µg) of antigen together with adjuvant to induce specific secretory IgA immune responses at the mucosal effector sites. In the present study, we show that a single administration of 0.4µg of oligopeptide complexed with an agonistic anti-chicken CD40 (chCD40) monoclonal antibody (Mab) effectively targets antigen-presenting cells of the bird's mucosa-associated lymphoid tissue in vivo, and induces peptide-specific secretory IgA (sIgA) in the trachea 7days post administration. Anti-chCD40 Mab-peptide complex was administered once to four-week old male Leghorns via various mucosal routes (orally, via cloacal drinking, or oculo-nasally) or via subcutaneous (s.c.) immunization. Immunization through any of the three mucosal induction routes induced significant peptide-specific mucosal sIgA responses 7 and 14days after immunization. Interestingly, s.c. injection of the complex also induced mucosal sIgA. Our data suggest in vivo targeting of CD40 as a potential adjuvant platform, particularly for the purpose of enhancing and speeding up mucosal vaccine responses in chickens, and potentially other food animals. This is the first study able to elicit specific sIgA immune responses in remote mucosal sites with a single administration of only 0.4µg of antigen.