Expression of VP1 protein of serotype A and O of foot-and-mouth disease virus in transgenic sunnhemp plants and its immunogenicity for guinea pigs

Recently, transgenic plants expressing immunogenic proteins of foot-and-mouth disease virus (FMDV) have been used as oral or parenteral vaccines against foot-and-mouth disease (FMD). They exhibit advantages like cost effectiveness, absence of processing, thermostability, and easy oral application. FMDV VP1 protein of single serotype has been mostly used as immunogen. Here we report the development of a bivalent vaccine with tandem-linked VP1 proteins of two serotypes, A and O, present in transgenic forage crop Crotalaria juncea. The expression of the bivalent protein in the transgenic plants was confirmed by Western blot analysis. Guinea pig reacted to orally or parenterally applied vaccine by humoral as well as cell-mediated immune responses including serum antibodies and stimulated lymphocytes, respectively. The vaccine protected the animals against a challenge with the virus of serotype A as well as O. This is the first report on the development of a bivalent FMD vaccine using a forage crop.

Stabilizing the Native Trimer of HIV-1 Env by Destabilizing the Heterodimeric Interface of the gp41 Postfusion Six-Helix Bundle

The HIV-1 envelope glycoprotein (Env) is a trimer of gp120-gp41 heterodimers and is essential for viral entry. The gp41 subunit in native, prefusion trimeric Env exists in a metastable conformation and attains a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers, that drives the fusion of viral and cellular membranes. We attempted to stabilize native Env trimers by incorporation of mutations at the NHR-CHR interface that disrupt the postfusion 6-HB of gp41. The mutations V570D and I573D stabilize native Env of the HIV-1 JRFL strain and occlude nonneutralizing epitopes to a greater extent than the previously identified I559P mutation that is at the interface of the NHR trimers in the 6-HB. The mutations prevent soluble-CD4 (sCD4)-induced gp120 shedding and 6-HB formation. In the context of cell surface-expressed JRFL Env, introduction of a previously reported additional disulfide between residues A501 and T605 perturbs the native conformation, though this effect is partially alleviated by furin coexpression. The data suggest that positions 570 and 573 are surface proximal in native Env and that the NHR homotrimeric coiled coil in native Env terminates before or close to residue 573. Aspartic acid substitutions at these positions stabilize native trimers through destabilization of the postfusion 6-HB conformation. These mutations can be used to stabilize Env in a DNA vaccine format. IMPORTANCE The major protein on the surface of HIV-1 is the envelope (Env) glycoprotein. Env is a trimer of gp120-gp41 heterodimers. gp120 is involved in receptor/coreceptor binding and gp41 in the fusion of viral and cellular membranes. Like many other viral fusion proteins, the gp41 subunit in native trimeric Env exists in a metastable conformation. gp41 readily forms a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers that drives fusion of viral and cellular membranes. While it is expected that native Env is a good immunogen, its metastability results in exposure of immunodominant nonneutralizing epitopes. In the present study, we stabilize native Env trimers by incorporation of a number of different mutations at the NHR-CHR interface that disrupt the postfusion 6-HB of gp41. The stabilized constructs described here can be incorporated into DNA vaccine candidates.

Structural Characterizations of the Dimeric Anti-HIV Antibody 2G12 and the HIV-2 Envelope Glycoprotein

More than thirty years after the discovery that Human Immunodeficiency Virus (HIV) was the causative agent of Acquired Immunodeficiency Syndrome (AIDS), the disease remains pandemic as long as no effective universal vaccine is found. Over 34 million individuals in the world are infected with the virus, and the vast majority of them have no access to the antiretroviral therapies that have largely reduced HIV to a chronic disease in the developed world. The first chapter of this thesis introduces the history of the virus. The key to the infectious mechanism of the virus lies in its envelope glycoprotein (Env), a trimeric spike on the viral surface that utilizes host T cell receptors for entry. Though HIV-1 Env is immunogenic, most infected patients do not mount an effective neutralizing antibody response against it. Broadly-neutralizing anti-Env antibodies (bNAbs) present in the serum of a minority of infected individuals are usually sufficient to prevent the progression to full blown AIDS. Thus, the molecular details of these bNAbs as well as the antibody-antigen interface are of prime interest for structural studies, as insight gained would contribute to the design of a more effective immunogen and potential vaccine candidate. The second chapter of this thesis describes the low-resolution crystal structure of one such antibody, 2G12 dimer, which targets a high mannose epitope on the surface of Env. Patients infected with HIV-2, a related virus with ~35% sequence identity in the Env region, can generally mount a robust antibody response sufficient for viral control for reasons still unknown. The final two chapters of this thesis focus on the first reported structural studies of HIV-2 Env, the molecular details of which may inform HIV-1 therapy and immunogen design.

Enzyme-linked immunosorbent assay of changes in serum levels of growth hormone (cGH) in common carps (Cyprinus carpio)

The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH from pituitaries. Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma cell lines (FMU-cGH 1-14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1-6, 12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1-7, 9 and 10) were used in fluorescent staining and immunohistochemistry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes. A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally, we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9-and 5.8-fold, respectively, when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions.

Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain

A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50 degrees C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.

Immunoprotective analysis of VhhP2, a Vibrio harveyi vaccine candidate

VhhP2 is an Outer membrane protein identified in a pathogenic Vibrio harveyi strain, T4, isolated from diseased fish. When used as a Subunit Vaccine, purified recombinant VhhP2 affords high level of protection upon Japanese flounder against V harveyi challenge. Vaccination with VhhP2 induced the expression of a number of immune-related genes, especially those encoding immunoglobulin M (IgM) and major histocompatibility complex (MHC) II alpha. A VhhP2 surface display system, in the form of the fish commensal strain FIR harboring the vhhP2-expressing plasmid pJVP, was constructed. PF3/pJVP is able to produce and present recombinant VhhP2 on cell surface. Vaccination of fish with live PF3/pJVP via intraperitoneal injection elicited Strong immunoprotection. Vaccination of fish orally with live PF3/pJVP embedded in alginate microspheres also induced effective immunoprotection. In addition, a VhhP2-based surface display system was created, in which VhhP2 serves as a carrier for the Surface delivery of a heterologous Edwardsiella tarda immunogen, Et18, that is fused in-frame to VhhP2. DH5 alpha/pJVP18, which expresses and surface-displays the VhhP2-Et18 chimera, proved to be an effective vaccine that call protect fish against infections by V. harveyi and E. tarda to the extents comparable to those produced by vaccination with purified recombinant VhhP2 and Et18, respectively. These data suggest that VhhP2 may be applied as a vaccine and a vaccine carrier against infections by V. harveyi and other pathogens such as F. tarda. (C) 2009 Elsevier Ltd. All rights reserved.

Identification of an Edwardsiella tarda surface antigen and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a surface-anchored subunit vaccine expressed by a fish commensal strain

Edwardsiella tarda is the etiological agent of edwardsiellosis, a systematic disease that affects a wide range of marine and freshwater fish cultured worldwide. In order to identify E. tarda antigens with vaccine potential, we in this study conducted a systematic search for E. tarda proteins with secretion capacity. One of the proteins thus identified was Esa1, which contains 795 amino acid residues and shares extensive overall sequence identities with the D15-like surface antigens of several bacterial species. In silico analyses indicated that Esa1 localizes to outer membrane and possesses domain structures that are conserved among bacterial surface antigens. The vaccine potential of purified recombinant Esa1 was examined in a Japanese flounder (Paralichthys olivaceus) model, which showed that fish vaccinated with Esa1 exhibited a high level of survival and produced specific serum antibodies. Passive immunization of naive fish with antisera raised against Esa1 resulted in significant protection against E. tarda challenge. Taking advantage of the secretion capacity of Esa1 and the natural gut-colonization ability of a fish commensal strain, we constructed an Esa1-expressing recombinant strain, FP3/pJsa1. Western immunoblot and agglutination analyses showed that FP3/pJsa1 produces outer membrane-localized Esa1 and forms aggregates in the presence of anti-Esa1 antibodies. Vaccination analyses showed that FP3/pJsa1 as an intraperitoneal injection vaccine and an oral vaccine embedded in alginate microspheres produced relative percent survival rates of 79% and 52%, respectively, under severe challenging conditions that resulted in 92-96% mortality in control fish. Further analyses showed that following oral vaccination, FP3/pJsa1 was able to colonize in the gut but unable to disseminate into other tissues. Together these results indicate that Esa1 is a protective immunogen and an effective oral vaccine when delivered by FP3/pJsa1 as a surface-anchored antigen. (c) 2010 Elsevier Ltd. All rights reserved.

Adenovirus F protein as a delivery vehicle for botulinum B.

BACKGROUND: Immunization with recombinant carboxyl-terminal domain of the heavy chain (Hc domain) of botulinum neurotoxin (BoNT) stimulates protective immunity against native BoNT challenge. Most studies developing a botulism vaccine have focused on the whole Hc; however, since the principal protective epitopes are located within beta-trefoil domain (Hcbetatre), we hypothesize that immunization with the Hcbetatre domain is sufficient to confer protective immunity. In addition, enhancing its uptake subsequent to nasal delivery prompted development of an alternative vaccine strategy, and we hypothesize that the addition of targeting moiety adenovirus 2 fiber protein (Ad2F) may enhance such uptake during vaccination. RESULTS: The Hcbetatre serotype B immunogen was genetically fused to Ad2F (Hcbetatre/B-Ad2F), and its immunogenicity was tested in mice. In combination with the mucosal adjuvant, cholera toxin (CT), enhanced mucosal IgA and serum IgG Ab titers were induced by nasal Hcbetatre-Ad2F relative to Hcbetatre alone; however, similar Ab titers were obtained upon intramuscular immunization. These BoNT/B-specific Abs induced by nasal immunization were generally supported in large part by Th2 cells, as opposed to Hcbetatre-immunized mice that showed more mixed Th1 and Th2 cells. Using a mouse neutralization assay, sera from animals immunized with Hcbetatre and Hcbetatre-Ad2F protected mice against 2.0 LD50. CONCLUSION: These results demonstrate that Hcbetatre-based immunogens are highly immunogenic, especially when genetically fused to Ad2F, and Ad2F can be exploited as a vaccine delivery platform to the mucosa.

Genetic signatures in the envelope glycoproteins of HIV-1 that associate with broadly neutralizing antibodies.

A steady increase in knowledge of the molecular and antigenic structure of the gp120 and gp41 HIV-1 envelope glycoproteins (Env) is yielding important new insights for vaccine design, but it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies. To help bridge this gap, we used phylogenetically corrected statistical methods to identify amino acid signature patterns in Envs derived from people who have made potently neutralizing antibodies, with the hypothesis that these Envs may share common features that would be useful for incorporation in a vaccine immunogen. Before attempting this, essentially as a control, we explored the utility of our computational methods for defining signatures of complex neutralization phenotypes by analyzing Env sequences from 251 clonal viruses that were differentially sensitive to neutralization by the well-characterized gp120-specific monoclonal antibody, b12. We identified ten b12-neutralization signatures, including seven either in the b12-binding surface of gp120 or in the V2 region of gp120 that have been previously shown to impact b12 sensitivity. A simple algorithm based on the b12 signature pattern was predictive of b12 sensitivity/resistance in an additional blinded panel of 57 viruses. Upon obtaining these reassuring outcomes, we went on to apply these same computational methods to define signature patterns in Env from HIV-1 infected individuals who had potent, broadly neutralizing responses. We analyzed a checkerboard-style neutralization dataset with sera from 69 HIV-1-infected individuals tested against a panel of 25 different Envs. Distinct clusters of sera with high and low neutralization potencies were identified. Six signature positions in Env sequences obtained from the 69 samples were found to be strongly associated with either the high or low potency responses. Five sites were in the CD4-induced coreceptor binding site of gp120, suggesting an important role for this region in the elicitation of broadly neutralizing antibody responses against HIV-1.

The production and characterisation of an antibody to detect the coccidiostat toltrazuril and its metabolite ponazuril.

The production of an antibody to detect toltrazuril or its metabolite ponazuril is complicated due to structural constraints of conjugating these coccidiostats to a carrier protein. Therefore a search was carried out for a compound that shared a common substructure to use as an antigen mimic. The chosen compound, trifluoraminoether, was conjugated to two carrier proteins (HSA and BTG) and used in the immunisation of six rabbits. Two immunogen doses (1 mg and 0.1 mg) were also used. All six rabbits produced an immunological response to the hapten regardless of the carrier protein or immunogen dose used. The most sensitive polyclonal antibody produced, designated R609, was subsequently characterised. This antiserum exhibited an IC50 of 18 ng ml-1 using a competitive ELISA format. Cross reactivity studies show that this serum is specific for toltrazuril and its metabolites (toltrazuril sulfoxide and toltrazuril sulfone) but does not cross-react with other coccidiostats such as halofuginone, nitroimidazoles or nicarbazin. This is the first reported production of an antibody capable of specifically binding toltrazuril and ponazuril.

Langerhans Cells Prime IL-17-Producing T Cells and Dampen Genital Cytotoxic Responses following Mucosal Immunization

Langerhans cells (LCs) are dendritic cells (DCs) localized in stratified epithelia, such as those overlaying skin, buccal mucosa, and vagina. The contribution of LCs to the promotion or control of immunity initiated at epithelial sites remains debated. We report in this paper that an immunogen comprising OVA linked to the B subunit of cholera toxin, used as delivery vector, was efficient to generate CTLs after vaginal immunization. Using Lang-EGFP mice, we evaluated the contribution of distinct DC subsets to the generation of CD4 and CD8 T cell responses. We demonstrate that the vaginal epithelium, unlike the skin epidermis, includes a minor population of LCs and a major subset of langerin(-) DCs. Intravaginally administered Ag is taken up by LCs and langerin(-) DCs and carried up to draining lymph nodes, where both subsets prime CD8 T cells, unlike blood-derived DCs, although with distinct capabilities. LCs prime CD8 T cells with a cytokine profile dominated by IL-17, whereas Lang(-) DCs induce IFN-gamma-producing T cells. Using Lang-DTR-EGFP mice to ensure a transient ablation of LCs, we found that these cells not only are dispensable for the generation of genital CTL responses but also downregulate these responses, by a mechanism that may involve IL-10 and IL-17 cytokines. This finding has implications for the development of mucosal vaccines and immunotherapeutic strategies designed for the targeting of DCs.

Hapten synthesis and antibody production for the development of a melamine immunoassay

The incorporation of melamine into food products is banned but its misuse has been widely reported in both animal feeds and food. The development of a rapid screening immunoassay for monitoring of the substance is an urgent requirement. Two haptens of melamine were synthesized by introducing spacer arms of different lengths and structures on the triazine ring of the analyte molecular structure. 6-Aminocaproic acid and 3-mercaptopropionic acid were reacted with 2-chloro-4,6-diamino-1,3,5-triazine (CAAT) to produce hapten 1[3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylamino) hexanoic acid] and hapten 2[3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylthio) propanoic acid]. respectively. The molecular structures of the two haptens were identified by I H nuclear magnetic resonance spectrometry, mass spectrometry and infrared spectrometry. An immunogen was prepared by coupling hapten 1 to bovine serum albumin (BSA). Two plate coating antigens were prepared by coupling both haptens to egg ovalbumin (OVA). A competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed to evaluate homogeneous and heterogeneous assay formats. The results showed that polyclonal antibodies with high titers were obtained, and the heterogeneous immunoassay format demonstrated a better performance with an IC50 of 70.6 ng mL(-1), a LOD of 2.6 ng mL(-1) and a LOQ of 7.6 ng mL(-1). Except for cyromazine, no obvious cross-reactivity to common compounds was found. The data showed that the hapten synthesis was successful and the resultant antisera could be used in an immunoassay for the rapid and sensitive detection of this banned chemical. (C) 2010 Elsevier B.V. All rights reserved.

Development of a high-throughput enzyme-linked immunosorbent assay for the routine detection of the carcinogen acrylamide in food, via rapid derivatisation pre-analysis

The spontaneous formation of the neurotoxic carcinogen acrylamide in a wide range of cooked foods has recently been discovered. These foods include bread and other bakery products, crisps, chips, breakfast cereals, and coffee. To date, the diminutive size of acrylamide (71.08Da) has prevented the development of screening immunoassays for this chemical. In this study, a polyclonal antibody capable of binding the carcinogen was produced by the synthesis of an immunogen comprising acrylamide derivatised with 3-mercaptobenzoic acid (3-MBA), and its conjugation to the carrier protein bovine thyroglobulin. Antiserum from the immunised rabbit was harvested and fully characterised. it displayed no binding affinity for acrylamide or 3-MBA but had a high affinity for 3-MBA-derivitised acrylamide. The antisera produced was utilised in the development of an ELISA based detection system for acrylamide. Spiked water samples were assayed for acrylamide content using a previously published extraction method validated for coffee, crispbread, potato, milk chocolate and potato crisp matrices. Extracted acrylamide was then subjected to a rapid 1-h derivatisation with 3-MBA, pre-analysis. The ELISA was shown to have a high specificity for acrylamide, with a limit of detection in water samples of 65.7 mu g kg(-1), i.e. potentially suitable for acrylamide detection in a wide range of food commodities. Future development of this assay will increase sensitivity further. This is the first report of an immunoassay capable of detecting the carcinogen, as its small size has necessitated current analytical detection via expensive, slower, physico-chemical techniques such as Gas or Liquid Chromatography coupled to Mass Spectrometry. (c) 2007 Elsevier B.V. All rights reserved.

The potential of polymeric nanoparticles to increase the immunogenicity of the hapten clenbuterol

Micro-and nanoparticles prepared front the biodegradable and biocompatible polymers poly(lactide-co-glycolide) (PLGA) and polymetylmethacrylate (PMMA) have been successfully used as immunopotentiating antigen delivery systems. In our study, this approach was used to improve polyclonal antibody production to clenbuterol (CBL), a model hapten. PLGA and PMMA nanoparticles were loaded with either CBL alone or with a clenbuterol-transferrin conjugate (CBL-Tfn) and administered subcutaneously to mice. PLGA nano-particles were administered with or without the saponin adjuvant Quil A. The anti-CBL titres present in experimental sera were determined by an enzyme immunoassay (ELISA). CBL-Tfn-loaded PLGA nanoparticles co-administered with Quil A had obvious advantages immmunologically over the currently used method of raising antibodies to CBL (the positive control). The combined adjuvanticity of Quil A and PLGA nanoparticles resulted in a positive response in all four of the mice tested and in higher antibody titles than were seen in the positive control group. Furthermore, the sustained release of immunogen from the nanoparticles permitted a reduction in immunizing frequency over the 15-week study period.