Use Of Antigen Research Articles

Identification of antigenic Sarcoptes scabiei proteins for use in a diagnostic test and of non-antigenic proteins that may be immunomodulatory.

BackgroundScabies, caused by the mite, Sarcoptes scabiei, infects millions of humans, and many wild and domestic mammals. Scabies mites burrow in the lower stratum corneum of the epidermis of the skin and are the source of substances that are antigenic or modulate aspects of the protective response of the host. Ordinary scabies is a difficult disease to diagnose.ObjectiveThe goal of this project was to identify S. scabiei proteins that may be candidate antigens for use in a diagnostic test or may be used by the mite to modulate the host’s protective response.MethodsAn aqueous extract of S. scabiei was separated by 2-dimensional electrophoresis and proteins were identified by mass spectrometry. A parallel immunoblot was probed with serum from patients with ordinary scabies to identify IgM and/or IgG-binding antigens. The genes coding for 23 selected proteins were cloned into E. coli and the expressed recombinant proteins were screened with serum from patients with confirmed ordinary scabies.ResultsWe identified 50 different proteins produced by S. scabiei, 34 of which were not previously identified, and determined that 66% were recognized by patient IgM and/or IgG. Fourteen proteins were screened for use in a diagnostic test but none possessed enough sensitivity and specificity to be useful. Six of the 9 proteins selected for the possibility that they may be immunomodulatory were not recognized by antibodies in patient serum.ConclusionsThirty-three proteins that bound IgM and/or IgG from the serum of patients with ordinary scabies were identified. None of the 14 tested were useful for inclusion in a diagnostic test. The identities of 16 proteins that are not recognized as antigens by infected patients were also determined. These could be among the molecules that are responsible for this mite’s ability to modulate its host’s innate and adaptive immune responses.

Engineering Mycobacteria for the Production of Self-Assembling Biopolyesters Displaying Mycobacterial Antigens for Use as a Tuberculosis Vaccine.

ABSTRACTTuberculosis (TB) is a disease caused by Mycobacterium tuberculosis or Mycobacterium bovis and still remains one of the world's biggest global health burdens. Recently, engineered polyhydroxyalkanoate (PHA) biobeads that were produced in both Escherichia coli and Lactococcus lactis and displayed mycobacterial antigens were found to induce significant cell-mediated immune responses in mice. We observed that such PHA beads contained host cell proteins as impurities, which we hypothesized to have the potential to induce immunity. In this study, we aimed to develop PHA beads produced in mycobacteria (mycobacterial PHA biobeads [MBB]) and test their potential as a TB vaccine in a mouse model. As a model organism, nonpathogenic Mycobacterium smegmatis was engineered to produce MBB or MBB with immobilized mycobacterial antigens Ag85A and ESAT-6 on their surface (A:E-MBB). Three key enzymes involved in the poly(3-hydroxybutyric acid) pathway, namely, β-ketothiolase (PhaA), acetoacetyl-coenzyme A reductase (PhaB), and PHA synthase (PhaC), were engineered into E. coli-Mycobacterium shuttle plasmids and expressed in trans. Immobilization of specific antigens to the surface of the MBB was achieved by creating a fusion with the PHA synthase which remains covalently attached to the polyester core, resulting in PHA biobeads displaying covalently immobilized antigens. MBB, A:E-MBB, and an M. smegmatis vector control (MVC) were used in a mouse immunology trial, with comparison to phosphate-buffered saline (PBS)-vaccinated and Mycobacterium bovis BCG-vaccinated groups. We successfully produced MBB and A:E-MBB and used them as vaccines to induce a cellular immune response to mycobacterial antigens.IMPORTANCE Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis or Mycobacterium bovis and still remains one of the world's biggest global health burdens. In this study, we produced polyhydroxyalkanoate (PHA) biobeads in mycobacteria and used them as vaccines to induce a cellular immune response to mycobacterial antigens.

Novel polyclonal antibodies as a useful tool for expression studies in amphioxus embryos.

Cephalochordates, commonly called amphioxus or lancelets, are widely regarded as a useful proxy for the chordate ancestor. In recent decades, expression patterns of important developmental genes have been used extensively to infer homologies between cephalochordate and vertebrate embryos. Such comparisons provided important insight into cephalochordate biology and the origin of vertebrate traits. Most of the developmental expression data are collected using whole-mount in situ hybridization that allows the distributions of specific transcripts to be detected in fixed embryos. Here, we describe an experimental pipeline for production of small amounts of functional antibodies directed against amphioxus antigens for use in immunohistochemical labelling. In this pilot study, we generated antibodies against β-catenin and the transcription factors FoxA, Lhx1, Lhx3 and Pax6. We demonstrate the usefulness of antibodies by performing immunostainings on fixed specimens of B. lanceolatum and B. floridae. We anticipate that amphioxus-specific antibodies will provide a useful tool for high-resolution labelling of individual cells within the embryo and for determining the subcellular localization of the corresponding proteins.

Differing rates of antibody acquisition to merozoite antigens in malaria: implications for immunity and surveillance.

Antibodies play a key role in acquired human immunity to Plasmodium falciparum (Pf) malaria and target merozoites to reduce or prevent blood-stage replication and the development of disease. Merozoites present a complex array of antigens to the immune system, and currently, there is only a partial understanding of the targets of protective antibodies and how responses to different antigens are acquired and boosted. We hypothesized that there would be differences in the rate of acquisition of antibodies to different antigens and how well they are boosted by infection, which impacts the acquisition of immunity. We examined responses to a range of merozoite antigens in 2 different cohorts of children and adults with different age structures and levels of malaria exposure. Overall, antibodies were associated with age, exposure, and active infection, and the repertoire of responses increased with age and active infection. However, rates of antibody acquisition varied between antigens and different regions within an antigen following exposure to malaria, supporting our hypothesis. Antigen-specific responses could be broadly classified into early response types in which antibodies were acquired early in childhood exposure and late response types that appear to require substantially more exposure for the development of substantial levels. We identified antigen-specific responses that were effectively boosted after recent infection, whereas other responses were not. These findings advance our understanding of the acquisition of human immunity to malaria and are relevant to the development of malaria vaccines targeting merozoite antigens and the selection of antigens for use in malaria surveillance.

Aspergillus vaccines: Hardly worth studying or worthy of hard study?

Vaccines rank among the greatest advances in the history of public health. Yet, despite the need, there are no licensed vaccines to protect humans against fungal diseases, including aspergillosis. In this focused review, some of the major scientific and logistical challenges to developing vaccines to protect at-risk individuals against aspergillosis are discussed. Approaches that have shown promise in animal models include vaccines that protect against multiple fungal genera and those that are specifically directed to Aspergillus Advances in proteomics and glycomics have facilitated identification of candidate antigens for use in subunit vaccines. Novel adjuvants and delivery systems are becoming available that can skew vaccine responses toward those associated with protection. Immunotherapy consisting of adoptive transfer of Aspergillus-specific T cells to allogeneic hematopoietic transplant recipients has advanced to human testing but is technically difficult and of unproven benefit. While progress has been impressive, much work still needs to be done if vaccines against aspergillosis are to become a reality.

Abstract 3995: pVAC-Seq: A genome-guided in silico approach to identify tumor neoantigens for personalized immunotherapy

Abstract Despite the recent surge in high-throughput sequencing of cancer genomes, the challenge of translating these data into clinically actionable information remains. One promising approach involves the identification of tumor-specific mutant antigens (‘TSMAs’ or neoantigens) via massively parallel sequencing and analysis of matched cancer and normal samples that can be used to create personalized vaccines. In the past, this effort has primarily focused on targeting selection of ‘shared’ tumor antigens, found across many patients. Here, we advocate a more ‘personalized’ approach. These unique antigenic markers or TSMAs arise from numerous genetic changes, acquired somatically that are present exclusively in tumor (mutant) and not in normal (wild- type) cells. Vaccines incorporate these short, antigen-derived peptides (called epitopes) that aim to enhance the immune system's anti-tumor activity by selectively increasing the frequency of anti-tumor specific CD8+ T-cell antigens, and hence expand the ability of the immune system to recognize and destroy cancerous cells. Selecting the best/most immunogenic epitopes from a large number of mutations is an important challenge, in particular in cases of high mutational load such as melanoma and lung cancer. To address this need, we have developed an in silico based sequence analysis method for identification and subsequent refinement of patient-specific antigens for use in personalized vaccines. This flexible and streamlined computational workflow for identification of personalized variant antigens by cancer sequencing (pVAC-Seq) integrates tumor mutation and expression data (DNA- and RNA-Seq) to shortlist candidate neoantigen peptides for a personalized vaccine. Harnessing existing class I prediction algorithms, high-affinity neoantigens over varying peptide lengths are evaluated. To demonstrate the workings of pVAC-Seq, we applied it to four metastatic melanoma patients, the clinical results for three of whom were described previously. These patients were enrolled in a phase 1 vaccine clinical trial employing autologous, functionally mature, interleukin (IL)-12p70-producing dendritic cells (DC). Since melanoma patients harbor hundreds of mutations, it can be challenging to filter down and target the best set of potentially immunogenic neoantigens for vaccine design. By implementing the methods developed in pVAC-Seq, we were able to rapidly streamline the screening and identification of a smaller number of potentially immunogenic neoepitopes within the landscape of all neoepitopes. Citation Format: Jasreet Hundal, Beatriz M. Carreno, Allegra A. Petti, Gerald P. Linette, Obi L. Griffith, Malachi Griffith, Elaine R. Mardis. pVAC-Seq: A genome-guided in silico approach to identify tumor neoantigens for personalized immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3995.

Bivalent vaccine platform based on Japanese encephalitis virus (JEV) elicits neutralizing antibodies against JEV and hepatitis C virus

Directly acting antivirals recently have become available for the treatment of hepatitis C virus (HCV) infection, but there is no prophylactic vaccine for HCV. In the present study, we took advantage of the properties of Japanese encephalitis virus (JEV) to develop antigens for use in a HCV vaccine. Notably, the surface-exposed JEV envelope protein is tolerant of inserted foreign epitopes, permitting display of novel antigens. We identified 3 positions that permitted insertion of the HCV E2 neutralization epitope recognized by HCV1 antibody. JEV subviral particles (SVP) containing HCV-neutralization epitope (SVP-E2) were purified from culture supernatant by gel chromatography. Sera from mice immunized with SVP-E2 inhibited infection by JEV and by trans-complemented HCV particles (HCVtcp) derived from multi-genotypic viruses, whereas sera from mice immunized with synthetic E2 peptides did not show any neutralizing activity. Furthermore, sera from mice immunized with SVP-E2 neutralized HCVtcp with N415K escape mutation in E2. As with the SVP-E2 epitope-displaying particles, JEV SVPs with HCV E1 epitope also elicited neutralizing antibodies against HCV. Thus, this novel platform harboring foreign epitopes on the surface of the particle may facilitate the development of a bivalent vaccine against JEV and other pathogens.

Modulation of innate immunity in the tumor microenvironment.

A recent report from the Center for Disease Control identified melanoma as being among the highest causes of cancer-related mortalities in the USA. While interventions such as checkpoint blockade have made substantial impact in terms of improving response rates and overall survival, a significant number of patients fail to respond to treatment or become resistant to therapy. A better understanding of the tumor microenvironment in these patients becomes imperative for identifying immune suppressive mechanisms that impact the development of effective anti-tumor immune responses. We have investigated innate immune cells (dendritic cells, NK cells) in the tumor microenvironment (TME) in order to devise effective targeted anticancer immune therapies. We find that matrix metalloproteinase-2 (MMP-2), secreted from melanoma cells and stromal cells, cleaves IFNAR1 and stimulates TLR-2 on dendritic cells (DC) within the TME. Both these events independently culminate in programing the DCs to promote pro-tumorigenic TH2 T cell differentiation. In addition, we have shown that NK cells become functionally exhausted in melanoma patients. We identified the expression of Tim-3 as one of the factors responsible for NK cell exhaustion and showed that anti-Tim3 antibodies partially reversed this exhaustion. We have initiated local intervention strategies such as intra-tumoral administration of DC activating Poly-ICLC and compared the efficacy of different TLR agonists and melanoma antigens for use as combination tumor vaccine in clinical trials. Such approaches will provide a unique insight into tumor biology and will facilitate in development of highly effective and cell type-specific immune therapies.

PRODUCTION OF MYCOBACTERIUM TUBERCULOSIS TB10.4 RECOMBINANT PROTEIN IN ESCHERICHIA COLI

Nowadays tuberculosis is considered one of the most dangerous infectious diseases occurring everywhere, and it remains a cause of death of millions of people around the world. According to the World Health Organization data, in 2013 tuberculosis caused more than 9 million cases worldwide and about 1.5 million of infected people died. The causative agent of tuberculosis in most cases is Mycobacterium tuberculosis. But sometimes it can be Mycobacterium bovis or Mycobacterium africanum. Mainly as a result of infection, a bacterial infection affects the lungs, but the disease may develop in other organs and tissues. Now for the prevention of tuberculosis vaccination of newborns with attenuated vaccine BCG is widely used. The production of this vaccine is cheap and it is safe to use. Thus today, vaccination is the primary means of prevention of tuberculosis. However dubious efficacy and a number of side effects observed after vaccination, makes the scientific community to develop new effective methods for the treatment of tuberculosis. One of the ways to develop new vaccines against tuberculosis is to provide a subunit vaccine based on recombinant proteins. Advantages of subunit vaccines are that the preparation containing the purified protein is stable and secure, its chemical properties are known, it does not contain additional proteins and nucleic acids, which could cause undesirable effects in the human body. One of the most promising antigens for use as components in new vaccines is considered a low molecular weight secreted protein TB10.4. TB10.4 protein is recognized at an early stage of tuberculous infection and contributes to the proliferation of lymphocytes responsible for the production of IFNγ. TB10.4 protein also possesses an adjuvant effect when administered in combination with mycobacterial proteins. Given these properties, the recombinant protein TB10.4 can be used to generate new candidate vaccines against tuberculosis. During the study was created high-yield E. coli strain, which produces the recombinant protein TB10.4, selected the optimal protocol of induction of the gene encoding the protein. The protein was purified using metal affinity chromatography. The purity of the final preparation reached 98%.

Development of multiplex serological assay for the detection of human African trypanosomiasis

Human African trypanosomiasis (HAT) is a disease caused by Kinetoplastid infection. Serological tests are useful for epidemiological surveillance. The aim of this study was to develop a multiplex serological assay for HAT to assess the diagnostic value of selected HAT antigens for sero-epidemiological surveillance.We cloned loci encoding eight antigens from Trypanosoma brucei gambiense, expressed the genes in bacterial systems, and purified the resulting proteins. Antigens were subjected to Luminex multiplex assays using sera from HAT and VL patients to assess the antigens' immunodiagnostic potential. Among T. b. gambiense antigens, the 64-kDa and 65-kDa invariant surface glycoproteins (ISGs) and flagellar calcium binding protein (FCaBP) had high sensitivity for sera from T. b. gambiense patients, yielding AUC values of 0.871, 0.737 and 0.858 respectively in receiver operating characteristics (ROC) analysis. The ISG64, ISG65, and FCaBP antigens were partially cross-reactive to sera from Trypanosoma brucei rhodesiense patients. The GM6 antigen was cross-reactive to sera from T. b. rhodesiense patients as well as to sera from VL patients. Furthermore, heterogeneous antibody responses to each individual HAT antigen were observed. Testing for multiple HAT antigens in the same panel allowed specific and sensitive detection. Our results demonstrate the utility of applying multiplex assays for development and evaluation of HAT antigens for use in sero-epidemiological surveillance.

Progress with viral vectored malaria vaccines: A multi-stage approach involving “unnatural immunity”

Viral vectors used in heterologous prime-boost regimens are one of very few vaccination approaches that have yielded significant protection against controlled human malaria infections. Recently, protection induced by chimpanzee adenovirus priming and modified vaccinia Ankara boosting using the ME-TRAP insert has been correlated with the induction of potent CD8+ T cell responses. This regimen has progressed to field studies where efficacy against infection has now been reported. The same vectors have been used pre-clinically to identify preferred protective antigens for use in vaccines against the pre-erythrocytic, blood-stage and mosquito stages of malaria and this work is reviewed here for the first time. Such antigen screening has led to the prioritization of the PfRH5 blood-stage antigen, which showed efficacy against heterologous strain challenge in non-human primates, and vectors encoding this antigen are in clinical trials. This, along with the high transmission-blocking activity of some sexual-stage antigens, illustrates well the capacity of such vectors to induce high titre protective antibodies in addition to potent T cell responses. All of the protective responses induced by these vectors exceed the levels of the same immune responses induced by natural exposure supporting the view that, for subunit vaccines to achieve even partial efficacy in humans, “unnatural immunity” comprising immune responses of very high magnitude will need to be induced.

Burkholderia pseudomallei Capsular Polysaccharide Recognition by a Monoclonal Antibody Reveals Key Details toward a Biodefense Vaccine and Diagnostics against Melioidosis.

Burkholderia pseudomallei is the bacterium responsible for melioidosis, an infectious disease with high mortality rates. Since melioidosis is a significant public health concern in endemic regions and the organism is currently classified as a potential biothreat agent, the development of effective vaccines and rapid diagnostics is a priority. The capsular polysaccharide (CPS) expressed by B. pseudomallei is a highly conserved virulence factor and a protective antigen. Because of this, CPS is considered an attractive antigen for use in the development of both vaccines and diagnostics. In the present study, we describe the interactions of CPS with the murine monoclonal antibody (mAb) 4C4 using a multidisciplinary approach including organic synthesis, molecular biology techniques, surface plasmon resonance, and nuclear magnetic spectroscopy. Using these methods, we determined the mode of binding between mAb 4C4 and native CPS or ad hoc synthesized capsular polysaccharide fragments. Interestingly, we demonstrated that the O-acetyl moiety of CPS is essential for the interaction of the CPS epitope with mAb 4C4. Collectively, our results provide important insights into the structural features of B. pseudomallei CPS that enable antibody recognition that may help the rational design of CPS-based vaccine candidates. In addition, our findings confirm that the mAb 4C4 is suitable for use in an antibody-based detection assay for diagnosis of B. pseudomallei infections.

Use of ELISA based on NcSRS2 of Neospora caninum expressed in Pichia pastoris for diagnosing neosporosis in sheep and dogs.

Neosporosis is a disease caused by the protozoon Neospora caninum that leads to significant economic losses in many countries. In the present study, we report on use of the recombinant protein NcSRS2 of N. caninum expressed in Pichia pastoris in an indirect immunoenzymatic assay (ELISA) for diagnosing neosporosis infection in sheep and dogs. We observed that the ELISA test yielded specificity of 94.5% and sensitivity of 100% for sheep and specificity of 93.3% and sensitivity of 100% for dogs. We observed that the sensitivity was higher than shown by the indirect fluorescent antibody test, and this was confirmed by means of Western blot. The results from this study suggest that the recombinant protein expressed in P. pastoris is a suitable antigen for use in immunodiagnosis to detect N. caninum in two important species exposed to this parasitosis.

Molecular mimicry between dengue virus and coagulation factors induces antibodies to inhibit thrombin activity and enhance fibrinolysis.

Dengue virus (DENV) is the most common cause of viral hemorrhagic fever, and it may lead to life-threating dengue hemorrhagic fever and shock syndrome (DHF/DSS). Because most cases of DHF/DSS occur in patients with secondary DENV infection, anti-DENV antibodies are generally considered to play a role in the pathogenesis of DHF/DSS. Previously, we have found that antithrombin antibodies (ATAs) with both antithrombotic and profibrinolytic activities are present in the sera of dengue patients. However, the mechanism by which these autoantibodies are induced is unclear. In this study, we demonstrated that antibodies induced by DENV immunization in mice and rabbits could bind to DENV antigens as well as to human thrombin and plasminogen (Plg). The binding of anti-DENV antibodies to thrombin and Plg was inhibited by preadsorption with DENV nonstructural protein 1. In addition, affinity-purified ATAs from DENV-immunized rabbit sera could inhibit thrombin activity and enhance Plg activation both in vitro and in vivo. Taken together, our results suggest that molecular mimicry between DENV and coagulation factors can induce the production of autoantibodies with biological effects similar to those of ATAs found in dengue patients. These coagulation-factor cross-reactive anti-DENV antibodies can interfere with the balance of coagulation and fibrinolysis, which may lead to the tendency of DHF/DSS patients to bleed. Dengue virus (DENV) infection is the most common mosquito-borne viral disease in tropical and subtropical areas. Over 50 million DENV infection cases develop each year, and more than 2.5 billion people are at risk of dengue-induced hemorrhagic fever and shock syndrome. Currently, there is no vaccine or drug treatment for DENV. In the present study, we demonstrated that DENV immunization could induce thrombin and plasminogen (Plg) cross-reactive antibodies, which were able to inhibit thrombin activity and enhance Plg activation. These results suggest that molecular mimicry between DENV antigens, thrombin, and Plg may elicit antibodies that disturb hemostasis. The selection of appropriate candidate antigens for use in DENV vaccines should prevent these potentially dangerous autoimmune responses.

Antigen characterization from second instars of oestrid bot flies for the detection of anti-Cephenemyia stimulator antibodies by ELISA in roe deer (Capreolus capreolus).

A study to determine the most appropriate antigen for use in the serodiagnosis of Cephenemyia (Diptera: Oestridae) infestation in roe deer (Capreolus capreolus) was carried out using immunoenzymatic tests. Serum samples from 43 roe deer from northern Spain were obtained post-mortem and corresponding numbers of bot fly larvae established. Three antigen complexes were tested, including Cephenemyia stimulator Clark excretory/secretory antigens (CsES), C. stimulator somatic antigens (CsSA) and Oestrus ovis L. (Diptera: Oestridae) excretory/secretory antigens (OoES). In addition, the composition of each antigen was analysed using an electrophoresis system. Cephenemyia stimulator larvae were found in 25% of roe deer; the mean intensity of infection was 24.3 larvae per infested animal. In the antigen analysis, CsSA showed four exclusive bands of molecular weight (17-19, 62, 65 and 67-70 kDa). A positive correlation between immunoglobulin G (IgG) values and total number of larvae was found with CsES and CsSA. The highest sensitivity value, negative predictive value and negative likelihood ratio were obtained using CsES. The highest specificity value, positive likelihood ratio and kappa value were achieved with CsSA. The predictive values of the enzyme-linked immunosorbent assay (ELISA) using CsES and CsSA reached statistical significance and seroprevalence values were 26-44%. The use of ELISA with CsES and CsSA seems promising in the non-invasive diagnosis of Cephenemyia infestation in roe deer.

Evaluation of Mdh1 Protein as an Antigenic Candidate for a Vaccine against Candidiasis

Candida albicans malate dehydrogenase (Mdh1p) has been screened by previous proteome studies as a candidate for a vaccine against candidiasis. In this study, recombinant Mdh1 protein with a His-tag was produced in Escherichia coli and evaluated as an immunogenic protein against candidiasis. Mdh1p was administrated to mice by two methods subcutaneous injection and intranasal administration before challenging them with a lethal dose of C. albicans. After vaccination of Mdh1p, antibody responses were observed. To evaluate the vaccination effect of Mdh1p, survival tests were performed after 35 d. Although all control mice died within 24 d or 25 d, 100% and 80% of mice survived with subcutaneous and intranasal administration, respectively. Therefore, our results indicate that, among C. albicans antigens examined thus far, Mdh1p is currently the most effective antigen for use as a vaccine for C. albicans.

Combining Proteomic Strategies and Molecular Display Technology for Development of Vaccines against Candida albicans

Recent developments in pharmaceutical technology allow systematic investigation of molecules operating in a living cell, facilitating identification of key players, which show potential as novel drug targets. One of the tools that has emerged for identifying potential antigens for use as vaccines against infectious diseases is time-course proteome analysis. The model virulent microorganism Candida albicans has been well studied in terms of both physiological characteristics and clinical aspects for developing pharmaceutics in treatments. In this article, we review a time-course proteome study of C. albicans during adaptation to a serum. Furthermore, we introduce a novel biotechnological strategy for developing vaccines using characteristic proteins that have been identified as virulence-related molecules.

Invasion-inhibitory antibodies elicited by immunization with Plasmodium vivax apical membrane antigen-1 expressed in Pichia pastoris yeast.

In a recent vaccine trial performed with African children, immunization with a recombinant protein based on Plasmodium falciparum apical membrane antigen 1 (AMA-1) conferred a significant degree of strain-specific resistance against malaria. To contribute to the efforts of generating a vaccine against Plasmodium vivax malaria, we expressed the ectodomain of P. vivax AMA-1 (PvAMA-1) as a secreted soluble protein in the methylotrophic yeast Pichia pastoris. Recognized by a high percentage of sera from individuals infected by P. vivax, this recombinant protein was found to have maintained its antigenicity. The immunogenicity of this protein was evaluated in mice using immunization protocols that included homologous and heterologous prime-boost strategies with plasmid DNA and recombinant protein. We used the following formulations containing different adjuvants: aluminum salts (Alum), Bordetella pertussis monophosphoryl lipid A (MPLA), flagellin FliC from Salmonella enterica serovar Typhimurium, saponin Quil A, or incomplete Freund's adjuvant (IFA). The formulations containing the adjuvants Quil A or IFA elicited the highest IgG antibody titers. Significant antibody titers were also obtained using a formulation developed for human use containing MPLA or Alum plus MPLA. Recombinant PvAMA-1 produced under "conditions of good laboratory practice" provided a good yield, high purity, low endotoxin levels, and no microbial contaminants and reproduced the experimental immunizations. Most relevant for vaccine development was the fact that immunization with PvAMA-1 elicited invasion-inhibitory antibodies against different Asian isolates of P. vivax. Our results show that AMA-1 expressed in P. pastoris is a promising antigen for use in future preclinical and clinical studies.

Antemortem and postmortem examinations of the cattle calf naturally infected withMycobacterium aviumsubsp.paratuberculosis

A male cattle calf was detected as subclinically and naturally infected with Mycobacterium avium subspecies paratuberculosis (MAP) by a series of antemortem and postmortem tests. The MAP infection was identified by strong antibody and cell-mediated immune (CMI) response by a commercial ELISA kit and an intradermal Johnin test, respectively, in the initial antemortem examination. The antemortem status of the calf was further confirmed by MAP-specific interferon gamma (IFN-γ) response. For detection of IFN-γ response, MAP-specific IFN-γ release assays (IGRAs): (a) immuno capture ELISA (IC-ELISA) and (b) ELISPOT was employed. In addition, the presence of intracellular cytokine IFN-γ was detected by flow cytometry. For all cytokine assays, MAP-specific recombinant antigens HSP65 and 35 kDa were employed to overcome the poor sensitivity and specificity resulting from the use of Johnin, the crude protein purified derivative of MAP. Postmortem examination of the MAP-infected/suspected cattle calf did not reveal any pathognomonic gross lesions in the gastro-intestinal tract. Histopathological examination of multiple organs showed the presence of epithelioid cells/macrophages and edematous lesions in the mesenteric lymph nodes suggestive of MAP; however, no granulomas were observed in the intestinal tract. The necropsy samples of rectum and mesenteric lymph nodes were positive for isolation of MAP by culture in the BACTEC™ MGIT™ 960 system, and acid fast bacilli were demonstrated by fluorescence microscopy confirming the infection. Due to differential and complex expression patterns of MAP antigens reported in literature, a combination of assays such as those based on IGRAs and antibody detection is essential. Therefore, the current experimental evidence confirms the efficacy of the approach adopted. However, further studies will be needed to understand the optimal combination MAP-specific antigens for use in IGRAs or antibody assays that can be used for detecting MAP infection in every stage of the disease.

Booster responses by oral vaccination with transgenic plants against chicken leucocytozoonosis

We developed a transgenic potato (TrP/R7) expressing the recombinant R7 (rR7) antigen for use as an oral vaccine to protect against a chicken protozoan disease, chicken leucocytozoonosis. The TrP/R7 potato was produced by Agrobacterium tumefaciens-mediated transformation and regeneration, and the R7 gene insertion into potato chromosomes was confirmed by genomic polymerase chain reaction and Southern hybridization. rR7 antigen expression in TrP/R7 potato was also confirmed by sandwich enzyme-linked immunosorbent assay and western blotting using an antibody against the second-generation schizont of Leucocytozoon caulleryi. A transgenic potato clone with the highest rR7 antigen expression (3 µg rR7 antigen per gram of fresh-weight potato leaves) was selected, cultivated, and used in oral administration experiments to examine its ability to boost immunity. Chickens were immunized with chicken leucocytozoonosis vaccine “Hokken” by injection, and chickens that developed moderate levels of antibody titres were fed with TrP/R7 leaves. Chickens fed with TrP/R7 leaves showed increased antibody responses. In contrast, chickens fed with non-transgenic potato leaves showed a continuous decrease in antibody titres. Furthermore, chickens fed with TrP/R7 potato leaves showed strong resistance against experimental challenge with L. caulleryi infection. This study demonstrates the use of a plant-based oral vaccine to boost immunity against a protozoan disease.