Antigenic Polypeptides Research Articles

Putative new combination vaccine candidates identified by reverse vaccinology and genomic approaches to control enteric pathogens

ObjectivesThe pathogenic microorganisms that cause intestinal diseases can significantly jeopardize people's health. Currently, there are no authorized treatments or vaccinations available to combat the germs responsible for intestinal disease.MethodsUsing immunoinformatics, we developed a potent multi-epitope Combination (combo) vaccine versus Salmonella and enterohemorrhagic E. coli. The B and T cell epitopes were identified by performing a conservancy assessment, population coverage analysis, physicochemical attributes assessment, and secondary and tertiary structure assessment of the chosen antigenic polypeptide. The selection process for vaccine development included using several bioinformatics tools and approaches to finally choose two linear B-cell epitopes, five CTL epitopes, and two HTL epitopes.ResultsThe vaccine had strong immunogenicity, cytokine production, immunological properties, non-toxicity, non-allergenicity, stability, and potential efficacy against infections. Disulfide bonding, codon modification, and computational cloning were also used to enhance the stability and efficacy of expression in the host E. coli. The vaccine's structure has a strong affinity for the TLR4 ligand and is very durable, as shown by molecular docking and molecular modeling. The results of the immunological simulation demonstrated that both B and T cells had a heightened response to the vaccination component.ConclusionsThe comprehensive in silico analysis reveals that the proposed vaccine will likely elicit a robust immune response against pathogenic bacteria that cause intestinal diseases. Therefore, it is a promising option for further experimental testing.

Abstract 4091: A novel cancer vaccine based on hyaluronic acid nanogel combined with adoptive T cell therapy induces complete regression of established tumors and long-lasting memory CD8+ T cells

Abstract Background: We have developed nanosized hydrogel particles (nanogels) to create new nanomaterials for biomedical applications. In particular, Hyaluronic Acid, partially hydrophobized by chemical modification with cholesteryl groups, has a distinguished characteristic to form physically cross-linked NanoGel particles. HANG efficiently forms a stable complex with an antigenic polypeptide (HANG-Vaccine) through hydrophobic interactions. In this study, we investigated the efficacy of HANG-V against tumors resistant to immune checkpoint inhibitors (ICIs) and adoptive TCR-T therapy. Methods: C57BL/6-derived B16F10 melanoma and BALB/c-derived CMS5a sarcoma are both known to be resistant to ICIs and adoptive TCR-T therapy. The efficacy of HANG-V was tested by using each tumor-bearing mice treatment model. HANG-V was prepared by mixing gp100 or CMS5a neoantigen (Ag) with HANG, respectively. Results: HANG-V was subcutaneously injected into B16F10-bearing C57BL/6 on days 7, 11, and 15, and CD8+ T cells from Pmel-1 mice were intravenously administered on days 8 and 12. Surprisingly, complete regression of established B16F10 tumors with more than 100 mm2 were observed. Vaccinated mice survived with changing hair color of mice into white, while non-vaccinated mice were all dead by day 25. Moreover, gp100-specific CTLs was observed abundantly in systemic including tumor site. Even two months after the treatment, transferred T cells with memory phenotype were persistently detected in peripheral bloods. Furthermore, booster vaccination elicited robust expansion of these memory T cells persisting for more than one year. In similar experimental settings, we treated CMS5a tumor-bearing mice by HANG-V combined with adoptive transfer of CD8+ T cells from DUC18 mice, whose T cells are specific for mutated ERK2 neoAg. All mice survived as well with complete reduction of established CMS5a tumors, rejected a rechallenge with CMS5a and induced an Ag-specific CTL responses on more than 2 years. Notably, by using neoantigen-knockout CMS5a, we obtained results suggesting that HANG-V with TCR-T therapy potently induced the so-called “antigen spreading”. Furthermore, induction of Ag-specific CTLs was associated with CD44 hyaluronic acid receptor which expressed in APCs crucial for cross-presentation to CD8+ T cells. Conclusions: HANG-V strongly enhanced the efficacy of adoptive TCR-T cell therapy against ICI refractory tumors leading to total tumor suppression. Furthermore, HANG-V induced potent and persistent Ag-specific CTLs systemically through interaction with CD44 hyaluronic receptor, allowing long-term protection of tumor recurrence with memory CD8+ T cells. Our studies may propose crucial insights for clinical application of HANG vaccine with adoptive T cell therapy in patient with ICI-resistant tumors with poor prognosis. Citation Format: Fumiyasu Momose, Takashi Nakai, Kohei Yabuuchi, Makiko Yamane, Tae Hayashi, Linan Wang, Yoshiyuki Nakagawa, Shogo Aso, Toru Katsumata, Tsuyoshi Shimoboji, Yoshihiro Miyahara. A novel cancer vaccine based on hyaluronic acid nanogel combined with adoptive T cell therapy induces complete regression of established tumors and long-lasting memory CD8+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4091.

Development of a novel multi-epitope oral DNA vaccine for rabies based on a food-borne microbial vector

Rabies has been with humans for a long time, and its special transmission route and almost 100 % lethality rate made it once a nightmare for humans. In this study, by predicting the rabies virus glycoprotein outer membrane region and nucleoprotein B-cell antigenic epitopes, the coding sequence of the predicted highly antigenic polypeptide region obtained was assembled using the eukaryotic expression vector pcDNA3.1(-), and then E. coli was used as the delivery vector.The immunogenicity and protective properties of the vaccine were verified by in vivo and in vitro experiments, which demonstrated that the vaccine could produce antibodies in mice and prolong the survival time of mice exposed to the strong virus without any side effects. This study demonstrated that the preparation of an oral rabies DNA vaccine using food-borne microorganisms as a transport vehicle is feasible and could be a new strategy to eradicate rabies starting with wild animals.

The use of ORF2 and ORF3 proteins for creating an enzyme-linked immunosorbent assay for viral hepatitis E

Due to the lack of national analogies a prototype of enzyme-linked immunosorbent assay (ELISA) test system was developed for the qualitative determination of antibodies class G to hepatitis E virus (HEV) based on the using of antigenic polypeptides ORF2 and ORF3 of the 3rd genotype (FSBSI «I.I. Mechnikov Research Institute of Vaccines and Serums», Russia). The results of experimental ELISA conducted in parallel with the reference test system «DS-ELISA-ANTI-HEV-G» (NPO Diagnostic Systems, Russia) are presented. The working concentrations of ORF2 and ORF3 antigens were 2.5 µg/mL and 0.25 µg/mL, respectively. They were tested with 200 donors and patients’ serum samples. The article reflects the determination of sensitivity, specificity and reproducibility results. The developed test system demonstrated high efficiency in the detection of specific anti-HEV-IgG.

Construction of a cDNA library for Sparganum mansoni and screening of diagnostic antigen cadidates

To construct a cDNA library of Sparganum mansoni and immunoscreen antigen candidates for immunodiagnosis of sparganosis mansoni. Total RNA was extracted from S. mansoni, and reversely transcribed into cDNA, which was ligated into the phage vector. These recombinant vectors were packaged in vitro to construct the SMART cDNA library of S. mansoni. Then, the cDNA library was immunoscreened with sera from patients with sparganosis mansoni to yield positive clones. The inserted fragments of positive clones were sequenced and subjected to homology analyses, and the structure and functions of the coding proteins were predicted. The SMATR cDNA library of S. mansoni was successfully constructed. The titer of the cDNA library was 6.25 × 106 pfu/mL, with a recombinant efficiency of 100%, and the mean length of the inserted fragments in the library was larger than 1 100 bp. A total of 12 positive clones were obtained by immunoscreening, and were categorized into Sm-I (Sm60-1), Sm-II (Sm58-1), Sm-III (Sm20-1) and Sm-IV (Sm22-3), with 1 134, 1 063, 883 bp and 969 bp long inserted fragments. Their coding proteins were highly homologous with the Spirometra erinaceieuropaei antigenic polypeptide, cytoplasmic antigen, ribosomal protein S4-like protein and unnamed protein product, respectively. A SMART cDNA library of S. mansoni has been successfully constructed and 4 categories of positive clones have been identified, which provides a basis for further studies on diagnostic antigens for sparganosis mansoni.

Expression of Concern: Synthesized multiple antigenic polypeptide vaccine based on B-cell epitopes of human heparanase could elicit a potent antimetastatic effect on human hepatocellular carcinoma in vivo.

Expression of Concern: Synthesized multiple antigenic polypeptide vaccine based on B-cell epitopes of human heparanase could elicit a potent antimetastatic effect on human hepatocellular carcinoma in vivo.

Treatment Effect of Tuftsin and Antigen Peptide Combined with Immune Cells on Colorectal Cancer.

BackgroundThe aim of this study was to investigate the effect of antigenic peptides on dendritic cell maturation and activation as well as the role of dendritic cell induced cell function. The tumor-specific cytotoxic T lymphocytes induced by activation of the dendritic cells were also evaluated.Material/MethodsSW-480 cell lysate and peptide antigens were selected as adjuvants in dendritic cell sensitization, and tuftsin was used to induce the phagocytosis of dendritic cells. Immature dendritic cells were stimulated with the antigen and adjuvant as follows: group A was negative control; group B was SW-480 (20 μg/mL); group C was SW-480 (20 μg/mL)+tumor necrosis factor (TNF)-α (10 μg/mL); group D was SW-480 (20 μg/mL)+tuftsin (20 μg/mL); group E was antigen peptide (2 μg/mL); group F was antigen peptide (2 μg/mL)+TNF-α (10 μg/mL); group G was antigen peptide (2 μg/mL)+tuftsin (20 μg/mL). Cytotoxic T lymphocytes activation and in vitro anti-tumor effects were examined by detecting the maturation marks of dendritic cells as well as interleukin (IL)-10 and IL-12 levels secreted by dendritic cells. Cells with the strongest immunizing effects were injected into nude mice and tumor suppression status was evaluated.ResultsGroup D (SW-480+tuftsin), group E (antigen peptides), group F (antigen peptide+TNF-α), and group G (antigen peptides+tuftsin) displayed significant differences compared to the control group (P<0.05). Group G (antigen peptides+tuftsin) could also promote the secretion of cytokines IL-12, as well as inhibit cytokine IL-10 secretion, compared to the other experimental groups (P<0.05). In the in vivo experiments of tumor inhibitions, antigenic polypeptide+tuftsin was the most effective (P<0.05).ConclusionsCombination of cytotoxic T lymphocytes and T peptide therapy in treating human colorectal cancer might be used as a new treatment strategy based on adoptive cellular immunotherapy.

Screening HCV genotype-specific epitope peptides based on conserved sequence analysis and B cell epitope prediction in HCV E2 region.

The high mutation rate of the hepatitis C virus (HCV) genome increases the genotype diversity and renders the detection of the virus more difficult. Therefore, prediction and assessment of highly conserved and strongly antigenic epitope polypeptide sequences have become a focus of current research. The E2 region is the target binding region of neutralizing antibodies. HCV genomics, especially the high mutation rate of E2 region sequence, makes its genotyping more and more diverse, and the detection of HCV and genotype is becoming more and more strict. In this study, four HCV B cell epitope polypeptides were constructed based on assessment of conserved sequences in the HCV E2 region and prediction of B cell epitopes, including sequences specific to genotype 1A (DC-13: 434-DTGWLAGLFYYHK-446), genotype 1B (HC-13: 434-HTGFLAALFYAKS-446), genotype 4D (NC-13: 434-NTGFLASLFYTHK-446), and a consensus sequence (FC-9: 447-FNSSGCPER-455). Epitope polypeptides combined with serum from 29 HCV-infected or 25 non-HCV-infected individuals were assayed by enzyme-linked immunosorbent assay (ELISA), and differences were analyzed by T/T' test methods in SPSS v20.0 software. Binding levels of genotype 1A, 4D, and consensus epitope polypeptides with sera of HCV-infected patients were higher than those of non-infected individuals. Moreover, binding of genotype 1B epitope polypeptides with serum of HCV 1B-infected patients was higher than that of HCV 2A-infected patients. While the screening results of HCV genotype-specific epitope polypeptides were preliminary, these findings indicated that we successfully established an HCV and genotype serological ELISA detection method. Such an approach would facilitate the discovery of epitope polypeptides which may become new antigen candidates in peptide vaccine development for the prevention of HCV infection.

Compositional differences between Copaxone and Glatopa are reflected in altered immunomodulation ex vivo in a mouse model.

Copaxone (glatiramer acetate, GA), a structurally and compositionally complex polypeptide nonbiological drug, is an effective treatment for multiple sclerosis, with a well-established favorable safety profile. The short antigenic polypeptide sequences comprising therapeutically active epitopes in GA cannot be deciphered with state-of-the-art methods; and GA has no measurable pharmacokinetic profile and no validated pharmacodynamic markers. The study reported herein describes the use of orthogonal standard and high-resolution physicochemical and biological tests to characterize GA and a U.S. Food and Drug Administration-approved generic version of GA, Glatopa (USA-FoGA). While similarities were observed with low-resolution or destructive tests, differences between GA and USA-FoGA were measured with high-resolution methods applied to an intact mixture, including variations in surface charge and a unique, high-molecular-weight, hydrophobic polypeptide population observed only in some USA-FoGA lots. Consistent with published reports that modifications in physicochemical attributes alter immune-related processes, genome-wide expression profiles of ex vivo activated splenocytes from mice immunized with either GA or USA-FoGA showed that 7-11% of modulated genes were differentially expressed and enriched for immune-related pathways. Thus, differences between USA-FoGA and GA may include variations in antigenic epitopes that differentially activate immune responses. We propose that the assays reported herein should be considered during the regulatory assessment process for nonbiological complex drugs such as GA.

Analysis of genes encoding high-antigenicity polypeptides in three serotypes of Miamiensis avidus

The ciliate Miamiensis avidus causes scuticociliatosis in Japanese flounder Paralichthys olivaceus. We previously reported three serotypes of this ciliate distinguishable by serotype-specific antigenic polypeptides (serotype I, 30kDa; serotype II, 38kDa; serotype III, 34kDa). In this study, we determined the localization site of the serotype-specific polypeptides in the ciliate and determined the genes encoding the polypeptides, using the isolates IyoI (serotype I), Nakajima (serotype II), and Mie0301 (serotype III). SDS-PAGE and immunoblot analysis of cilia, membrane proteins, and cytoskeletal elements of the ciliates revealed that the polypeptides were abundant in the former two. Scanning electron microscopy of ciliates immobilized by homologous antiserum showed morphological changes in the cilia. These evidences suggested that the polypeptides were ciliary membrane immobilization antigens. The ciliary genes identified showed low identity scores—<51.5% between serotypes. To differentiate the serotypes, we designed serotype-specific PCR primer sets based on the DNA sequences. The PCR-based serotyping results were completely consistent with conventional serotyping methods (immobilization assay and immunoblot analysis). Twenty of 21 isolates were classified as either serotype I or II, and one isolate was undistinguishable. The combination of species-specific PCR previously reported and three serotype-specific PCR could be useful for identifying, serotyping, and surveillance for occurrences of new serotypes of M. avidus.

Antigen-loaded polymeric hybrid micelles elicit strong mucosal and systemic immune responses after intranasal administration

Increasing attention has been paid to nasal delivery. Subunit vaccines based on antigenic proteins or polypeptides offer good safety. However, lack of delivery efficiency, particularly for nasal immunization, is a big issue. Here we designed a highly tunable polymeric hybrid micelle (PHM) system offering good vaccine efficacy after nasal administration. PHMs are formulated from two amphiphilic diblock copolymers, polycaprolactone-polyethylenimine (PCL-PEI) and polycaprolactone-polyethyleneglycol (PCL-PEG), the ratio of which determines PHM physicochemical properties. Citraconic anhydride-modified ovalbumin (Cit-OVA), as model antigen, was incorporated into PHMs via electrostatic interaction, giving antigen-loaded micelles of around 150nm in size. Their surface characteristics which are found closely related to their in vivo kinetics can be modulated by adjusting the mass ratio of PCL-PEG and PCL-PEI. PHM/Cit-OVA complexes containing PCL-PEI and PCL-PEG in a 1:1 mass ratio induced strong immune responses in nasal mucosa and serum in vivo without causing obvious toxicity, and Cit-OVA was efficiently internalized by dendritic cells. These results demonstrate the promise of this multifunctional polymeric delivery system for nasal vaccination.

Prediction and identification of B-cell epitopes for tumor necrosis factor-α

The aim of the present study was to predict and identify B‑cell epitopes for mouse tumor necrosis factor‑α (mTNF‑α). DNAStar and BcePred software were used to predict B‑cell epitopes for mTNF‑α. A predicted eight‑branch multiple antigenic polypeptide (MAP) was synthesized and used to immunize BALB/c mice, combined with a promiscuous helper interleukin‑1β epitope (VQGEESNDK, amino acids 163‑171). The serum titer was measured. The specificity and avidity were determined by western blotting and indirect enzyme‑linked immunosorbent assay (ELISA). Amino acids 54‑65 (MAP1) and 78‑92 (MAP2) of mTNF‑α were predicted as most likely to be B‑cell epitopes. Dynamic monitoring of antibody concentration demonstrated that MAP1 and MAP2 may induce the production of specific antibodies with a higher antibody level for MAP2. Furthermore, MAP1 and MAP2 were confirmed to induce mTNF‑α‑specific antibodies by western blotting. Indirect ELISA was used to confirm that MAP2 had the highest affinity with commercial anti‑mTNF‑α antibody. Amino acids 54‑65 and 78‑94 of mTNF‑α are B‑cell epitopes, wherein amino acids 78‑94 have the strongest immunogenicity. The present study provides a theoretical basis for further research into the mTNF‑α polypeptide antibody and a B‑cell MAP vaccine.

Effect of heat, pH and shear on digestibility and antigenic characteristics of wheat gluten

Processing-induced conformational changes may affect digestibility and antigenic potential of food proteins. In vitro gastrointestinal digestibility of gluten proteins was established after treatment at various pH (3 or 7), temperature (room temperature or 100 °C) and shear (0 or 1500 s−1). Electrophoretic patterns (SDS-PAGE) of the resulting hydrolysates and antigenicity (ELISA and immunoblotting) of gliadin fraction of the proteins were also studied. Digestibility was positively correlated with α-helix/β-sheet ratio. Lower antigenic reaction shown by digested hydrolysate of gluten samples treated at pH 3 and room temperature was ascribed to its acidic deamidation and improved digestibility. Further heating to 100 °C slightly increased antigenicity. In contrast, hydrolysate of gluten at pH 7 and room temperature exhibited highest antigenicity, attributed to partial resistance of α-/β-gliadin to digestion and appearance of some new potentially antigenic polypeptides. However, heating at 100 °C caused heat-induced protein aggregation, consequently lowered digestibility and availability of antigenic components resulting in a minimum (60 % reduction) antigenicity. Overall, shear had no effect on digestibility and antigenicity irrespective of pH and temperature. Thus, antigenic potential of gliadins can be minimized by selecting appropriate parameters during processing.

Cross antigenicity of immunodominant polypeptides of somatic antigen of Oesophagostomum columbianum with other helminths by western blotting.

Aim:Oesophagostomum columbianum in small ruminants in India is found as mixed infection commonly in sheep and goat. Haemonchus contortus, an abomasal nematode is found as concurrent infection with it. Eggs of Haemonchus and O. columbianum cannot be easily distinguished. Diagnosis of O. columbianum may only be possible if a non-cross antigenic polypeptide was available for immunodiagnosis.Materials and Methods:Somatic antigen (SoAg) of O. columbianum was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodominant polypeptides were identified by western blotting with homologous hyperimmune serum (HIS) and experimental sera of sheep or goat infected with other helminths.Results:SoAg of O. columbianum was immunoaffinity purified. Sharp polypeptide bands of 130, 72 and 68 KDa were observed along with several faint bands of lower molecular weight. Western blot of purified SoAg of O. columbianum with homologous HIS showed reaction with all the protein bands of 17, 28, 30, 32, 35, 38, 50, 68, 100, 130, 150, and 170 kDa. For identification of non-cross antigenic polypeptide, immunoaffinity purified SoAg of O. columbianum was reacted to heterologous HIS against H. contortus, Paramphistomum epiclitum, and Fasciola gigantica in western blotting utilizing completely dry method (i-blot). Among high molecular weight polypeptides 100 and 150 kDa were non-cross antigenic and among low molecular weight except 50 kDa polypeptide, 17, 30, 32, 35, and 38 kDa of O. columbianum were not cross antigenic with other helminths.Conclusions:Hence, polypeptides of 17, 30, 32, 35 and 38 kDa as well as 100 and 150 kDa polypeptides of O. columbianum may be exploited for immunodiagnosis of the infection in sheep and goat with extensive studies on cross antigenicity.

Development and Characterization of a New Antipeptide Monoclonal Antibody Directed to Human CD20 Antigen.

The rapid expansion of immunotherapeutic approaches for treatment of various diseases, including cancers, has been greatly facilitated by the invention of new generation of antibodies. Clinical studies have indicated that anti-CD20 mAb-based therapies represent an effective treatment for various diseases with overexpression of CD20 on their cell surface, such as non-Hodgkin's lymphoma, hemolytic anemia, as well as autoimmune diseases like rheumatoid arthritis. Technically, due to a short extra membrane domain, the recombinant CD20 protein is a difficult antigen to raise immune responses. In search for new monoclonal antibodies, the authors used an antigenic polypeptide, which yielded numbers of new binders that may lead to production of anti-CD20 antibodies, with improved diagnostic or clinical attributes. Mice were immunized with extra membrane loop of human CD20 (exCD20) polypeptide. The exCD20 antigen showed a desired immune response and was able to develop a monoclonal antibody, 3B4C10, which reacted well with peptide antigen as well as native antigen on the surface of Raji B-cell line. The antibody 3B4C10 with a balanced K(on) and K(off) may be applicable in the construction of affinity columns or beads for isolation and purification of CD20-positive cells and cancer stem cells.

Molecular chaperoning facilitates the inflammasome activation and an innate immune response to microbial pathogens (IRC4P.455)

Abstract While certain molecular chaperones are known to regulate an adaptive immune response by shutting antigenic polypeptides and facilitating cross-presentation, its potential impact on innate immune responses to pathogens is not well understood. Assembly and activation of NLRP3 inflammasome plays a central role in sensing microbial infections and the host innate immunity. We report that extracellular glucose-regulated protein 170 (Grp170), a large molecular chaperone, profoundly enhances microbial CpG oligodeoxynucleotides (CpG-ODN) induced NLRP3 inflammasome activation, which is independent of ATP. Mechanistic studies reveal that Grp170 potentiates the endocytosis of CpG-ODN, followed by lysosomal damage and the cathepsin B release. Further, the elevation of intracellular calcium and calcium-dependent activation of calpain are crucial for Grp170-potentiated lysosomal instability and the inflammasome activation. Lastly, chaperoning-enhanced inflammasome activation results in more efficient bacterial elimination in mice compared with CpG-ODN alone. These results indicate that molecular chaperoning of pathogen-associated molecules (e.g., microbial DNA) by Grp170 facilitates the immune recognition of invading pathogens via modulation of NLRP3 inflammasome activation. Our findings underscore an important role of extracellular chaperone molecules, possibly released from tissues injury following pathogen infection, in amplifying an innate immune response.

Preparation and characterization of polyclonal antibody against Kaposi's sarcoma-associated herpesvirus lytic gene encoding RTA.

Replication and transcription activator (RTA) is a critical lytic protein encoded by Kaposi's sarcoma-associated herpesvirus (KSHV). To prepare rabbit polyclonal antibody against RTA, three antigenic polypeptides of KSHV RTA were initially synthesized. The fragment of RTA was cloned into p3FlagBsd to construct the recombinant plasmid, pRTA-Flag. 293T and EA.hy926 cells were transfected with pRTA-Flag to obtain RTA-Flag fusion protein, which was detected using anti-Flag antibody. Next, New Zealand white rabbits were immunized with keyhole limpet hemocyanin-conjugated peptides to generate polyclonal antibodies against RTA. Enzyme-linked immunosorbent assays were performed to characterize the polyclonal antibodies, and the titers of the polyclonal antibodies against RTA were greater than 1:11,000. Western blotting and immunofluorescence assay revealed that the prepared antibody reacted specifically with the RTA-Flag fusion protein as well as the native viral protein in KSHV-infected primary effusion lymphoma cells. Collectively, our work successfully constructed the recombinant expression vector, pRTA-Flag, and prepared the polyclonal antibody against RTA, which was valuable for investigating the biochemical and biological functions of the critical KSHV lytic gene.

Exploring early and late Toxoplasma gondii strain RH infection by two-dimensional immunoblots of chicken immunoglobulin G and M profiles.

Toxoplasma gondii is an intracellular apicomplexan parasite infecting warm-blooded vertebrate hosts, with only early infection stage being contained with drugs. But diagnosis differencing early and late infection was not available. In the present investigation, 2-dimensional immunobloting was used to explore early and late infections in chickens. The protein expression of T. gondii was determined by image analysis of the tachyzoites proteome separated by standard-one and conventional two-dimentional gel polyacrylamide electrophoresis (2D- PAGE). Pooled gels were prepared from tachyzoites of T. gondii. A representative gel spanning a pH range of 3-10 of the tachyzoite proteome consisted of 1306 distinct polypeptide spots. Two-dimensional electrophoresis (2-DE) combined with 2-DE immunoblotting was used to resolve and compare immunoglobulins (Igs) M & G patterns against Toxoplasma gondii strain RH (mouse virulent strain). Total tachyzoite proteins of T. gondii were separated by two-dimensional gel electrophoresis and analyzed by Western blotting for their reactivity with the 7 and 56 days post-infection (dpi) SPF chicken antisera. Different antigenic determinant patterns were detected during analysis with M and G immunoglobulins. Of the total number of polypeptide spots analyzed (1306 differentially expressed protein spots), 6.97% were identified as having shared antigenic polypeptide spots on immunoblot profiles with IgG and IgM antibodies regardless the time after infection. Furthermore, some of the immunoreactive polypeptide spots seemed to be related to the stage of infection. Interestingly, we found natural antibodies to toxoplasmic antigens, in addition to the highly conserved antigenic determinants that reacted with non-specific secondary antibody; goat anti-chicken IgG antibodies conjugated with horseradish peroxidase. In conclusion, unique reactive polypeptide spots are promising candidates for designation of molecular markers to discriminate early and late chicken infection.

Multiple antigenic polypeptide composed of heparanase B‑cell epitopes shrinks human hepatocellular carcinoma in mice.

The purpose of this study was to evaluate the anti‑growth effect of the self‑designed multiple antigenic polypeptide (MAP) vaccine comprising B‑cell epitopes of heparanase (HPSE) on HCC97‑H hepatocellular carcinoma (HCC) in mice. The polyclonal antibodies against the B‑cell epitopes of HPSE were prepared by immunizing rabbits with freshly synthesized MAP vaccine. HCC‑bearing models were constructed on BALB/c nude mice. Anti‑MAP antibodies were administrered to the models to assess the effects on HPSE activity, HCC growth, the expression of VEGF/bFGF and the value of micro‑vessel density (MVD). The anti‑MAP antibodies were harvested, purified and identified. These antibodies were able to specifically bind with the dominant epitopes of the precursor protein and large subunit monomer of HPSE, decrease HPSE activity, suppress the expressions of VEGF and bFGF, reduce the MVD, and markedly shrink the HCC volume. Based on these findings, MAP vaccine based on the B‑cell epitopes of HPSE seemed to provide theoretical evidence for further study of the synthesized HPSE MAP vaccine in the treatment of HCC.

Molecular characterization of a Spirometra mansoni antigenic polypeptide gene encoding a 28.7 kDa protein.

The Spirometra mansoni antigenic polypeptide (SmAP) gene was expressed in Escherichia coli, and its characteristics and value as an antigen for the serodiagnosis of sparganosis were investigated. The recombinant SmAP protein (rSmAP) has the molecular weight of 28.7 kDa. On Western blotting analysis, the rSmAP strongly reacted with the sera of mice infected with spargana, but not with normal sera; the anti-rSmAP serum obviously recognized the 28.7-kDa band in the crude antigens and excretory-secretory (ES) antigens of spargana. The immunofluorescence test (IFT) results showed that the positive staining was observed at different stages of spargana from the infected frogs and mice, but not adult worm of S. mansoni. An immunolocalization analysis identified SmAP in the teguments and parenchymal tissues of spargana. ELISA with rSmAP antigen or sparganum ES antigens were evaluated for the serodiagnosis of sparganosis. The results showed that the sensitivity of rSmAP-ELISA and ES-ELISA was 83.3% (25/30) and 100% (30/30), respectively, for the detection of anti-sparganum IgG antibodies in sera of the experimentally infected mice (P > 0.05), the specificities of both ELISA were 100% (67/67). It is suggested that the rSmAP might be a potential candidate antigen for serodiagnosis of sparganosis.