Characterization of physicochemical and immunogenic properties of allergenic proteins altered by food processing:a review

Food allergens are mainly naturally-occurring proteins with immunoglobulin E(IgE)-binding epitopes.Understanding the structural and immunogenic characteristics of allergenic proteins is essential in assessing whether and how food processing techniques reduce allergenicity.We here discuss the impacts of food processing technologies on the modification of physicochemical,structural,and immunogenic properties of allergenic proteins.Detection techniques for characterizing changes in these properties of food allergens are summarized.Food processing helps to reduce allergenicity by aggregating or denaturing proteins,which masks,modifies,or destroys antigenic epitopes,whereas,it cannot eliminate allergenicity completely,and sometimes even improves allergenicity by exposing new epitopes.Moreover,most food processing techniques have been tested on purified food allergens rather than food products due to potential interference of other food components.We provide guidance for further development of processing operations that can decrease the allergenicity of allergenic food proteins without negatively impacting the nutritional profile.

Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein

Enterovirus （EV71） can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 （VP1）, plays a critical role in the pathogenicity of EVT1. High level expression and secretion ofVP 1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP 1 DNA vaccines, including wt-VP 1, tPA-VP 1, VP l-d, VP 1-hFc and VP 1 - mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different （P〈0.05）. In this study, we further investigated the protein lev- els of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc （hFc） to VP1. VP 1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VPI-hFc protein for additional studies.

Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

Flavobacterium columnare causes columnaris disease in freshwater fi sh. In the present study, the antigenic regions of fi ve outer membrane proteins(OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 k Da, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purifi ed recombinant protein was used to vaccinate the grass carp, C tenopharyngodon idella. Following vaccination of the fi sh their Ig M antibody levels were examined, as was the expression of I g M, Ig D and Ig Z immunoglobulin genes and other genes such as MHC Iα and MHC I I β, which are also involved in adaptive immunity. Interleukin genes( IL), including I L- 1β, IL- 8 and I L- 10, and type I and type II interferon(I FN) genes were also examined. At 3 and 4 weeks post-vaccination(wpv), signifi cant increases in Ig M antibody levels were observed in the fi sh vaccinated with the recombinant fusion protein, and an increase in the expression levels of I g M, Ig D and Ig Z genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fi sh. At four wpv, the fi sh were challenged with F. column a re, and the vaccinated fi sh showed a good level of protection against this pathogen, with 39% relative percent survival(RPS) compared with the control group. It can be concluded, therefore, that the fi ve OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fi sh and protection against F. columnare.

Immunogenicity of recombinant attenuated Salmonella typhimurium expressing a 45-peptide hybrid antigen gene of Plasmodium falciparum

A synthetic hybrid 45-peptide gene of Plasmodium falciparum (Pf), which encoded two CSP repeated peptides NANP and three merozoite peptides SPf83. 1, SPf55. 1 and SPf35. 1, was cloned in an expression vector pWR450-1, then the recombinant plasmid pWRA was introduced into the attenuated Salmonella typhimurium SL3261. When used as a live vaccine and administered orally (po), intravenously (iv) or intraperitoneally (ip),the recombinant strain was able to live in vivo and elicit specific humoral and cellular immunity in BALB/c mice and rabbits. As oral immunization is safe and effective, it is thought that the live recombinant Salmonella tyPhimurium vaccine may bring the Pf oral live vaccine a step nearer.

Immunogenicity of Lyophilized MVA Vaccine for HIV-1 in Mice Model

Highly attenuated modified vaccinia Ankara（MVA） is sensitive to repeat freeze-thaw cycle and easy to lose activity. In order to make the activity of MVA vaccine remain stable during its manufacturing, storage, and administration, the lyophilization as a good option could be resorted to; through screening, the right stabilizer composition and its production procedure were obtained. The final moisture content of freezing-dried recombinant MVA-HIV vaccine was lower than 3%. It can be reconstituted quickly and shows regular physical appearance and stable potency. In vivo functional experiment, mice were divided randomly into the liquid vaccination group, the lyophilized vaccination group, and the control group. Having been DNA vaccine priming, the mice were boosted with a dose of 10^7 pfu MVA- HIV vaccine, which produced indistinguishable antibody titer and cytotoxic T-lymphocyte（CTL） level compared with those of liquid vaccination group （ P 〉 0.05 ）. These results demonstrate that lyophilized MVA vaccine can induce high immunogenicity in mice.

Generation and Immunogenicity of a Recombinant Adenovirus Co-Expressing the E2 Protein of Classical Swine Fever Virus and the GP5 Protein of Porcine Reproduction and Respiratory Syndrome Virus

Classical swine fever （CSF） and porcine reproduction and respiratory syndrome （PRRS） are both economically important, highly contagious diseases of swine worldwide. To develop an effective vaccine to control these two diseases, we constructed a recombinant adenovirus rAdV-GP52AE2, using a replication-defective human adenovirus serotype 5 as a delivery vector, to co-express the GP5 protein of highly pathogenic porcine reproduction and respiratory syndrome virus （PRRSV） and the E2 protein of classical swine fever virus （CSFV）. Foot-and-mouth disease virus （FMDV） 2A peptide was used as a linker between the GP5 and E2 proteins to allow automatic self-cleavage of the polyprotein. The GP5 and E2 genes were expressed as demonstrated by immunofluorescence assay and Western blotting. Immunization of mice resulted in a CSFV-neutralizing antibody titer of 1：128 and a PRRSV-neutralizing antibody titer of 1：16. The lymphoproliferative responses were detected by Cell Counting Kit-8 assay and the stimulation index of CFSV-specific and PRRSV-specific lymphocytes in the rAdV-GP52AE2 group was significantly higher than that in the negative control group. The results show that rAdV-GP52AE2 can induce both effective humoral and cell-mediated immune responses in mice. The protective efficacy of the recombinant virus against CSF was evaluated in immunized rabbits, which were protected from fever induced by challenge with C-strain. Our study provides supporting evidence for the use of FMDV 2A to develop a bivalent genetically-engineered vaccine.

Comparative Study on the Immunogenicity between Hsp70 DNA Vaccine and Hsp65 DNA Vaccine in Human Mycobacterium Tuberculosis

The BALB/c mice were immunized with Hsp70 DNA and Hsp65 DNA vaccines in human Mycobacterium tuberculosis. Eight weeks after immunization, the eyeballs were removed, blood and spleen taken, and intraperitoneal macrophages were harvested. The lymphocytic stimulating index (SI) was used to measure the cellular proliferating ability and NO release to measure the phagocytic activity of the macrophages. With ELISA kit, the levels of interleukin-2 (IL-2) and interferon-γ (IFN-γ) in serum and the splenic lymphocytic cultured supernatant were detected. The results showed that after the mice were immunized with 100 μg/mouse of Hsp70 DNA vaccine intramuscularly, the splenic lymphocytic proliferating ability in the mice was significantly increased as compared with that in the control group, vector group and Hsp65 DNA vaccine group (P<0.01); The contents of NO in the intraperitoneal macrophages of the mice were significantly lower than in the control group and Hsp65 DNA vaccine group (P<0.01); The levels of serum IL-2 in the mice were significantly higher than in the control group, but there was no statistical difference between Hsp65 DNA group and vector group (P>0.05); The contents of serum IFN-γ in the mice were significantly higher than in the control group, but significantly lower than in the Hsp65 DNA vaccine group (P<0.05). It was indicated that immunization with Hsp70 DNA vaccine could obviously enhance the immune response, but its intensity seemed inferior to Hsp65 DNA vaccine. The anti-infection mechanisms and clinical use in the future of the vaccines of Hsp70 DNA and Hsp65 DNA are worth further studying.

Prokaryotic Expression and Immunogenicity of Major Antigen Epitope of Rabbit Haemorrhagic Disease Virus VP60

[ Objective] To investigate the immunogenicity of main antigen epitope of RHDV （ Rabbit haemorrhagic disease virus） VP60 expressed in a prokaryotic system. [ Method] The major antigen epitope gene of RHDV VP60 was amplified by RT-PCR. It was cloned into pET-28b （ ＋ ） and expressed in E. coli Rosetta strain. The recombinant protein was detected by Western blot. The pudfied recombinant protein was used to immunize rabbits in order to observe its immunogenicity. [ Result] Western blot analysis revealed a clear band at approximately 24.0 kDa. The purified recom- binant protein reacted with the purified RHDV in ELISA. [ Conclusion] The prokaryotically expressed main antigen epitope of RHDV VP60 shows good immunogenicity.

Immunogenicity and immunoprotection of recombinant PEB1 in Campylobacter-jejuni-infected mice

AIM: To construct a prokaryotic expression vector carrying Campylobacter jejuni peb1A gene and express it in Escherichia coli.Immunoreactivity and antigenicity of rPEB1 were evaluated.The ability of rPEB1 to induce antibody responses and protective efficacy was identified.METHODS: peb1A gene was amplified by PCR,target gene and prokaryotic expression plasmid pET28a (+) was digested with BamHI and XhoI,respectively.DNA was ligated with T4 DNA ligase to construct recombinant plasmid pET28a(+)-peb1A.The rPEB1 was expressed in E.coli BL21 (DE3) and identified by SDS-PAGE.BALB/c mice were immunized with rPEB1.ELISA was used to detect the specific antibody titer and MTT method was used to measure the stimulation index of spleen lymphocyte transformation.RESULTS: The recombinant plasmid pET28a (+)-peb1A was correctly constructed.The expression output of PEB1 protein in pET28a (+)-peb1A system was approximately 33% of total proteins in E.coli.The specific IgG antibody was detected in serum of BALB/c mice immunized with rPEB1 protein.Effective immunological protection with a lower sickness incidence and mortality was seen in the mice suffering from massive C.jejuni infection.CONCLUSION: rPEB1 protein is a valuable candidate for C.jejuni subunit vaccine.

Expression and Immunoreactivity of a Human Group A Rotavirus Vp4

Rotavirus capsid protein Vp4 plays an important role in the virus adhering and entering the cells. In this study, a Vp4 gene cloned from a rotavirus strain TB-Chen was highly expressed in E.coli BL21 (DE3). The results of the Western blot showed that the protein possesses specific immuno-reactivities and can be specifically recognized by guinea pig antibodies against rotavirus strain SA11 or Wa. Some Vp4 dimers were formed during renaturation. These data obtained from this study provide a strong basis for further study on the structure and function of the Vp4.

Immunogenicity Analysis of Prokaryotic Expression Products of Kaposi's Sarcoma Associated Herpesvirus orf65

To purify the protein encoding the small capsid protein (SCP) of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi's sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf65 was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and the fusion protein was purified by chromatography. The expressed protein and its purified product were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and showed that 9 kDa was the expected size of the purified orf65 protein. The antiserum was produced in rabbit which was immunized by purified orf65 protein. An ELISA assay was established to analyze the immunogenicity of the purified orf65 protein. The ELISA analysis demonstrated that orf65 protein has strong immune activity, and the immune activity of polyclonal antibody against orf65 was more than 4 fold higher than that in the serum of the non-immunized rabbit. These results demonstrate that purified orf65 protein has very strong immunogenicity and can be used in screening KSHV infection in the general population using ELISA.

Investigation of bone reconstruction using an attenuated immunogenicity xenogenic composite scaffold fabricated by 3D printing

Bone is known to have a natural function to heal itself.However,if the bone damage is beyond a critical degree,intervention such as bone grafting may be imperative.In this work,the fabrication of a novel bone scaffold composed of natural bone components and polycaprolactone(PCL)using 3D printing is put forward.α1,3-galactosyltransferase deficient pigs were used as the donor source of a xenograft.Decellularized porcine bone(DCB)with attenuated immunogenicity was used as the natural component of the scaffold with the aim to promote bone regeneration.The 3D printed DCB-PCL scaffolds combined essential advantages such as uniformity of the interconnected macropores and high porosity and enhanced compressive strength.The biological properties of the DCB-PCL scaffolds were evaluated by studying cell adhesion,viability,alkaline phosphatase activity and osteogenic gene expression of human bone marrow-derived mesenchymal stem cells.The in vitro results demonstrated that the DCB-PCL scaffolds exhibit an enhanced performance in promoting bone differentiation,which is correlated to the DCB content.Furthermore,critical-sized cranial rat defects were used to assess the effect of DCB-PCL scaffolds on bone regeneration in vivo.The results confirm that in comparison with PCL scaffolds,the DCB-PCL scaffolds can significantly improve new bone formation in cranial defects.Thus,the proposed 3D printed DCB-PCL scaffolds emerge as a promising regeneration alternative in the clinical treatment of large bone defects.

Expression and immunoreactivity of an epitope of HCV in a foreign epitope presenting system

AIM: To construct and highly express an epitope of hepatitis C virus (HCV) in a foreign epitope presenting vectorbased on an insect virus, and to study the antigenicity of the epitope.METHODS: The HCV epitope sequence (amino acidresidues 315 to 328: EGHRMAWDMMMNWS) of the E1 region was constructed at different positions of a foreign epitope presenting vector based on an insect virus, flock house virus (FHV) capsid protein encoding gene as a vector, and expressed in E. coli cells. Western blottingand ELISA were used to detect the immunoreactivity of these recombinant proteins.RESULTS: The gene encoding of the concerned B-cell epitope of HCV E1 envelope protein was expressed on FHV capsid carrier protein at positions I1 (aa 106), I2 (aa153) and I3 (aa 305), respectively, on the surface of FHV capsid protein. The recombinant proteins in this system could be highly expressed in more than 40% of total cell protein of E. coli BL21. All the expressed recombinant proteins were in inclusion body form, and showed obvious immunoreactivity by Western blotting. Further purified recombinant proteins were detected by indirect ELISA as coating antigen respectively. All recombinant proteins could still show immunoreactivity.CONCLUSION: The epitope of HCV E1 envelope protein can be highly expressed in FHV carrier system as a chimeric protein with high immunoreactivity. This system has multiple entry sites conferring many possible conformations closer to the native one for a given sequence.

Expression of Outer Capsid Protein VP5 of Grass Carp Reovirus in E.coli and Analysis of its Immunogenicity

Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.

Immunogenicity of recombinant hepatitis B virus vaccine in patients with and without chronic hepatitis C virus infection:A case-control study

AIM: To compare the response of standard hepatitis B virus (HBV) vaccination between patients with chronic hepatitis C virus (HCV) infection and healthy individuals. METHODS: This is a prospective case-control study. A total of 38 patients with chronic HCV infection and 40 healthy controls were included. Vaccination was performed by injection of 20 μg recombinant HBsAg into the deltoid muscle at mo 0, 1 and 6. Anti-HBs concentration was determined 3 mo after the last dose and compared between the two groups. The response pattern was characterized as (1) high-response when the anti-HBs antibody titer was > 100 IU/L, (2) low-response when the titer was 10-100 IU/L and (3) no-response when the titer was < 10 IU/L. RESULTS: In the patient group, there were 10/38 (26.3%) non-responders, 8/38 (21.1%) low-responders and 20/38 (52.6%) high-responders. The corresponding values in the control group were 2/40 (5.0%), 7/40 (17.5%) and 31/40 (77.5%), respectively. The response pattern was statistically different between the two groups. In multivariate analysis, smoking was a significant confounder, while HCV infection lost its significant correlation with lower antibody response. CONCLUSION: Patients with chronic HCV infection tend to respond weakly to HBV vaccination compared to healthy individuals, though this correlation is not independent according to multivariate analysis.

Immunogenicity and protective efficacy of Vibrio harveyi pcFlaA DNA vaccine in Epinephelus awoara

The FlaA gene from Vibrio harveyi,with a short nucleotide sequence encoding the Flag marker,was cloned into the eukaryotic expression vector pcDNA3.1(+) (designated as pcFlaA).Ninety grouper (Epinephelus awoara) were separated into three equal size groups.An experimental group was immunized with pcFlaA,Control I group was immunized with the vector pcDNA3.1(+),and Control II group was immunized with PBS.The expression of pcFlaA mRNA and protein was examined using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry.We also evaluated the immunogenicity and protective efficacy of pcFlaA against V.harveyi by measuring the lymphocyte proliferation response and serum levels of specific antibody and conducting a bacterial challenge test.We successfully transfected the fish muscle with pcFlaA.The pcFlaA mRNA and protein was expressed in the muscle cells for up to one month following injection.The proliferation response of lymphocytes in fish immunized with pcFlaA was significantly higher than in control group II.Furthermore,the immunized fish generated specific antibody.The vaccination also resulted in significantly higher survival during the bacterial challenge test.

Efficacy,effectiveness,immunogenicity- are not the same in vaccinology

Manuscript of Carrera et al is devoted to immunization in inflammatory bowel disease(IBD)that is very important issue in gastroenterology.However,some specific definitions used in the article need clarification.Efficacy of vaccine is measured in a randomised,placebo-controlled studies,that are expensive and difficult to plan.Moreover,it is unethical to offer a placebo instead of vaccine.For all of these reasons,efficacy of vaccine is measured in IBD patients rarely.Effectiveness of vaccine is measured as an epidemiological affect from observational studies.These studies are also uncommon in IBD because it would be difficult to perform a study that assess the prevalence of one rare disease(vaccine-preventable)in patients with a chronic rare condition,such as IBD.Immunogenicity of vaccine refers to the ability of a vaccine to induce an immune response in a vaccinated individual that is,in fact,the matter of the article.

Preparation and immunogenicity of tag-free recombinant human eppin

Human epididymal protease inhibitor （eppin） may be effective as a male contraceptive vaccine. In a number of studies, eppin with an engineered His6-tag has been produced using prokaryotic expression systems. For production of pharmaceutical-grade proteins for human use, however, the His6-tag must be removed. This study describes a method for producing recombinant human eppin without a His6-tag. We constructed plasmid pET28a （＋）-His6-tobacco etch virus （TEV）-eppin for expression in Escherichia coli. After purification and refolding, the fusion protein His6-TEV-eppin was digested with TEV protease to remove the His6-tag and was further purified by NTA-Ni2＋ affinity chromatography. Using this procedure, 2 mg of eppin without a His6-tag was isolated from 1 I of culture with a purity of 〉95%. The immunogenicity of the eppin was characterized using male Balb/c mice.

Investigation of immunogenicity of cryopreserved limbal stem cells

AIM: To investigate changes in immunogenicity of cryopreserved limbal stem cells. METHODS: Cryopreserved limbal stem cells, fresh primary limbal stem cells and blank controls were inoculated subcutaneously in C57BL-6 mice and the percentage of CD25 cells in limbal explants was determined by flow cytometry at day 21 post inoculation. Morphological studies were performed by light and electron microscopy of limbal explant sections. RESULTS: The number of regional and systemic lymphocytes derived from cryopreserved limbal stem cells was lower than that from fresh primary limbal stem cells. CONCLUSION: Lymphocytes derived from cryopreserved limbal stem cells showed changes in immunogenicity, but the significance is unknown. The cryopreservation and thawing methods await further study.

Low immunogenicity of endothelial derivatives from rat embryonic stem cell-like cells

胚胎的干细胞(转换字符) 被建议给予免疫者优惠，但是他们带不受管束的扩大和恶意的风险。在区别之上，他们失去他们的形成肿瘤的能力，但是他们由 MHC 分子的一个正常集合的表示变得产生免疫性。这 immunogenicity 可能在再生治疗在申请以后触发拒绝。在这学习 MHC 表情和对老鼠的 endothelial 衍生物的有免疫力的回答胚胎的茎在煽动性的条件下面的像房间的房间(RESC ) 与主要老鼠相比被决定大动脉的 endothelial 房间(EC ) 。细胞以及体液紧密相联识别在 vitro 被分析。另外，在 vivo 的有免疫力的反应被干扰素的紧密相联抗体的生产和决心估计 -- 纬(IFN 纬)-secreting 紧密相联反应的 T 房间。RESC 衍生物表示了 MHC 一级，和没有 MHC 班 II 的低却重要的层次。响应 IFN 纬 MHC 一级，表达式被提高，当类 II transactivator 正式就职在这些房间完全失败了时；MHC 班 II 表示仍然保持一致地不在。机能上地， RESC 衍生物显示出一个减少的 allo-stimulatory 能力，保护对体液在到细胞毒素的 T 房间细胞溶解的 vitro 和稍微减少的危险性紧密相联识别。而且，在 vivo，实验证明这些细胞不触发宿主免疫者反应，由没有紧密相联抗体的生产和紧密相联反应的记忆 T 细胞的没有正式就职描绘了。我们的结果证明 RESC 的 endothelial 衍生物甚至在煽动性的条件下面有特殊减少的产生免疫性的力量。