Antigenicity of tissues and organs from GGTA1/CMAH/β4GalNT2 triple gene knockout pigs

Clinical xenotransplantations have been hampered by human preformed antibody-mediated damage of the xenografts.To overcome biological incompatibility between pigs and humans,one strategy is to remove the major antigens[Gal,Neu5 Gc,and Sd(a)]present on pig cells and tissues.Triple gene(GGTAI,CMAH,and β4 GalNT2)knockout(TKO)pigs were produced in our laboratory by CRISPR-Cas9 targeting.To investigate the antigenicity reduction in the TKO pigs,the expression levels of these three xenoantigens in the cornea,heart,liver,spleen,lung,kidney,and pancreas tissues were examined.The level of human IgG/IgM binding to those tissues was also investigated,with wildtype pig tissues as control.The results showed that aGal,Neu5 Gc,and Sd(a)were markedly positive in all the examined tissues in wildtype pigs but barely detected in TKO pigs.Compared to wildtype pigs,the liver,spleen,and pancreas of TKO pigs showed comparable levels of human IgG and IgM binding,whereas corneas,heart,lung,and kidney of TKO pigs exhibited significantly reduced human IgG and IgM binding.These results indicate that the antigenicity of TKO pig is significantly reduced and the remaining xenoantigens on porcine tissues can be eliminated via a gene targeting approach.

Study on enzymatic hydrolysis of soybean β-conglycinin using alkaline protease from Bacillus subtilis ACCC 01746 and antigenicity of its hydrolysates

Due to its beneficial health effects,the use of soybean protein has shown a continuous increase,but concerns regarding the allergenicity of soybean antigenic protein have also increased.This study aimed to evaluate the hydrolytic effects of a non-commercial alkaline protease isolated from the Bacillus subtilis ACCC 01746 on soybeanβ-conglycinin and the allergenicity of its hydrolysates.Alkaline protease of the strain was separated by precipitation method of organic solvents,and theβ-conglycinin was separated by alkali-solution and acid-isolation and purified by use of gel column.Using the degree of hydrolysis(DH)and inhibition rate as evaluation indexes,the enzymatic hydrolysis parameters ofβ-conglycinin was optimized by single factor and L_(9)(3^(4))orthogonal tests,so as to explore the effect of the protease on the hydrolysis degree and the antigenicity ofβ-conglycinin hydrolysates.The results showed that the native enzyme existed as an 18.3 kDa monomer with a 430 U/g maximum activity.The purity ofβ-conglycinin was 84.8%.The single-factor test results showed that DH showed the oppostie trendency with the inhibition rate,and the increase of protein concentration causedmonotone increasing and monotone decreasing of the inhibition rate and the DH,and the optimal protein concentration was 30 mg/mL.The optimization results showed that pH had the largest impacts on both DH and the inhibition rate,followed by enzyme dosage,hydrolysis temperature and hydrolysis time.Under the optimum hydrolysis conditions of protein concentration 30mg/mL,enzymedosage0.7%,hydrolysis time40min,temperature 55°C and pH8.5,the DH reached the highest of 76.28%,and the inhibition rate was the lowest of 27.03%,which was reduced greatly compared with that before optimization.These results suggested that alkaline protease appeared to show a relatively high effeciency in lowering soybean allergenicity,making it possible to produce low-allergenicity soybean protein.

Effect of extrusion on the structure and antigenicity of soybean β-conglycinin

β-Conglycinin,the main component of 7S globulin in soybean protein,is also a key soybean antigen protein that causes allergic reactions.Extrusion technologies have received considerable attention as amethod for modifying soybean protein allergens.This study investigated the changes inβ-conglycinin structure and antigenicity upon extrusion.Isoelectric precipitation,ammoniumsulfate precipitation,and sepharose CL-6B gel filtration were used to isolate and purifyβ-conglycinin from soybean powder,and single-factor and orthogonal tests were used to study the effects of water content,extrusion temperature,screw rotation speed,and feeding speed on the antigenicity ofβ-conglycinin after extrusion.Fourier transforminfrared spectrometry(FTIR)was then employed to analyze the structure ofβ-conglycinin after extrusion under the optimal conditions determined by the orthogonal test.The results showed that extrusion significantly reduced the antigenicity ofβ-conglycinin(P<0.05),and the degree of influence of the factors studied may be ordered as extrusion temperature>feeding speed>screw rotation speed>water content.The optimal parameters were temperature at 130°C,screwrotation speed at 140 r/min,and feeding speed at 35 g/min.Under these conditions,the contents ofα-helix,β-pleated sheet,andβ-turn structures inβ-conglycinin were significantly reduced(P<0.05),while the contents of random coils were significantly increased(P<0.05).The peak absorption intensity of amides I,II,and III also decreased.Taken together,the findings suggest that extrusion could be an effective method for reducing the antigenicity ofβ-conglycinin.

Enzymatic kinetics ofβ-conglycinin using alkaline protease from Bacillus subtilis ACCC 01746 and analysis of antigenicity of hydrolyzed peptide

β-Conglycinin,the main protein of soybean,is a key allergen that causes soybean allergies,and hydrolysis is usually applied to lower its antigenicity.We evaluated the enzymolysis characters ofβ-conglycinin from the perspective of enzymolysis kinetics using alkaline protease from B.subtilis ACCC 01746.A dynamic model describing the hydrolysis ofβ-conglycinin was proposed using the initial substrate concentration,enzyme dosage(enzyme to substrate ratio)and hydrolysis time as variables to illustrate the kinetic behavior of enzymatic hydrolysis.The hydrolysis of soybeanβ-conglycinin was carried out at 60 g/L protein concentration,0.6%enzyme dosage,55℃ and pH 8.5 to observe the peptides with anti-enzymatic activities.The hydrolysates were gradually fractionated by ultrafiltration through cut-off membranes with molecular weights of 40,30,20,and 10 kDa,and their antigenicities were evaluated using indirect competitive enzyme-linked immunosorbent assay.The results showed that the degree of hydrolysis(DH)ofβ-conglycinin decreased as theβ-conglycinin concentration(S0)increased,but increased with enzyme dosage(E0)increasing.Thus,the enzymatic hydrolysis ofβ-conglycinin followed the first-order kinetics model.The hydrolysis rate(V)was(527.89C_(E0)-2.5533C_(S0))exp(-0.022DH),the DH-hydrolysis time was 45.454ln[1+(11.614C_(E0)/C_(S0)-0.0562)t],and the correlated kinetic constants k2 and kd were 527.89 min^(−1)and 8.6126 min^(−1),respectively.The hydrolysis behavior ofβ-conglycinin varied considerably among theα',α,andβsubunits.Faster hydrolysis rates were observed for theα'andαsubunits compared to theβsubunit.The relative molecular weights of the intercepted peptides from the hydrolysates were 14.8-40.1 kDa,and the antigenicity of the peptides with smaller molecular weight was reduced,but not removed completely.However,the alkaline protease from the strain appeared to effectively reduce the allergenicity ofβ-conglycinin.Therefore,it is possible to produce less allergenic soybean proteins using enzymatic hydrolysis.Additionally,the microbial alkaline protease may serve as a potential novel food enzyme and should be evaluated for the development of hypoallergenic foods.

Antigenicity of the HCV HVR1 Peptide Analyzed by Computer Modeling

To find out the protective polypeptide epitopes of HCV HVR1, the antigenicity of the synthetic peptide was predicted by computer modeling and verified by ELISA and lymphocyte transformation test. It was found that the outcome of the computer modeling was in accord with the experimental results. The method by using computer modeling would be a economic approach by which the protective peptides could be identified quickly.

Differentiation induced by physiological and pharmacological stimuli leads to increased antigenicity of human neuroblastoma cells

Sympathetic neuronal differentiation is associated with favorable prognosis of neuroblastoma （NB）, the most common extra-cranial solid tumor of early childhood. Differentiation agents have proved useful in clinical protocols of NB treatment, but using them as a sole treatment is not sufficient to induce tumor elimination in patients. Therefore, complementary approaches, such as immunotherapy, are warranted. Here we demonstrate that differentiation of NB cell lines and ex vivo isolated tumor cells in response to physiological or pharmacological stimuli is associated with acquisition of increased antigenicity. This manifests as increased expression of surface major histocompatibility class I complexes and ICAM-1 molecules and translates into increased sensitivity of NB cells to lysis by cytotoxic T lymphocytes （CTLs） and natural killer （NK） cells. The latter is paralleled by enhanced ability of differentiated cells to form immune conjugates and bind increased amounts of granzyme B to the cell surface. We demonstrate, for the first time, that, regardless of the stimulus applied, the differentiation state in NBs is associated with increased tumor antigenicity that enables more efficient elimination of tumor cells by cytotoxic lymphocytes and paves the way for combined application of differentiation-inducing agents and immunotherapy as an auxiliary approach in NB patients.

The Secondary Structure of Heated Whey Protein andIts Hydrolysates Antigenicity

Fourier transform infrared spectroscopy(FTIR) and circular dichroism(CD) were used to investigate the conformational changes of heated whey protein(WP) and the corresponding changes in the hydrolysates immunoreactivity were determined by competitive enzyme-linked immunosorbent assay(ELISA).Results showed that the contents of α-helix and β-sheet of WP did not decrease much under mild heating conditions and the antigenicity was relatively high;when the heating intensity increased(70 ℃ for 25 min or 75 ℃ for 20 min),the content of α-helix and β-sheet decreased to the minimum,so was the antigenicity;However,when the WP was heated at even higher temperature and for a longer time,the β-sheet associated with protein aggregation begun to increase and the antigenicity increased correspondingly.It was concluded that the conformations of heated WP and the antigenicity of its hydrolysates are related and the optimum structure for decreasing the hydrolysates antigeniity is the least content of α-helix and β-sheet.Establishing the relationship between the WP secondary structure and WP hydrolysates antigenicity is significant to supply the reference for antigenicity reduction by enzymolysis.

Antigenicity and Hemaglutination Activity of a Recombinant Hemagglutinin-Neuraminidase of Paramyxovirus Tianjin Strain

Paramyxovirus Tianjin strain, a new genotype of Sendai virus, was isolated from the lungs of common cotton-eared marmoset that died of severe respiratory infection in the marmoset colonies. The 19.28% IgM positive rate in the young children with acute respiratory tract infection suggested a close relationship between Tianjin strain and humans. Hemagglutinin-neuraminidase (HN) is its major transmembrane glycoprotein responsible for viral attachment, penetration and release. To clear the relationship between HN structure and function of paramyxovirus Tianjin strain, rHN1, rHN2 and rHN3 overlapping the ectodomain of HN protein were expressed. Their antigenicity and hemaglutination activity, as well as cross reactivity to standard antisera against influenza virus type A, type B were analyzed. The results indicated expressed rHNs have the natural antigenicity. The segment rHN2 possesses more linear epitopes exposed on the surface of the native HN protein than found in segments rHN3 and rHN1. The hemagglutination activity of segment rHN3 is higher than that of segments rHN2 and rHN1, and partially dependent on the three-dimensional conformation of HN3 protein. Cross-reactivity between rHNs and standard antisera against influenza virus type A, type B suggested that rHNs might not be the best alternative as specific antigens to detect virus in clinical serum specimens.

Cloning, Prokaryotic Expression, and Antigenicity Analysis of NS1 Gene of H9N2 Swine Influenza Virus

To obtain the NS1 gene of swine influenza virus H9N2 subtype expressed efficiently in E. coli, to develope the effective diagnostic methods for swine influenza virus H9N2 subtype, the NS 1 gene of swine influenza virus H9N2 subtype was amplified by reverse transcriptase polymerase chain reaction （RT-PCR） and cloned into a prokaryotic expression vector, pET-28a（＋）, and overexpressed in E. coli BL21-DE3 after induction with 5 mmol L-1 lactose. The recombinant protein was purified by Ni-NTA and identified by western-blotting. An indirect enzyme-linked immunosorbent assay （ELISA） was used to analyze the antigenicity of the recombinant protein. The recombinant protein of NS1 was about 26 kD. The Western-blotting test showed that the recombinant protein reacted specifically with positive sera. The results of the ELISA test showed that the recombinant protein had good antigenicity.

EXPERIMENTAL STUDY OF INFLUENCE OF LIQUID NITROGEN PRESERVATION ON ANTIGENICITY OF HUMAN HOMOGRAFT AORTIC VALVE

To illustrate the effect of liquid nitrogen preservation on antigenicity of human homograft aortic valve (HAV), human aortic valve tissue were cocultured together with peripheral blood mononuclear cells (PBMCs). Following detections were done to show the difference of antigenicity of HAV indirectly at different period after being preserved in liquid nitrogen: ① 3H TdR incorporation (cpm) was observed after tissue cell cocultured and stimulation index (SI) was calculated;② Density of IL 2 in medium was measured by MTT means. The results indicated that antigenicity of fresh HAV was the strongest; with the prolong of being preserved, antigenicity decreased gradually. It decreased significantly within first 48 h preserved at 4℃, then decreased significantly after preserved in liquid nitrogen for 24 h. 2 weeks later, antigenicity decreased to the lowest level.

Antigenicity of Synthetic Peptides Derived from Plasmodium Apoptosis-Linked Pathogenicity Factors

Background： Plasmodium falciparum malaria remains a major life-threatening disease. Recently, the Plasmodium apoptosis-linked pathogenicity factors （PALPF） have been identified. These antigens PALPF are expressed only by P falciparum-infected erythrocytes triggering endothelial cell apoptosis （apoptogenic）. Methods： We designed ten synthetic peptides （PI to P10） from PALPF： PF07 0032, PF10_0226, PFI0130c, PFD0875c and MAL13P1.206, and analyzed their antigenicity with an ELISA method using plasma samples from subjects living in Dienga, Gabon. Results： Four peptides showed good reactivity with human antibodies. The prevalence rate of specific IgG was 61%, 51%, 44% and 34% for P5, P6, P4 and P2, respectively. The median optical density of total IgG anti-P2 was higher than that directed against P4 and P6 （P = 0.009; P = 0.012 respectively）. The prevalence rate oflgG subclasses determined with plasma samples recognizing peptide 5 for IgGl, 2, 3 and 4 isotypes was 69%, 45%, 76% and 62%, respectively. All the subjects had at least one immunoglobulin subclass, while 13 （44%） had both IgG1 and IgG3 antibodies. There was no significant difference in the prevalence rate of anti-P5 IgG1, IgG3 and IgG4. Conclusion： These results warrant further immunogenicity studies of peptides 2, 4, 5 and 6 with a view of a tentative to antimalarial vaccine development.

The study of differences of antigenicity in rabbit intervertebral disc

Objective To reveal and compare the immunogenicity of glycoprotein from nucleus pulposus and collagen from anulus fibrosus in rabbit intervertebral disc.Methods According to the similarity principle of genome biology and the amount

Prediction, Synthesis and Antigenicity of the Antigenic Peptides of 26kDa Glutathione S-Transferasc of Schistosoma Japonicum (Sj26)

Six antigenic peptides of 26 kDa glutathione S-transferase of Schistosoma japonicum(Sj26) have been predicted according to their hydrophilicity, flexibility. accessibility. chargedistribution and β -turn in the secondary structure by the determination of its primary structure andsynthesized by solid phase method. All of them showed antigenicity with anti-schistosomajaponicum immunoglobulin polyclonal antibody, anti-Sj-lgG PcAb by Dot-ELISA. Three of themshowed good antigenicity. They would serve as candidates of synthetic anti-schistosomal vaccine.

The development of a recombinant hybrid protein of Plasmodium falciparum and analysis of its antigenicity and protectivity

A hybrid gene encoding several putative protective epitopes from erythrocytic stage antigens (MSA1,MSA2 and RESA) of P. falciparum as well as exgenous T cell enhancer epitopes from interleukin-1 and tetanus toxin was synthesized chemically. This gene (named HGFC) was cloned and connected with another hybrid gene (HPFA) synthesized previously to make a bigger hybrid gene (HGFCAC). HGFC and HGFCAC were cloned in an expression vector pWR450-1 and transformed into E. Coli JM109. The engineered bacteria could express fusion proteins with molecular weights of 65 and 77 kDa after inducing with isopropylthio-β-D-galactoside (IPTG). The expression rate was about 35% of total bacterial proteins. The expressed products showed sepcific immunological reaction with rabbit antibodies against P. falciparum peptide Glu-Glu-Asn-Val-Glu-His-Asp-Ala (EENVEHDA)by Western blotting. The fusion proteins were pruified by precipitation with amonium sulfate. gel filtration and ion-exchange chromatography and the purity was 82%. The purified protein reacted specifically with mouse immune serum against falciparum blood stage antigens by dot enzyme-linked immunosorbent assay (dot-ELISA).The fusion protein was emulsified with Freund's complete adjuvant (FCA) and used to immunize rabbits. The immune serum can recognize P. falciparum erythrocytic stage antigens of Fcc-1/HN strain and Yunnan strain and had weak cross reaction with P. vivax,but had no reaction with P. cynomlogi and P. berghei antigens. The protective effect of the antibody was tested by in vitro inhibition test to cultured falciparum parasites. Preliminary results indicated that the immune sera could effectively reduce the invasion rates of the parasites to red blood cells and inhibit the growth of the in vitro cultured falciparum parasites. The inhibitory capacity of the immune sera to parasite invasion is enhanced as the amount of the sera increases and the incubation time of the sera with the parasites is prolonged.It was shown that after 72 h incubation at 20% concentration with the parasites, the serum can suppress the multiplication of P.falciparum growth in vitro to a level of 80%.The immune sera caused dispersion of the parasite cytoplasm,atrophy of parasites,agglutination of free merozoites and degeneration of schizonts.

Elementary study on the antigenicity of SARS-CoV Mc region

To test the antigenic activity of M protein （Mc protein） in the inner membrane of SARS-CoV, SARS-CoV Me protein＇s bases locating inside the membrane were cloned, the His-fusion protein was expressed in E. coli and analyzed for its antigenic activity. Among those 7 clinically diagnosed patients＇ sera, there were 5 positive and 2 negative in reaction with His-fusion protein. All of the 20 healthy persons＇ sera and rabbit anti-OC43 and 229E were of negative reaction with His-fusion protein. The animals immunized with His-fusion protein have produced muhi-clonal antibody. The His-fusion protein could specially react with clinically diagnosed SARS patients＇ sera and the animals immunized with His-fusion protein could produce specifically multi-clonal antibody, but it could not react with the sera of healthy persons and the rabbit anti-OC43 and 229E.

The Structural Characterization and Antigenicity of the S Protein of SARS-CoV

The corona-like spikes or peplomers on the surface of the virion under electronic microscope are the most striking features of coronaviruses. The S (spike) protein is the largest structural protein, with 1,255 amino acids, in the viral genome. Its structure can be divided into three regions: a long N-terminal region in the exterior, a characteristic transmembrane (TM) region, and a short C-terminus in the interior of a virion. We detected fifteen substitutions of nucleotides by comparisons with the seventeen published SARS-CoV genome sequences, eight (53.3%) of which are non-synonymous mutations leading to amino acid alternations with predicted physiochemical changes. The possible antigenic determinants of the S protein are predicted, and the result is confirmed by ELISA (enzyme-linked immunosorbent assay) with synthesized peptides. Another profound finding is that three disulfide bonds are defined at the C-terminus with the N-terminus of the E (envelope) protein, based on the typical sequence and positions, thus establishing the structural connection with these two important structural proteins, if confirmed. Phyloge-netic analysis reveals several conserved regions that might be potent drug targets.

The C-Terminal Portion of the Nucleocapsid Protein Demonstrates SARS-CoV Antigenicity

In order to develop clinical diagnostic tools for rapid detection of SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.

Evaluation of the infectivity,gene and antigenicity persistence of rotaviruses by free chlorine disinfection

The effects of free chlorine disinfection of tap water and wastewater effluents on the infectivity, gene integrity and surface antigens of rotaviruses were evaluated by a bench-scale chlorine disinfection experiments. Plaque assays, integrated cell culture-quantitative RT- PCR （ICC-RT-qPCR）, RT-qPCR, and enzyme-linked immunosorbent assays （ELISA）, respectively, were used to assess the influence of the disinfectant on virus infectivity as well as genetic and antigenic integrity of simian rotavirus SA11 as a surrogate for human rotaviruses. The ICC-RT-qPCR was able to detect rotaviruses survival from chlorine disinfection at chlorine dose up to 20 mg/L （60 min contact）, which suggested a required chlorine dose of 5 folds （from 1 to 5 mg/L） higher than that indicated by the plaque assay to achieve 1.8 log10 reductions in tap water with 60 rain exposing. The VP7 gene was more resistant than the infectivity and existed at chlorine dose up to 20 mg/L （60 min contact）, while the antigencity was undetectable with chlorine dose more than 5 mg/L （60 min contact）. The water quality also impacted the inactivation efficiencies, and rotaviruses have a relatively higher resistant in secondary effluents than in the tap water under the same chlorine disinfection treatments. This study indicated that rotaviruses have a higher infectivity, gene and antigencity resistance to chlorine than that previously indicated by plaque assay only, which seemed to underestimate the resistance of rotaviruses to chlorine and the risk of rotaviruses in environments. Present results also suggested that re-evaluation of resistance of other waterborne viruses after disinfections by more sensitive infectivity detection method （such as ICC-RT-qPCR） may be necessary, to determine the adequate disinfectant doses required for the inactivation of waterborne viruses.

Parallel profiling of antigenicity alteration and immune escape of SARS-CoV-2 Omicron and other variants

SARS-CoV-2 variants have evolved a variety of critical mutations,leading to antigenicity changes and immune escape.The recent emerging SARS-CoV-2 Omicron variant attracted global attention due to its significant resistance to current antibody therapies and vaccines.