Synthesis of modified tuftsins containing monosaccharides or monosaccharide derivatives

Synthesis of some modified tuftsins is described in which a monosaccharide or a monosaccharide derivative was incorporated in the molecule. Acylation of H-Thr-Lys(Z)-Pro-Arg(NO₂)-OBzl with D(+)-gluco-1,5-lactone followed by catalytic hydrogenation gave Nα-gluconyl-tuftsin. Glycosylation of the carboxyl function of the C-terminal arginine has been achieved by reacting, through the mixed anhydride procedure, Boc-Thr-Lys(Z)-Pro-OH with 2-deoxy-2-(N^G-nitroargininamido)-D-glucopyranose followed by catalytic hydrogenation and trifluoroacetic acid treatment. O-Glucosyl-tuftsin has been prepared by reacting o-nitrophenyl N-benzyloxycarbonyl-O-[(α + β)2,3,4,6-tetra-O-benzyl-D-grucopyranosyl]-threoninate with H-Lys(Z)-Pro-Arg(NO₂)-OBzl in the presence of 1-hydroxybenzotriazole. Flash chromatography on silica gel allowed a partial separation of the diastereoisomers, one of which has been isolated in a reasonable yield. The single diasteroisomer and the α + β anomeric mixture were separately deblocked by catalytic hydrogenation and purified by RP-HPLC.     

中山市新生儿乙肝疫苗计划免疫管理情况的调查分析

目的通过调查分析中山市乙肝疫苗纳入计划免疫管理工作的开展情况,为制定本市切实可行的乙肝免疫预防措施提供依据。方法抽查乙肝疫苗接种登记表及月报表,接种点现场询问承担新生儿乙肝疫苗接种工作人员有关乙肝疫苗接种知识、安全接种及资料转报情况。结果市、镇两级乙肝疫苗专项经费落实率、及时率100％,专项经费专项使用率100％。常住新生儿首针接种率、及时率和三针完整接种率均达98％以上。暂住新生儿首针接种率和三针完整接种率也达98％以上,但首针接种及时率只有88．4％,与常住新生儿有显著统计学差异（Χ^2=533．06,P〈0．01）。常住新生儿与暂住新生儿的第一针和第三针接种率亦有统计学差异（Χ^2=8．70,Χ^2=11．18,P〈0．01）。接种工作人员对乙肝疫苗接种的知识、安全接种基本掌握,工作规范。结论中山市新生儿乙肝疫苗纳入计划免疫管理工作已全面启动,新生儿免费接种乙肝疫苗工作进展顺利,但暂住新生儿的首针接种率和及时率还有待继续提高,对承担新生儿乙肝疫苗接种的单位还要加强管理,提高工作质量。     

Mucoadhesive glycol chitosan nanoparticles for intranasal delivery of hepatitis B vaccine: enhancement of mucosal and systemic immune response

In this study, for the first time, glycol chitosan (GC) nanoparticles (NPs) were prepared and evaluated to obtain systemic and mucosal immune responses against nasally administered hepatitis B surface antigen (HBsAg). Size, zeta potential and morphology of the NPs were investigated as a function of preparation method. NPs with high loading efficacy (?>?95%) and positively charged surface were obtained with an average particle size of approximately 200?nm. The structural integrity of HBsAg in NPs was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and further confirmed by measuring the in vitro antigenicity using an enzyme immunoassay. During in vivo studies, GC NPs showed the lowest nasal clearance rate and better mucosal uptake when compared with chitosan (CS) NPs. The immunogenicity of NPs-based delivery system(s) was assessed by measuring anti-HBsAg antibody titer in mice serum and secretions after intranasal administration. The alum-based HBsAg vaccine injected subcutaneously was used as positive control. Results indicated that alum-based HBsAg induced strong humoral but negligible mucosal immunity. However, GC NPs induced stronger immune response at both of the fronts as compared to generated by CS NPs. This study demonstrates that this newly developed system has potential for mucosal administration of vaccines.     

Studies on the delineation of the antigenic determinants of chicken riboflavin carrier protein (cRCP)

As riboflavin carrier proteins play a critical role in the maintenance of pregnancy, studies on the immunology of these proteins have become very relevant. This paper describes our attempts to identify the sequential antigenic determinants of chicken riboflavin carrier protein (cRCP). Potential surface oriented regions of cRCP were identified by constructing acrophilicity and hydrophilicity profiles of the protein. Of the three regons identified, the pentadecapeptide 10–24 of cRCP forms the subject of the present study. The pentadecapeptide amide was synthesized by solid phase synthesis using Fmoc chemistry. Immunization of rabbits with the peptide-diphtheria toxoid conjugate elicited high titres of the antipeptide antibodies, revealing the high immunogenicity of the peptide. The antipeptide antibodies bound to both cRCP and reduced carboxymethylated RCP (RCM-RCP). Antisera to RCM-RCP showed significant binding to the peptide showing thereby that the region 10–24 of cRCP is perhaps one of the major epitopes of RCM-RCP. Thus, the studies have resulted in the identification of the region 10–24 as an antigenic determinant of cRCP.     

新型乙型肝炎病毒表面抗原中蛋白核酸疫苗的免疫原性研究

目的 观察新型乙型肝炎病毒（HBV）表面抗原中蛋白（MHBs)核酸疫苗的免疫原性。方法 以新型人体应用载体质粒pSW3891构建MHBs核酸疫苗（pSW3891/MHBs/adr)，对照组和实验组Balb/c小鼠分别基因枪法免疫对照载体质粒（pSW3891)和MHBs核酸疫苗，采用酶联免疫吸附试验检测抗HBs,LDH释放测定法检测小鼠特异性细胞毒T淋巴细胞（CTL）杀伤活性。结果 MHBs核酸疫苗可在体外高效表达，免疫小鼠后可产生高滴度抗HBs，血清阳性终点滴度达1：97200,小鼠脾细胞HBs特异性细胞毒性T淋巴细胞（CTL）杀伤活性达78.81%。结论 新型MHBs核酸疫苗在Balb/c小鼠实验中表现出良好的体液和细胞免疫原性。     

Systemic and mucosal immune response induced by transcutaneous immunization using Hepatitis B surface antigen-loaded modified liposomes

We have evaluated the efficiency of novel modified liposomes (ethosomes) for transcutaneous immunization (TCI) against Hepatitis B. Antigen-loaded ethosomes were prepared and characterized for shape, lamellarity, fluidity, size distribution, and entrapment efficiency. Spectral bio-imaging and flow cytometric studies showed efficient uptake of Hepatitis B surface antigen (HBsAg)-loaded ethosomes by murine dendritic cells (DCs) in vitro, reaching a peak by 180 min. Transcutaneous delivery potential of the antigen-loaded system using human cadaver skin demonstrated a much higher skin permeation of the antigen in comparison to conventional liposomes and soluble antigen preparation. Topically applied HBsAg-loaded ethosomes in experimental mice showed a robust systemic and mucosal humoral immune response compared to intramuscularly administered alum-adsorbed HBsAg suspension, topically applied plain HBsAg solution and hydroethanolic (25%) HBsAg solution. The ability of the antigen-pulsed DCs to stimulate autologous peripheral blood lymphocytes was demonstrated by BrdU assay and a predominantly TH1 type of immune response was observed by multiplex cytometric bead array analysis. HBsAg-loaded ethosomes are able to generate a protective immune response and their ability to traverse and target the immunological milieu of the skin may find a potential application in the development of a transcutaneous vaccine against Hepatitis B virus (HBV).     

Chemical synthesis and immunological properties of a cyclic eicosapeptide (Gly88,90) 82–101 of the beta subunit of human chorionic gonadotrophin

The eicosapeptide (Gly^88,90) 82–101 hCG-β was synthesized by the fragment condensation of the nonapeptide (Gly^88,90) 82–90 and the undecapeptide 91–101 followed by iodine oxidation to make the disulfide loop. Intramolecularity of the disulfide linking cysteines at 93 and 100 was confirmed. Anti-peptide antibodies were elicited in rabbits upon immunization with a conjugate of the peptide with tetanus toxoid. The repertoire of these antibodies was directed solely against the undecapeptide 91–101. These antibodies showed no recognition for hCG, hCG-β or ARCM hCG-β, suggesting that the conformational epitopes of hCG-β in the region 82–101 may not be accessible to antibodies.     

Synthesis of new head-activator analogues and their application for improved radioimmunoassays

New analogues of head activator were produced for receptor and radioimmunoassay studies. The precursor molecules [(4′-I)Phe¹¹] head activator and [Tyr¹¹] head activator were synthesised for catalytic tritiation and iodination, respectively. With the tracer [(3,5-¹²⁵ I₂)Tyr¹¹] head activator the sensitivity range of the radioimmunoassay was 5–100 fmol.     

Synthesis of O-glycosylated tuftsins by utilizing threonine derivatives containing an unprotected monosaccharide moiety

Synthesis is described of four tuftsin derivatives containing a D-ghcopyranosyl or a D-galactopyranosyl unit covalently linked to the hydroxy side chain function of the threonine residue through either an α or βO-glycosidic linkage. Fmoc-threonine derivatives containing the suitable unprotected sugar were used for incorporating the O-glycosylated amino acid residue. Z-Thr[α-Glc(OBzl)₄]-OBzl and Z-Thr[α-Gal(OBzl)₄]-OBzl were prepared from the tetra-O-benzylated sugar and Z-Thr-OBzl by the trichloroacetimidate method in the presence of trimethylsilyl trifluoromethane sulfonate. The α glycosylated threonine derivatives were converted into Fmoc-Thr(α-G1c)-OH and Fmoc-Thr(α-Gal)-OH by catalytic hydrogenation followed by acylation with Fmoc-OSu. β-Glucosylation and β-galactosylation of threonine were carried out by reacting the proper per-O-acetylated sugar with Z-Thr-OBzl and boron trifluoride ethyl etherate in dichloromethane. Catalytic hydrogenation of the β-O-glycosylated threonine derivatives followed by acylation with Fmoc-OSu and deacetylation with methanolic hydrazine yielded Fmoc-Thr(β-Glc)-OH and Fmoc-Thr(β-Gal)-OH, respectively. The O-glycosylated threonine derivatives were then reacted with H-Lys(Z)-Pro-Arg(NO₂)-OBzl in the presence of DCC and HOBt and the resulting glycosylated tuftsin derivatives were fully deblocked by catalytic hydrogenation, purified by HPLC, and characterized by optical rotation, amino acid analysis, and ¹H NMR. The β-galactosylated tuftsin was also prepared by the continuous flow solid phase procedure.     

A new synthetic functionalized antigen carrier

A new synthetic functionalized antigen carrier is described. It consists of a core of seven branched lysine residues, of which each of the four N-terminal lysine residues contains two N-(S-acetylmercaptoacetyl)-glutamyl residues. After removal of the protecting S-acetyl groups affording eight thiol functions, the carrier can easily be conjugated to a properly functionalized antigen, e.g. an S-(Npys)-cysteinyl peptide, thus affording a high molecular weight conjugate with an unusually high antigen content.     

Research Article: Synthetic Peptide Vaccine Development: Designing Dual Epitopes into a Single Pilin Peptide Immunogen Generates Antibody Cross-reactivity between Two Strains of Pseudomonas aeruginosa

One of the main challenges of Pseudomonas aeruginosa vaccine development is the design of an antigen that elicits cross-reactive antibodies against multiple virulent strains. Using a rational design approach, we have developed a single 17-residue peptide immunogen that generates antibodies that target the receptor-binding domain of the type IV pilus of more than one strain of P. aeruginosa. Using the receptor-binding domain sequence, of native strain PAO as a template, we have systematically changed up to five residues in the PAO sequence of the peptide immunogen into that of the PAK sequence. We show by indirect and competitive ELISA that the mutant peptide immunogens elicit the development of polyclonal sera that is cross-reactive to both native strain PAO and PAK pilin. We further show that there are at least two separate antibody populations in the polyclonal sera that possess closely related epitopes but which are each strain specific. Moreover, part of the epitope for the PAO-specific antibodies consists of several residues outside the disulfide loop of the receptor-binding domain. This allows us to create two unique epitopes within the same receptor-binding domain sequence.     

Synthesis and biological activity of [L-hydroxyproline]3-tuftsin analogue and its α- or β-O-D-glucosylated derivatives

Syntheses are described of the Hyp³-tuftsin analogue and of its derivatives α- or β-O-glycosylated at the side chain function of the hydroxyproline residue. The carbohydrate-free tetrapeptide was prepared by reacting Z-Thr-Lys(Z)-OH with H-Hyp-Arg(NO₂)-OBzl by the mixed anhydride procedure. In the synthesis of the α-glycosylated analogue the O-glycosyl amino acid was incorporated by reacting Boc-(Glcα+β)Hyp-OH with H-Arg(NO₂)-OBzl through the same procedure. The α-glucosylated dipeptide was isolated from the diastereomeric mixture, selectively deblocked, and acylated with Z-Thr-Lys(Z)-OH by the mixed anhydride procedure. In the preparation of the β-glucosylated analogue the BOP procedure was used for reacting Boc-[Glc(Ac)₄β]Hyp-OH with H-Arg(NO)₂-OBzl was well as for the final coupling to tetrapeptide. Removal of protecting groups from crude tetrapeptides was achieved by catalytic hydrogenation. Deacetylation of the sugar moiety of the β-glucosylated tetrapeptide was achieved by treatment with sodium methoxide in methanol. The synthetic compounds were isolated by ion exchange chromatography, and characterized by elemental analysis, amino acid analysis, optical rotation and proton NMR. Their capacity to evoke the release of interleukin 1 from mouse peritoneal macrophages and to modulate immunogenic activity of antigen-fed cells was evaluated, in comparison with tuftsin and rigin. All of the analogues were found to possess tuftsin-like activity.     

Synthesis of glycosylated tuftsins and tuftsin-containing IgG fragment undecapeptide

Syntheses are described of two new tuftsin derivatives containing a 2-acetamido-2-deoxy-D-galac-topyranosyl unit α- or β-glycosidically linked to the threonine's hydroxy side chain function and of the glycosylated undecapeptide corresponding to the tuftsin region of the heavy chain of IgG (amino acid sequence 289–299). The glycosylated tuftsins were synthesized by the solution procedure. Fmoc-[Gal NAc(Ac)₃α]Thr-OH and Fmoc-[GalNAc(Ac)₃β]Thr-OH were allowed to react with H-Lys(Z)-Pro-Arg(NO₂)-OBzl by the mixed anhydride procedure and the resulting glycosylated tetrapeptides were fully deblocked by catalytic hydrogenation followed by treatment with potassium cyanide, purified by ion exchange chromatography and characterized by analytical HPLC, elemental and amino acid analyses, optical rotation, and proton NMR spectroscopy. Synthesis of the glycosylated undecapeptide was achieved by the continuous flow solid phase procedure on 4-hydroxymethylphenoxyacetyl-norleucyl derivatized Kieselguhr-supported resin. Fmoc-amino acid symmetrical anhydrides or pentafluorophenyl esters, in the presence of N-hydroxybenzotriazole, were used as the acylating agents. To mimic the native sequence of the tuftsin region at the Fc-domain of immunoglobulin G a 2-acetam～do-2-deoxy-β-D-glucopyranosyl unit was N-glycosidically linked to the amide side chain of Asn 297. The glycosylated asparagine residue was introduced as N²-fluorenylmethyloxycarbonyl-N⁴-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopy-ranosyl)-asparagine pentafluorophenyl ester. After cleavage from the resin the glycopeptide was deprotect-ed, purified by ion exchange chromatography, and characterized by analytical HPLC, amino acid analysis, high voltage electrophoresis, and proton NMR. The conformational features of the glyco-undecapeptide were determined by circular dichroism measurements both in water and in 98% trifluoroethanol. Results of biological assays will be published elsewhere.     

经皮免疫用微针疫苗研究进展

疫苗接种是降低传染性疾病患病率及死亡率的有效方法。微针(microneedles, MNs)作为一种无针经皮免疫(needle-free transcutaneous immunization, NF-TCI)接种技术可以克服传统注射接种疫苗存在的免疫效率低、顺应性差和资源浪费等问题。微针疫苗能够直接突破皮肤角质层屏障,将疫苗有效地递送到皮肤组织内的免疫细胞而启动机体免疫应答,同时不会伤及真皮层内的神经和血管,具有安全性高、免疫效果佳和实用经济等优势,在传染性疾病的防治和肿瘤治疗中应用前景广阔。本文针对MNs在经皮免疫技术中的发展背景、疫苗递送类型、免疫效果影响因素、未来亟需解决的问题和发展方向等方面进行了综述和展望。     

Advantages of a Synthetic Peptide Immunogen Over a Protein Immunogen in the Development of an Anti‐Pilus Vaccine for Pseudomonas aeruginosa

The type IV pilus is an important adhesin in the establishment of infection by Pseudomonas aeruginosa. We have previously reported on a synthetic peptide vaccine targeting the receptor‐binding domain of the main structural subunit of the pilus, PilA. The receptor‐binding domain is a 14‐residue disulfide loop at the C‐terminal end of the pilin protein. The objective of this study was to compare the immunogenicity of a peptide‐conjugate to a protein subunit immunogen to determine which was superior for use in an anti‐pilus vaccine. BALB/c mice were immunized with the native PAK strain pilin protein and a synthetic peptide of the receptor‐binding domain conjugated to keyhole limpet haemocyanin. A novel pilin protein with a scrambled receptor‐binding domain was used to characterize receptor‐binding domain‐specific antibodies. The titres against the native pilin of the animals immunized with the synthetic peptide‐conjugate were higher than the titres of animals immunized with the pilin protein. In addition, the affinities of anti‐peptide sera for the intact pilin receptor‐binding domain were significantly higher than affinities of anti‐pilin protein sera. These results have significant implications for vaccine design and show that there are significant advantages in using a synthetic peptide‐conjugate over a subunit pilin protein for an anti‐pilus vaccine.     

吉水县2010年麻疹疫苗强化免疫的效果分析

目的 总结评价吉水县2010年麻疹疫苗强化免疫的效果,探讨控制麻疹的策略.方法 按照《2010年江西省麻疹疫苗强化免疫活动实施方案》的要求实施,采用描述性方法对相关数据进行流行病学分析.结果 本次强化免疫8个月～4岁应种儿童23 256名,实种儿童22 896名,实际接种率达98.45％,快速评估接种率97.75％.强化免疫期间,全县共报告麻疹相关疑似预防接种异常反应1例,发生率4.37/10万.没有发生因麻疹疫苗强化免疫引起的群体事件,无疫苗质量事故和实施差错事故报告,无死亡病例发生.实施麻疹疫苗强化免疫后麻疹发病率大幅下降.结论 吉水县2010年麻疹疫苗强化免疫活动目标儿童麻疹疫苗接种率＞95％,达到预期目的.     

Factors associated with long-term antibody production induced by hepatitis B vaccine in patients undergoing hemodialysis: a retrospective cohort study

STUDY OBJECTIVE: To assess the influence of various clinical factors on antibody production induced by hepatitis B vaccine in patients receiving hemodialysis up to 24 months after vaccination. DESIGN: Retrospective cohort study. SETTING: Outpatient dialysis center. PATIENTS: Adult patients undergoing hemodialysis who received a three-dose series of intramuscular hepatitis B vaccine 40 microg at time 0, 1, and 6 months, according to protocol. MEASUREMENTS AND MAIN RESULTS: Antibody to hepatitis B surface antigen (anti-HBs) titers were monitored quarterly, and booster doses were given according to protocol. Patients with anti-HBs of at least 10 mIU/ml were considered seropositive. Clinical variables--age, diabetes mellitus status, serum albumin level, and equilibrated Kt/V (eKt/V; Kt/V is a measure of urea clearance during dialysis, used to quantify the delivered dose of hemodialysis)--were compared between seropositive and seronegative patients 12 months (cohort 1) and 24 months (cohort 2) after vaccination. In cohort 1 (66 patients), 24 (36.4%) were seropositive at 12 months. In cohort 2 (40 patients), 15 (37.5%) were seropositive at 24 months. Comparison of seropositive and seronegative patients revealed no statistically significant differences in mean age, sex, serum albumin level, or eKt/V. However, at 24 months, patients with diabetes were 2.5 times more likely to demonstrate seropositivity than those without diabetes (60% vs 24%, respectively, p=0.02). CONCLUSION: Long-term seroprevalence induced by hepatitis B vaccine was low in our patients 12 and 24 months after vaccination. These results were comparable to previously reported long-term results. Larger, prospective studies would be needed to confirm the finding that patients with diabetes had superior hepatitis B vaccine-induced antibody production at 24 months.     

Synthesis and biological investigations of new tuftsin analogs with elongated peptide chain

In this paper the synthesis of the following elongated tuftsin analogs: Thr-Lys-Pro-Lys-Thr-Lys-Pro-Lys (I), Thr-Lys-Pro-Lys-Thr-Lys-Pro-Arg (II) and Ala-Lys-Thr-Lys-Pro-Arg-Glu-Gln (III) by the classical method is described. The compound II markedly inhibited the growth of murine sarcoma viruses (MSV).     

Adverse events following immunization: real causality and myths

ABSTRACT

Introduction: To assure the highest safety of immunization programs, detect adverse events following immunization (AEFIs), eliminate concerns, and reduce the risk of low vaccination coverage, authorities in industrialized countries have collected years of reports of suspected AEFIs and have systematically assessed their clinical importance.

Areas covered: In this paper, the methods used to assess vaccine safety and the results obtained by the analysis of reports, studies, and meta-analyses are discussed.

Expert opinion: Severe AEFIs are rare, and all evaluations of safety of vaccines recommended for both children and adults have demonstrated that the advantages of vaccines are always significantly higher than the problems that they cause, and there is no need to modify recommendations. However, the definition of AEFI is dependent on the vaccines themselves, complicating the definition of an AEFI and explaining why doubts and concerns have been raised. Presently, disease epidemiology data collected in healthy people and in subjects with underlying disease, general vaccine coverage, and the vaccination status of subjects with AEFIs are managed by many independent institutions. Only strict co-operation between these institutions will lead to the successful identification of AEFIs and to a reduction of the weight of anti-vaccine arguments.     

Synthesis of tritiated tuftsin and macrophage inhibitory tripeptide via acetylenic intermediates

Acetylenic analogues of tuftsin (Thr-Dah-Pro-Arg) and of a macrophage inhibitory tripeptide (Thr-Dah-Pro) have been synthesized by conventional procedures in solution (Dah = 2,6-diamino-4-hexynoic acid). These acetylenic derivatives are intermediates for the preparation of structurally unmodified, tritiated peptides. Catalytic tritiation of Thr-Dah-Pro-Arg and of Thr-Dah-Pro has afforded the radioactive tetra- and tripeptides with specific activities of 11.4 Ci/mmol and 37 Ci/mmol, respectively.