用人源肠毒素性大肠杆菌CS3菌毛构建新型呈现载体

根据对人源大肠杆菌菌毛CS3亚单位亲水性、表位、二级结构和柔韧性计算机预测结果,以CS3亚单位第72位氨基酸残基之后作为外源表位的插入位点,构建了一种新的表面呈现载体pCSX72。用该载体分别表达口蹄疫病毒（FMDV）的VP1表位和C-myc的十肽表位（410-419aa）。SDS-PAGE结果以及电镜和免疫电镜观察证明,插入的外源表位和CS3亚单位以杂合菌毛的形式呈现在菌体表面。ELISA检测结果表明,pCSX72表达的杂合蛋白的抗原性较之其他插入位点的载体要高得多。用重组细菌腹腔免疫小鼠,能够诱发机体对杂合蛋白的双重免疫应答。构建的表面呈现载体可望成为构建多价疫苗的有力工具。     

A conserved coronavirus epitope, critical in virus neutralization, mimicked by internal-image monoclonal anti-idiotypic antibodies.

Monoclonal antibody (MAb) 6A.C3 neutralizes transmissible gastroenteritis coronavirus (TGEV) and is specific for a conserved epitope within subsite Ac of the spike (S) glycoprotein of TGEV. Six hybridomas secreting anti-idiotypic (Ab2) MAbs specific for MAb 6A.C3 (Ab1) have been selected. All six MAbs inhibited the binding of Ab1 to TGEV and specifically cross-linked MAb1-6A.C3. Four of these hybridomas secreted gamma-type anti-idiotypic MAbs. The other two Ab2s (MAbs 9A.G3 and 9C.E11) were recognized by TGEV-specific antiserum induced in two species. This binding was inhibited by viruses of the TGEV group but not by serologically unrelated coronaviruses. These results indicate that MAb2-9A.G3 and MAb2-9C.E11 mimic an antigenic determinant present on the TGEV surface, and they were classified as beta-type ("internal-image") MAbs. TGEV-binding Ab3 antiserum was induced in 100% of mice immunized with the two beta-type MAb2s and in 25 to 50% of mice immunized with gamma-type MAb2. Both beta- and gamma-type Ab2s induced neutralizing Ab3 antibodies in mice that were mainly directed to antigenic subsite Ac of the S protein.     

Expression of heterologous peptides at two permissive sites of the MalE protein: antigenicity and immunogenicity of foreign B-cell and T-cell epitopes.

We previously determined a number of 'permissive' sites in the periplasmic maltose-binding protein (MalE) from Escherichia coli. These sites accept the insertion of heterologous peptides without major deleterious consequences for the activities, structure and cellular location of the protein. This study explores the versatility of two such permissive sites for the synthesis of foreign peptides, and examines the antigenicity and the immunogenicity of the inserts. One site is located after amino acid 133 (aa133) of MalE, and the other after aa303. Both sites tolerate inserts of up to at least 70 aa and accept sequences of different natures. Hydrophobic aa sequences are accepted, although strongly hydrophobic sequences, such as the Sendai virus F protein membrane anchor, affected export. We compared the antigenic and the immunogenic properties of peptides derived from the coat proteins of HBV and poliovirus which contain well defined B-cell epitopes. Specific monoclonal antibodies show that the antigenic properties of the inserted B-cell epitopes were different at the two sites. Despite these differences, the inserted peptides elicited strong and comparable antibody responses in mice against the corresponding synthetic peptides. In this case, and with these criteria, the molecular context of the peptides did not affect the immunogenicity of B-cell epitopes. We show for the first time that when a foreign peptide carrying a T-cell epitope was inserted in MalE, the hybrid proteins can elicit a T-cell response against the foreign peptide both in vivo and in vitro. Furthermore, the MalE hybrid was as efficient as free peptide in stimulating T-cell hybridomas in vitro. The MalE vectors provide a powerful genetic system to study how the position and the conformation of a peptide within a protein affect the B-cell and T-cell responses.     

Analysis of murine B-cell epitopes on Eastern equine encephalitis virus glycoprotein E2

The Eastern equine encephalitis virus (EEEV) E2 protein is one of the main targets of the protective immune response against EEEV. Although some efforts have done to elaborate the structure and immune molecular basis of Alphaviruses E2 protein, the published data of EEEV E2 are limited. Preparation of EEEV E2 protein-specific antibodies and define MAbs-binding epitopes on E2 protein will be conductive to the antibody-based prophylactic and therapeutic and to the study on structure and function of EEEV E2 protein. In this study, 51 EEEV E2 protein-reactive monoclonal antibodies (MAbs) and antisera (polyclonal antibodies, PAbs) were prepared and characterized. By pepscan with MAbs and PAbs using enzyme-linked immunosorbent assay, we defined 18 murine linear B-cell epitopes. Seven peptide epitopes were recognized by both MAbs and PAbs, nine epitopes were only recognized by PAbs, and two epitopes were only recognized by MAbs. Among the epitopes recognized by MAbs, seven epitopes were found only in EEEV and two epitopes were found both in EEEV and Venezuelan equine encephalitis virus (VEEV). Four of the EEEV antigenic complex-specific epitopes were commonly held by EEEV subtypes I/II/III/IV (1-16aa, 248-259aa, 271-286aa, 321-336aa probably located in E2 domain A, domain B, domain C, domain C, respectively). The remaining three epitopes were EEEV type-specific epitopes: a subtype I-specific epitope at amino acids 108–119 (domain A), a subtype I/IV-specific epitope at amino acids 211–226 (domain B) and a subtype I/II/III-specific epitope at amino acids 231–246 (domain B). The two common epitopes of EEEV and VEEV were located at amino acids 131–146 and 241–256 (domain B). The generation of EEEV E2-specific MAbs with defined specificities and binding epitopes will inform the development of differential diagnostic approaches and structure study for EEEV and associated alphaviruses.     

Identification of linear B‐cell epitopes within Tarp of Chlamydia trachomatis

Chlamydia trachomatis is one of the most prevalent sexually transmitted pathogens. There is currently no commercially available vaccine against C. trachomatis. Chlamydial translocated actin‐recruiting phosphoprotein (Tarp) can induce cellular and humoral immune responses in murine models and has been regarded as a potential vaccine candidate. In this report, the amino acid sequence of Tarp was analyzed using computer‐assisted techniques to scan B‐cell epitopes, and six possible linear B‐cell epitopes peptides (aa80–95, aa107–123, aa152–170, aa171–186, aa239–253 and aa497–513) with high predicted antigenicity and high conservation were investigated. Sera from mice immunized with these potential immunodominant peptides was analyzed by ELISA, which showed that epitope 152–170 elicited serum immunoglobulin G (IgG) response and epitope 171–186 elicited both serum IgG and mucosal secretory immunoglobulin A response. The response of immune sera of epitope 171–186 to endogenous Tarp antigen obtained from the Hela229 cells infected with C. trachomatis was confirmed by Western blot and indirect fluorescence assay. In addition, binding of the antibodies against epitope 171–186 to endogenous Tarp was further confirmed by competitive ELISA. Our results demonstrated that the putative epitope (aa171–186) was an immunodominant B‐cell epitope of Tarp. If proven protective and safe, this epitope, in combination with other well‐documented epitopes, might be included into a candidate epitope‐based vaccine against C. trachomatis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

A SmD peptide induces better antibody responses to other proteins within the small nuclear ribonucleoprotein complex than to SmD protein via intermolecular epitope spreading

Autoantibody response against the small nuclear ribonucleoprotein (snRNP) complex is a characteristic feature of systemic lupus erythematosus. The current investigation was undertaken to determine whether activation of SmD-reactive T cells by synthetic peptides harboring T cell epitopes can initiate a B cell epitope spreading cascade within the snRNP complex. T cell epitopes on SmD were mapped in A/J mice and were localized to three regions on SmD, within aa 26-55, 52-69, and 86-115. Immunization with synthetic peptides SmD(31-45), SmD(52-66), and SmD(91-110) induced T and B cell responses to the peptides, with SmD(31-45) inducing the strongest response. However, only SmD(52-66) immunization induced T cells capable of reacting with SmD. Analysis of sera by immunoprecipitation assays showed that intermolecular B cell epitope spreading to U1RNA-associated A ribonucleoprotein and SmB was consistently observed only in the SmD(52-66)-immunized mice. Surprisingly, in these mice, Ab responses to SmD were at low levels and transient. In addition, the sera did not react with other regions on SmD, indicating a lack of intramolecular B cell epitope spreading within SmD. Our study demonstrates that T cell responses to dominant epitope on a protein within a multiantigenic complex are capable of inducing B cell responses to other proteins within the complex. This effect can happen without generating a good Ab response to the protein from which the T epitope was derived. Thus caution must be taken in the identification of Ags responsible for initiating autoimmune responses based solely on serological analysis of patients and animals with systemic autoimmune disorders.     

表达轮状病毒SA11株Vp4的抗原表位诱导病毒中和抗体生成

以昆虫病毒Flockhousevirus（FHV）外壳蛋白为载体的外源抗原表位表达系统（FHV-RNA2载体系统）．在重组杆状病毒和重组pET系统中构建和表达了SA11Vp4胰酶切割位点两侧和重叠切割位点3个抗原表位氨基酸序列（抗原表位A,aa223~242;抗原表位B,aa243～262;抗原表位C,aa234～251）,并对其免疫原性进行了研究。结果表明：这3个抗原表位能诱导动物产生抗同源氨基酸序列的抗体和抗同源病毒（SA11）感染性的血清中和抗体。研究结果提示：RVVp4胰酶切割位点区氨基酸序列除了具有胰酶切割增强病毒感染力外,还具有诱导动物机体产生血清中和抗体的能力,是RV重组抗原表位亚单位疫苗研究中重要的抗原表位氨基酸序列。     

Priming biologically active antibody responses against an isolated,conformational viral epitope by DNA vaccination

The immunodominant, conformational "a" determinant of hepatitis B surface Ag (HBsAg) elicits Ab responses. We selectively expressed the Ab-binding, glycosylated, native a determinant (residue 120-147) of HBsAg in a fusion protein containing C-terminally the HBsAg fragment SII (residue 80-180) fused to a SV40 T-Ag-derived hsp73-binding 77 aa (T(77)) or non-hsp-binding 60 aa (T(60)) N terminus. A DNA vaccine encoding non-hsp-binding secreted T(60)-SII fusion protein-stimulated murine Ab responses with a similar efficacy as a DNA vaccine encoding the secreted, native, small HBsAg. A DNA vaccine encoding hsp73-binding, intracellular T(77)-SII fusion protein-stimulated murine Ab responses less efficiently but comparable to a DNA vaccine encoding the intracellular, native, large HBsAg. HBsAg-specific Abs elicited by either the T(60)-SII-expressing or the T(77)-SII-expressing DNA vaccine suppressed HBsAg antigenemia in transgenic mice that produce HBsAg from a transgene in the liver; hence, a biologically active B cell response cross-reacting with the native, viral envelope epitope was primed by both DNA vaccine constructs. HBsAg-specific Ab and CTL responses were coprimed when an S(20-50) fragment (containing the immunodominant, L(d)-binding epitope S(28-39)) of HBsAg was fused C-terminally to the pCI/T(77)-SII sequence (pCI/T(77)-SII-L(d) DNA vaccine). Chimeric, polyepitope DNA vaccines encoding conformational, Ab-binding epitopes and MHC class I-binding epitopes can thus efficiently deliver antigenic information to different compartments of the immune system in an immunogenic way.     

Identification of B‐cell epitopes of Borrelia burgdorferi outer surface protein C by screening a phage‐displayed gene fragment library

Outer surface protein C (OspC) of Borrelia stimulates remarkable immune responses during early infection and is therefore currently considered a leading diagnostic and vaccine candidate. The sensitivity and specificity of serological tests based on whole protein OspC for diagnosis of Lyme disease are still unsatisfactory. Minimal B‐cell epitopes are key in the development of reliable immunodiagnostic tools. Using OspC fragments displayed on phage particles (phage library) and anti‐OspC antibodies isolated from sera of naturally infected patients, six OspC epitopes capable of distinguishing between LD patient and healthy control sera were identified. Three of these epitopes are located at the N‐terminus (OspC E1 aa19–27, OspC E2 aa38–53, OspC E3 aa62–66) and three at the C‐terminal end (OspC E4 aa155–163, OspC E5 aa184–190 and OspC E6 aa201–207). OspC E1, E4 and E6 were highly conserved among LD related Borreliae. To our knowledge, epitopes OspC E2, E3 and E5 were identified for the first time in this study. Minimal B‐cell epitopes may provide fundamental data for the development of multi‐epitope‐based diagnostic tools for Lyme disease.     

Epitope specificity of protective lactogenic immunity against swine transmissible gastroenteritis virus.

The epitope specificity of the protective immune response against swine transmissible gastroenteritis (TGE) has been investigated by using circulating and secretory antibodies. This study was carried out with sows vaccinated with TGEV or the antigenically related porcine respiratory coronavirus (PRCV). TGEV vaccination of sows resulted in greater lactogenic protection of suckling piglets against TGEV challenge and a higher secretory immune response than PRCV vaccination did. These differences in the immune response were conditioned by the route of antigen presentation as a result of the different tropism of each virus. Epitopes on S protein, and in particular those contained in its antigenic site. A, were more immunogenic than epitopes on N and M proteins in both groups of vaccinated sows, as determined by a competitive radioimmunoassay. Minor differences in antibody response against the previously defined antigenic subsites Aa, Ab, and Ac were also detected, with subsite Ab being the most antigenic in both TGEV- and PRCV-immune sows. These findings suggest that antigenic site A on S protein, involved in virus neutralization, is the immunodominant site in pregnant sows that confer lactogenic protection. They also validate, in experiments with secretory antibodies, the antigenic maps made with murine monoclonal antibodies. Therefore, this antigenic site should be considered for vaccine or diagnostic development.     

Characterization of the B cell epitopes associated with a truncated form of Pseudomonas exotoxin (PE38) used to make immunotoxins for the treatment of cancer patients

Recombinant immunotoxins composed of an Ab Fv fragment joined to a truncated portion of Pseudomonas exotoxin A (termed PE38) have been evaluated in clinical trials for the treatment of various human cancers. Immunotoxin therapy is very effective in hairy cell leukemia and also has activity in other hemological malignancies; however, a neutralizing Ab response to PE38 in patients with solid tumors prevents repeated treatments to maximize the benefit. In this study, we analyze the murine Ab response as a model to study the B cell epitopes associated with PE38. Sixty distinct mAbs to PE38 were characterized. Mutual competitive binding of the mAbs indicated the presence of 7 major epitope groups and 13 subgroups. The competition pattern indicated that the epitopes are discrete and could not be reproduced using a computer simulation program that created epitopes out of random surface residues on PE38. Using sera from immunotoxin-treated patients, the formation of human Abs to each of the topographical epitopes was demonstrated. One epitope subgroup, E1a, was identified as the principal neutralizing epitope. The location of each epitope on PE38 was determined by preparing 41 mutants of PE38 in which bulky surface residues were mutated to either alanine or glycine. All 7 major epitope groups and 9 of 13 epitope subgroups were identified by 14 different mutants and these retained high cytotoxic activity. Our results indicate that a relatively small number of discrete immunogenic sites are associated with PE38, most of which can be eliminated by point mutations.     

脊髓灰质炎病毒C3表位和C—myc＋肽表位在细菌表面的表达

为了进一步验证载体pCSB136和pCSX72作为表面呈现载体的可行性,化学合成脊髓灰质炎病毒C3表位和C-myc十肽有位的基因片段,并插入到上述载体的相应位点间,采用全菌PCR筛选到正向插入的重组子,全细胞ELISA和电镜观察显示,重组蛋白以杂和菌毛的形式得到了表达,并保持CS3和外源位的抗原性,表明脊髓灰质炎病毒C3表位和C-myc十肽捕位在细菌表面得到了表达,证实载体pCSB136和pCSX72可用于外源表位的呈现,为构建基因工程活苗疫苗奠定了基础。     

Positive selection of hepatitis delta antigen in chronic hepatitis D patients

Liver disease may become ameliorated in some patients with chronic hepatitis D virus (HDV) infection. We present here a study based on longitudinal sampling to investigate the viral dynamics in chronic HDV infection. We examined the HDV variants from different time points, especially those before and after the elevation of serum aminotransferase levels. The datasets from each patient were tested for positive selection by using maximum-likelihood methods with heterogeneous selective pressures along the nucleotide sequence. An average of 4.9%, ranging from 3.1 to 6.8%, of the entire delta antigen sites was regulated by a diversifying selection. Most of the positively selected sites were associated with immunogenic domains. Likelihood ratio tests revealed a significant fitness of positive selection over neutrality of the hepatitis delta antigen gene in all patients. We further adapted a neural network method to predict potential cytotoxic T ligand epitopes. Among the HLA-A*0201 cytotoxic T ligand epitopes, three consistent epitopes across all three genotypes were identified: amino acids (aa) 43 to 51, 50 to 58, and 114 to 122. Three patients (60%) had sites evolving under positive selection in the epitope from aa 43 to 51, and four patients (80%) had sites evolving under positive selection in the epitope from aa 114 to 122. The discovery of immunogenic epitopes, especially cytotoxic-T-lymphocyte ligands, associated with chronic HDV infection may be crucial for further development of novel treatments or designs in vaccine for HDV superinfection.     

猪传染性胃肠炎病毒S蛋白抗原位点分子特征分析

为了进一步研究猪传染性胃肠炎病毒S蛋白抗原位点的分子特征,选取GenBank中22株TGEV分离株,采用生物信息学方法对抗原位点氨基酸序列进行同源性比对分析。结果表明,不同分离株的A和C位点高度保守,B和D位点则有一些变化。     

Topological mapping of the P1-adhesin of Mycoplasma pneumoniae with adherence-inhibiting monoclonal antibodies.

Five adherence-inhibiting monoclonal antibodies (mAbs) were used for topological mapping of the binding sites of the 169 kDa membrane-integrated adhesin of Mycoplasma pneumoniae. Antibody binding sites were characterized using overlapping synthetic octapeptides. Three regions of the protein seem to be involved in adherence: the N-terminal region [N-reg, epitopes beginning at amino acid (aa) 1 to aa 14 and aa 231 to aa 238, respectively]; a domain (D1) approximately in the middle of the molecule (beginning at aa 851 to aa 858 and aa 921 to aa 928); and a domain (D2) closer to the C-terminus (beginning at aa 1303 to aa 1310, aa 1391 to aa 1398 and aa 1407 to aa 1414). Each of the mAbs P1.26 and P1.62 reacted with two primary amino acid sequences. Both antibodies bound to the D1 region, but mAb P1.62 showed additional binding to a sequence (aa 231 to aa 238) near the N-terminus, and mAb P1.26 reacted with a second epitope in the D2 domain (aa 1303 to aa 1310). Such dual binding by the two antibodies suggests that in the native protein the epitopes are composed of two sequences which are located on two different sites of the molecule (D1/N-reg and D1/D2, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Permissive sites and topology of an outer membrane protein with a reporter epitope.

We are developing a genetic approach to study with a single antibody the folding and topology of LamB, an integral outer membrane protein from Escherichia coli K-12. This approach consists of inserting the same reporter foreign antigenic determinant (the C3 epitope from poliovirus) at different sites of LamB so that the resulting hybrid proteins have essentially kept the in vivo biological properties of LamB and therefore its cellular location and structure; the corresponding sites are called permissive sites. A specific monoclonal antibody can then be used to examine the position of the reporter epitope with respect to the protein and the membrane. We present an improved and efficient procedure that led us to identify eight new permissive sites in LamB. These sites appear to be distributed on both sides of the membrane. At one of them (after residue 253), the C3 epitope was detected on intact bacteria, providing the first direct argument for exposure of the corresponding LamB region at the cell surface. At this site as well as at four others (after residues 183, 219, 236, and 352), the C3 epitope could be detected with the C3 monoclonal antibody at the surface of the extracted trimeric LamB-C3 hybrid proteins. We provide a number of convergent arguments showing that the hybrid proteins are not strongly distorted with respect to the wild-type protein so that the conclusions drawn are also valid for this protein. These conclusions are essentially in agreement with the proposed folding model for the LamB protein. They agree, in particular, with the idea that regions 183 and 352 are exposed to the periplasm. In addition, they suggest that region 236 is buried at the external face of the outer membrane and that region 219 is exposed to the periplasm. Including the 3 sites previously determined, 11 permissive sites are now available in LamB, including 3 at the cell surface and most probably at least 3 in the periplasm. We discuss the nature of such sites, the generalization of this approach to other proteins, and possible applications.     

Identification of epitopes of trichosanthin by phage peptide library

The phage displayed random peptide library has recently emerged as a powerful technique for analyzing Ab-Ag interactions. In this study, the method was employed to identify epitopes of trichosanthin. Two monoclonal Abs (4B5, 2E9) which recognized different epitopes of trichosanthin (TCS) were selected and a phage-peptide library with nine amino acids (9 aa) was used to screen the positive phage clones that have high affinity to the mAbs. Two groups of phage clones that carried peptide-specific binding to mAbs were identified by the screen. The identified phage clones carried peptide-specific binding to 4B5 and 2E9 mAbs were immunized in mice. To evaluate mimotope of selected phages, the specific binding activity to TCS was measured in the serum from phage-immunized mice. They all showed positive results. The conserved interaction motifs were deduced from the peptide sequences of each group of selected phage clones. When compared the motif sequence with the sequence of TCS, it was predicted that 4B5-corresponding epitope was located at 27-37 aa of TCS protein and 2E9-corresponding epitope was located at 41-48 aa of TCS. The predicted sequence of 4B5-corresponding epitope was further confirmed by site-directed mutation of TCS protein. The data showed that the expressed TCS protein mutated in 4B5-corresponding epitope was unable to bind 4B5 mAb. The results suggested that the phage display peptide library is useful to identify Ag epitopes and to raise Ab in disease diagnosis and treatment.     

Potent Neutralization of Staphylococcal Enterotoxin B In Vivo by Antibodies that Block Binding to the T-Cell Receptor

To develop an antibody (Ab) therapeutic against staphylococcal enterotoxin B (SEB), a potential incapacitating bioterrorism agent and a major cause of food poisoning, we developed a “class T" anti-SEB neutralizing Ab (GC132) targeting an epitope on SEB distinct from that of previously developed “class M" Abs. A systematic engineering approach was applied to affinity-mature Ab GC132 to yield an optimized therapeutic candidate (GC132a) with sub-nanomolar binding affinity. Mapping of the binding interface by hydrogen–deuterium exchange coupled to mass spectrometry revealed that the class T epitope on SEB overlapped with the T-cell receptor binding site, whereas other evidence suggested that the class M epitope overlapped with the binding site for the major histocompatibility complex. In the IgG format, GC132a showed ∼ 50-fold more potent toxin-neutralizing efficacy than the best class M Ab in vitro, and fully protected mice from lethal challenge in a toxic shock post-exposure model. We also engineered bispecific Abs (bsAbs) that bound tetravalently by utilizing two class M binding sites and two class T binding sites. The bsAbs displayed enhanced toxin neutralization efficacy compared with the respective monospecific Ab subunits as well as a mixture of the two, indicating that enhanced efficacy was due to heterotypic tetravalent binding to two non-overlapping epitopes on SEB. Together, these results suggest that class T anti-SEB Ab GC132a is an excellent candidate for clinical development and for bsAb engineering.     

An Unstable Th Epitope of P. falciparum Fosters Central Memory T Cells and Anti-CS Antibody Responses

Malaria is transmitted by Plasmodium-infected anopheles mosquitoes. Widespread resistance of mosquitoes to insecticides and resistance of parasites to drugs highlight the urgent need for malaria vaccines. The most advanced malaria vaccines target sporozoites, the infective form of the parasite. A major target of the antibody response to sporozoites are the repeat epitopes of the circumsporozoite (CS) protein, which span almost one half of the protein. Antibodies to these repeats can neutralize sporozoite infectivity. Generation of protective antibody responses to the CS protein (anti-CS Ab) requires help by CD4 T cells. A CD4 T cell epitope from the CS protein designated T* was previously identified by screening T cells from volunteers immunized with irradiated P. falciparum sporozoites. The T* sequence spans twenty amino acids that contains multiple T cell epitopes restricted by various HLA alleles. Subunit malaria vaccines including T* are highly immunogenic in rodents, non-human primates and humans. In this study we characterized a highly conserved HLA-DRβ1*04:01 (DR4) restricted T cell epitope (QNT-5) located at the C-terminus of T*. We found that a peptide containing QNT-5 was able to elicit long-term anti-CS Ab responses and prime CD4 T cells in HLA-DR4 transgenic mice despite forming relatively unstable MHC-peptide complexes highly susceptible to HLA-DM editing. We attempted to improve the immunogenicity of QNT-5 by replacing the P1 anchor position with an optimal tyrosine residue. The modified peptide QNT-Y formed stable MHC-peptide complexes highly resistant to HLA-DM editing. Contrary to expectations, a linear peptide containing QNT-Y elicited almost 10-fold lower long-term antibody and IFN-γ responses compared to the linear peptide containing the wild type QNT-5 sequence. Some possibilities regarding why QNT-5 is more effective than QNT-Y in inducing long-term T cell and anti-CS Ab when used as vaccine are discussed.     

Monoclonal antibodies directed against epitopes within the core protein structure of the large aggregating proteoglycan (aggrecan) from the swarm rat chondrosarcoma.

The core protein of the large hyaline cartilage proteoglycan, aggrecan, is composed of six distinct domains: globular 1 (G1), interglobular, globular 2 (G2), keratan sulfate attachment, chondroitin sulfate (CS) attachment, and globular 3 (G3). Monoclonal antibodies that recognize epitopes in these domains were raised against Swarm rat chondrosarcoma aggrecan that was either denatured through reduction and alkylation or partially deglycosylated through chondroitinase ABC digestion or alkali elimination, the latter with or without sulfite addition. Monoclonal antibodies were further characterized for reactivity to purified aggrecan substructures including rat chondrosarcoma G1 and CS attachment domains, a recombinant rat chondrosarcoma G3 domain fusion protein, bovine articular cartilage G2 domain, and rat chondrosarcoma link protein (LP). Biochemical characterization of the specificities of these monoclonal antibodies indicated that one (1C6) recognized an epitope shared by both the G1 and the G2 domains; one (5C4) recognized an epitope shared by both LP and the G1 domain; one (7D1) recognized an epitope shared by both the G1 and the CS attachment domains; two (14A1 and 15B2) recognized epitopes in the CS attachment domain; one (14B4) recognized an epitope in the G3 domain; and one (13D1) recognized a ubiquitous epitope shared by the G1, G2, G3, and CS attachment domains of aggrecan and also LP. Collectively the specificities of these antibodies confirm the occurrence of multiple repeated epitopes (both carbohydrate and protein in nature) throughout the different domain structures of aggrecan. These antibodies have been proven to be useful for identifying aggrecan-like molecules in several connective tissues other than cartilage.