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PubMed2005

Expression of MUC1, MUC2 and oligosaccharide epitopes in breast cancer: prognostic significance of a sialylated MUC1 epitope.

MUC1 represents a promising marker in breast cancer. However, due to the structural complexity of the MUC1 glycoprotein, multiple epitopes can be detected by monoclonal antibodies. This fact may be responsible for the contradictory results of previous investigations regarding the clinical and prognostic relevance of MUC1 expression in breast cancer. Therefore, we tried to evaluate the role of different glycosylated and non-glycoslyated MUC1 epitopes as well as other mucin-associated peptides (MUC2) and carbohydrates (Thomsen-Friedenreich antigen, sialyl-Lewisa, sialyl-Lewisx) as predictors of the clinical course and prognosis in mammary carcinomas. An immunohistochemical study applying numerous monoclonal antibodies (mabs) was performed to characterize the expression of a selected panel of MUC1 epitopes, and of MUC2, Thomsen-Friedenreich antigen, sialyl-Lewisa, and sialyl-Lewisx in a series of 140 patients with breast cancer. The results were correlated with clinicopathological variables as well as overall survival. Generally, more than 90% of the mammary cancers, were strongly stained with the MUC1-specific mabs. Especially ductal and lobular carcinomas were strongly MUC1- and sialyl Lewisa-positive, whereas MUC2 binding was significantly elevated in mucinous neoplasms. Associations between the immunoreactivity of any mab under study and tumor progression as reflected by pTNM staging could not be observed. However, expression of the sialylated MUC1 epitope detected by mab MY1.E12 was revealed as a favourable independent prognostic factor. These results confirm that MUC1 is generally strongly expressed in mammary carcinomas. As an exception, mucinous carcinomas are significantly less MUC1 reactive, but strongly express MUC2. Our data suggest that only the presence of a sialylated short-chain MUC1 glycoform is associated with a better prognosis, whereas the other molecules under study are not correlated with the course of disease and survival probability.

Adult↗
PubMed1990

Mapping of epitopes on the La(SS-B) autoantigen of primary Sjögren's syndrome: identification of a cross-reactive epitope.

Autoepitopes on the ribonucleoprotein La(SS-B) were identified by using recombinant La(SS-B) polypeptides and sera from 166 patients with the antinuclear autoantibody anti-La(SS-B). The La(SS-B) polypeptides were encoded by polymerase chain reaction-derived overlapping or nonoverlapping fragments of the La(SS-B) gene, which encodes a protein of 408 amino acids (aa). Of the 166 sera tested, 99% reacted with a fusion protein comprising the first 107 N-terminal aa (LaA); 91% reacted with a fusion protein comprising aa 111 to 242 (LaC), and 91% reacted with a fusion protein comprising aa 346 to 408 (LaL2/3) at the C terminus of La(SS-B). The order of immunodominance as assessed by the number of sera reacting with each epitope and the strength of the reactivity was LaA (aa 1 to 107) greater than LaC (aa) 111 to 242) much greater than LaL2/3 (aa 346 to 408). Cross-reactivity was observed between antibodies eluted from LaC (aa 111 to 242) and LaL2/3 (aa 346 to 408), but there was no significant primary sequence homology between the two regions. The LaC region contained at least two epitopes, one encompassing a putative RNA-binding motif (aa 112 to 187) which was recognized by 83% of patient sera. Serial serum samples from three patients showed that the antibody response to La(SS-B) was initially directed to the N terminus (LaA, aa 1 to 107), but over a period of time all three major epitopes, including that encompassing the putative RNA-binding motif, were recognized. This result suggests that the primary immune response to La(SS-B) is restricted to an immunodominant epitope. As the specificity of the autoantibody response broadens, it includes the RNA-binding motif, which may have important implications for the expression of disease.

Autoantibodies↗
PubMed1987

Alzheimer's neurofibrillary tangles contain unique epitopes and epitopes in common with the heat-stable microtubule associated proteins tau and MAP2.

Ten monoclonal antibodies raised against Alzheimer's neurofibrillary tangles (ANTs) were characterized for reactivity with heat-stable microtubule fractions from bovine and human brain. Five of the antibodies showed very little reaction, but the other five reacted strongly with heat-stable microtubule associated proteins (MAPs). The proteins recognized by these antibodies have estimated molecular weights similar to those of known heat-stable MAPs, tau (52-68 kd) and MAP2 (200-250 kd). That the proteins are indeed tau and MAP2 is demonstrated by reaction of electroblotted proteins with antibodies raised in mouse and guinea pig against bovine brain tau and MAP2. One anti-ANT antibody reacts only with tau, two bind strongly to tau and weakly to MAP2, one recognizes both tau and MAP2 equally well, and one primarily stains MAP2. Extraction of ANT with 2% SDS does not remove tau or MAP2 epitopes from ANT, indicating that epitopes shared with heat-stable MAPs are integral components of ANT. The existence of tau epitopes in ANT is also demonstrated by immunoblotting of ANT-enriched fractions with anti-tau antibodies. Most of the material recognized by anti-tau antibodies in ANT-enriched fractions is present in large molecules excluded by 3% polyacrylamide gel upon electrophoresis. Anti-tau antibodies immunostain ANT in immunofluorescence and immunoperoxidase studies. The immunostaining can be blocked by absorption of anti-tau antibodies with purified tau proteins from bovine brain. Not all ANTs in any given tissue section or isolated Alzheimer perikarial preparations, however, are stained by anti-tau antibodies. These results are consistent with previous studies that have demonstrated heterogeneity of ANTs. Whether this heterogeneity is due to biochemical modification of MAPs or absence of MAPs in some ANTs is unknown. The significance of what appear to be shared epitopes recognized by monoclonal antibodies in tau and MAP2, and the implications this may have on the pathogenesis of ANT formation, requires further investigation.

Alzheimer Disease↗
PubMed1987

Epitopes associated with the MHC restriction site of T cells. I. Selective expression of Iat epitopes on H-2-restricted helper T cells.

We previously established monoclonal antibodies (mAb) that are putatively directed to the I region of H-2k but are reactive only with T cells. Because of their specificity to the unique epitopes different from class II antigens, they are designated as anti-Iat reagents. The present study demonstrated that these anti-Iat inhibit the H-2k-restricted helper T (Th) cell function by acting on the very H-2 restriction site of both H-2k and H-2kxb F1 T cells. This was determined by both the cytotoxic treatment and blocking of antigen-primed Th cells. In the F1 Th population, only those restricted to H-2k were eliminated, leaving the H-2b-restricted Th cells uninhibited. The inhibition of the response was not due to the induction of suppressor T cells, but to the elimination of the function of radioresistant Lyt-1+,2- Th cells. Iatk epitopes were also found on an H-2k-restricted but not on H-2b-restricted Th cell clone established from the same H-2kxb F1 animal. None of the anti-Iatk were reactive with class II antigens on B cells. These results indicate that Iat epitopes are not directly encoded by the I region genes, but are associated with the H-2 restriction site of T cells, which see the self class II polymorphism. Thus, Iat epitopes are expressed clonally in high frequency on H-2k-restricted Th cells of F1, being excluded from the H-2b-restricted Th population. The relationship between Iat and T cell receptor molecules is unknown.

Animals↗
PubMed1994

Identification of immunodominant epitopes in Trypanosoma cruzi trypomastigote surface antigen-1 protein that mask protective epitopes.

The gene that encodes trypomastigote surface Ag-1 (TSA-1), a major surface Ag of the bloodstream trypomastigote stage of Trypanosoma cruzi, was expressed in a baculovirus expression system. To determine the epitope(s) in TSA-1 that was recognized during T. cruzi infection and after immunization with TSA-1, subregions of the TSA-1 gene were expressed in a bacterial expression system. As seen by Western blotting, both mice and rabbits immunized with recombinant TSA-1 protein, as well as T. cruzi-infected mice, developed strong immune responses to the carboxyl-proximal region of TSA-1, but show no reaction to the amino-proximal portion of TSA-1. When mice were immunized with either recombinant TSA-1 protein or the carboxyl-proximal region of TSA-1, they did not survive challenge with 10(3) bloodstream trypomastigotes. However, 70% of the mice immunized with the amino-proximal portion of TSA-1 survived challenge with 10(3) bloodstream trypomastigotes. Thus, the immune responses elicited by recombinant TSA-1 or the carboxyl-proximal portion of TSA-1 are nonprotective during T. cruzi infection. In contrast, vaccination with the amino proximal region of TSA-1 elicits a protective immune response. These results suggest that responses to immunodominant epitope(s) within the carboxyl-proximal portion of TSA-1 mask epitopes within the amino-proximal portion that are capable of stimulating host-protective immune responses. It is suggested that immunodominant regions in surface molecules such as TSA-1 may provide a mechanism for the parasite to evade the host immune response by directing the response away from epitopes that have the potential to elicit a reaction that is damaging to the parasite.

Animals↗
PubMed1993

The B cell immune response to an idiotype-inducing peptide epitope can be inhibited by immunodominance of a neighboring epitope.

Three synthetic peptides, EEYEYE (peptide 1), EEYEYEEEYEYE (peptide 2), and YEEEEY (peptide 3), were tested for their ability to induce common Id that had been previously characterized for murine antibodies specific to random synthetic polymers of glutamic acid, alanine, and tyrosine ((Glu,Ala,Tyr)n) (cGAT Id). Protein conjugates of either peptide 2 or peptide 3, but not peptide 1, induced cGAT Id. This unique approach directly identified two peptides capable of inducing cGAT Id antibodies. Previous immunization with peptide 1-protein conjugate inhibited the cGAT Id response to peptide 2 conjugated to a different protein carrier. Thus, a neighboring or overlapping epitope can be used to inhibit a cGAT Id-inducing epitope without the participation of carrier-specific Ts and without using anti-Id antibodies. In contrast, previous immunization with peptide 1-protein conjugate did not inhibit cGAT Id induction by peptide 3-protein conjugate or by (Glu,Ala,Tyr)n. This ruled out the participation of Id-specific Ts cells. The effectiveness of inhibition coincided with the avid binding of anti-peptide 1 antibodies to peptide 2, which was > 10 and 100 times stronger than the binding to peptide 3 and (Glu,Ala,Tyr)n, respectively. We hypothesize that the primed peptide 1-specific B cells capture and process peptide 2-protein efficiently and act as APC to Th cells specific to the protein of the challenging Ag, resulting in the selective and dominant activation of peptide 1-specific B cells. Thus, our data suggest that epitope priming can inhibit an Id response to a neighboring epitope by a mechanism of clonal dominance.

Amino Acid Sequence↗
PubMed1998

Use of complete eluted peptide sequence data from HLA-DR and -DQ molecules to predict T cell epitopes, and the influence of the nonbinding terminal regions of ligands in epitope selection.

In diseases with a strong association with an HLA haplotype, identification of relevant T cell epitopes may allow alteration of the pathologic process. In this report we use a reverse immunogenetic approach to predict possible HLA class II-restricted T cell epitopes by using complete pool sequencing data. Data from HLA-DR2(B1*1501), -DR3(B1*0301), -DQ2(A1*0501, B1*0201), and -DQ8(A1*0301, B1*0302) alleles were used by a computer program that searches a candidate protein to predict ligands with a relatively high probability of being processed and presented. This approach successfully identified both known T cell epitopes and eluted single peptides from the parent protein. Furthermore, the program identified ligands from proteins in which the binding motif of the HLA molecule was unable to do so. When the information from the nonbinding N- and C-terminal regions in the pool sequence was removed, the ability to predict several ligands was markedly reduced, particularly for the HLA-DQ alleles. This suggests a possible role for these regions in determining ligands for HLA class II molecules. Thus, the use of complete eluted peptide sequence data offers a powerful approach to the prediction of HLA-DQ and -DR peptide ligands and T cell epitopes.

Amino Acid Sequence↗
PubMed1998

B cell responses to a peptide epitope. V. Kinetic regulation of repertoire discrimination and antibody optimization for epitope.

The influence of imposing various conformational constraints on immune responses to a model epitope within a synthetic peptide immunogen was examined in mice. Although overall immunogenicity was affected, the model epitope (sequence DPAF) remained the predominant recognition site regardless of the conformation in which it was presented. A comparison of anti-DPAF mAbs obtained in response to two analogue peptides, PS1CT3 and CysCT3, in which the DPAF segment was either unconstrained or held within a cyclic loop, respectively, revealed a significant homology in the paratope composition. At one level a subset of anti-PS1CT3 and anti-CysCT3 mAbs was found to share a common heavy chain variable region. In addition, nucleotide sequence homology comparisons of both heavy and light chain variable regions identified the presence of anti-PS1CT3 and anti-CysCT3 mAbs that collectively appeared to derive from a common progenitor, but with nonidentical somatic mutations. Interestingly, however, no bias toward homologous Ag could be discerned on measurement of relative affinities of the mAbs for the two peptides. In contrast, mAb binding on-rates clearly discriminated between peptides representing the homologous vs the heterologous confomer of the DPAF epitope. Thus, it would appear that the kinetics of Ag recognition dominate over equilibrium binding criteria both in epitope-driven repertoire selection and Ab maturation in a humoral response.

Amino Acid Sequence↗
PubMed1999

Development of a polyclonal antibody with broad epitope specificity for advanced glycation endproducts and localization of these epitopes in Bruch's membrane of the aging eye.

PURPOSE: To develop an antibody that recognizes a variety of advanced glycation endproduct (AGE) epitopes. METHODS: Glycolaldehyde was used to modify bovine serum albumin and HPLC analysis was used to measure pentosidine formation as an indicator of AGE formation. A polyclonal anti-AGE antibody was synthesized by injecting glycolaldehyde-incubated keyhole limpet hemocyanin into rabbits, affinity purified using AGE modified bovine serum albumin coupled to an affinity resin column, and characterized by immunoblot analysis. RESULTS: HPLC analysis of glycolaldehyde treated bovine serum albumin detected high levels of pentosidine formation, suggesting that glycolaldehyde is a potent precursor for pentosidine. By immunoblot analysis, our antibody recognized carboxymethyllysine and pentosidine, two well-characterized AGEs, as well as other AGE epitopes. Immunohistochemical evaluation showed evidence of AGEs in Bruch's membrane (including basal laminar deposits and drusen), choroidal extracellular matrix, and vessel walls in an 82 year old nondiabetic globe. A similar staining pattern was observed in an age-matched diabetic control. In contrast, no staining was seen with the antibody in a 20 month old nondiabetic globe. CONCLUSIONS: A unique anti-AGE antibody was synthesized that recognizes a variety of AGE epitopes including carboxymethyllysine and pentosidine. Its best use might be in broad surveys of the age-dependent accumulation of a large number of AGE epitopes that might not be revealed by antibodies to pentosidine or CML.

Acetaldehyde↗
PubMed1996

Epitope recognition of recoverin in cancer associated retinopathy: evidence for calcium-dependent conformational epitopes.

Recoverin, a photoreceptor-specific calcium-binding protein, is a target for antibodies in human cancer-associated retinopathy. We have studied the binding properties of antirecoverin human autoantibodies and rat monoclonal antibodies (MAbs). The majority of antibodies to recoverin, both in human disease and in animals immunized with recoverin, are directed against the same major immunodominant region, the sequence within the residue 64-70 in proximity to the calcium-binding domain EF-hand 2. The immunodominant epitopes consist of linear and conformational components. Our data demonstrate that the reactivity of autoantibodies was dependent on conformational changes induced by the binding of calcium to recoverin. Some patients' antibodies could be detected by immunocytochemical methods and enzyme linked immunosorbent assay (ELISA); however, their binding was abolished on Western blots; their epitopes were sensitive to SDS treatment. Immunization of Lewis rat with purified recoverin induces antibodies with properties strikingly similar to human autoantibodies. Comparison between the monoclonal antibodies (MAbs) and native autoantibodies showed that the calcium-induced changes in conformation were critical for specificities of the antibody generated. The recoverin region within the sequence 61-82 is a key region for antibody binding; it also contains a major T-cell epitope, and it is highly uveitogenic in Lewis rats.

Amino Acid Sequence↗
PubMed2004

Epitope mapping and features of the epitope for monoclonal antibodies inhibiting enzymatic activity of Helicobacter pylori urease.

Two characteristic monoclonal antibodies (HpU-2 and -18) out of 26 monoclonal antibodies (HpU-1 approximately 26) produced against Helicobacter pylori (H. pylori) urease showed a strong inhibitory effect against the enzymatic activity of the urease. Epitope mapping about some monoclonal antibodies of the HpU-series inhibiting enzymatic activity was performed by using a surface plasmon resonance apparatus and by digesting H. pylori urease with trypsin, followed by mass spectroscopy. The sequences of the epitopes recognized by HpU-2 and -18 were SVELIDIGGNRRIFGFNALVDR (22 mer) and IFGFNALVDR (10 mer), respectively. The former sequence is present as a part of a loop structure at a position close to the C-terminal of the alpha-subunit of H. pylori urease, although it has been suggested that the active site of the urease resides in the beta-subunit. The above peptide (22 mer) was chemically synthesized in a linear and cyclic form, and its conjugate with BSA was immunized in rabbits. The resultant serum induced by the linear form could specifically bind to H. pylori infecting human gastric mucosa. These results suggest that the above sequence (22 mer) must be an important epitope, although it locates in the alpha-subunit but not in the beta-subunit.

Amino Acid Sequence↗
PubMed1999

Identification of an epitope on the dengue virus membrane (M) protein defined by cross-protective monoclonal antibodies: design of an improved epitope sequence based on common determinants present in both envelope (E and M) proteins.

The protective capacity of monoclonal antibodies (MAbs) generated to the dengue-2 virus envelope (E) and premembrane (prM) proteins was tested in vivo. Two anti-E MAbs, 2C5.1 and 4G2 and two anti-prM MAbs, 2A4.1 and 2H2 provided cross-protection against all four dengue virus serotypes. Overlapping sets of synthetic peptides spanning amino-acid sequence 301-401 (domain III) of the E protein and the entire prM protein were then used to locate their epitopes. The anti-E MAbs strongly reacted with the peptide sequence 349-GRLITVNPIVT-359 (E349-359) from domain III and the immunodominant epitope, 274-SGNLLFTGHL-283 (E274-283) from the hinge region between domains I and II. The anti-prM MAbs strongly reacted with the sequence, 40-PGFTVMAAIL-49 (M40-49) from the first membrane-spanning domain of the M protein. These anti-prM MAbs also reacted with peptides E274-283 and E349-359, while the anti-E MAbs reacted with a peptide sequence, 1-FHLTTRNGEP-10 from the prM protein and these cross-reactions with both proteins were confirmed using immunoblot assays. MAbs 2C5.1, 4G2 and 2H2 more strongly reacted with an MEH1 peptide GLFTPNLITI, which was designed as an antigenic hybrid between these E and prM peptide sequences, than with any of these natural peptide sequences. These peptide sequence will now be tested for their ability to generate cross-protective antibodies against each dengue virus serotype when delivered with appropriate T-helper epitopes.

Amino Acid Sequence↗
PubMed2006

Identification and characterization of HIV-1 epitopes presented by HLA-A*2603: comparison between HIV-1 epitopes presented by A*2601 and A*2603.

Human leukocyte antigen (HLA)-A*26 is one of the alleles associated with a slow progression to AIDS. Identification and characterization of HIV-1-specific epitopes presented by this allele are necessary for studies on the immunopathogenesis of AIDS and vaccine development in Asia, where three HLA-A*26 subtypes are frequently found. In the present study, we sought to identify HLA-A*2603-restricted HIV-1 epitopes by using reverse immunogenetics and to compare them with HLA-A*2601-restricted ones recently identified. We found that 31 of 110 HIV-1 peptides bound to HLA-A*2603 and that only two peptides (Gag169-177 and Env63-72) induced specific CD8+T cells by stimulating peripheral blood mononuclear leukocytes from HIV-1-infected individuals carrying HLA-A*2603. The specific cytotoxic T lymphocyte clones killed HIV-1 recombinant vaccinia-infected cells, indicating that these two peptides were naturally occurring peptides presented by HLA-A*2603. Gag169-177-specific CD8+T cells were frequently detected in both HLA-A*2601+ and -A*2603+ individuals with chronic HIV-1 infection, whereas Env63-72-specific ones were frequently detected only in the HLA-A*2603+ individuals. Gag169-177 peptide bound equally to both HLA-A*26 antigens, whereas Env63-72 peptide bound to A*2603 much more strongly than to A*2601. These findings suggest that the relative affinity of these peptides for the HLA-A*26 subtypes determines whether these peptides are recognized as epitopes in HIV-1-infected individuals carrying these alleles.

Animals↗
PubMed1999

Investigation of the epitopic structure of thymosin beta10 by epitope mapping experiments.

We present here a study on the epitopic structure and the immunochemical characteristics of thymosin beta10 (Tbeta10), a 43 aminoacid peptide involved in important cellular mechanisms, by using the epitope mapping Multipin method. Octapeptides overlapping by one amino acid so as to represent the whole sequence of Tbeta10 were synthesized on polystyrene pins and screened, using an ELISA method, with a polyclonal antiserum raised against intact recombinant Tbeta10. The octapeptides were also tested with anti-peptide oligoclonal antisera raised against the synthetic fragments Tbeta10[1-16] and Tbeta10[31-43], with polyclonal antisera raised against natural thymosin gamma4 (Tbeta4) or thymosin beta9 (Tbeta9), and with anti-peptide oligoclonal antisera raised against various fragments of Tbeta4 (i.e. Tbeta4[1-11], Tbeta4[30-43] and Tbeta4[16-38]). Four distinct epitopic fragments were revealed, namely the sequences 1-13, 19-30, 29-40 and 36-43. Among them, the sequence 36-43 appears to offer unique immunochemical characteristics to the Tbeta10 molecule.

Epitope Mapping↗
PubMed2001

Epitope mapping of the transforming growth factor-beta superfamily protein, macrophage inhibitory cytokine-1 (MIC-1): identification of at least five distinct epitope specificities.

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the transforming growth factor-beta (TGF-beta) superfamily whose increased expression is associated with macrophage activation and which is expressed highly in placenta as compared to other tissues. There are two known allelic forms of human MIC-1 due an amino acid substitution at position 6 of the mature protein. We have raised four monoclonal antibodies (MAbs) and one polyclonal antiserum to the mature protein region of human MIC-1 and have used an extensive panel of MIC-1 relatives, mutants, and chimeras to map their epitopes. None of the MAbs were able to cross-react with either the murine homologue of MIC-1 or with hTGF-beta1, and all of the MAb epitopes were conformation-dependent. A distinct cross-reactivity pattern with the various antigens was observed for each of the monoclonal and polyclonal antibodies suggesting the presence of at least five immunogenic regions on the MIC-1 surface. One of the MAbs is directed against the amino terminus of the protein and can distinguish between the two allelic forms of MIC-1. The epitopes for the other three MAbs were located near the tips of the so-called "fingers" of the protein and appeared to be partially overlapping as each involved amino acids in the region 24-37. In one case, it was possible to mutate murine MIC-1 so that it could be recognized by one of the MAbs. Finally, the use of another mutant in which Cys 77 was replaced by serine enabled confirmation of the location of the MIC-1 interchain disulfide bond.

Amino Acid Sequence↗
PubMed2003

Recognition of mycobacterial epitopes by T cells across mammalian species and use of a program that predicts human HLA-DR binding peptides to predict bovine epitopes.

Bioinformatics tools have the potential to accelerate research into the design of vaccines and diagnostic tests by exploiting genome sequences. The aim of this study was to assess whether in silico analysis could be combined with in vitro screening methods to rapidly identify peptides that are immunogenic during Mycobacterium bovis infection of cattle. In the first instance the M. bovis-derived protein ESAT-6 was used as a model antigen to describe peptides containing T-cell epitopes that were frequently recognized across mammalian species, including natural hosts for tuberculosis (humans and cattle) and small-animal models of tuberculosis (mice and guinea pigs). Having demonstrated that some peptides could be recognized by T cells from a number of M. bovis-infected hosts, we tested whether a virtual-matrix-based human prediction program (ProPred) could identify peptides that were recognized by T cells from M. bovis-infected cattle. In this study, 73% of the experimentally defined peptides from 10 M. bovis antigens that were recognized by bovine T cells contained motifs predicted by ProPred. Finally, in validating this observation, we showed that three of five peptides from the mycobacterial antigen Rv3019c that were predicted to contain HLA-DR-restricted epitopes were recognized by T cells from M. bovis-infected cattle. The results obtained in this study support the approach of using bioinformatics to increase the efficiency of epitope screening and selection.

Animals↗
PubMed2000

Linear PADRE T helper epitope and carbohydrate B cell epitope conjugates induce specific high titer IgG antibody responses.

Linear carbohydrate-peptide constructs based on the 13 amino acid nonnatural pan DR epitope (PADRE) and carbohydrate B cell epitopes are demonstrated to be potent immunogens. These data support our belief that PADRE should be considered as an alternative to more complex carriers for use in prophylaxis and therapeutic vaccines. Two model carbohydrate-PADRE glycoconjugates were used to demonstrate that PADRE could effectively provide T cell help for carbohydrate-specific Ab responses. Conjugates of PADRE covalently linked to the human milk oligosaccharide, lacto-N-fucopentose II or a dodecasaccharide derived from Salmonella typhimurium O-Ag induced high titer IgG Ab responses in mice, which were comparable to glycoconjugates employing human serum albumin (HSA) as the carrier protein. Different adjuvants, in combination with PADRE conjugates, allowed for the modulation of the isotype profile with alum supporting an IgG1 profile; QS-21 an IgG2a, 2b profile, while an alum/QS-21 mixture generated a balanced IgG1/IgG2b isotype profile. As defined by binding to synthetic glycoconjugates, dodecasaccharide-specific Abs exhibited fine specificity similar to protective polyclonal Ab responses previously reported for dodecasaccharide-protein conjugates. The same Abs bound to intact S. typhimurium cells, suggesting that biologically relevant specificities were produced. The affinity of the dodecasaccharide-specific Abs was further shown to be comparable to that of a well-characterized, high affinity monoclonal anti-carbohydrate Ab recognizing the same epitope.

Adjuvants, Immunologic↗
PubMed2006

[Construction and immunogenicity of hCG chimeric peptides (CP1 and CP10) containing three self- B cell epitopes and six foreign T cell epitopes].

Two bio-synthesizing chimeric peptide (CP) immunogens named CP1 and CP10 have been designed, which consist of three linear B cell epitopes (BCE) of human chorionic gonadotropin beta subunit (beta-hCG) and six foreign T cell epitopes including two "promiscuous" TCEs from hepatitis B surface antigen and tetanus toxoid. Two artificial genes encoding CP1 and its derivative CP10 were synthesized, which could be expressed in E. coli at the level of about 1% of the total cell proteins when inserted into the thermo-induction vector respectively. In Western blot tests, the expressed CP1 and CP10 proteins with about 16.5 kD shown on the SDS-polyacrylamide gel electrophoresis (PAGE) gel can be recognized by the monoclonal or polyclonal antibodies specific to each linear epitope of beta-hCG. Each of expressed proteins can be purified with 95% relative homogeneity using our improved method of preparative gel PAGE. Their yields were about 1-2 mg per 12 L culture. Also, the CPl and CP10 immunogens can induce antibodies in mice that recognize recombinant CP1 betar CP10 and natural beta-hCG, and there are three anti-beta5, beta9 and beta8 BCE antibodies in their antisera. The construction and expression of beta-hCG CP1 and CP10 will provide new immunogens for developing an ideal and superior hCG birth control and/or tumor therapeutic vaccine.

Animals↗