HLAMatchmaker: a molecularly based algorithm for histocompatibility determination. I. Description of the algorithm

This report describes an algorithm for identifying acceptable HLA antigens for highly alloimmunized patients without the need for extensive serum screening. This algorithm is based on the concept that immunogenic epitopes are represented by amino acid triplets on exposed parts of protein sequences of human leukocyte antigen chains (HLA-A, HLA-B, and HLA-C) accessible to alloantibodies. A computer program (HLAMatchmaker) has been developed to determine class I HLA compatibility at the molecular level. It makes intralocus and interlocus comparisons of polymorphic triplets in sequence positions to determine the spectrum of non-shared triplets on donor HLA antigens. In most cases is it possible to identify certain mismatched HLA antigens that share all their polymorphic triplets with the patient's HLA antigens and could therefore, be considered fully compatible. HLAMatchmaker permits also the identification of additional mismatches that are acceptable as determined from the triplet information on HLA-typed panel cells that do not react with patient's serum.HLAMatchmaker provides an assessment of donor-recipient HLA compatibility at the structural level and this algorithm is different from conventional methods based on the mere counting of numbers of mismatched HLA antigens or CREGs. This donor selection strategy is suitable especially for allosensitized patients in need of a compatible transplant or platelet transfusion.     

HLAMatchmaker: a molecularly based algorithm for histocompatibility determination. V. Eplet matching for HLA-DR, HLA-DQ, and HLA-DP

This report describes the design of the eplet version of HLAMatchmaker to determine class II compatibility at the structural level. This matching algorithm is based on the hypothesis, developed from molecular modeling of crystallized antigen-antibody complexes, that functional epitopes are represented by patches of surface-exposed nonself-amino acid residues surrounded by residues within a 3-A radius. Patch determinations with a molecular viewer of crystalline structural models downloaded from the Entrez Molecular Modeling Database Web site led to the identification of 44 DRB, 33DQB, 29 DQA, 20 DPB, and 9 DPA unique combinations of polymorphic positions. The residue compositions of these patches were then determined from amino acid sequences. This analysis resulted in a repertoire of 146 DRB, 74 DQB, 58 DQA, 45 DPB, and 19 DPA eplets. In many eplets, the residues are in short linear sequences, but many other eplets have discontinuous sequences of residues that cluster on or near the molecular surface. This analysis has also shown that all serologically defined DR and DQ antigens detectable by monospecific antibodies have unique eplets. Other eplets are present in groups of class II antigens, many of which appear as cross-reacting. The eplet version of HLAMatchmaker should be considered as a hypothetical model for the structural assessment of donor-recipient compatibility and the determination of mismatch acceptability for sensitized patients. This computer algorithm can be downloaded from the HLA Matchmaker Webside at http://tpis.upmc.edu.     

Modification of the inhibitory amino acid for epitope peptide binding onto major histocompatibility complex class II molecules enhances immunogenicity of the antigen

Previously, the arginine at hen egg-white lysozyme 61 (HEL 61) was characterized as inhibiting T-lymphocyte stimulation due to the inefficient binding of the arginine-containing epitope peptide to the corresponding major histocompatibility complex class II molecules in C57BL/6 mice. In this study, we produced recombinant HEL, with arginine or alanine at HEL 61, and compared its ability to induce immune responses in mice to see whether modification of an inhibitory amino acid could enhance the immunogenicity of an inefficient antigen. Immunization of the mice with modified HEL induced strong antibody and T-cell immune responses against the native antigen. The enhanced T-cell immune response was due to a more specific elevation of the T-cell responses to the HEL 46-61 epitope region than to other epitope regions, although recognition of the other epitope peptides of HEL was generally increased. Mass spectrometric analyses of the epitope peptides generated by splenic antigen-presenting cells indicated that production of the epitope peptides encompassing HEL 46-61 was efficient using the modified antigen. These results suggest that modification of the critical amino acid residue(s) involved in hampering induction of an efficient immune response is an effective method to improve the immunogenicity of an inefficient antigen.     

人类肝素酶B细胞表位的预测和免疫原性鉴定

目的 预测并鉴定肝素酶(heparanase)蛋白B细胞表位免疫原性.方法 以肝素酶蛋白的氨基酸序列为基础,采用DNAStar分析软件以及Bcepred在线二级结构分析工具分析其蛋白二级结构并预测B细胞表位.根据预测结果 ,采用8分支多抗原肽结构合成针对该表位的抗原肽,将后者与通用型T辅助表位人IL-1β线性短肽(VQGEESNDK,氨基酸163～171)联合免疫日本白毛黑眼兔,检测免疫血清效价,鉴定其特异性和免疫原性.结果 软件预测显示,肝素酶蛋白大亚基的第1～15位(MAP1)、第279～293位(MAP2)及175～189位(MAP3)氨基酸序列最可能为其优势B细胞表位.间接酶联免疫吸附试验、免疫印迹及免疫组化分析,证实MAP1、MAP2及MAP3均能诱导机体产生高滴度抗体,但仅MAP1、MAP2抗体具有高特异性,MAP2抗体与肝癌组织的结合力最强.结论 肝素酶大亚基的第1～15位、第279～293位氨基酸为其优势B细胞表位,其中第279～293位氨基酸的免疫原性最强,这为肝素酶多肽抗体及B细胞优势短肽疫苗研制提供了理论依据.     

Identification of the amino acid residues contributing to monoclonal antibody-defined DQw1 epitopes.

The reactivity of monoclonal antibodies (mAbs) R1, S1, and S5, shown previously to recognize polymorphic epitopes on HLA-DQ molecules, have been found to correlate with the presence of certain DQB1 alleles. mAb S5 reacts with cells expressing DQB1*0503, 0601, 0602, 0603, or 0604 alleles while R1 and S1 react with all DQB1 alleles except *0201 and 0301. In the case of R1 and S1, sequence comparison of these chains suggests the involvement of residues 45-47 (GVY) in formation of the epitopes. This prediction has been confirmed by showing that a G----E mutation in position 45 of the DQB1*0302 gene eliminates binding of both mAbs.     

The structure of influenza viruses: II. C-terminal amino acid analyses

Reaction of influenza B virus (LEE strain) with carboxypeptidase resulted in the rapid and simultaneous release of leucine and tyrosine in the approximate molar ratio of 1.5:1, followed by smaller amounts of other, unidentified amino acids. This result was obtained only when the virus was denatured with formamide or cuprammonium sulphite; practically no amino acids were released from native virus under similar conditions of digestion.Leucine and tyrosine were released only from the fraction of cuprammonium sulphite-treated LEE virus previously shown to be lacking N-terminal aspartic acid. The protein which possessed an N-terminal aspartic acid residue was much less susceptible to the action of carboxypeptidase, and its C-terminal residue was not identified. Fractions obtained by electrophoresis of dodecyl sulphate-disrupted LEE virus were reacted with carboxypeptidase. Equimolar amounts of leucine and tyrosine were split off from the fastest fraction, but from the slowest, the ratio of leucine to tyrosine released was 7.5:1. These results suggest that particles of LEE virus contain at least three different kinds of polypeptide chains.Reaction with carboxypeptidase of the MEL, BEL, and PR8 strains of influenza A virus (denatured by cuprammonium sulphite), resulted in the slow and simultaneous release of about five amino acids from each strain. None of these could be unequivocally identified as C-terminal.     

Immunological cross-reactivity between mimicking epitopes on a virus protein and a human autoantigen depends on a single amino acid residue.

Recently we identified a novel autoantigenic region in the alpha chain of the human acetylcholine receptor (HuAChR), residues 160-167 (P. L. Schwimmbeck, T. Dyrberg, D. B. Drachman, and M. B. A. Oldstone, J. Clin. Invest., in press). Antibodies to that sequence appeared in the sera of a subset of patients with myasthenia gravis (MG) and affinity-purified antibody was biologically active for an in vitro assay of the HuAChR. Sequence homology between the autoantigen and two separate regions of herpes simplex virus glycoprotein D (HSV-GpD) have been identified. These components are, respectively, the HuAChR, residues [160-167] (PESDQPDL), and residues [286-293] (PNATQPEL) and [381-388] (PEDDQPSS) of HSV-GpD. Antisera from rabbits immunized with synthetic peptides representing HSV-GpD [286-293] bound strongly to the AChR peptide. In contrast, antiserum to HSV-GpD [381-388] bound minimally, if at all, even though both virus peptides shared four amino acids with the receptor sequence. To investigate the molecular basis for the differential binding, we tested the reactivity of the HSV-GpD antisera to analogs of the [381-388] virus peptide containing single amino acid substitutions. The results demonstrated that the cross-reactivity between HuAChR [160-167] and HSV-GpD [286-293] predominantly depends on a single residue, the C-terminal leucine in HSV-GpD, position 293.     

Metabolism of sulfur-containing amino acids in the dermatophyte Microsporum gypseum. II. Acidic amino acid derivatives

The dermatophyte Microsporum gypseum was cultivated on a glucose-arginine medium supplemented with five strongly acidic derivatives of cysteine (L-cysteine sulfinic acid, L-cysteic acid, L-serine-O-sulfate and taurine at a concentration of 5 mmol/l, and L-S-sulfocysteine at a concentration of 2.5 mmol/l). The addition of these substances did not stimulate the growth as compared with the control containing 0.5 mmol/l cystine. Cysteine sulfinic acid and cysteic acid showed rather inhibitory effects. A strong inhibition of the growth was caused by the presence of serine sulfate. During the growth, all substances investigated were gradually consumed and utilized not only as a source of sulfur but of nitrogen and carbon as well. Cysteine sulfinic acid and S-sulfocysteine were utilized most rapidly. Cysteic acid was also rapidly utilized but after a certain adaptation. Taurine was utilized slowly and serine sulfate very slowly. Excess sulfur contained in the substances used was excreted into the medium in the form of sulfate. Sulfate excretion was most rapid with cysteine sulfinic acid and slowest with taurine. With cysteine sulfinic acid, S-sulfocysteine and cysteic acid, small amounts of sulfite were found in the medium. The results obtained are in accordance with the presumption that cysteine sulfinic acid (but not cysteic acid and taurine) is an intermediate of cysteine catabolism in dermatophytes.     

A simplified method for determination of peptide-protein molar ratios using amino acid analysis.

In this report, we have described methods to improve the efficiency of coupling synthetic peptides to keyhole limpet hemocyanin (KLH) and for the analysis of the composition of the resulting peptide-protein conjugates. KLH was first dissolved in buffers containing 3 M guanidine hydrochloride to maintain solubility and derivatized with either of two water soluble, heterobifunctional crosslinkers, m-maleimido-benzoyl-N-hydroxy-sulfosuccinimide ester (SMBS), or sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SSMCC) (300:1 molar excess over KLH). Synthetic peptides containing an amino terminal cysteine were then crosslinked to the modified KLH via sulfhydryl reaction with the crosslinker maleimide groups. Following dialysis to remove free peptide, the amino acid composition of the conjugate was determined. The molar ratio of peptide to protein within the conjugate was obtained by comparing the conjugate composition with that of both the KLH and peptide analyzed separately, and by a multiple regression, least squares analysis of the data. This method is generally applicable to the analysis of the molar ratios of protein-protein conjugates of unknown sequence or composition, and requires only the prior determination of the experimental amino acid composition of each component of the conjugate separately.     

Mucosal immunogenicity of polysaccharides conjugated to a peptide or multiple-antigen peptide containing T- and B-cell epitopes.

In this study we investigated the mucosal and systemic responses to two T-cell-independent polysaccharides, a serogroup f polysaccharide (formed of rhamnose glucose polymers [RGPs]) from Streptococcus mutans OMZ 175 and a mannan from Saccharomyces cerevisiae, covalently conjugated either to a linear peptide (peptide 3) or to a multiple-antigen peptide (MAP), both derived from S. mutans protein SR, an adhesin of the I/II protein antigen family of oral streptococci. Peptide 3 and MAP, which contained at least one B- and one T-cell epitope, were tested as carriers for the polysaccharides and as protective immunogens. Intragastric intubation of rats with the conjugates (RGPs-peptide 3, RGPs-MAP, mannan-peptide 3, and mannan-MAP) associated with liposomes produced salivary immunoglobulin A (IgA) antibodies which reacted with RGPs or mannan, peptide 3 or MAP, protein SR, and S. mutans or S. cerevisiae cells. Administration of conjugate boosters to the animals showed that both carriers conjugated to the polysaccharides were able to induce, in immunized animals, a salivary antipolysaccharide IgA memory. In contrast, animals primed and challenged with unconjugated polysaccharide showed no anamnestic response. Rats orally immunized with the conjugates also developed systemic primary antipolysaccharide and antipeptide IgM antibody responses which were characterized by a switch from IgM to IgG during the course of the secondary response. Data presented here demonstrated that both peptide 3 and the MAP construct can act as good carriers for orally administered polysaccharides. Unexpectedly, the use of a MAP did not further improve the immunogenicity of polysaccharides at the mucosal level; nevertheless, such a construct should be of great interest in overcoming the problem of genetic restriction induced by linear peptides.     

The amino acid sequence determination of a granulin and polyhedrin from two baculoviruses infecting Agrotis segetum.

The amino acid sequence of Agrotis segetum Granulosis virus (AsGV) granulin and A. Segetum nuclear polyhedrosis virus (AsNPV) polyhedrin was determined by sequencing tryptic and chymotryptic peptides from reduced and carboxymethylated proteins and tryptic fragments of oxidized and maleylated granulin. The comparison of the established peptide structures with the primary structures of other occlusion body proteins from related baculoviruses was also used for the polypeptide chains' reconstruction. The polypeptide chains of AsGV granulin and AsNPV polyhedrin comprise 247 and 246 amino acid residues, respectively. The proteins possess a high percentage of homology in their primary structures (63%).     

Possible conformation of interferons: a prediction based on amino acid composition and sequence

Using the method of estimation of alpha-helical and beta-folded types of secondary structure from amino acid composition, it has been established that interferons contain about 60-70% of alpha-helices and not more than 10-20% of beta-structure. The same result was obtained using empirical and stereochemical rules for the prediction of protein secondary structure of amino acid sequence. The hydrophobic clusters on the surfaces of the alpha-helical segments were determined. Using the method for packing of alpha-helical segments into the globule suggested by Efimov, and taking into account the disulfide bond arrangement in leucocyte interferon, two alternative globular conformations with prevailing alpha-helix were obtained. The analysis of these conformations shows an anomalous distribution of charged amino acid residues conserved in the primary structure of leucocyte and fibroblasts interferons. Nine positively charged amino acid residues are concentrated in one site on the surface of protein globule. It is possible that this site is responsible for some general biological activity of the interferons.     

In Vitro Work Load and Rat Heart Metabolism: II. Effect on amino acid transport

In vitro pressure overload was found to accelerate protein synthesis in the isolated working rat heart (Hjalmarson and Isaksson 1972 a). Since membrane transport of amino acids is considered to be one rate-limiting step in protein synthesis, the amino acid transport in the isolated rat heart was investigated using the non-utilizable amino acids α-aminoisobutyric acid (AIB) and 1-aminocyclopentane carboxylic acid (cycloleucine). Increased pressure load (afterload) accelerated amino acid uptake after a perfusion period of 15 ruin, and a 50—100% increase in the intracellular to extracellular distribution ratio of the amino acids was seen after 60 min of perfusion. This accelerated uptake was not inhibited by cycloheximide, suggesting that the work load effect was not dependent upon a continuous synthesis of proteins. The accumulation rate continued to be stimulated for some time after normalization of the work load and coronary flow, indicating that the work load effect was not directly linked to coronary flow. Increased preload did not stimulate amino acid uptake. Hearts from hypophysectomized rats showed a decreased concentrative uptake but the amino acid uptake was still accelerated by pressure overload. It is suggested that an increased uptake of amino acids could be of physiological significance in relation to the increased protein synthesis under these conditions.     

Development and characterisation of molecularly imprinted polymers based on methacrylic acid for selective recognition of drugs

Specific molecularly imprinted polymers (MIPs) for the drug reserpine (RES) using methacrylic acid (MAA) as the functional monomer were developed and characterised for the first time in this study. Evaluation of the various polymers by binding assays indicated that the optimum ratio of functional monomer to template was 4:1. Furthermore, the imprinting effect of the MIPs was assessed by the chromatographic method, which demonstrated that the MIPs had better chromatographic behavior and selectivity than those of the corresponding NIPs. A combination of BET, NMR, UV spectroscopy, and MISPE analyses for investigation of the imprinting and recognition properties revealed that strong specific interactions between the functional monomer and RES in the prepolymerization solutions and the aqueous solutions were probably responsible for RES recognition. The preparation of RES MIPs and elucidation of imprinting and recognition mechanisms may serve as useful references for other drug MIPs.     

Selective amino acid substitutions of a subdominant Epstein-Barr virus LMP2-derived epitope increase HLA/peptide complex stability and immunogenicity: implications for immunotherapy of Epstein-Barr virus-associated malignancies.

The latent membrane protein 2 is an immunogenic antigen expressed in Epstein-Barr virus (EBV)-associated tumors and consequently it may represent a target for specific cytotoxic T lymphocyte (CTL)-based immunotherapies. However, the efficacy of such a therapy is limited by the poor immunogenicity of the protein that induces weak CTL responses directed to the CLGGLLTMV (CLG) epitope only in the minority of EBV-seropositive donors. We have now demonstrated that selective peptide stimulation of peripheral blood lymphocytes induced CLG-specific CTL in all donors, suggesting that this epitope can be a suitable target for specific immunotherapies. We found that the CLG peptide has a low affinity for HLA-A*0201 and does not produce stable complexes, both factors that are likely to determine the strength of CTL responses to this epitope. Therefore, we synthesized and tested CLG analogues carrying single or combined amino acid substitutions to increase HLA/peptide stability. Among the analogues tested we identified two peptides which, compared to the natural epitope, showed higher affinity for HLA-A*0201 molecules, and produced stable complexes. These peptides demonstrated a potent, specific stimulatory capacity and could be used for selective CTL-based therapies.     

Immunoglobulin gamma chains of a monotreme mammal, the echidna (Tachyglossus aculeatus): amino acid composition and partial amino acid sequence.

The echidna represents the lowest stage in phylogeny at which molecules clearly homologous to IgG antibodies appear to occur. We provide evidence that a fraction of gamma chains possess an unblocked N terminal sequence comparable to the VHIII sub-group of human gamma chains and that glycine is the C-terminal residue. Statistical comparison of amino acid composition of the component chains with other immunoglobulin heavy chains suggests that echidna gamma chains are more closely related to eutherian gamma chains than to the 7S Ig heavy chains from amphibia or aves. The results are consistent with our view that true gamma-type heavy chains did not appear in evolution until after the mammalian line diverged from the stem reptiles.     

On the relative immunogenicity of DR alloantigens: T cell recognition of HLA-DR2a and HLA-DR2b

The HLA-DR2 haplotype encodes two highly polymorphic DR molecules, DR2a and DR2b. Because little is known regarding the relative immunogenicity of different HLA-DR molecules, we have studied the T-cell recognition of DR2a and DR2b molecules from the DRw15, Dw2 haplotype. A series of DR2-specific alloreactive T-cell clones were analyzed with murine L-cell transfectants expressing either the DR2a or the DR2b molecules as stimulator cells in proliferation assays. Somewhat surprisingly, both DR2a and DR2b were capable of stimulating DR2-specific T-cell clones with equal magnitude and similar frequency. In addition, DR2a and DR2b are functionally distinct, that is, no clone was identified which was stimulated by both DR2a and DR2b molecules.     

A single amino acid is critical for the expression of B-cell epitopes on the helicase domain of the pestivirus NS3 protein

Truncated NS3 proteins, expressed by recombinant baculoviruses, were used to investigate the location of conserved B-cell epitopes on this non-structural bovine viral diarrhoea virus (BVDV) protein. A goat anti-pestivirus antiserum, and a panel of anti-NS3 monoclonal antibodies, including the BVDV-1 specific antibody P1D8, were used to verify the presence or absence of the epitopes. Interestingly, the monoclonal antibodies reacted only with the truncated protein encompassing the helicase domain of NS3. Expression of the B-cell epitopes was dependent on, but not within, a 57 amino acid sequence at the carboxy-terminal end of this protein, supporting observations that these conserved epitopes are conformational in nature. A comparison of deduced amino acid sequences of the helicase domain from BVDV-1, BVDV-2, BDV and CSFV isolates highlighted a single amino acid that appeared to be unique to P1D8-reactive BVDV-1 isolates. Site-directed mutagenesis studies confirmed that this amino acid is critical for the expression of the BVDV-1 specific NS3 epitope recognised by the P1D8 monoclonal antibody. Surprisingly, the amino acid was also important for an epitope recognised by two group-specific monoclonal antibodies, P1H11 and P4A11. Protein modelling studies, based on the structure of the hepatitis C NS3 helicase domain, indicated that this amino acid occupies a prominent position on the surface of the protein.     

In vitro mutagenesis of HLA-B27: single and multiple amino acid substitutions at consensus B27 sites identify distinct monoclonal antibody-defined epitopes.

The consensus HLA-B27 sequence includes a unique constellation of amino acid residues along the peptide-binding cleft. To investigate the potential role of this region in the antigenic structure of HLA-B27, a panel of transfected cell lines was produced expressing 24 mutant B27 molecules with single or multiple substitutions within this constellation of residues. The cells were analyzed by flow cytometry with a panel of four anti-B27 mAb: ME1, GSP5.3, GS145.2, and B27M2. Previous studies have suggested that position 67 exerts a conformational effect on the ME1, GSP5.3, and GS145.2 epitopes. This was further supported in these studies by the observation that additional substitutions at the flanking residues 63 and 70 could reverse the disruption of these mAb epitopes by large residues at 67. Substitutions at positions 69-71 disrupted the binding of ME1 and GSP5.3, apparently by a direct effect. Individual substitutions at either of the two positions bearing residues unique to B27, 70 and 97, had no significant influence on the binding of any of the four mAb. The region of amino acid positions 63-71 in HLA-B27 thus appears to participate in the formation of at least three distinct epitopes shared by B27 and B7, identified by ME1, GSP5.3, and GS145.2.     

Monoclonal antibodies specific for O-antigenic polysaccharides of Shigella flexneri: clones binding to II, II:3,4, and 7,8 epitopes.

Hybrid cells producing monoclonal antibodies against the O-antigens of Shigella flexneri were obtained by polyethylene glycol-mediated fusion of myeloma cells and lymphocytes from BALB/c mice immunized with whole heat-killed S. flexneri bacteria of serotypes 2a and 2b. Clones were selected for their binding specificity to structurally defined S. flexneri lipopolysaccharides (LPS). The following three groups were identified as recognizing three different epitopes: monoclonal antibodies binding to (i) S. flexneri LPS with the II:3,4 antigens, (ii) S. flexneri LPS with the II:3,4 antigens and the II:7,8 antigens, and (iii) S. flexneri LPS with the 7,8 group antigen only. Of cloned and characterized antibodies, more than 90% had either the mu or gamma 3 heavy chain and 98% had the kappa light chain. The exquisite specificity of each monoclonal antibody preparation was in complete contrast to the polyclonal specificities seen in sera from immunized rabbits. Even absorbed rabbit S. flexneri typing sera contained antibodies reacting with several different LPS, i.e., they were not type antigen specific. Ascites from immunoglobulin G monoclonal antibody preparations representing the three different specificities were used for sensitizing Staphylococcus aureus Cowan 1 bacteria and were used in coagglutination. In testing 211 clinical isolates of all different serotypes of S. flexneri, the reagents were shown to be sensitive and specific in correctly identifying all S. flexneri II and 7,8 antigen-containing strains with no false positives. Two isolated immunoglobulin M antibody clones specific for the II:3,4 and 7,8 antigens were used as successfully for identification by direct slide agglutination. These results suggest that the monoclonal reagents are superior to conventional typing antisera.