Valine 11 and phenylalanine 13 have a greater impact on the T-cell response to citrullinated peptides than the 70-74 shared epitope of the DRB1 molecule in macaques.

OBJECTIVES: Various rheumatoid arthritis (RA) HLA-DRB-1 risk haplotypes have been regrouped under the shared epitope (SE) in position 70-74. The presence of Valine in position 11 (Val11) and phenylalanine in position 13 (Phe13) are also associated with RA, but it is impossible to differentiate their role compared with the SE since they are in strong linkage disequilibrium (LD) in humans. Similar to humans, certain macaques express the SE (H6). We analysed the effect of various DRB1 haplotypes on T-cell response to citrullinated peptides (Cit-P) in macaques. METHODS: Six H6 and six non-H6 macaques were immunized with four Cit-P. T-cell response was assessed using Interferon γ enzyme-linked immunospot. RESULTS: Animals developed a specific anti-Cit-P T-cell response. Surprisingly, H6 animals had a significantly lower T-cell response than non-H6. In macaques, the 70-74 SE and the Val11 are on separate haplotypes. Presence of Val11 was strongly associated with the anti-Cit-P T-cell response, whatever the 70-74 sequence was. This response was amplified in case of presence of Phe13. CONCLUSION: The absence of LD between Val11 and SE in macaques allowed us to demonstrate that the most important HLA positions to induce a T-cell response against Cit-P were Val11 and Phe13 and not the 70-74 SE.

Protective Effect of Val129-PrP against Bovine Spongiform Encephalopathy but not Variant Creutzfeldt-Jakob Disease.

Bovine spongiform encephalopathy (BSE) is the only known zoonotic prion that causes variant Creutzfeldt-Jakob disease (vCJD) in humans. The major risk determinant for this disease is the polymorphic codon 129 of the human prion protein (Hu-PrP), where either methionine (Met129) or valine (Val129) can be encoded. To date, all clinical and neuropathologically confirmed vCJD cases have been Met129 homozygous, with the exception of 1 recently reported Met/Val heterozygous case. Here, we found that transgenic mice homozygous for Val129 Hu-PrP show severely restricted propagation of the BSE prion strain, but this constraint can be partially overcome by adaptation of the BSE agent to the Met129 Hu-PrP. In addition, the transmission of vCJD to transgenic mice homozygous for Val129 Hu-PrP resulted in a prion with distinct strain features. These observations may indicate increased risk for vCJD secondary transmission in Val129 Hu-PrP-positive humans with the emergence of new strain features.

Generation of a novel monoclonal antibody that recognizes the alpha (α)-amidated isoform of a valine residue.

BACKGROUND: Alpha (α)-amidation of peptides is a mechanism required for the conversion of prohormones into functional peptide sequences that display biological activities, receptor recognition and signal transduction on target cells. Alpha (α)-amidation occurs in almost all species and amino acids identified in nature. C-terminal valine amide neuropeptides constitute the smallest group of functional peptide compounds identified in neurosecretory structures in vertebrate and invertebrate species. METHODS: The α-amidated isoform of valine residue (Val-CONH2) was conjugated to KLH-protein carrier and used to immunize mice. Hyperimmune animals displaying high titers of valine amide antisera were used to generate stable hybridoma-secreting mAbs. Three productive hybridoma (P15A4, P17C11, and P18C5) were tested against peptides antigens containing both the C-terminal α-amidated (-CONH2) and free α-carboxylic acid (-COO(-)) isovariant of the valine residue. RESULTS: P18C5 mAb displayed the highest specificity and selectivity against C-terminal valine amidated peptide antigens in different immunoassays. P18C5 mAb-immunoreactivity exhibited a wide distribution along the neuroaxis of the rat brain, particularly in brain areas that did not cross-match with the neuronal distribution of known valine amide neuropeptides (α-MSH, adrenorphin, secretin, UCN1-2). These brain regions varied in the relative amount of putative novel valine amide peptide immunoreactive material (nmol/µg protein) estimated through a fmol-sensitive solid-phase radioimmunoassay (RIA) raised for P18C5 mAb. CONCLUSIONS: Our results demonstrate the versatility of a single mAb able to differentiate between two structural subdomains of a single amino acid. This mAb offers a wide spectrum of potential applications in research and medicine, whose uses may extend from a biological reagent (used to detect valine amidated peptide substances in fluids and tissues) to a detoxifying reagent (used to neutralize exogenous toxic amide peptide compounds) or as a specific immunoreagent in immunotherapy settings (used to reduce tumor growth and tumorigenesis) among many others.

Fc gamma RIIIa expression is not increased on natural killer cells expressing the Fc gamma RIIIa-158V allotype.

The presence of a valine (V) versus a phenylanaline (F) at position 158 of Fc gamma RIIIa/CD16a improves the affinity for IgG and is associated with higher therapeutic response to rituximab. Increased CD16 expression on natural killer (NK) cells from donors with the VV or VF versus FF genotype has recently been reported. We indeed observed higher binding of the anti-CD16 monoclonal antibody (mAb) 3G8 on NK cells from V carriers (VV = VF > FF). However, the binding of two other anti-CD16 mAbs, LNK16 and DJ130c, decreased with the number of V allele (VV < VF < FF). CD16 transcript levels were independent on the genotype. Rituximab binding to NK cells from V carriers was higher than its binding to FF NK cells at low concentrations (10 and 100 microg/mL). However, the difference was nearly completely abolished at saturating concentrations (>or=1,000 microg/mL). Finally, nearly 100% of CD16-expressing NK cells displayed a complete down-modulation of the receptor after optimal engagement by plate-bound 3G8, whatever the genotype. By contrast, the percentages of NK cells down-modulating CD16 after competitive engagement of the receptor by plate-bound rituximab increased with the number of V allele (FF, 18.2 +/- 8.6%; VF, 32.0 +/- 4.9%; and VV, 42.4 +/- 9.9%). These results are in discrepancy with the expected increased competition that would result from an increased expression of CD16 on VV and VF NK cells. We conclude that increased binding and functional and clinical responses associated with the high-affinity Fc gamma RIIIa-158V are unrelated to an increased expression of this allotype.

Development of an ELISA for the detection of the residues of the fungicide iprovalicarb.

A competitive indirect enzyme-linked immunosorbent assay was developed for the fungicide iprovalicarb, using a polyclonal antibody produced against a hapten conjugated through the carboxyl group on the benzene ring to keyhole limpet hemocyanin. Under an optimized condition using a heterologous format, an IC(50) of 3.51 ng/mL and the lowest detection limit of 0.065 ng/mL were obtained. When the isopropoxy group was removed from the iprovalicarb structure for the synthesis of a hapten, the resulting hapten was not successful as an immunogen, indicating that the isopropyl moiety was an important epitope, as evidenced by the cross-reactivities of some structurally related compounds. When applied to the real crop and water samples, the recoveries were in the range of 80.52-144.70% (n = 4) and 72.11-100.43% (n = 4), respectively. Accordingly, this ELISA can be used as a useful method for monitoring iprovalicarb residues in crop and water samples.

Antibodies directed against L and D isovaline using a chemical derivatizing reagent for the measurement of their enantiomeric ratio in extraterrestrial samples: first step-production and characterization of antibodies.

Determining the enantiomeric ratio of amino acids in meteorites requires very sensitive and precise measurements. In this study, an immunochemical approach, combined with new chemical derivatizing agents, was investigated for the measurement of the enantiomeric ratio of isovaline. In the initial step, L and D isovaline were derivatized with epsilon-benzyloxycarbonyl-L-lysine-(t-butyl ester)-chloroethylnitrosourea (Z-L-Lys-(OtBu)-CENU). The Z group was hydrolyzed and the resulting isovaline derivatives (L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline) were conjugated with protein using glutaraldehyde and reduced with sodium borohydride. Rabbits were immunized with the immunogenic conjugates thus obtained. Antibodies were characterized using many compounds, both derivatized and underivatized, in competitive ELISA tests. These competition experiments performed enabled us to establish the following results: 1) unconjugated L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline were poorly recognized; 2) all related L-Lys(OtBu)-alpha-hydrogenated amino acids (L and D) were not recognized at all, which eliminates the possibility of the measurements being distorted by contamination; 3) only conjugated L-Lys(OtBu)-alpha-amino-isobutyric acid (AIB) was recognized by the antibody, 4) the enantiomeric discrimination of L and D isovaline through their derivatives (diastereoisomeric L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline) was in accordance with the measurement of their enantiomeric ratio. Immunopurification was shown to enhance antibody specificity. The strategy employed shows potential for the quantification of meteoritic amino acids.

The effect of amino acid spacers on the antigenicity of dimeric peptide--inducing cross-reacting antibodies to a cell surface protein antigen of Streptococcus mutans.

In the course of developing a synthetic peptide vaccine for dental caries, we identified a unique 13-mer peptide named PAc(365-377), TYEAALKQYEADL, as a minimum peptide inducing cross-inhibiting antibodies to a cell surface protein antigen (PAc) of Streptococcus mutans. However, the peptide could hardly induce the production of antibody in the absence of adjuvant. Thus using this peptide as a unit peptide, tandem constructs of dimeric unit peptide with or without spacer amino acid residues were synthesized, and their antigenicities were examined in B10.D2 mice. Significant augmentation of antigenicity was obtained in all of the dimeric unit peptides with spacers, especially for lysine spacers. In addition, analysis for cross-reactivity of anti-construct antibodies against a set of double valine-substituted analogues of the unit peptide revealed that the di-lysine spacer might be more effective in inducing the cross-reacting antibodies to rPAc.

Identification and characterization of a DR4-restricted T cell epitope within chlamydia heat shock protein 60.

An epitope within the 60 kD Chlamydia trachomatis heat shock protein (hsp) 60, recognized by a HLA-DRB1*0401-restricted T cell clone from a reactive arthritis patient, has been characterized. Stimulatory peptides contained a nine amino acid sequence (residues 38-46) predicted by algorithm to confer strong binding to DRB1*0401, with valine in the P1 position. The overall length of the peptide was critical for efficient recognition; peptides with at least one residue N-terminal to the putative P1 position were markedly more stimulatory than a peptide whose N-terminal is the P1 valine. Optimal responses were seen with 14mer peptides having two to three amino acids N- and C-terminal to the core 9mer. The sequence of the defined epitope is identical in hsp60 from both C. trachomatis and C. pneumoniae. Since the latter is a common respiratory pathogen, patients infected with C. trachomatis may already be primed for responses to hsp60 by prior infection with C. pneumoniae. Such secondary responses are important in the pathogenesis of chlamydia-induced inflammatory diseases such as trachoma. Priming by infection with enteric organisms was considered because of the similarity of the epitope sequence in Escherichia coli hsp60. However, although an E. coli-related peptide was recognized, intact E. coli hsp60 was not, suggesting that the epitope is cryptic in E. coli hsp60. Human hsp60 has six amino acid differences from chlamydial hsp60 in the epitope sequence and was not recognized. Thus cross-reactive recognition of self hsp60 could not be implicated in the pathogenesis of chlamydia-induced reactive arthritis in this patient.

Virus and cytotoxic T lymphocytes: crucial role of viral peptide secondary structure in major histocompatibility complex class I interactions.

Viral antigens are presented to cytotoxic T lymphocytes (CTLs) by H-2-restricted major histocompatibility complex (MHC) glycoproteins. Binding of the endogenously processed viral peptides (epitopes) to their specific MHC molecules is an early intracellular event in the recognition process and is necessary for subsequent killing of virus-infected cells by virus-specific CTLs. It is now well established that interaction between a viral antigenic peptide and MHC is dependent on the primary structure (length and amino acid sequence) of that antigen. Here we show, using the H-2Db-restricted epitope GP277-286 of lymphocytic choriomeningitis virus as a model, that the secondary structure (conformation) of the viral sequence also plays a crucial role in the binding of a viral antigen to MHC glycoprotein and in its subsequent presentation to virus-specific CTLs.

Idiotypic cross-reactivity between antibodies of different specificities.

Cross-idiotypic specificity has been demonstrated between antibody populations of different specificities using antibodies directed toward human sickle cell hemoglobin (HbS). A site-specific antibody directed toward the beta6-position of HbS, anti-Val, was used to elicit an anti-idiotypic response in rabbits. Using this anti-idiotypic serum, idiotypic cross-reactivity was demonstrated between antibody populations that bind to human adult hemoglobin (HbA). It was demonstrated that in the case of the goat antibodies, these idiotypically cross-reacting antibodies are directed towards the beta6-position of the hemoglobin molecule. However, they differ in their specificity, binding to this site on HbA, whereas anti-Val binds only to HbS. The sheep antibody populations directed toward HbS differ qualitatively from those of the goat. Using rabbit anti-idiotypic serum specific for sheep anti-Val, cross-reactivity could be demonstrated with antibodies directed toward the alpha-chain of the hemoglobin molecule, as well as the beta-chain. There was also a low level of cross-reactivity with antibodies from a goat immunized with HbA.

Specific antibodies to hemoglobin A1 (anti-Glu) and hemoglobin S (anti-Val) in the guinea pig: immunologic and structural correlations.

Like goats and sheep, guinea pigs can produce, in response to human sickle cell hemoglobin (beta6 Glu leads to Val), an antibody population (anti-Val) that will bind sickle cell hemoglobin but not normal hemoglobin HbA. Unlike goats and sheep, guinea pigs can produce in response to human hemoglobin A1 an antibody fraction, anti-Glu, that will not react with human sickle cell hemoglobin. These anti-Glu antibodies have been isolated by affinity chromatography and their specificity confirmed by fluorescence-quenching titrations. The sequence of the first 10 amino acids of the beta-chain of guinea pig hemoglobin has been determined. This sequence differs from those of both hemoglobin HbA and sickle cell hemoglobin by two residues, those at positions 5 and 6. This explains the similarity of the immunogenicity of this site on the two human hemoglobins when administered to guinea pigs. Both goats and sheep are identical to hemoglobin A1 at the beta-6 position.