Differential antigenicity of recombinant polyepitope-antigens based on loop- and helix-forming B and T cell epitopes.

To investigate a strategy for the design of chimeric antigens based on B cell epitopes (BCEs) we have genetically recombined multiple copies of loop- (L) and helix-forming (H) sequential and protective BCEs of the measles virus hemagglutinin protein (MVH) in a number of high-molecular-weight polyepitope constructs (24.5-45.5 kDa). The BCE cassettes were combined semi-randomly together with a promiscuous T cell epitope (TCE; tt830-844) to yield 13 different permutational constructs. When expressed in mammalian cells, all constructs were detectable by Western blot as distinct bands of predicted molecular weight. Flow cytometry with conformation-specific antibodies revealed the Cys-loop in two [(L(4)T(4))(2) and (L(2)T(2))(4)] and the helix conformation in one [(H(2)T(2))(4)] of the different permutational constructs. The larger constructs, containing 16 epitope cassettes, seemed more likely to express the BCEs in their native conformation than the 8-mers. In the T cell proliferation assay, constructs with a higher copy number of TCEs, such as (L(2)T(2))(4), were more antigenic, as long as tandem repeats were separated by spacers. Since the conformation of even sequential BCEs and the processing of TCEs are both sensitive to their molecular environment it is difficult to predict the antigenic properties of polyepitopes. However, with the permutational approach we have developed several polyepitope constructs [(L(4)T(4))(2), (L(2)T(2))(4), (H(2)T(2))(4)] based on complex sequential BCEs that are antigenic for both T and B cells. Several constructs induced sera that reacted with reporter peptides, demonstrating that the sequential nature of the viral epitopes was conserved in the polyepitopes. Although several sera contained antibodies directed against amino acids critical for neutralization, only one construct induced antibodies that cross-reacted with the virus. Our results show the difficulty of designing chimeric antigens based on B cell epitopes mimicking their antigenic and immunologic properties even when these are sequential in nature.

Neutralization of measles virus wild-type isolates after immunization with a synthetic peptide vaccine which is not recognized by neutralizing passive antibodies.

The sequence H379-410 of the measles virus haemagglutinin (MV-H) protein forms a surface-exposed loop and contains three cysteine residues (Cys-381, Cys-386 and Cys-394) which are conserved among all measles isolates. It comprises the minimal sequential B cell epitope (BCE) (H386-400) of the neutralizing and protective MAb BH6 that neutralizes all wild-type viruses tested. The aim of this study was to design synthetic peptides which induce neutralizing antibodies against MV wild-type isolates. Peptides containing one or two copies of T cell epitopes (TCE) and BCEs of different lengths (H386-400, B(CC); H379-400, B(CCC)), in different combinations and orientations were produced and iteratively optimized for inducing neutralizing antibodies. Peptides with the shorter BCE induced sera that cross-reacted with MV but did not neutralize. The longer BCE containing the three cysteines (B(CCC)) and two homologous TCE were required for neutralization activity. These sera neutralized wild-type strains of different clades and geographic origins. Neutralizing serum was also obtained after immunization with human promiscuous TCEs. Furthermore B(CCC)-based peptides were fully immunogenic even in the presence of pre-existing MV-specific antibodies. The results suggest that subunit vaccines based on such peptides could potentially be used to actively protect infants against wild-type viruses irrespective of persisting maternal antibodies.

Field test evaluation of aerobic, anaerobic, and wheelchair basketball skill performances.

Forty-six male wheelchair basketball players performed a set of field tests to evaluate aerobic capacity (25 m shuttle run), anaerobic capacity (30s sprint), and six specific wheelchair basketball skills. Overall test-retest reliability (n = 20) ranged from r = 0.65 to r = 0.97. To study the validity (criterion related evidence) of the shuttle run test, heart rate (HR) was recorded for 15 subjects, who also performed a continuous, multistage arm cranking exercise until volitional fatigue. Moderate to high correlations were calculated between shuttle run distances covered (1375 243,6 m) and VO2max (2208+/-461.6 mL/min) and POmax (93.8+/-17.97 W), measured during maximal arm cranking (respectively r = 0.64 and r = 0.87). Maximal HR during shuttle run (174.9+/-16.6 B/min) and arm cranking (169+/-14.21 B/min) were correlated (r = 0.78). High correlations between shuttle run test and anaerobic field tests, however, indicate high implication of anaerobic and wheelchair maneuverability performances. The 30 s sprint test was validated (n = 15) against a Wingate Anaerobic Test (WAnT) on a roller ergometer. Comparing distance (field test: 90+/-6.7 m) with mean power output (WAnT: 852.1+/-234.9 W) the correlation was r = 0.93. Principal components factor analysis identified 'wheelchair propulsion dynamics' and 'eye-hand-coordination' as the underlying constructs of the six skill proficiency measurements, accounting for 80.1% of the variance. In conclusion, the newly developed field test battery is a reliable and valid tool for anaerobic capacity and skill proficiency assessment in wheelchair basketball players.

Crossreactivity of mimotopes and peptide homologues of a sequential epitope with a monoclonal antibody does not predict crossreactive immunogenicity.

The sequence H236-256 of the measles virus (MV) hemagglutinin (H) contains the sequential epitope of the neutralizing and protective monoclonal antibody (mAb) BH129 with the minimal epitope E(245)L-QL(249). Using this mAb, we have recently developed 7mer mimotopes binding up to 135x better than the corresponding 7mer epitope H244-250. In this study, we combined T cell epitopes (TCE) with either highly crossreactive 7mer mimotopes, 13mer mimotopes or less crossreactive MV-derived B cell epitopes (BCE). Antigenicity of these TBB, TTB and TTBB peptides was determined with BH129 in a competition ELISA against MV. We found that chimeric peptides including mimotopes were up to 80x better binders to the mAb than peptides containing the original BCEs. All peptides irrespective of their antigenicity were used for immunization to compare their virus- crossreactive immunogenicity. Unexpectedly, none of the highly antigenic mimotope-based peptides induced MV-crossreactive antibodies. In contrast, a number of peptides with the viral BCE sequence that did not bind to the mAb, induced MV-crossreactive and even neutralizing antibodies. This report describes a striking example of disparity between antigenicity and crossreactive immunogenicity and casts considerable doubt on the predictive value of antigenicity in immunogenicity studies, considerably complicating the selection of potential vaccine candidates.