Identification of the neutralizing epitopes of Merkel cell polyomavirus major capsid protein within the BC and EF surface loops.

Merkel cell polyomavirus (MCPyV) is the first polyomavirus clearly associated with a human cancer, i.e. the Merkel cell carcinoma (MCC). Polyomaviruses are small naked DNA viruses that induce a robust polyclonal antibody response against the major capsid protein (VP1). However, the polyomavirus VP1 capsid protein epitopes have not been identified to date. The aim of this study was to identify the neutralizing epitopes of the MCPyV capsid. For this goal, four VP1 mutants were generated by insertional mutagenesis in the BC, DE, EF and HI loops between amino acids 88-89, 150-151, 189-190, and 296-297, respectively. The reactivity of these mutants and wild-type VLPs was then investigated with anti-VP1 monoclonal antibodies and anti-MCPyV positive human sera. The findings together suggest that immunodominant conformational neutralizing epitopes are present at the surface of the MCPyV VLPs and are clustered within BC and EF loops

Characteristics of 14 anti-MCPyV mAbs produced with VP1 from MCC350 and MKT21 strains.

<p>BL, buried linear, C, conformational.</p

ELISA reactivity of MCPyV monoclonal antibodies against MCPyV insertional mutants BC, DE, EF and HI.

<p>In order to characterize the epitopes of the MCPyV VP1, the reactivity of mAbs was analyzed using the MCPyV wt VP1 VLPs and the four mutants with insertion within the BC, DE, EF and HI VP1 loops. ELISAs were performed using hybridoma culture supernatants diluted 1:3. The results are presented as relative binding defined as the reactivity of mAb to mutant VLPs divided by the reactivity of the same mAb observed with wild-type VLPs. The data presented are the means of three determinations (+/- SEM).</p

Monoclonal antibodies neutralization mechanism after pre (black) or post (white) MCPyV pseudovirions attachment.

<p>For the detection of neutralizing antibodies, COS-7 cells (10⁴/well) and MCPyV luciferase pseudovirions (0.2 RLU) were used. For the pre attachment determination, pseudovirions were mixed with monoclonal antibodies supernatants diluted 1:3 during 1 h and then added to the cells for 3 h at 37°C. The mixture was removed and 100 μl of DMEM-FCS were added. For investigation of post-attachment neutralization, pseudovirions were bound to cells for 1 h at 4°C. Unbound virions were removed and then antibodies diluted 1:3 were added during 1h. The antibodies were removed and 100 μl of DMEM-FCS were added. After incubation for 48 h at 37°C the luciferase activity was measure. The results were expressed as the percentage of inhibition of luciferase activity. The data presented are the means of three determinations performed in duplicate (+/- SEM).</p

ELISA reactivity of 10 neutralizing anti-MCPyV positive human sera (MCC patients) against MCPyV insertional mutants BC, DE, EF and HI.

<p>In order to characterize the epitopes of the MCPyV VP1, the reactivity of the human sera was analyzed from ten patients using the MCPyV wt VP1 VLPs and the four mutants with insertion within the BC, DE, EF and HI VP1 loops. ELISAs were performed using neutralizing anti-MCPyV positive human sera diluted 1:1000. The results are presented as relative binding defined as the reactivity of human serum to mutant VLPs divided by the reactivity of the same human serum observed with wild-type VLPs. The data presented are the means of three determinations (+/- SEM).</p

Sequence of primers used for generation of MCPyV VP1 mutants^a

<p>^a Restriction site sequences are italicized</p><p>Sequence of primers used for generation of MCPyV VP1 mutants<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121751#t001fn001" target="_blank">^a</a></p

MCPyV VP1 insertional mutants BC, DE, EF and HI.

<p><b>A)</b> MCPyV surface exposed loops model generated using the MKT21 sequence with the VP1 structure information of MCPyV w162 strain (4FMG pdb file) by Swiss-Model. The StreptagII motif (WSHPQFEK) coding sequence was inserted into each predicted surface exposed loop, after S88 of BC, after H150 of DE, after T189 of EF and after T296 of HI to generate four insertional mutants, BC, DE, EF and HI, respectively. <b>B)</b> MCPyV VP1 mutant particles observed by transmission electron microscopy after recombinant baculovirus expression and CsCl gradient purification.</p