The modified surface killing assay distinguishes between protective and nonprotective antibodies to PspA

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Pneumococcal surface protein A (PspA) elicits antibody protective against lethal challenge by Streptococcus pneumoniae and is a candidate noncapsular antigen for inclusion in vaccines. Evaluation of immunity to PspA in human trials would be greatly facilitated by an in vitro functional assay able to distinguish protective from nonprotective antibodies to PspA. Mouse monoclonal antibodies (MAbs) to PspA can mediate killing by human granulocytes in the modified surface killing assay (MSKA). To determine if the MSKA can distinguish between protective and nonprotective MAbs, we examined seven MAbs to PspA. All bound recombinant PspA, as detected by enzyme-linked immunosorbent assay and Western blotting; four gave strong passive protection against fatal challenge, two were nonprotective, and the seventh one only delayed death. The four that were able to provide strong passive protection were also most able to enhance killing in the MSKA, the two that were not protective in mice were not effective in the MSKA, and the MAb that was only weakly protective in mice was weakly effective in the MSKA (P < 0.001). One of the four most protective MAbs tested reacted to the proline-rich domain of PspA. Two of the other most protective MAbs and the weakly protective MAb reacted with a fragment from PspA’s a-helical domain (aHD), containing amino acids (aa) 148 to 247 from the N terminus of PspA. The fourth highly protective MAb recognized none of the overlapping 81- or 100-aa fragments of PspA. The two nonprotective MAbs recognized a more N-terminal aHD fragment (aa 48 to 147). IMPORTANCE The most important finding of this study is that the MSKA can be used as an in vitro functional assay. Such an assay will be critical for the development of PspA-containing vaccines. The other important findings relate to the locations and nature of the protection-eliciting epitopes of PspA. There are limited prior data on the locations of protection-eliciting PspA epitopes, but those data along with the data presented here make it clear that there is not a single epitope or domain of PspA that can elicit protective antibody and there exists at least one region of the aHD which seldom elicits protective antibody. Moreover, these data, in concert with prior data, strongly make the case that protective epitopes in the aHD are highly conformational (=100-amino-acid fragments of the aHD are required), whereas at least some protection-eliciting epitopes in the proline-rich domain are encoded by =15-amino-acid sequences.
Genschmer KR., Vadesilho CFM, McDaniel LS., Park S-S, Hale Y, Miyaji EN, et al. The modified surface killing assay distinguishes between protective and nonprotective antibodies to PspA. mSphere. 2019 Dec;4(6):e00589-19. doi:10.1128/mSphere.00589-19.
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