CMV specific cytokine release assay in whole blood is optimized by combining synthetic CMV peptides and toll like receptor agonists.

BACKGROUND

Interferon gamma release assays (IGRAs) are widely used to detect pathogen specific cellular immunity. Cytomegalovirus (CMV) is the foremost problematic viral infection in immunocompromised patients such as transplant or HIV infected patients. CMV antibody ELISAs are not able to predict CMV specific cellular immunity during immunosuppression. We developed a CMV specific IGRA comparing synthetic CMV peptides, native lysate and recombinant antigen. In addition, TLR agonists were tested to enhance CMV antigen immunogenicity.

METHODS

397 healthy controls (HC) were stratified according to CMV IgM and IgG serostatus and subsequently tested for IFNγ- and IL2-secretion in whole blood after challenge with synthetic, native or recombinant CMV antigens and TLR agonists by ELISA. The selected TLR agonists were lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN), zymosan (Zym), polyinosinic-polycytidylic acid (Poly(I:C)), flagellin (Fla), R848, loxoribine (Lox) and bropirimine (Bro).

RESULTS

Synthetic pp65 peptides elicited strong IFNγ responses in CMV seropositive, but not seronegative HC (6418 vs. 13 pg/ml). Native lysates and recombinant pp65 induced equally high IFNγ responses in seropositive (35,877 and 26,428 pg/ml) and increased background IFNγ expression in seronegative HC (43 and 1148 pg/ml). Diagnostic sensitivity and specificity with regard to anti-CMV serology reached 100% for synthetic pp65 and native CMV lysate, but 57% and 100% for recombinant pp65, respectively. TLR agonists LTA and Poly(I:C) augmented IFNγ responses after challenge with synthetic pp65 peptide, native lysate or recombinant pp65 in seropositive HC. Seronegative HC remained unaffected. IL2 production was negligible compared to IFNγ.

CONCLUSION

IGRAs using synthetic CMV peptides or native lysate showed the best cytokine signal to noise ratio compared to recombinant antigen and TLR agonists LTA and Poly(I:C) constitute potential costimulating reagents.