Cytomegalovirus vaccines fail to induce epithelial entry neutralizing antibodies comparable to natural infection

Abstract

Antibodies that neutralize cytomegalovirus (CMV) entry into fibroblasts are predominantly directed against epitopes within virion glycoproteins that are required for attachment and entry. However, the mechanism of CMV entry into epithelial and endothelial cells differs from fibroblast entry. Using assays that simultaneously measured neutralizing activities against CMV entry into fibroblasts and epithelial cells, we found that human immune sera and CMV-hyperimmuneglobulins have on on average 48-fold higher neutralizing activities against epithelial cell entry compared to fibroblast entry, suggesting that natural CMV infections elicit neutralizing antibodies that are epithelial entry-specific. This activity could not be adsorbed with recombinant gB. The Towne vaccine and the gB/MF59 subunit vaccine induced epithelial entry-specific neutralizing activities that were on on average 28-fold (Towne) or 15-fold (gB/MF59) lower than those observed following natural infection. These results suggest that CMV vaccine efficacy may be enhanced by the induction of epithelial entry-specific neutralizing antibodies.

Figure 1

A GFP-based virus neutralization assay. (A) A stock of BADrUL131-Y4 was two-fold serially diluted and equal aliquots of each dilution were added to ARPE-19 retinal pigmented epithelial cells (E) or MRC-5 fibroblasts (F). (B) Two-fold serial dilutions of human sera or CMV-hyperimmuneglobulin were incubated with a fixed amount of virus for one hour, then replicate volumes were used to infect the two cell types. Samples include a seronegative serum (Neg.) and a convalescent serum (Pos.) obtained 11 years after seroconversion, representative sera from Towne and gB/MF59 vaccine recipients, and CytoGam® pre-diluted 20-fold. Negative controls (ϕ) contained virus but no sera. Representative micrographs were taken with a fixed exposure three days post infection.

Figure 2

Fibroblast and epithelial neutralizing titers compared between different subject groups. The GFP-based assay shown in Fig. 1 was used to measure fibroblast (F) and epithelial (E) IC₅₀ neutralizing titers for sera from human subjects or two CMV-hyperimmuneglobulin (CMV-HIG) products. (A) Ten seronegative random blood donor sera (note: four sera having no neutralizing activity were assigned a value of 1:2 to permit presentation on the graph); (B) eight naturally seropositive random blood donor sera; (C) CytoGam® (squares) and Cytotect CP (triangles); (D) sera from 23 Towne vaccine recipients; (E) sera from ten gB/MF59 vaccine recipients. Lines indicate logarithmic means; brackets represent one standard deviation above and below means.

Figure 3

Ratios of reciprocal epithelial to fibroblast neutralizing titers were calculated from data shown in Fig. 2. CytoGam® (square); Cytotect CP (triangle). Lines indicate logarithmic means; brackets represent one standard deviation above and below means.

Figure 4

Fibroblast and epithelial IC₅₀ neutralizing titers were measured for serial sera from a subject that underwent natural CMV infection after receiving the Towne vaccine. The pre-vaccination sample was seronegative, post-vaccination sample was drawn 2 months after Towne vaccination, and the post-natural infection sample was drawn 12 months post vaccination. A urine sample collected concurrent with the 12 month serum was culture positive for wild type CMV. Lines indicate logarithmic means of triplicate assays on each serum; brackets represent one standard deviation above and below means.