PMC

BALB/c mice (n = 5) were i.p. immunized with either MVA-PC alone (5x10⁷ PFU), purified PC protein alone (1 μg, admixed with AddaVax), or the indicated prime-boost regimen. Shown are NAb titers (NT50) following two (A and C) or three (B and C) immunizations that were measured against HCMV TB40/E on ARPE-19 EC. Black triangles indicate immunizations. Bars represent 95% confidence interval of the geometric mean. Floating bars in C represent minimum, maximum, and mean NT50 titers measured on EC at week 16 in the two- and three-immunization regimens. Shown is multiple t-test statistical analysis of NAb titers. (** = p≤0.01, * = p≤0.05).

Balb/c mice (n = 4) were immunized three-times by intramuscular (i.m.), intraperitoneal (i.p.), or subcutaneous (s.c.) route with 1 μg of purified PC protein admixed with AddaVax adjuvant. Control animals (n = 3) were immunized i.p. using AddaVax alone. Serum NAb titers (NT50) were evaluated against TB40/E on ARPE-19 EC (A) and MRC-5 FB (B) by microneutralization assay at multiple time points over a period of 28 weeks. Black triangles indicate immunizations. Bars represent 95% confidence interval of the geometric mean. PC-specific binding antibody titers (EC50) at week 11 and 28 (C) were analyzed by ELISA. Differences between groups were evaluated using multiple t-test (* = p≤0.05, ** = p≤0.01).

A. Size exclusion chromatography (SEC) on HCMV PC purified from cell supernatant with a 12E2 IgG resin. B. SDS-PAGE analysis of PC 12E2 affinity purification eluate and SEC fractions collected in (A) under reducing conditions. kDa = kilo Dalton; FT = flow through, T = test eluate, P = final prep eluate. C. ELISA binding of PC- and gH-specific NAb to the purified PC. NAb targeting different epitopes of the UL128/130/131A subunits (1B2, 54E11, 21F6, 13B5) or gH (18F10, 62–11, 62–100, AP86) were tested in saturating amounts to bind the purified PC protein via ELISA. Error bars represent difference in binding of three independent experiments. D. Inhibition of EC entry by purified PC protein. Serial dilutions of purified PC protein or, anti-PC NAb 1B2, or anti-gH NAb 62–11 were pre-incubated with HCMV TB40/E and subsequently evaluated via microneutralization assay using ARPE-19 EC to determine the protein/antibody concentration at which 50% HCMV infection was neutralized (NT50). E and F. Antibody depletion by purified PC. Commercially available serum products of HCMV seropositive (Seracare 71 and 60) and seronegative (Seracare neg.) individuals or intravenous hyperimmune globulins (IvIg) were serially incubated in ELISA plate wells coated with the purified PC or with mock (1% BSA/PBS). Depleted and mock-depleted samples were tested by ELISA and microneutralization to determine PC-specific binding antibodies (E) and NAb titer (NT50) that block TB40/E infection of ARPE-19 EC (F), respectively.