Abstract
Human cytomegalovirus (HCMV) can cause severe clinical disease in immunocompromised individuals, such as allograft recipients and infants infected in utero. Neutralizing activity of antibodies, measured as the ability to prevent the entry of cell-free virus, has been correlated with the reduction in HCMV transmission and the severity of HCMV-associated disease. However, in vivo HCMV amplification may occur mainly via cell-to-cell spread. Thus, quantifying the inhibition of cell-to-cell transmission could be important in the evaluation of therapeutic antibodies and/or humoral responses to infection or immunization. Here, we established a quantitative plaque reduction assay, which allowed for the measurement of the capacity of antibodies to limit HCMV spread in vitro. Using an automated fluorescence spot reader, infection progression was assayed by the expansion of viral plaques during the course of infection with various GFP-expressing viruses. We found that in contrast to non-neutralizing monoclonal antibodies (mAbs), neutralizing mAbs against both glycoprotein B and H (gB and gH) could significantly inhibit viral plaque expansion of different HCMV strains and was equally efficient in fibroblasts as in epithelial cells. In contrast, an anti-pentamer mAb was active only in epithelial cells. Taken together, our data demonstrate that specific anti-HCMV mAbs can significantly limit cell-associated virus spread in vitro.
Highlights
For direct comparison of the data obtained for different target cell types of Human cytomegalovirus (HCMV), the Plaque Reduction Assay (PRA) results were expressed as % reduction of mean plaque size relative to the no inhibitor control (0% inhibition) after subtraction of the initial fluorescent spot size, determined as mean spot size with no inhibitor measured at 4 dpi of the respective experiment
Statistical analysis was performed by ordinary one-way analysis of variance (ANOVA). n.s., not significant; *, p ≤ 0.1; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; p values refer to antibodies vs. no antibody control
Deletion of the pentamer increased plaque size without antibody about two-fold (Figure 3a,e, compare plaque sizes at 8 dpi), so the cell-associated spread is clearly enhanced by this mutation and the mutation somehow contributes to sensitivity to neutralizing antibodies
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Whether the results using virus isolates which are restricted to replication via the cell-to-cell route reflects the situation found in vivo remains to be determined as to the best of our knowledge unbiased studies analyzing a representative number of recent virus isolates with respect to their production of cell-free virus and resistance to antibody neutralization are lacking In light of this imponderability, we have chosen for our study a more comprehensive but less specific term of “cell-associated virus spread” that defines the spread from an infected cell to adjacent cells under a semi-solid overlay containing virus-neutralizing antibodies. To this end, we have developed an observer bias-independent automated quantification system for the systematic analysis of antibody-mediated inhibition of plaque formation. The inhibitory activity of a pentamerspecific mAb was restricted to epithelial cells, while all analyzed anti-gB and anti-gH mAbs limited cell-associated HCMV spread in HFF fibroblasts and ARPE-19 epithelial cells with comparable efficiency
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