Cell-to-Cell Spread Blocking Activity Is Extremely Limited in the Sera of Herpes Simplex Virus 1 (HSV-1)- and HSV-2-Infected Subjects

Abstract

Herpes simplex virus 1 (HSV-1) and HSV-2 can evade serum antibody-mediated neutralization through cell-to-cell transmission mechanisms, which represent one of the central steps in disease reactivation. To address the role of humoral immunity in controlling HSV-1 and HSV-2 replication, we analyzed serum samples from 44 HSV-1 and HSV-2 seropositive subjects by evaluating (i) their efficiency in binding both the purified viral particles and recombinant gD and gB viral glycoproteins, (ii) their neutralizing activity, and (iii) their capacity to inhibit the cell-to-cell virus passage in vitro All of the sera were capable of binding gD, gB, and whole virions, and all sera significantly neutralized cell-free virus. However, neither whole sera nor purified serum IgG fraction was able to inhibit significantly cell-to-cell virus spreading in in vitro post-virus-entry infectious assays. Conversely, when spiked with an already described anti-gD human monoclonal neutralizing antibody capable of inhibiting HSV-1 and -2 cell-to-cell transmission, each serum boosted both its neutralizing and post-virus-entry inhibitory activity, with no interference exerted by serum antibody subpopulations.IMPORTANCE Despite its importance in the physiopathology of HSV-1 and -2 infections, the cell-to-cell spreading mechanism is still poorly understood. The data shown here suggest that infection-elicited neutralizing antibodies capable of inhibiting cell-to-cell virus spread can be underrepresented in most infected subjects. These observations can be of great help in better understanding the role of humoral immunity in controlling virus reactivation and in the perspective of developing novel therapeutic strategies, studying novel correlates of protection, and designing effective vaccines.

Keywords: cell-to-cell virus spread; herpes simplex virus; human monoclonal antibodies; humoral immunity; neutralizing activity; serum neutralizing antibodies.

FIG 1

Binding analysis of 87 human sera to HSV-1 and -2. A threshold OD₄₅₀ of 0.5 was set to stratify binding activity of 87 human sera against HSV-1 HF and HSV-2 MS purified virions coated on an ELISA plate. In detail, a panel of 5 sera (A to E) from healthy uninfected subjects was tested for HSV-1 and -2 binding activity. The cutoff corresponding to an OD₄₅₀ of 0.5 was set at 2.5 times the higher OD signal registered for the negatives in order to minimize the binding contribution of possible antibody cross-reactivity. Serum E represents the nonneutralizing (non-NT) control for the experiments. Blue boxes indicate positive sera, and white boxes indicate negative ones. Moreover, sera were stratified into six categories: sera able to bind only HSV-1 (light red) or only HSV-2 (light green), sera that bind both viruses equally (gray), sera that preferentially bind HSV-1 over HSV-2 (red) and vice versa (green), and negative sera (black). Positive and negative control sera were added as well.

FIG 2

Soluble protein purification. His-tagged soluble forms of HSV-1 glycoprotein D (gD1) and B (gB1) were affinity purified. Soluble HCV envelope protein E2 (HCV E2) was purified as a control. Protein stocks were validated using human monoclonal antibodies specific for the glycoproteins and a primary antibody specific for His-tagged proteins. Means ± standard deviations (SD) are reported.

FIG 3

Characterization of binding to purified virions and soluble glycoproteins. Graphs show the results from binding analysis of 44 sera to HSV-1 (▲) and -2 virions (●) (A) and soluble glycoprotein D (■) and B (◆) of HSV-1 (B). Sera were used at a 1:200 dilution. The median optical density (OD₄₅₀), with interquartile range, is reported for each binding analysis (***, P < 0.001; ****, P < 0.0001). Each data point is represented by its stratification color: sera able to bind only HSV-1 (light red) or only HSV-2 (light green), sera that bind both viruses equally (gray), sera that preferentially bind HSV-1 over HSV-2 (red) and vice versa (green), and negative sera (black).

FIG 4

Evaluation of serum activity against HSV-1 and -2 infections. (A) Neutralizing (NT) activity of 44 sera was assessed on Vero E6 cells, and results show that HSV-1 (▲) was more efficiently blocked than HSV-2 (●). (B) When tested in PEI assay on the same cells, no samples (except one) impaired virus replication efficiently, although higher activity again was observed against HSV-1 (▲) than HSV-2 (●). Sera were used at 1:200 dilution. Median values with interquartile ranges are reported for each experiment (****, P < 0.0001). Each data point is represented by its stratification color: sera able to bind only HSV-1 (light red) or only HSV-2 (light green), sera that bind both viruses equally (gray), sera that preferentially HSV-1 over HSV-2 (red) or vice versa (green), and negative sera (black).

FIG 5

Evaluation of purified IgG activity against HSV-1 and -2 infections. PEI assay was performed on Vero E6 cells using lower dilutions of the neutralizing (NT) serum (1:100) control and purified total IgGs obtained from the same serum sample (100 μg/ml). Nonneutralizing (non-NT) serum and its purified total IgG fraction were used as a negative control. Mean values ± SD are reported for each experimental condition (***, P < 0.001; ****, P < 0.0001). Titration results for anti-gD and anti-gB present in NT and non-NT sera and their corresponding purified IgG fractions are indicated under the graph. Median values are reported.

FIG 6

Titration of specific serum IgGs. (A) IgG titers specific for gB (●) and gD (△) were obtained for 44 serum samples. Neutralizing (NT) and nonneutralizing (non-NT) serum controls are included as well. Median values are reported. Anti-gB (B) and anti-gD (C) antibodies were quantified in ELISA by performing titration curves on purified soluble glycoproteins (gB1 and gD1) in order to evaluate the amount of serum IgG specific for these glycoproteins in the 44 selected sera. Control sera (NT serum and non-NT serum) are included as well. Means are reported; SD are omitted for clarity.

FIG 7

Graph of correlation matrix in R. Matrix heatmap plot corresponding to ρ correlation values between categories corresponding to parameters analyzed for the sera: binding to gD and gB, neutralizing activity, anti-gD and gB antibody titers, and postentry inhibition of the infection. Color scale indicates correlation values, with positive correlations assigned to red colors. X indicates P_adj value of <0.05. *, P_adj = 0.053. NT, neutralization assay. Roman numbers indicate 1:200 (I) and 1:400 (II) serum dilutions. Dotted boxes highlight how overall biological activities correlate with the binding to gD but not to gB.

FIG 8

Competition evaluation. Dose-dependent binding competitions between sera and IgG#33 were performed. FACS analyses were performed using IgG#33 combined with serial dilutions (1:100 to 1:800) of both neutralizing (NT) serum (A) and nonneutralizing (non-NT) (B) serum to evaluate the possible competitive binding of antibodies to gD1-transfected cells. As shown by the graphs, no competition resulted even with the lower serum dilution tested. NT (C) and PEI (D) assays next were performed on Vero E6 cells with NT serum and a non-NT serum supplemented by IgG#33 against both HSV-1 and -2 infections. In NT assays, IgG#33 was used at its 50% inhibitory concentration (1.092 μg/ml against HSV-1 and 0.731 μg/ml against HSV-2 tested isolates), while in PEI assays it was used at 25 μg/ml and 100 μg/ml against HSV-1 and -2, respectively. Sera were used at 1:100 dilution, and the MAb alone control is indicated by white columns. Mean values ± SD are reported for each experimental condition (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).