# Long-Term Administration of Valacyclovir Reduces the Number of Epstein-Barr Virus (EBV)-Infected B Cells but Not the Number of EBV DNA Copies per B Cell in Healthy Volunteers

## ABSTRACT

*P*= 0.02) but not in controls (half-life of 31 years;

*P*= 0.86). The difference in the slopes of the lines for the number of EBV-infected B cells over time for the valacyclovir group versus the control group approached significance (

*P*= 0.054). In contrast, the number of EBV DNA copies per B cell remained unchanged in both groups (

*P*= 0.62 and

*P*= 0.92 for the control and valacyclovir groups, respectively). Valacyclovir reduces the frequency of EBV-infected B cells when administered over a long period and, in theory, might allow eradication of EBV from the body if reinfection does not occur.

^{5}peripheral B cells are infected with EBV (14). The virus establishes latency in memory B cells. The level of the latent EBV load in healthy individuals remains stable over time, maintaining a “set point” for each individual (19). It is uncertain how this “set point” is maintained, but the latent EBV load is thought to reflect a balance between removal of EBV-infected cells due to the half-life of memory B cells and reinfection of new memory B cells during virus reactivation. The EBV genome replicates when B cells latently infected with EBV divide using the host DNA polymerase, which is not sensitive to the action of acyclovir. However, when the virus reactivates in latently infected B cells, EBV replicates using the viral DNA polymerase, which is inhibited by the phosphorylated form of acyclovir. Therefore, blocking production of new virus with acyclovir should decrease the latent EBV load at a rate equivalent to the half-life of memory B cells. Patients with zoster who were treated with oral acyclovir for 28 days showed no reduction in the EBV load in the blood, despite complete inhibition of EBV shedding in the saliva (23). These results suggested that antiviral therapy for longer than 28 days is necessary to detect a reduction in the EBV load in the blood.

## MATERIALS AND METHODS

### Subjects.

### Isolation of DNA from B cells and real-time PCR.

^{4}cells but mild to modest inhibition with more than 5 × 10

^{4}cells/well. Therefore, the concentration of cells was adjusted to 4,000 cells per 5 μl (some experiments used 1,500 to 8,000 cells per 5 μl), and 5 μl of B cells was plated into wells of a 96-well real-time PCR plate and stored at −20°C. For all specimens, a small portion of leftover cell suspension was stained with anti-CD20 monoclonal antibody conjugated with phycoerythrin (BD Pharmingen, San Diego, CA), and the purity of B cells was determined by flow cytometry. In most experiments, the fraction of B cells in each cell suspension was >80%. All plates from the same subject were thawed at the same time, and 5 μl of buffer containing proteinase K and Tween 20 was added to each well to disrupt cells as previously described (13). Quantitative real-time PCR amplification was performed by adding 15 μl of PCR master mix (Eurogentec North America, San Diego, CA) containing EBV BamHI W primers and probes (12) to each well of B-cell DNA. The limit of detection using this PCR system is ∼4 copies per reaction, based on experiments using standard curves obtained with serial dilutions of a plasmid with a single copy of BamHI W. Since EBV genomes contain 5 to 12 copies of BamHI W repeats (1) and each infected cell contains at least one EBV genome, this system has the potential to detect a single EBV-infected cell per well. Since we did not know the number of BamHI W repeats for each patient's virus, we expressed the data as the number of EBV DNA copies per infected cell. We compared each patient to him- or herself over time, and the number of copies of BamHI W repeats per viral genome is not likely to change in a given patient.

### Calculations to determine the number of EBV-infected cells and average EBV genome copy number per infected cell.

*n*and the number of EBV-positive wells as

*y*. We assume that the number of EBV-infected cells per well follows a Poisson distribution with parameter

*a*. Assuming a Poisson distribution for the number of EBV-positive cells within a well, the probability that a well has at least one positive cell is 1 − e

^{−}

^{a}(where e is the base of the natural logarithm). Thus, an estimate of

*a*is obtained by equating the observed proportion of positive wells, or

*y*/

*n*, to 1 − e

^{−}

^{a}. Solving this equation for

*a*gives

*a*= −ln(1 −

*y*/

*n*). For plates without any positive wells, i.e.,

*y*= 0, this estimate is 0 regardless of the value for the number of total wells (

*n*). However, plates with many wells have a more reliable 0 than plates with few wells. We thus smoothed the data to address this issue and pretended that each plate had one additional well that was 0.5 positive and estimated

*a*as follows:

*a*= −ln[1 − (

*y*+ 0.5)/(

*n*+ 1)]. Instead of estimating the proportion of positive wells by the ratio

*y*/

*n*, we used (

*y*+ 0.5)/(

*n*+ 1), which is the Bayesian posterior mean of the proportion of negative wells under a Jeffreys noninformative prior (5). Therefore, the estimation of the number of EBV-positive cells/well is as follows:

*a*= −ln[1 − (

*y*+ 0.5)/(

*n*+ 1)]. This can be viewed loosely as the “middle” of a confidence interval for the true proportion of positive wells. This smoothing method gives modestly higher estimates for plates with positive wells and has values closer to zero for totally negative plates with many wells compared to totally negative plates with few wells.

*q*=

*a*/(

*m*×

*p*/100), where

*m*is the number of cells per well and

*p*is the purity of B cells (%). We thus estimate

*q*as follows:

*q*=

*a*/(

*m*×

*p*/100) = −ln[1 − (

*y*+ 0.5)/(

*n*+ 1/)]/(

*m*×

*p*/100). To estimate the average number of EBV DNA copies per infected cell, the sum of all EBV genomes in a plate (

*S*) was determined directly from real-time PCR results and was divided by an estimate of the number of EBV-positive B cells in the plate (

*a*×

*n*). For example, if a plate contained three EBV-positive wells, with 110, 58, and 152 EBV copies,

*S*equaled 320. See Table 1 for an example.

## RESULTS

### Development of a technique to estimate the frequency of EBV-positive cells and the average number of EBV DNA copies/B cell in healthy persons.

^{−0.25}) × 100]. Therefore, we expected only 1 EBV-positive cell per well when the cells were plated at a concentration of 0.25 EBV-positive cells/well. Since we were able to detect EBV DNA in some wells with an average of 0.25 EBV-positive cells per well, our real-time PCR system was able to detect single EBV-positive cells in a well.

*a*= −ln[1 − (8 + 0.5)/(56 + 1)] = 0.161 (Table 2, column 4 and footnote

*a*). Based on the estimate of the number of EBV-positive cells/well (0.161), the number of cells added to each well (2,500), and the purity of the B cells (87.9%) (Table 2, columns 4, 5, and 6, respectively, and footnote

*a*), there was an estimated (0.161 × 10

^{7})/(2,500 × 87.9) = 7.35 EBV-positive cells/10

^{5}B cells (Table 2, column 7 and footnote

*b*). Using the total number of wells (56), the estimate of the number of EBV-positive cells/well (0.161), and the sum of EBV DNA copies in the plate (2,062) (Table 2, columns 2, 4, and 8, respectively), there was an average of 2,062/(0.161 × 56) = 229 EBV DNA copies/infected B cell (Table 2, column 9 and footnote

*c*), since our primers and probe for real-time PCR detect each copy of the BamHI W repeat. In a second experiment using cells from the same donor drawn on the same day, 9 of 32 wells were positive for EBV, with an estimated 11.98 EBV-positive cells/10

^{5}B cells and an average of 132 EBV DNA copies/infected B cell. In experiments with cells from blood bank donors B, C, and D, the numbers of EBV-infected B cells and copies of EBV DNA per B cell were similar for a given donor from the same day in independent experiments, with different numbers of wells and purities of B cells (Table 2). The mean number (± standard deviation) of EBV-positive cells per 10

^{5}B cells for donors A, B, C, and D was 9.7 ± 3.3, 2.2 ± 0.6, 16.1 ± 1.2, and 15.7 ± 4.3, respectively. The mean number (± standard deviation) of EBV DNA copies per infected cell for donors A, B, C, and D was 181 ± 69, 147 ± 46, 97 ± 36, and 112 ± 27, respectively. Thus, this method provides an estimation of the frequency of EBV-positive cells and the average number of EBV DNA copies/B cell at low viral loads with reasonable reproducibility.

### The number of EBV-infected B cells declines during valacyclovir treatment in healthy persons.

^{5}B cells was plotted against time, in months, for the first three patients enrolled in each group (Fig. 1). While there was variation during the five time points, we were able to plot a linear regression line for each subject. We then determined linear regression lines and slopes for the number of EBV-positive cells per 10

^{5}B cells over time for all of the subjects in the study (Fig. 2; data used to generate regression lines are shown in the supplemental material). There was considerable variability in the starting value (

*y*intercept) of each line as well as in the slope of the regression line among subjects even within the same group. The average of the regression lines (thick gray lines in Fig. 2A) was relatively flat for the control group but had a negative slope for the valacyclovir group. The mean of the slopes for the valacyclovir-treated group was −2.8 × 10

^{−2}± 1.2 × 10

^{−2}/month and was statistically different from a slope of 0 (

*P*= 0.02), while the mean of the slopes for the control group was −0.1 × 10

^{−2}± 0.8 ×10

^{−2}/month, which was not statistically different from 0 (

*P*= 0.86) (Fig. 2B). Comparison of the slopes of regression lines between the two treatment groups showed that the difference approached statistical significance (

*P*= 0.054; Wilcoxon test). The slopes of the control and valacyclovir groups were significantly different using regression analysis, with the viral load (log

_{10}) as outcome and month as the covariate (

*P*= 0.03 for common delta), and when analyzed using analysis of covariance, with slope as outcome and intercept as the covariate (

*P*= 0.04 for common delta). These findings indicate that the number of EBV-infected B cells declined over time in the valacyclovir treatment group.

### The EBV DNA copy number per infected B cell does not decline during valacyclovir treatment in healthy persons.

*P*= 0.62 and

*P*= 0.92 for the control and valacyclovir groups, respectively), and the difference in mean slopes between the groups was not significant (

*P*= 0.66). Thus, the mean EBV DNA copy number per infected cell (log

_{10}) is stable over time and is not affected by 1 year of valacyclovir therapy.

## DISCUSSION

^{−2}± 1.2 ×10

^{−2}/month) was 11 months, and the half-life calculated from median slopes was 12 months. In the control group, the half-life of EBV-infected B cells calculated from mean slopes (−0.1 × 10

^{−2}± 0.8 ×10

^{−2}/month) was 31 years, and that calculated from the median slopes was 41 years. There are several possibilities that may explain the discrepancy between our results and those of the prior study. First, Hadinoto et al. (11) measured the half-life of EBV in the blood during convalescence from mononucleosis, whereas our subjects presumably had been infected many years earlier. Balfour and colleagues (3) and Fafi-Kremer et al. (9) showed that the level of EBV in PBMCs was still elevated 6 weeks after primary infection. Second, one dose of valacyclovir each day is unlikely to completely block EBV reactivation and virus production. While the bioavailability of valacyclovir is about three to five times higher than that of acyclovir, valacyclovir is converted into acyclovir in the body, and the mean plasma elimination time of acyclovir is 1.5 to 6.3 h (4, 15). Third, it is possible that latent infection of memory B cells with EBV prolongs the half-life of these cells or that EBV may establish latency preferentially in a subset of memory B cells that has a longer life span. Fourth, compared with EBV-infected B cells from patients convalescing after infectious mononucleosis, EBV-infected B cells in our subjects might be more heterogeneous, with some infected cells resting, others actively proliferating, and a small minority undergoing lytic replication. Since valacyclovir affects only lytic replication of EBV, the marked differences in the decline of EBV copy numbers in different valacyclovir recipients (Fig. 2) might reflect differences in the relative proportions of resting, proliferating, and lytically infected cells. Nonetheless, the general reduction of EBV-infected B cells over time in the valacyclovir group suggests that infection by EBV of previously uninfected B cells is one of the mechanisms that maintains the latent EBV pool in healthy adults.

No. of X50-7 cells/well | Total no. of wells | No. of EBV- positive wells | Observed frequency of EBV-positive wells^{a} | Predicted frequency of EBV-positive wells^{b} | No. of EBV DNA copies/infected cell^{c} |
---|---|---|---|---|---|

0.00 | 16 | 0 | 0.00 | 0.00 | NA |

0.25 | 24 | 3 | 0.13 | 0.22 | 19.3 |

1.00 | 24 | 12 | 0.50 | 0.63 | 28.4 |

4.00 | 24 | 23 | 0.96 | 0.98 | 18.9 |

^{a}

^{b}

^{c}

*c*, and in the text. NA, not applicable.

Donor | Total no. of wells (n) | No. of EBV-positive wells (y) | Estimation of no. of EBV-positive cells/well^{a} (a) | No. of cells added to well (m) | Purity of B cells (%) (p) | No. of EBV- positive cells/10^{5} B cells^{b} (q) | Sum of EBV DNA copies (S) | No. of EBV DNA copies/infected cell^{c} |
---|---|---|---|---|---|---|---|---|

A | 56 | 8 | 0.161 | 2,500 | 87.9 | 7.35 | 2,062 | 229 |

32 | 9 | 0.340 | 3,000 | 94.5 | 11.98 | 1,432 | 132 | |

B | 44 | 2 | 0.057 | 3,500 | 95.6 | 1.71 | 449 | 179 |

88 | 8 | 0.100 | 4,000 | 96.4 | 2.60 | 1,003 | 114 | |

C | 88 | 38 | 0.567 | 4,000 | 83.5 | 16.96 | 3,583 | 71.8 |

38 | 14 | 0.465 | 4,000 | 76.2 | 15.25 | 2,168 | 123 | |

D | 44 | 18 | 0.530 | 3,310 | 94.0 | 17.02 | 3,136 | 135 |

44 | 13 | 0.357 | 3,430 | 92.4 | 11.25 | 1,909 | 122 | |

88 | 34 | 0.491 | 3,570 | 92.4 | 14.87 | 2,354 | 123 | |

85 | 29 | 0.420 | 3,620 | 89.7 | 12.94 | 1,317 | 66 | |

88 | 50 | 0.838 | 4,000 | 94.3 | 22.22 | 3,862 | 114 |

^{a}

*a*= −ln[1 − (

*y*+ 0.5)/(

*n*+ 1)], where

*n*is the total number of wells in the plate (second column) and

*y*is the number of EBV-positive wells (third column).

^{b}

^{5}B cells,

*q*= (

*a*× 10

^{7})/(

*m*×

*p*), where

*a*is the estimation of the number of EBV-positive cells/well (fourth column),

*m*is the number of cells in a well (fifth column), and

*p*is the percent purity of B cells (sixth column). Each well contains (

*m*×

*p*)/100 B cells.

^{c}

*S*/(

*a*×

*n*), where

*S*is the sum of all EBV genome numbers from a plate (eighth column; obtained directly from real-time PCR results),

*a*is the estimation of the number of EBV-positive cells/well (fourth column), and

*n*is the number of wells in the plate (second column). Therefore, (

*a*×

*n*) is the number of EBV-positive cells in the plate.

## Acknowledgments

## Supplemental Material

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