Figures and data in Rapid localized spread and immunologic containment define Herpes simplex virus-2 reactivation in the human genital tract

Version of Record: April 16, 2013

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Joshua T Schiffer

David Swan

Ramzi Al Sallaq

Amalia Magaret

Christine Johnston

Karen E Mark

Stacy Selke

Negusse Ocbamichael

Steve Kuntz

Jia Zhu

Barry Robinson

Meei-Li Huang

Keith R Jerome

Anna Wald

Lawrence Corey

(2013)
Rapid localized spread and immunologic containment define Herpes simplex virus-2 reactivation in the human genital tract

eLife 2:e00288.

https://doi.org/10.7554/eLife.00288
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Figures
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Tables

6 figures, 9 videos and 6 tables

Figures

Figure 1 with 3 supplements

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HSV-2 levels in the genital tract are stable over minutes, expand and decay markedly over hours, and fluctuate rapidly and unpredictably over days.

(A) Shedding quantity in a participant, who performed genital swabs every 5 min over 4 hr during a lesion, reveals low swab-to-swab variation in viral quantity. Using data from panel (A and B), …
https://doi.org/10.7554/eLife.00288.007

Figure 1—source data 1 Source data for Figure 1, Figure 1—figure supplement 1, Figure 1—figure supplement 2 and Figure 1—figure supplement 3.: https://doi.org/10.7554/eLife.00288.008
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Figure 1—figure supplement 1

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Dynamics of HSV-2 shedding over 5-min time intervals.

(A) and (B) Shedding quantity in two participants, who performed genital swabs every 5 min over 4 hr during a lesion, reveals low swab-to-swab variation in viral quantity. (C) Using data from panels …
https://doi.org/10.7554/eLife.00288.009

Figure 1—figure supplement 2

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Dynamics of HSV-2 shedding with every 2-4 hr sampling.

Shedding quantity in four participants, who performed 10 genital swabs per day over 4–5 days during a lesion reveal a characteristic saw-tooth pattern; arrows denote re-expansion. Participants had …
https://doi.org/10.7554/eLife.00288.010

Figure 1—figure supplement 3

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Dynamics of HSV-2 shedding with every 6-hr sampling over 8 days.

Shedding quantity in episodes detected in a participant who performed four genital swabs per day over 60 days demonstrates that viral re-expansion allows for shedding prolongation.

https://doi.org/10.7554/eLife.00288.011

Figure 2 with 3 supplements

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HSV-2 replicates and is contained in widely dispersed microenvironments across the genital tract.

(A) HSV shedding quantity in a participant, who underwent daily swabs in 23 regions across the genital tract for 30 days; days without sampling are marked with an X; stars denote days with a lesion; …
https://doi.org/10.7554/eLife.00288.013

Figure 2—source data 1 Source data for Figure 2 and Figure 2—figure supplement 1.: https://doi.org/10.7554/eLife.00288.014
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Figure 2—figure supplement 1

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Spatial features of HSV genital tract shedding.

HSV shedding quantity in a study participant, who underwent daily swabs in 23 regions across the genital tract for 30 days; days without sampling are marked with an X. The participant had three …
https://doi.org/10.7554/eLife.00288.015

Figure 2—figure supplement 2

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Spatial features of HSV-2 lesions.

A genital lesion consists of numerous round ulcers or vesicles (black dotted circle), clustered in space.

https://doi.org/10.7554/eLife.00288.016

Figure 2—figure supplement 3

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Spatial features of CD8+ T-cell response in genital skin.

Immunofluorescent staining of a biopsy performed (A) at the edge, and (B) 1 cm away from an ulcer 3 days post-healing. CD8+ T-cells (red) and CD4+ T-cells (green) at the dermal epidermal junction …
https://doi.org/10.7554/eLife.00288.017

Figure 3 with 1 supplement

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Mathematical model.

(A) Microregions are linked virally because cell-free HSV-2 can seed surrounding regions, and immunologically based on overlapping CD8+ T-cell densities between regions (not shown). (B) Schematic …
https://doi.org/10.7554/eLife.00288.019

Figure 3—figure supplement 1

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Spatial mathematical model.

Viruses produced from neurons (green), cell-associated viruses from epidermal cells (yellow), and cell-free viruses (orange) that form after rupture of epidermal cells, are distinguished in the …
https://doi.org/10.7554/eLife.00288.020

Figure 4 with 1 supplement

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The spatial model reproduces all shedding episode characteristics.

Colored bars represent results from (A) 14,685 genital swabs and (B–G) 1020 shedding episodes from 531 study participants. The model simulation, represented with black bars in each panel, continued …
https://doi.org/10.7554/eLife.00288.021

Figure 4—source data 1 Source data for Figure 4.: https://doi.org/10.7554/eLife.00288.022
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Figure 4—figure supplement 1

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Continuous sampling of spatial model output reveals more accurate measures of episode characteristics.

We subjected a 30-year simulation to daily and continuous sampling. (A) Median initiation to peak slope, and (B) peak to termination slopes increased substantially with continual sampling. (C) …
https://doi.org/10.7554/eLife.00288.023

Figure 5

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Containment of infected cells within a single ulcer is extremely rapid, although secondary ulcers explain prolonged episodes.

(A) Episode duration was a function of the number of ulcers before episode termination during 500 simulated episodes. (B)–(E) A 10-day simulated episode consisting of 24 ulcers: (B) Total cell-free …
https://doi.org/10.7554/eLife.00288.026

Figure 6 with 2 supplements

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Random spatial dispersion of viral particles from neurons reproduced the full diversity of episode characteristics if particles were released continuously, daily, or weekly from neurons.

White circles represent results from (A) 14,685 genital swabs and (B–G) 1020 shedding episodes from 531 study participants (Figure 4). The model simulations represented with colored bars in each …
https://doi.org/10.7554/eLife.00288.036

Figure 6—figure supplement 1

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Random spatial dispersion of viral particles from neurons reproduced the full diversity of episode characteristics during simulations in which particles were released into only a minority of the 300 modeled regions.

White circles represent results from (A) 14,685 genital swabs and (B–G) 1020 shedding episodes from 531 study participants (Figure 4). The model simulations represented with colored bars in each …
https://doi.org/10.7554/eLife.00288.037

Figure 6—figure supplement 2

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Dispersion of viral particles from neurons reproduced the full diversity of episode characteristics during simulations in which particles were released into a minority of modeled regions, provided that dispersion was random rather than clustered within a single geographic region.

White circles represent results from (A) 14,685 genital swabs and (B–G) 1020 shedding episodes from 531 study participants (Figure 4). The model simulations represented with colored bars in each …
https://doi.org/10.7554/eLife.00288.038

Videos

Video 1

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posterframe for video — Spatiotemporal demonstration of a 10-day episode.

The left panel represents total cell-free HSV DNA copies per milliliter present over time. The right panel represents spatial spread of virus during the episode, each hexagon contains one region of …
https://doi.org/10.7554/eLife.00288.025

Video 2

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Video 3

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Video 4

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Video 5

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Video 6

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Video 7

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Video 8

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Video 9

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Tables

Table 1

Five cohorts of HSV-2 genital tract shedding

https://doi.org/10.7554/eLife.00288.003

Cohort	Subjects	Total swabs	Swabbing frequency	Total episodes	Swabbing duration	Anatomic swabbing region	Purpose
A	3	96	Every 5 min	3	4 hr when lesion present	Total genital tract	Swab-to-swab sampling/assay variability
B	5	200	10 times/day (every 2 hr during the days and 4 hr overnight)	5	4–5 days when lesion present	Total genital tract	Episode expansion, clearance and re-expansion kinetics
C	25	4706	4 times/day	109	30–60 days without or with a lesion	Total genital tract	Accurate estimates for expansion/decay slopes for clinical and subclinical episodes
D	2	216	Daily	4	30 days without or with a lesion	23 separate regions	Spatial dispersion of HSV
E	531	14,685	Daily	1020	>30 days with or without a lesion	Total genital tract	Model fit

Table 2

Parameter ranges that result in accurate reproduction of model outcomes

https://doi.org/10.7554/eLife.00288.024

Parameter	Units	Symbol	Best fit value	Good fit		Average fit
Parameter	Units	Symbol	Best fit value	Lower limit	Upper limit	Lower limit	Upper limit
Cell-associated HSV infectivity	DNA copy days/cell (viruses needed per day to infect one adjacent cell)	β_i	5.4e⁻⁸ (111)	4.86e⁻⁸ (123)	7.83e⁻⁸ (76)	3.78e⁻⁸ (158)	1.32e⁻⁷ (45)
Cell-free HSV infectivity	DNA copy days/cell (viruses needed per day to initiate one new ulcer)	β_e	2.65e⁻¹¹ (2.26e⁵)	1.73e⁻¹¹ (3.46e⁵)	2.78e⁻¹¹ (2.15e⁵)	3.98e⁻¹² (1.50e⁶)	5.04e⁻¹¹ (1.19e⁵)
Epidermal HSV replication rate	HSV DNA copies per cell per day	p	1.03e⁵	7.21e⁴	1.7e⁵	5.15e⁴	1.96e⁵
Neuronal release rate	HSV DNA copies per day per genital tract	ϕ	82	45	90	41	123
Free-viral decay rate	Per day (half-life, hours)	c	8.8 (1.9)	7.0 (2.4)	9.7 (1.7)	6.2 (2.7)	12.3 (1.4)
Maximal CD8+ T-cell expansion rate	Per day	θ	2.84	1.85	3.27	1.85	5.25
CD8+ T-cell decay rate	Per day (half-life, days)	δ	1.47e⁻³ (471)	1.12e⁻³ (619)	1.69e⁻³ (409)	6.64e⁻⁴ (1040)	2.21e⁻³ (314)
CD8+ T-cell local recognition	Infected cells at which θ is half maximal	r	42	30	47	4	74
CD8+ regional codependence	0 = no codependence, 1 =full codependence	ρ	0.69	0.59	0.86	0.38	0.86
Viral production lag	Days	ε	0.96	0.53	1.1	0.34	1.1

Table 3

Predictive model parameters for key model outcomes

https://doi.org/10.7554/eLife.00288.031

	Single episode features		Long-term shedding features
	Peak viral load	Duration	Shedding rate	Episode rate
CD8+ T-cell density at reactivation site	−0.56	−0.47	NA	NA
Cell-associated HSV infectivity	—	0.12	—	0.13
Cell-free HSV infectivity	—	—	—	—
Epidermal cell replicate rate	0.13	0.14	−0.25	−0.31
Neuronal release rate	—	—	0.43	0.55
Free-viral decay rate	—	—	−0.2	—
Maximal CD8+ T-cell expansion rate	−0.09	—	0.37	0.51
CD8+ T-cell decay rate	—	0.09	—	−0.16
CD8+ T-cell local recognition	—	—	—	—
CD8+ regional co-dependence	—	—	0.32	0.34
Viral production lag	—	—	0.24	0.23

Partial correlation coefficients are listed only for parameters that are found to improve predictive effect on outcomes using Akaike information criteria models. Episode features are from 500 single episode simulations. Long-term shedding outcomes were measured over 10-years during 500 simulations.

Table 4

Spatial model simulations that varied only according to duration of sampling (30 days, 60 days, 365 days, and 10 years)

https://doi.org/10.7554/eLife.00288.039

Simulation duration		Percent of time with HSV DNA > 150 copies per mL	Percent of time with lesions* present	Episodes per year	Lesions per year
30 day	Mean	13.7	7.6	11	3.2
	Median	3.4	0	12	0
	Range	0–82.8	0–58.8	0–36.5	0–24.3
60 day	Mean	19.0	9.8	13.4	4.4
	Median	18.6	3.4	12	6
	Range	0–54.2	0–46.9	0–42.6	0–18
365 day	Mean	19.6	10.1	14.3	4.6
	Median	19.9	9.9	14	4
	Range	7.1–36.8	2.3–22	8–24	1–9
10 year	Mean	17.5	9.1	14.5	4.3
	Median	17.6	9.0	14.5	4.2
	Range	14.8–19.8	1.9–12.8	11.5–17.1	1.4–6

*

Lesions were defined as > 1 mm diameter ulcers.
Sixty simulations were performed at each of the sampling durations. Within shorter sampling duration simulations, lesion, and shedding frequency varied significantly, while ranges narrowed with prolonged sampling.

Table 5

Mathematical models of HSV-2 pathogenesis

https://doi.org/10.7554/eLife.00288.040

Model	Equations (additions to previous model are denoted in bold)	Variables (model fitting variable)	New features
1 (Schiffer et al., 2009; Schiffer et al., 2010)	$\begin{array}{l} • Δ S = [λ - (β_{i} \times S \times V) - (β i \times S \times V_{neu})] Δ t \\ • Δ I = [(β_{i} \times S \times V_{i}) + (β_{i} \times S \times V_{neu}) - (a \times I) - (f \times I \times E)] Δ t \\ • Δ E = [(F (I) \times θ \times E) - (δ \times E)] Δ t \\ • Δ V_{i} = [(p \times I) - (c \times V_{i}) - (β_{i} \times S \times V_{i})] Δ t \\ • Δ V_{neu} = [φ - (c \times V_{neu}) - (β_{i} \times S \times V_{neu})] Δ t \\ • F (I) = I / (I + r) \\ • V_{tot} = V_{i} + V_{neu} \\ • λ = d (S - S0) \end{array}$	S, I, E, V_i V_neu,	—
2	$\begin{array}{l} • Δ S = [λ - (β_{i} \times S \times V) - (β_{i} \times S \times V_{neu})] Δ t \\ • Δ I = [(β_{i} \times S \times V_{i}) + (β_{i} \times S \times V_{neu}) - (a \times I) - (f \times I \times E)] Δ t \\ • Δ E = [(F (I) \times θ \times E) - (δ \times E)] Δ t \\ • Δ V_{neu} = [φ - (c \times V_{neu}) - (β_{i} \times S \times V_{neu})] Δ t \\ • Δ V_{i} = [(p \times I) - (a \times V_{i}) - (β_{i} \times S \times V_{i})] Δ t \\ • Δ V_{e} = [(a \times V_{i}) - (c \times V_{e})] Δ t \\ • F (I) = I / (I + r) \\ • V_{tot} = V_{i} + V_{e} + V_{neu} \\ • λ = d (S - S0) \end{array}$	S, I, E, V_i, V_neu, (V_e)	Cell-free and cell-associated particles
3	$\begin{array}{l} • Δ S (i \dots 300) = = [λ - (β_{i} \times S \times V_{i}) - (β_{i} \times S \times V_{neu}) - (β_{e} \times S \times V_{e})] Δ t \\ • Δ I (i \dots 300) = [β_{i} \times S \times V_{i}) + (β_{i} \times S \times V_{neu}) - (β_{e} \times S \times V_{etot}) - (a \times l) - (f \times l \times E] Δ t \\ • Δ S (i \dots 300) = [(F (I) \times θ \times E) - (δ \times E)] Δ t \\ • Δ V_{neu} (i \dots 300) = [φ - (c \times V_{neu}) - (β_{i} \times S \times V_{neu})] Δ t \\ • Δ V_{i} (i \dots 300) = [(p \times I) - (a \times V_{i}) - (β_{i} \times S \times V_{i}) Δ t \\ • Δ V_{e} (i \dots 300) = [(a \times V_{i}) - (c \times V_{e})] Δ t \\ • F (I) = I / (I + r) \\ • I_{tot} = I_{1} + I_{2} + \dots + I_{e300} \\ • V_{etot} = V_{e 1} + V_{e 2} + \dots + V_{e 300} \\ • V_{itot} = V_{i 1} + V_{i 2} + \dots + V_{i 300} \\ • λ = d (S - S0) \end{array}$	S, I, E, V_neu, V_itot, (V_etot)	Concurrent plaques from cell-free particles
4*	$\begin{array}{l} • S0 = 1.67 e5 per region \\ • Δ S (i \dots 300) = [λ - (β_{i} \times S \times V_{i}) - (β_{i} \times S \times V_{neu}) - (β_{e} \times S \times V_{e})] Δ t \\ • Δ I (i \dots 300) = [(β_{i} \times S \times V_{i}) + (β_{i} \times S \times V_{neu}) + (β_{e} \times S \times V_{eadj}) - (a \times I) - (f \times I \times E)] Δ t \\ • Δ E (i \dots 300) = [(F (I) \times θ \times E) - (δ \times E)] Δ t \\ • Δ V_{neu} (i \dots 300) = [φ - (c \times V_{neu}) - (β_{i} \times S \times V_{neu})] Δ t \\ • Δ V_{i} (i \dots 300) = [(p \times I) - (a \times V_{i}) - (β_{i} \times S \times V_{i})] Δ t \\ • Δ V_{e} (i \dots 300) = [(a \times V_{i}) - (c \times V_{e})] Δ t \\ • λ = d (S - S0) \\ • F (I) = I/ (I + r) \\ • V_{eadj} = V_{e} from 6 adjacent regions \\ • I_{tot} = I_{1} + I_{2} + \dots + I_{300} \\ • V_{etot} = V_{e 1} + V_{e 2} + \dots + V_{e 300} \\ • V_{itot} = V_{i 1} + V_{i 2} + \dots + V_{i 300} \end{array}$	S, I, E, V_neu, V_itot, (V_etot)	Spatial model

*Model 4 has a parameter of regional CD8+ co-dependence (ρ) within each plaque-forming region. At episode onset within a region, the CD8+ density is adjusted to infer the spatial co-dependence of CD8 density from surrounding regions: E_i (time + 0.001) = (E_i × (1 − ρ)) + (E_avg × ρ) where E_avg is average of E from the 6 surrounding regions.
Models are described in the ‘Methods’. Units and values of each parameter in the optimized model are listed in Table 2. Variables include: S (susceptible skin cells), I (infected skin cells), E (CD8+ T cells), V_i (cell-associated HSV DNA particles), and V_e (cell-free HSV DNA particles).

Table 6

Model fit to Cohort E

https://doi.org/10.7554/eLife.00288.041

Summary measure	Summed criteria scores								Best fitting score	AIC
Summary measure	Shedding frequency	Episode duration	First positive swab	Last positive swab	Peak positive swab	Median expansion	Median decay	Episode frequency	Best fitting score	AIC
Model 1	2.13	5.59	0.05	0.08	0.24	0.07	0.43	0.01	8.61	−50
Model 2	1.26	8.33	0.38	0.34	0.22	0.04	0.09	0.03	10.69	−41
Model 3	0.25	3.75	0.31	0.22	0.61	0.05	0.50	0.22	5.92	−62
Model 4 solved for 10 parameters	0.25	0.13	0.29	0.15	0.09	0.01	0.01	0.03	0.96	−139
Model 4 solved for 5 parameters	0.39	0.32	0.44	0.21	0.09	0.04	0	0.18	1.67	−125

Summed criteria scores measure the degree of fit for each model according the eight individual shedding episode features using a weighted sum of squares. Model 4 is the spatial model. Models 1–3 are described in the ‘Methods’. Best fitting score is a sum of all summed criteria scores for a particular model with lower scores indicating better fit. AIC: Akaike information criteria with lower scores indicating better fit.

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Figures

HSV-2 levels in the genital tract are stable over minutes, expand and decay markedly over hours, and fluctuate rapidly and unpredictably over days.

Figure 1—source data 1

Dynamics of HSV-2 shedding over 5-min time intervals.

Dynamics of HSV-2 shedding with every 2-4 hr sampling.

Dynamics of HSV-2 shedding with every 6-hr sampling over 8 days.

HSV-2 replicates and is contained in widely dispersed microenvironments across the genital tract.

Figure 2—source data 1

Spatial features of HSV genital tract shedding.

Spatial features of HSV-2 lesions.

Spatial features of CD8+ T-cell response in genital skin.

Mathematical model.

Spatial mathematical model.

The spatial model reproduces all shedding episode characteristics.

Figure 4—source data 1

Continuous sampling of spatial model output reveals more accurate measures of episode characteristics.

Containment of infected cells within a single ulcer is extremely rapid, although secondary ulcers explain prolonged episodes.

Random spatial dispersion of viral particles from neurons reproduced the full diversity of episode characteristics if particles were released continuously, daily, or weekly from neurons.

Random spatial dispersion of viral particles from neurons reproduced the full diversity of episode characteristics during simulations in which particles were released into only a minority of the 300 modeled regions.

Dispersion of viral particles from neurons reproduced the full diversity of episode characteristics during simulations in which particles were released into a minority of modeled regions, provided that dispersion was random rather than clustered within a single geographic region.

Videos

Spatiotemporal demonstration of a 10-day episode.

Spatiotemporal demonstration of a 14-day episode according to viral production within each single region.

Spatiotemporal demonstration of infected cell and viral spread during a 6-day episode.

Spatiotemporal demonstration of 365 days of simulated shedding.

Spatiotemporal demonstration of 365 days of simulated shedding with noninfectious cell-free particles.

Spatiotemporal demonstration of immune response to a pair of 2-day episodes.

Spatiotemporal demonstration of immune response to a 7-day episode.

Spatiotemporal demonstration of immune response to a 14-day episode.

Spatiotemporal demonstration of immune response over 20 years.

Tables

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HSV-2 levels in the genital tract are stable over minutes, expand and decay markedly over hours, and fluctuate rapidly and unpredictably over days.

Figure 1—source data 1

Dynamics of HSV-2 shedding over 5-min time intervals.

Dynamics of HSV-2 shedding with every 2-4 hr sampling.

Dynamics of HSV-2 shedding with every 6-hr sampling over 8 days.

HSV-2 replicates and is contained in widely dispersed microenvironments across the genital tract.

Figure 2—source data 1

Spatial features of HSV genital tract shedding.

Spatial features of HSV-2 lesions.

Spatial features of CD8+ T-cell response in genital skin.

Mathematical model.

Spatial mathematical model.

The spatial model reproduces all shedding episode characteristics.

Figure 4—source data 1

Continuous sampling of spatial model output reveals more accurate measures of episode characteristics.

Containment of infected cells within a single ulcer is extremely rapid, although secondary ulcers explain prolonged episodes.

Random spatial dispersion of viral particles from neurons reproduced the full diversity of episode characteristics if particles were released continuously, daily, or weekly from neurons.

Random spatial dispersion of viral particles from neurons reproduced the full diversity of episode characteristics during simulations in which particles were released into only a minority of the 300 modeled regions.

Dispersion of viral particles from neurons reproduced the full diversity of episode characteristics during simulations in which particles were released into a minority of modeled regions, provided that dispersion was random rather than clustered within a single geographic region.

Spatiotemporal demonstration of a 10-day episode.

Spatiotemporal demonstration of a 14-day episode according to viral production within each single region.

Spatiotemporal demonstration of infected cell and viral spread during a 6-day episode.

Spatiotemporal demonstration of 365 days of simulated shedding.

Spatiotemporal demonstration of 365 days of simulated shedding with noninfectious cell-free particles.

Spatiotemporal demonstration of immune response to a pair of 2-day episodes.

Spatiotemporal demonstration of immune response to a 7-day episode.

Spatiotemporal demonstration of immune response to a 14-day episode.

Spatiotemporal demonstration of immune response over 20 years.