Fig 1.
IVIG protects B6 but not 129-Rag-/- mice from HSE.
129-Rag, B6-Rag, and 129.B6F1-Rag mice infected with 3200 PFU HSV1 were given 4 mg IVIG (red) or PBS (open) by ip injections at 24 h pi and observed for survival (2–6 experiments, n = 10–51 mice). The following data sets were statistically significant: B6-Rag+IVIG vs B6-Rag and B6-Rag+IVIG vs 129.B6F1-Rag: ***p = 0.0003; 129-Rag vs 129-Rag+IVIG: ****p<0.0001.
Fig 2.
IVIG protection of Rag mice is HSV1 dose dependent.
(A) B6-Rag or (B) 129-Rag mice infected with HSV1 at 10x, 32x or 100x LD50 were treated with either PBS or 4 mg IVIG at 24 h pi and monitored for survival (n = 12–28 mice / treatment group). (C) 129-Rag mice infected with HSV1 at 100x LD50 were treated with PBS or IVIG (1x) at 24 h pi and given either a second dose of IVIG at day 12 pi (2x), or a 7-day course of ACV treatment beginning day 10 pi (blue line) or day 12 pi (red line); (2–4 experiments, n = 12–32 mice / group). (D) Mononuclear cells isolated from pooled BS of 4 mice treated with 1x IVIG (control) at day 12 pi, 1x IVIG + d12 ACV (d12 ACV) or 2x IVIG (d12 IVIG) at day 15 pi were analyzed for infiltrating cell subsets by flow cytometry. (E) HD infected B6-Rag or (F) 129-Rag mice were treated with IVIG at 24 h pi. After the first dose, some mice received 1 (1x), 2 (2x) or 3 (3x) additional doses of IVIG given every 12 days (n = 20–65 mice / group). (*p<0.05 **p<0.01, ***p<0.001, ****p<0.0001).
Table 1.
Tg virus titers and BS inflammation in Rag mice.
Fig 3.
IVIG protection against HD HSV1 infection is T cell dependent.
(A) HD infected B6-Rag or (B) 129-Rag mice treated with 4 cyclical doses of IVIG were euthanized (Eu) at different times pi for determination of virus titers in the right Tg (B6-Rag+IVIG: red squares, Eu-R.Tg; 129-Rag+IVIG: red squares). B6-Rag mice that died were assayed for infectious virus in the right Tg (Nec-R.Tg: green diamonds) and BS (Nec-BS: cyan circles). Virus titers in Tgs isolated from HD infected PBS treated 129-Rag mice (blue circles) are also included in B. At day 60 pi (**), the right Tgs from surviving IVIG treated 129-Rag mice were explanted for 5 days and assayed for infectious virus (B); (n = 3–4 mice/ group per time point). HD infected 129-Rag (C) or B6-Rag (D) mice treated with IVIG at 24 h pi were given splenocytes, purified CD3+ or CD3- cell fractions (1–2x107 cells) isolated from naïve 129 or B6 wt or B6 OT-I spleens on day 8 or 10 pi; 129-Rag controls treated 1x or 2x with IVIG or B6-Rag controls treated 1x with IVIG did not receive cells. All 129-Rag recipients received a second IVIG dose on day 10 pi except for one group that received only 1x IVIG (d8 129 WT Spl + 1x IVIG) (n = 6–8 for controls and 10–14 for recipients / group). Arrows = day of cell transfer, arrowheads = day of IVIG injection. ***p = 0.0007; ***p = 0.0005.
Fig 4.
HS induced HSV1 reactivation in HD mice is controlled by T cells.
Healthy LD and HD infected B6-Rag (A) and 129-Rag mice (B) at day 60 pi were subjected to HS to induce IVR and monitored for survival (data represents 2 experiments; n = 8–20 mice / group). HSV1 titers were determined in the right Tg and BS of dying B6-Rag (C) and 129-Rag mice (D); base line titers were determined in Tg and BS of 4 control (non-HS treated) mice at day 60 pi (d0 for IVR). (E) B6-Rag and (F) 129-Rag mice were transferred with 1–2x107 naïve or memory (mem) splenocytes or CD3+ T cells isolated from mock or HSV1 immunized B6 or 129 wt mice respectively, 3 days prior to HS and monitored for survival. Data represents 1–2 experiments (n = 8–15 / mice per group). ***p = 0.004, ****p< 0.0001.
Fig 5.
Acute and latent gene expression in HD infected B6-Rag trigeminal ganglia.
qRT-PCR analysis of RNA from Tg collected from acute (day 5) PBS (blue bars) or IVIG treated (red) and latent (day 60) IVIG treated (black) HD B6-Rag mice are shown as fold-change relative to LAT expression (A-D) for Immediate Early (A), Early (B), Early/Late (C) and Late (D) genes, and delta-Ct (dCt) values normalized to GAPDH expression for Early, Early/Late and Late genes (E, F, and G, respectively). Tg sections obtained from latently infected LD, HD, and a HD Rag mouse showing signs of HSE were processed for RNA-FISH using a LAT RNA probe (H). The blue signal stains for LAT, the green signal comes from neuronal cytoplasmic lipofuscin (aggregates) autofluorescence. The dotted lines outline the nuclei. Wide-field imaging. Scale bar = 10 μm. (I) GAPDH normalized dCt values for LAT expression in HD Rag mice determined using RT-PCR (I). Statistical analysis is described in methods.
Fig 6.
A schematic depicting the Rag-latency model.
Details of establishment and characterization of latency in B6-Rag mice, in vivo reactivation and adoptive transfer of T cells into latently infected Rag mice are presented in the text and methods.