Herpes murine model as a biological assay to test dialyzable leukocyte extracts activity

Abstract

Human dialyzable leukocyte extracts (DLEs) are heterogeneous mixtures of low-molecular-weight peptides that are released on disruption of peripheral blood leukocytes from healthy donors. DLEs improve clinical responses in infections, allergies, cancer, and immunodeficiencies. Transferon is a human DLE that has been registered as a hemoderivate by Mexican health authorities and commercialized nationally. To develop an animal model that could be used routinely as a quality control assay for Transferon, we standardized and validated a murine model of cutaneous HSV-1 infection. Using this model, we evaluated the activity of 27 Transferon batches. All batches improved the survival of HSV-1-infected mice, wherein average survival rose from 20.9% in control mice to 59.6% in Transferon-treated mice. The activity of Transferon correlated with increased serum levels of IFN-γ and reduced IL-6 and TNF-α concentrations. Our results demonstrate that (i) this mouse model of cutaneous herpes can be used to examine the activity of DLEs, such as Transferon; (ii) the assay can be used as a routine test for batch release; (iii) Transferon is produced with high homogeneity between batches; (iv) Transferon does not have direct virucidal, cytoprotective, or antireplicative effects; and (v) the protective effect of Transferon in vivo correlates with changes in serum cytokines.

Figures

Figure 1

Standardization of mouse cutaneous herpes model. (a) Three different experiments performed with 5.0 × 104 PFU of HSV-1 (closed symbols). In each experiment, infected mice showed significantly lower survival than controls (open circles; log-rank Mantel-Cox test; P < 0.001). (b) Frequency of deaths from the experiments shown in (a).

Figure 2

Validation of the murine herpes model for the evaluation of Transferon activity. (a) Survival curves for HSV-1 infected mice. Treatments with 1 μg (open triangles) or 0.125 μg (open squares) of Transferon improved survival over placebo (open circles; Log-rank Mantel-Cox test; P < 0.01). In contrast, 12.5 ng of Transferon (open diamonds) had no significant effect. A group of mice was left uninfected (closed circles) as control. (b) Weight changes in HSV-1 infected mice. Transferon partially protects mice from the weight loss induced by HSV-1 (symbols are as in (a)). (c) Survival induced by five different Transferon batches was evaluated during a validation protocol. All batches improved survival versus placebo (Bonferroni t test; ∗P < 0.05, ∗∗P < 0.01; ∗∗∗P < 0.001).

Figure 3

Evaluation of biological activity of 27 Transferon batches in the validated murine model of herpes. Statistical analysis (ANOVA, Bonferroni's Multiple Comparison Test) showed no differences between doses.

Figure 4

In vitro evaluation of antiviral activity of Transferon. (a) HSV-1 was preincubated for 60 min with Transferon (20 μg/mL) or medium before evaluation of visible cytopathic effect (CPE) on Vero cells. Viruses with no preincubation (t0) were used as control (ANOVA, Bonferroni's Multiple Comparison Test; ns: nonsignificant). (b) Effect of Transferon (10 pg/mL–10 μg/mL) on HSV-1-induced cytopathology, evaluated by MTS assay. Acyclovir (ACV; 10 μg/mL) was included as a positive control. The graph represents data from 2 independent experiments (ANOVA, Bonferroni's Multiple Comparison Test; ∗P < 0.0001). (c) TCID50 obtained from Vero cells preincubated for 24 h with Transferon (20 μg/mL) or medium prior to HSV-1 infection (Student's t test). (d) Titration of samples obtained from Vero cells infected 24 h with HSV-1 and simultaneously treated with medium (open circles) or 20 μg/mL of Transferon (closed circles). ACV (10 μg/mL; triangles) was included as a positive control. Sum-of-squares F test showed no differences between the TCID50 from medium- and Transferon-treated cells (P = 0.7527). The graph represents data from 2 independent experiments.

Figure 5

Quantitative analysis of blood (serum) cytokine levels. Transferon (Batch 12C04; 0.75 μg/mouse) significantly reduced the concentrations of TNF-α (a) and IL-6 (b) at day 9 postinfection, but it increased that of IFN-γ at day 7 (c). Transferon administration had no effect on uninfected mice. All measurements were normalized to placebo-treated, uninfected controls. The graphs represent data of 2 independent experiments (ANOVA, Bonferroni's Multiple Comparison Test; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001).