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.

Transferon™, a peptide mixture with immunomodulatory properties is not immunogenic when administered with various adjuvants

“…hDLE have a demonstrated value as coadjuvants in treating diseases with an inflammatory component (Pizza et al 1996;Berron-Perez et al 2007;Hernandez et al 2013), such as herpes zoster infections (Estrada-Parra et al 1998) and atopic dermatitis (Flores Sandoval et al 2005), based on their immunomodulatory properties. Though mechanisms of action of hDLE are not fully understood, they have been associated with: toll-like receptor (TLR) stimulation (Garcia-Hernandez et al 2014); induction of transcription factors and second messengers in cd T-lymphocytes (Herlin et al 1981); and, modulation of tumor necrosis factor (TNF)-a, interferon (IFN)-c, and interleukin (IL)-6 levels in human and mouse serum (Hernandez et al 2013;Salinas-Jazmin et al 2015).…”

“…Thus, the quality, efficacy, and safety of these products must be examined using well-designed physicochemical and biological analyses that describe their overall components. Though characterizing the physicochemical/biological properties of Transferon has been difficult, studies in our laboratories have determined the peptide nature of Transferon (Medina-Rivero et al 2016), its batch-to-batch consistency (Medina-Rivero et al 2014), and biological activity (Salinas-Jazmin et al 2015). Nevertheless, its immunogenic effects remain unknown.…”

Early Differentiation of Human CD11c+NK Cells withγδT Cell Activation Properties Is Promoted by Dialyzable Leukocyte Extracts

“…The quality control of Transferon comprised (A) endotoxin content, quantified using the Endosafe-Portable Test (Charles River Laboratories, Charleston, SC, USA) according to the manufacturer's instructions (the specification for endotoxin was established in Mexican Pharmacopeia, Section MGA-0316 (≤4.0 EU/mL)); (B) microbiological tests, according to Mexican Pharmacopeia, Section MGA-0571; and (C) physicochemical characterization by a validated ultra-performance liquid chromatography (UPLC) method that analyzes molecular weights and the time of retention of the main peaks compared with those of an internal batch pattern. Peptide content per final dose was measured by bicinchoninic acid (BCA) method using the Pierce BCA kit (Thermo Fisher Scientific, Waltham MA, USA) according to the manufacturer's instructions [22]. Batch-to-batch reproducibility is consistently analyzed by SE-UPLC chromatographic profiles, while IFN γ production from DLE-stimulated Jurkat cells is quantified as a biological activity test [14].…”

“…In fact, a herpes murine model has become a powerful biological assay to test the functional activity of DLE [22]. Of special interest, a direct effect on viral replication or infected target cell viability could not been recorded.…”

Evaluación inmunológica durante el tratamiento de un caso de lepra dimorfa lepromatosa

“…[12][13][14][15] En este paciente se utilizó Transferon® (Instituto Politécnico Nacional, México), mezcla heterogénea de péptidos de bajo peso molecular (<10 kDa) liberados por la ruptura de leucocitos de sangre periférica de donadores sanos y cuyas acciones inmunológicas demostradas son la inducción in vitro de INF-γ en una línea celular de linfocitos T, 16 en modelos animales in vivo 17 y en pacientes con inmunidad Th1 funcionalmente disminuida o alterada. 14,15 La producción de citocinas Th1 pudo reestablecer la inmunidad celular y la función bactericida de los macrófagos, como fue reportado en el modelo murino de lepra anérgica.…”