Pathology, organ distribution, and immune response after single and repeated intravenous injection of rats with clinical-grade parvovirus H1

Abstract

Parvovirus H1 (H1PV) is an autonomous parvovirus that is transmitted in rodent populations. Its natural host is rats. H1PV infection is nonpathogenic except in rat and hamster fetuses and newborns. H1PV infection of human cancer cells caused strong oncolytic effects in preclinical models. For a clinical trial of H1PV in patients with brain tumors, clinical-grade H1PV was produced according to Good Manufacturing Practices. This report focuses on results obtained after a single high-dose intravenous injection of highly purified H1PV in 30 rats and multiple (n = 17) intravenous injections at 3 dose levels in 223 rats. In both studies, no virus-related mortality or macroscopic organ changes related to H1PV occurred. Histopathology after multiple virus injections revealed minimal diffuse bile duct hyperplasia in livers of animals of the highest dose group and germinal center development in spleens of animals from the high-dose group. Liver changes were reversible within a 2-wk recovery period after the last injection. Hematology, blood chemistry, and coagulation analyses did not reveal significant toxicologic changes due to H1PV. Virus injection stimulated the production of IgG antibodies but did not alter mononuclear cell function or induce cytokine release. PCR analysis showed dose-dependent levels of viral genomes in all organs tested. The virus was excreted primarily through feces. These data provide important information regarding H1PV infection in its natural host. Due to the confirmation of the favorable safety profile of H1PV in a permissive animal model, a phase I/IIa clinical trial of H1PV in brain tumor patients could be initiated.

Figures

Figure 1.

Portal triads in liver at the end of treatment. (A) Normal number and epithelial morphology of bile ducts in female rat treated with vehicle only. (B) Minimally increased number and minimally activated epithelial cells of bile ducts (arrows) in female rat treated with 108 pfu H1PV. Magnification, 40×.

Figure 2.

Germinal centers in spleen at the end of recovery period. (A) Absence of germinal centers in splenic follicles of male rat treated with vehicle only. (B) Mild germinal center development (arrows) in male rat previously treated with 108 pfu H1PV. Magnification, 10×.

Figure 3.

Results of toxicokinetics: day 1, left columns; day 28, right columns; upper panels, female rats; lower panels, male rats. Values are given as mean ± 1 SD. Lower limit of quantification (50 VG/PCR reaction) is equivalent to 20 VG/µL blood. Only values above the lower limit of quantification (LLOQ) are included in the calculation of mean values.

Figure 4.

Mean virus excretion. Concentrations below the lower limit of quantification (LLOQ) are presented as the LLOQ. (A) Feces (no. of VG/mg). (B) Saliva (no. of VG/swab). (C) Urine (no. of VG/µL). The standard deviation was calculated (if applicable, more than one value above LLOQ) for the log of single values. Only values above the LLOQ were included in the calculation of mean values, because no definitive values are available for samples with a VG concentration below LLOQ. When all values of a group were lower than the LLOQ, the mean value is presented as the LLOQ.

Figure 5.

Proliferation of PBMC after mitogenic stimulation. Optical density (OD) was measured at 570 nm at 2 time points (0 and 48 h after stimulation). Statistical analysis was performed using a 2-way ANOVA and a Bonferroni post test. *, Value significantly (P < 0.05) different from those for control groups. Results from male rats, which were analyzed individually, are shown. PBMC from female rats had to be pooled due to low cell numbers and showed similar effects to those in male rats (data not shown).

Figure 6.

Measurement of antiH1PV antibodies (IgG) by specific ELISA. Raw data (blank reductions) are shown groupwise for individual rats; results are shown for female (upper panels) and male (lower panels) rats.