Data in support of toxicity studies of structurally modified plant virus to safety assessment

This data article is related to the research article entitled “Assessment of structurally modified plant virus as a novel adjuvant in toxicity studies” (Nikitin et al., 2018), devoted to the safety study of structurally modified plant virus - spherical particles (SPs). SPs are generated by thermally denatured tobacco mosaic virus (TMV) coat protein and act as effective adjuvant for development of new vaccine candidates. This article reports the additional results on the toxicity studies of TMV SPs. The weight coefficients of laboratory animals internal organs complements the data of the subchronic toxicity studies. Also plaque-forming cell assay, delayed-type hypersensitivity test and peritoneal macrophage assay as a part of immunotoxicity studies of TMV SPs are presented.


a b s t r a c t
This data article is related to the research article entitled "Assessment of structurally modified plant virus as a novel adjuvant in toxicity studies" (Nikitin et al., 2018), devoted to the safety study of structurally modified plant virus -spherical particles (SPs). SPs are generated by thermally denatured tobacco mosaic virus (TMV) coat protein and act as effective adjuvant for development of new vaccine candidates. This article reports the additional results on the toxicity studies of TMV SPs. The weight coefficients of laboratory animals internal organs complements the data of the subchronic toxicity studies. Also plaque-forming cell assay, delayed-type hypersensitivity test and peritoneal macrophage assay as a part of immunotoxicity studies of TMV SPs are presented.
& Value of the data These data are useful to demonstrate that TMV SPs, a potential vaccine adjuvant, is not toxic in laboratory animals.
The data demonstrate the absence of immunotoxicity or deep overload of immune functions in mice as well as alterations in weight after TMV SPs administration.
The data are useful for further estimation of novel adjuvant safety. These data are valuable to researchers interested in plant viruses and their derivatives for biotechnological and medical application.

Data
An essential step for the novel universal adjuvant development is to study their safety on laboratory animals. In this article we display additional data on the weight coefficients of the internal organs within the subchronic toxicity studies and immunotoxicity studies of spherical particles based on structurally modified helical plant virus, including plaque-forming cell assay, delayed-type hypersensitivity test and peritoneal macrophage assay.

Structural modification of plant virus (TMV)
TMV SPs were obtained according to Refs. [2,3]. TMV SPs size was characterized by transmission electron microscopy and nanoparticle tracking analysis, as described previously [4,5]. For all experiments, TMV SPs were buffered with apyrogenic PBS sterile solution at concentration of 1 mg/ml.

Analysis of internal organs weight
To evaluate the weight coefficients of the internal organs following three consecutive intramuscular injections, at two-week intervals, TMV SPs in low (20 mg per animal) and high (200 mg per animal) doses were administered on rats and rabbits as described in Ref. [1]. Phosphate buffer saline was used as a control. Each group of rodents contained five males and five females, while each group of rabbits consisted of three males and three females. The groups were numbered sequentially. Animals were euthanized on the 42nd day, and an autopsy was undertaken. Organs were weighed to the nearest mg with analytical balance HR-250AZ (AND, Japan) and organ/body weight ratio was calculated (Table 1).

Immunotoxicity
Immunotoxicity evaluation was performed in compliance with federal regulatory requirements. F1 hybrid mice (CBA x C57BL/6) in three groups (10 or 100 μg of SPs in PBS IM or 100 μl PBS IM as a control), consisting of five males and five females in each group, were used. There were three tests, thus 90 mice (20-22 g) were used. Humoral-mediated immunity was assessed through T-dependent antibody response. The plaque-forming cell (PFC) antibody response to sheep red blood cells (SRBC), or the plaque assay, was chosen to analyze the effects on T-dependent antigen response. Mice were administered with doses of SPs or PBS, as indicated above, one hour before SRBC immunization, and were euthanized five days after. Then, a standard PFC protocol was performed [6]. PFCs per spleen were calculated.
Delayed-type hypersensitivity (DTH) as an in vivo assay of cell-mediated immune function was used [7]. DTH reaction was induced by trinitrobenzenesulfonate (TNBS) treatment [8]. The study and control groups were treated with SPs and PBS respectively an hour after TNBS immunization (200 μl 10 μM sterile TNBS solution in isotonic sodium chloride subcutaneously). Six days later, the right forepaw footpads were injected with 50 μl of 10 μM sterile TNBS solution in isotonic sodium chloride, and the left footpads with isotonic sodium chloride solution alone. Footpad swelling was estimated by footpad Table 1 The weight coefficients of the internal organs in repeated dose toxicity study. Groups 1, 4 are controls (PBS), groups 2, 5 -low dose of TMV SPs (20 mg) and groups 3, 6 -high dose of TMV SPs (200 mg). Rats (groups 1, 2, 3); rabbits (groups 4, 5, 6). Mean value and SD were calculated with IBM SPSS Statistics (23.0.0.0 64 bit for Windows); no significant difference by one-way ANOVA was detected.

Sex
Group diameter measurement [9], and using cross-sectional area calculation. Footpad cross-sectional area was approximated by ellips area formula. The semi-major axis was half of the maximal width (measured with vernier caliper (ChIZ, Russia) to the nearest 0,02 mm) and the semi-minor axis was the half of perpendicular measurement. The swelling index was calculated as the right/left footpad cross-sectional area ratio, as a percentage ( Table 2). The peritoneal macrophage assay was used for phagocytic activity testing. Murine immune cells were isolated using a common protocol [10], and then their activity was studied with a Vybrant Phagocytosis Assay Kit (Thermo Fisher Scientific, USA). Five replicates on each animal were used. The number of harvested cells and phagocytosis assay result were measured.