Radiation effects on Toxoplasma antigens: different immune responses of irradiated intact tachyzoites or soluble antigens in experimental mice models

Abstract

Purpose: Purpose: Protein irradiation causes aggregation, chain breakage, and oxidation, enhancing its uptake by antigen-presenting cells. To evaluate if irradiated proteins participate on the protection, we studied the immune response induced in mice immunized with irradiated soluble extracts of T. gondii tachyzoites (STag) or irradiated intact T. gondii RH tachyzoites (RH^0.25 kGy).

Material and Methods: Soluble extracts of Toxoplasma gondii tachyzoites (STag) were irradiated at different dose by Cobalt-60 source. By polyacrylamide gel electrophoresis (SDS-Page) we evaluated the effects on primary structures of protein STags induced by irradiation. By Enzyme-linked Immunosorbent Assay (ELISA) we evaluated the difference between humoral immune response induced by irradiated STag or RH tachyzoites in immunized mice from the detection of specific immunoglobulin G (IgG) antibodies in the serum of immunized mice. From challenge with viable RH strain of T. gondii we evaluated the protection induced in the immunized animals. By cytometry we performed the phenotyping of T and B lymphocytes in the peripheral blood of the immunized animals.

Results: Irradiation dose of 1.5 kGy induced minimal changes in most proteins, without affecting their antigenicity or immunogenicity. Immunization showed saturation at the dose of 10 µg/mice, with worst response at higher doses. STag irradiated at 1.5  kGy (STag^1.5 kGy) induced higher survival and protection similar to T. gondii RH strain irradiated at 0.25 kGy (RH^0.25 kGy), with higher serum levels of high affinity IgG compared to STag native. Blood immune memory cells of mice immunized with STag^1.5 kGy had higher proportions of CD19⁺ (cluster of differentiation 19) and CD4⁺ (cluster of differentiation 14) cells, whereas mice RH^0.25 kGy had high proportion of memory CD8⁺ (cluster of differentiation 8) cells.

Conclusions: Our data suggest that major histocompatibility complex type I (MHCI) pathway, appears seem to be used by RH^0.25 kGy to generate cytotoxic cells while STag1.5 kGy uses a major histocompatibility complex type II (MHCII) pathway for B-cell memory, but both induce sufficient immune response for protection in mice without any adjuvant. Irradiation of soluble protein extracts enhances their immune response, allowing similar protection against T. gondii in mice as compared to irradiated intact parasites.

Keywords:

• Gamma irradiation
• Toxoplasma gondii
• vaccines
• protection
• subcomponent

Acknowledgments

The authors acknowledge Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for support to da Costa A. (project n° 2014/17029-4) and Conselho Nacional de Pesquisa e Desenvolvimento (CNPq). The authors gratefully thank the support of Biotechnology division of IPEN/CNEN-SP and LIMHCFMUSP.

Ethics approval

All animal procedures were performed with the approval of the Research Ethics Committee of the Institute of Tropical Medicine of São Paulo (Project 000274A) and in agreement with the Guidelines for the Care and Use of Laboratory Animals from the National Academy of Sciences.

Disclosure statement

The authors declare that they have no competing interests.

Additional information

Funding

The present study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), process n° 2014/17029-4 and the Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES), Brazil - Financing Code 001.

Notes on contributors

Andrea da Costa

Andrea da Costa, MSc, PhD, is a Postgraduate Student at the Protozoology Laboratory of the Tropical Medicine Institute of São Paulo/USP, SP-Brazil.

Nanci do Nascimento

Nanci do Nascimento, MSc, PhD is a Researcher at the Biotechnology Center at Instituto de Pesquisas Energ´ticas e Nucleares, São Paulo, SP- Brazil (in memoriam).

Andrés Jimenez Galisteo

Andrés Jimenez Galisteo Jr., MSc, PhD is a Scientific Researcher VI, of the Clinical Hospital of the University of São Paulo School of Medicine (HCFMUSP) at the Protozoology Laboratory of the Tropical Medicine Institute of São Paulo/USP.

Aline Bastos Dias dos Passos

Aline Bastos Dias dos Passos, BSc, is a Scientific Scholarship at the Protozoology Laboratory of the Tropical Medicine Institute of São Paulo/USP, SP-Brazil.

Heitor Franco de Andrade

Heitor Franco de Andrade Jr., is a Graduate in Medicine, PhD., and full professor in Infectious Diseases Pathology. Associate professor of Department Pathology FMUSP and chief of Protozoology laboratory at Tropical Medicine Institute of São Paulo/USP.