H₂O₂-Inactivated Salmonella typhimurium RE88 Strain as a New Cancer Vaccine Carrier: Evaluation in a Mouse Model of Cancer

Abstract

Purpose

This study aimed to describe a novel cancer vaccine developed using H₂O₂-inactivated Salmonella typhimurium RE88 [with deletions of AroA (the first enzyme in the aromatic amino acid biosynthesis pathway) and DNA adenine methylase] as the carrier.

Methods

The pVLT33 plasmid was used to engineer an RE88 strain induced to express ovalbumin (OVA) by isopropylthiogalactoside (RE88-pVLT33-OVA). The immune responses and anticancer effects of H₂O₂-inactivated RE88-pVLT33-OVA were compared with those of non-inactivated RE88-pVLT33-OVA and OVA (positive control) in mice carrying OVA-expressing tumors (EG7-OVA) cells.

Results

Anti-ovalbumin IgG (immunoglobulin G) titer following vaccination with H₂O₂-inactivated RE88-pVLT33-OVA was higher for subcutaneous than for intragastric vaccination. When subcutaneous administration was used, H₂O₂-inactivated RE88-pVLT33-OVA (2 × 10⁹ CFU (colony forming units)/mouse) achieved an anti-ovalbumin IgG titer higher than that for the same dose of RE88-pVLT33-OVA and comparable to that for 10 µg ovalbumin (positive control). The binding of mouse serum antibodies to EG7-OVA cells was stronger for H₂O₂-inactivated RE88-pVLT33-OVA (2 × 10⁹ CFU/mouse) than for 10 µg ovalbumin. Furthermore, subcutaneous vaccination with H₂O₂-inactivated RE88-pVLT33-OVA (2 × 10⁹ CFU/mouse) induced greater activation of splenic T cells and more extensive tumor infiltration with CD4⁺/CD8⁺ T cells compared with 10 µg ovalbumin (positive control). The mice vaccinated subcutaneously with H₂O₂-inactivated RE88-pVLT33-OVA at a dose of 2 × 10⁸ or 6 × 10⁸ CFU/mouse had smaller tumors compared with mice in the negative control groups. Tumor weight in mice vaccinated with H₂O₂-inactivated RE88-pVLT33-OVA at a dose of 2 × 10⁹ CFU/mouse was significantly lower than that in both negative control groups (P < 0.05) and decreased with the increasing dose of H₂O₂-inactivated RE88-pVLT33-OVA. H₂O₂-inactivated RE88-pVLT33-OVA was potentially safer than the non-inactivated strain, could carry exogenous antigens, and had specific epitopes that could be exploited as natural adjuvants to facilitate the induction of cellular and humoral immune responses.

Conclusion

It was anticipated that H₂O₂-inactivated RE88-pVLT33-OVA could be used as a novel delivery system for new cancer vaccines.

Keywords:

• cancer vaccine
• H₂O₂ inactivation
• RE88-pVLT33-OVA
• whole-cell bacterial vaccine

Acknowledgments

Yingzi Fan and Tingting Bai are co-first authors for this study.

Abbreviations

ANOVA, analysis of variance; BSA, bovine serum albumin; DAPI, 4ʹ,6-diamidino-2-phenylindole; H₂O₂, hydrogen peroxide; IFN-γ, interferon-gamma; IPTG, isopropyl β-d-1-thiogalactopyranoside; OVA, ovalbumin; PBS, phosphate-buffered saline; PBST, Tween-20 in PBS; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Data Sharing Statement

The datasets used and/or analyzed in the present study are available from the corresponding author on reasonable request.

Ethics Approval and Consent to Participate

The study was approved by the Ethics Review Committee for Animal Experimentation of Sichuan University. All procedures involving animal treatment were conducted in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (National Institutes of Health Publication Pub No 85-23, revised 1996).

Consent for Publication

Not applicable.

Disclosure

The authors declare no competing interests.

Additional information

Funding

This study was supported by the National Major Scientific and Technological Special Project for “Significant New Drugs Development (2018ZX09733001 and 2018ZX09201018) and 1.3.5 project for disciplines of excellence,” West China Hospital, Sichuan University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.