Engineered porous/hollow Burkholderia pseudomallei loading tumor lysate as a vaccine

Abstract

Due to its size, shape, and inherent expression of pathogen-associated molecular patterns and invasion-assistant adhesion proteins, Burkholderia pseudomallei can easily attach to, and then be internalized by, dendritic cells (DCs), leading to more efficient antigen cross-presentation if modified as carrier. Herein, we engineered Burkholderia pseudomallei as a porous/hollow carrier (SB) for loading tumor lysates (L) and adjuvant CpG (C) to be used as a tumor vaccine (SB-LC). We found that the adhesion proteins of Burkholderia pseudomallei promote internalization of the SB-LC vaccine by DCs, and result in enhanced DC maturation and antigen cross-presentation. SB-LC induces robust cellular and humoral antitumor responses that synergistically inhibit tumor growth with minimal adverse side effects in several tumor models. Moreover, SB-LC vaccination reverses the immunosuppressive tumor microenvironment, apparently as a result of CD8⁺-induced tumor ferroptosis. Thus, SB-LC is a potential model tumor vaccine for translating into a clinically viable treatment option.

Introduction

Currently, the most effective vaccines are those that are related to pathogenic microorganisms. Vaccination with such vaccines has led to the effective control or even complete eradication of many infectious diseases, such as tuberculosis, smallpox, measles, poliomyelitis, tetanus, and diphtheria, because the host immune system can evoke a stronger response against the pathogenic microenvironment [[1], [2], [3]]. The success of these vaccines can be attributed to the strong immunogenicity of the pathogenic microorganisms as a result of their expression of pathogen-associated molecular patterns (PAMPs), e.g., lipopolysaccharides (LPS) and cytidinephosphate-guanosine (CpG) oligodeoxynucleotides [4,5]. Due to the conserved molecular structures of PAMPs, the host immune system can rapidly distinguish PAMPs as “non-self” and activate a specific adaptive immune response [6]. Thus, PAMPs have been widely used as adjuvants to enhance immune responses during vaccination [7]. However, the effects using PAMPs (single or combination) as adjuvants of tumor vaccines are still unpredictable and elusive owing to the extreme complexity of the immune system and the suppressive tumor microenvironment [8,9]. In addition, the specific physical properties of the microorganisms are also known to influence the host immune response. A previous study showed that rod-like particles of 2–3 μm are recognized and internalized by macrophages [10,11]. A recent study also showed that a biomimetically-engineered Bacillus could potentiate vaccination against tumors [12]. These findings indicate that rod-shaped bacteria can be modified as carriers for loading tumor antigen and/or adjuvants as novel model vaccines.

Cross-presentation is the capability of antigen-presenting cells (APCs) to take up, process, and present extracellular antigens with MHC I molecules to CD8⁺ T cells. Cross-presentation is of particular importance for the development of cancer immunotherapy vaccines, because it can stimulate naive cytotoxic CD8⁺ T cells into activated cytotoxic CD8⁺ T cells [13,14]. Burkholderia pseudomallei (B. pseudomallei) is a Gram-negative rod-shaped bacterium [15]. Up to now, no successful vaccine has even been developed for preventing melioidosis, the human disease caused by Burkholderia pseudomallei, or glanders, a disease largely of horses caused by the very closely related pathogen Burkholderia mallei. Vaccine studies for these diseases have been undertaken numerous times over many years. As an intracellular bacterium, B. pseudomallei has developed various adhesion proteins, such as type IV pilus protein PilA, BoaA, and BoaB. Most of these adhesion proteins are specific ligands for protease-activated receptor-1 (PAR1), which is inherently expressed on the surface of dendritic cells (DCs), macrophages, endothelial cells, and platelets [16,17]. Once bound to the host cells, the bacteria enter through endocytosis [16,17]. Therefore, we hypothesized that using engineered porous/hollow B. pseudomallei as carriers may be superior to other rod-shaped bacteria for loading tumor antigen and/or adjuvants as vaccines.

It is well known that tumor lysates are the major sources of tumor antigens, by which all antigens can be targeted at once by immune system, and thereby trigger stronger specific antitumor immunities. Thus, whole tumor lysates as a source of antigens for loading DCs as vaccine strategies for treatment of tumor patients have been reported in clinics [[18], [19], [20]]. Therefore, in this study, we engineered B. pseudomallei as a porous/hollow carrier (SB) for loading tumor lysates (L) and CpG (C) as a tumor vaccine (SB-LC), and investigated its antitumor effects in murine models.