Background
Current advanced cancer immunotherapy treatments boost humoral and cellular immunity without the non-specific targets and toxic effects on normal cells as conventional cancer treatment [
1,
2]. However, despite these advances, the inability to predict treatment efficacy, the need for additional biomarkers, the development of resistance to cancer immunotherapies, and the high treatment costs continue to serve as a limitation in immunotherapeutic treatments [
3,
4]. Therefore, therapy with live tumor-targeting bacteria has received attention as a unique option to overcome these challenges [
5]. Studies have shown that bacteria-based therapies can serve as a monotherapy or complement to other anticancer therapies [
5‐
7]. Previously, we showed that live
Salmonella and cytokine combination therapy induced potent T-cell immunity and long-term tumor control in mice [
8]. We also demonstrated that heat-inactivated
Salmonella (S. typhimurium) could display tumor antigens to achieve tumor-specific immune responses [
9]. Nevertheless, a significant limitation of live bacteria lies in its off-target toxicity and lowered efficacy in inactivated bacteria [
10]. Therefore, further investigation is needed to develop an approach with bacteria-based therapies that is more potent than heat-inactivated bacteria therapies, while also addressing the safety concerns of using live attenuated bacteria.
In recent years, studies have shown that gram-negative bacteria can naturally release outer membrane vesicles (OMVs), which comprises of lipopolysaccharides (LPS), outer membrane proteins, periplasmic proteins, and phospholipids and can serve as carriers for various substances such as toxins, metabolites, enzymes, virulence factors, and genetic material (DNA and RNA) [
11‐
13]. Unlike attenuated bacteria, OMVs are considered safer and can effectively stimulate the immune system by delivering key immunogens from their parent bacteria [
13‐
15]. Genetic engineering techniques have shown that the construction of recombinant OMVs improves target precision through surface protein and carries exogenous proteins for improved immunogenicity [
16,
17]. As a neoantigen vaccine, OMVs can fuse multiple surface proteins and therefore simultaneously display various distinct tumor antigens to elicit a synergistic antitumor immune response in metastatic lung melanoma and subcutaneous colorectal cancer models [
18]. These studies have further underscored the impact of bacteria OMVs as a versatile immunotherapeutic approach in developing cancer vaccines, as it presents a balance between immunogenicity and safety [
15,
19‐
21].
Previously, we optimized a bacteria antigen-display strategy through modification of the Human Papillomavirus (HPV)-associated E7 antigen, incorporating nine arginine residues (9RE7) for enhanced E7 coating [
8]. Poly-l-arginine is a cell penetrating peptide (CPP), often used for mammalian cell uptake and delivery of drugs or macromolecules such as proteins and enzymes [
22,
23]. Here, we coated 9RE7 on
Salmonella OMV (SOMV), a naturally released OMV derived from
Salmonella SL7207, and synthesized SOMV-9RE7 which will be investigated to serve as a safer and more effective method to delivery HPV E7 antigen. By generating systemic E7-specific CD8+ T cells and recruiting them to the tumor microenvironment (TME), SOMV-9RE7 exhibited promising antitumor effects. These results demonstrate a broad application of 9RE7 peptide and an alternative to traditional bacteria immunotherapy.
Material and methods
Cell preparation
E7-expressing TC-1 tumor cells and dendritic cells were grown in vitro in RPMI 1640 media containing 10% (v/v) fetal bovine serum, 50 units/mL of penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 2 mM non-essential amino acids, and 0.1% (v/v) 2-mercaptoethanol under 37 °C with 5% CO2. E7-specific CD8+ splenocytes were isolated from mice vaccinated with E7 DNA and incubated with E7 expressing cells and maintained in culture.
Bacteria-derived outer membrane vesicle
Salmonella SL7207 was grown overnight in LB broth at 37 °C. On the next day, 1 mL of the overnight Salmonella culture was added to 9 mL of fresh LB medium and incubated until O.D. 600 reading of 0.5. This freshly cultured Salmonella was added to fresh LB medium at 1:100 dilution in 250 mL culture at 37 °C for 16 h. The supernatant was collected after centrifugation and filtered through a 0.45 µm MCE Membrane Filter (Millipore Sigma). Filtered medium was transferred to ultracentrifuge tubes and at 150,000 RPM for 3 h (Beckman). The supernatant was removed and particles at the bottom of the tubes were collected and suspended in 1 mL PBS and stored at − 80 °C. OMV yield is calculated using Bio-Rad protein assay with a concentration of approximately 1.5 mg/mL.
Peptide synthesis
Peptides used in this study include RRRRRRRRR-RAHYNIVTF (E7 protein amino acids 49–57), termed 9RE7, and was synthesized by GenScript (Piscataway, NJ, USA) at a purity of over 90%.
SOMV-9RE7 generation and characterization
SOMV-9RE7 is synthesized by combining SOMV and 9RE7 in PBS buffer to be vortexed for 30 min. Subsequent dialysis is performed using a 50kD Amicron Ultra Centrifugal Filter (Millipore Sigma) to remove unbound peptides. For characterization of SOMV-9RE7, 10 µg of SOMV was mixed with 1 µg of FITC conjugated peptides E7 or 9RE7 in the PBS buffer. Bacteria/peptide mixture was vortexed at room temperature for 30 min, followed by dialysis with the 50kD Amicron Ultra Centrifugal Filter (Millipore Sigma) to remove unbound peptides. FITC signals were measured by 13-color B-Y-R-V CytoFLEX S (Beckman Coulter). Particle size and charge was determined by Malvern Zetasizer (Worcestershire, UK). 40% of 9RE7 remained coated on SOMV after dialysis. This was determined by interpolating the standard curve of FITC-labeled SOMV-9RE7 at 500 nm with Nanodrop One (Thermo Fisher Scientific).
In vitro T cell activation
10 µg of SOMV and 1 µg 9RE7 are used to synthesize SOMV-9RE7 as described above. E7-specific CD8+ T cell activation follows previously established protocol [
24,
25]. SOMV-9RE7 is incubated with 1 × 10
5 dendritic cell line in 96 well plate cultured with complete RPMI media at 37 °C, 5% CO2 overnight. After aspirating culture medium and washing with PBS, 5 × 10
5 E7-specific CD8+ T cells were added to the dendritic cell line and blocked with Brefeldin A + Monensin Golgi Plug (Thermo Fisher Scientific) overnight. Cells were collected and stained with APC-A750-conjugated anti-mouse CD8α antibody (Biolegend) before permeabilization with eBioscience Fixation (Invitrogen) and intracellular staining FITC-conjugated IFNγ antibodies.
Mice vaccination
For tumor inoculation, 1 × 105 TC-1 cells in 50 µL of PBS were subcutaneously injected into 6–8 weeks old female C57BL/6 mice at the lower right abdomen. Largest length and width were measured by digital calipers twice per week. Tumor volumes were calculated by the formula: V = (Length × Width2)/2. At the indicated time points, TC-1 tumor-bearing mice were vaccinated subcutaneously in the tumor graft region with 10 µg of 9RE7 peptides, 10 µg SOMV, or SOMV-9RE7 (10 µg of SOMV and 10 µg of 9RE7).
Flow cytometry analyses
Blood samples were collected from vaccinated mice after final treatment. Red blood cell (RBC) lysis using RBC lysis buffer (eBioscience) collected peripheral blood mononuclear cells (PBMC). For tumor tissue sample preparation, tissue was collected from mice and transferred to FACS buffer in gentleMACS C tubes (Miltenyi Biotec). Tissue digestion enzymes including Collagenase I, Collagenase IV, and DNase I were added to samples. Samples were dissociated with gentleMACS Dissociator (Miltenyi Biotec) before incubating for 20 min. After centrifugation and buffer exchange, tumor samples were purified by loading onto Ficoll-Paque Plus (GE Healthcare Life Sciences, Marlborough, MA). Tubes were centrifuged for 20 min and Ficoll-RPMI interface was collected. Samples were then counted, plated at equal cell numbers, and prepared for flow cytometry. Spleen grinded through Corning® 70 μm Cell Strainer (Millipore Sigma) with syringe stopper and suspended in RPMI medium. Next, RBC lysis was performed and splenocytes were counted and plated at appropriate cell numbers.
For FACS analysis, live cells were identified with Zombie Aqua live/dead (BioLegend) and Fc Block to reduce nonspecific antibody binding. Peripheral antigen-reactive CD8+ T cell population in PBMC was identified with PE-conjugated HPV16 E7aa49–57 peptide loaded H-2 Db E7 tetramer and APC-A750-conjugated anti-mouse CD8α antibodies. For tumor infiltrating lymphocyte and splenocyte analyses, we used APC-A700-conjugated anti-mouse CD45 antibodies, BV421-conjugated anti-mouse CD3 antibodies, PE-Cy5-conjugated anti-mouse CD8 antibodies, PE-conjugated HPV16 E7 tetramer, and BV-650-conjugated anti-mouse IFNγ antibodies. FACS analysis was performed using CytoFLEX S (Beckman Coulter Life Sciences) and fluorescent compensation was generated using single-antibody controls. All flow cytometry data and gating strategies were performed by FlowJo software.
Statistical analysis
GraphPad Prism V.10 software was used to perform data statistical analysis. Data is represented as means and standard error of the mean. Kaplan–Meier survival plots were used to estimate the survival percentage and tumor-free rate. Long rank tests were used to compare the survival time between treatment groups. Comparison between individual data points were analyzed for variance with one-way ANOVA and the Tukey–Kramer multiple comparison test, *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns = not significant.
Discussion
In this study, we wanted to demonstrate the wide applicability of our efficient arginine peptide-enhanced antigen coating platform that is not only restricted to bacteria as carriers [
8], but also bacteria-derived OMVs. Here, we saw the importance of arginine residues in enhancing affinity between 9RE7 peptide and SOMV. In contrast, FITC-labeled E7 coating of SOMV resulted in heterogeneous populations of SOMV-E7-FITC, indicating non-uniform distribution of E7-FITC on SOMV (Fig.
1A). Further investigation of synthesized SOMV-9RE7 confirmed its 9RE7 presentation due to a positive surface charge increase from SOMV (Fig.
1E). These results suggest that the 9RE7 peptide coating strategy can also be applied to OMVs, demonstrating its diverse utility as an immunotherapeutic method.
By retaining the similar inflammatory components to parental gram-negative bacteria, OMVs can engage APCs through TLR4 recognition and cross-present antigens to T cells [
26]. Previously we saw that 9RE7 peptide could stimulate E7-specific through peptide loading on dendritic cells [
8]. This creates a potential confound of false positive activation from unbound 9RE7 in SOMV-9RE7 synthesis that wasn’t removed during the filtration procedure. To eliminate this artifact, the 9RE7 control group was dialyzed with a 50kD centrifugal filter to examine peptide removal efficiency. From the low T cell activation treated with filtrated 9RE7 (Fig.
1G), it can be confirmed that the increased IFNγ expression in SOMV-9RE7 treated group was not an artifact from unremoved free peptide.
Delivering SOMV-9RE7 subcutaneously in TC-1 tumor-bearing mice led to significant anti-tumor effects after the first dose. Development of E7 antigen-specific T cells was confirmed in PBMC after two dose treatments, further confirming the effectiveness of the 9RE7 antigen coating strategy. SOMV-9RE7’s therapeutic effect is potentially due to direct activation of APCs in the tumor-draining lymph-node for immediate onset of adaptive immunity [
27]. Due to the native inflammatory agents present on SOMV, it induced mild inflammatory responses including symptoms of redness at the injection site and weight loss of around 5%. These symptoms cleared quickly within one or two days after injection. They became much less pronounced following the second dose, meaning the mice have begun to tolerate the treatment. In future experiments, a reduced overall dose or a gradual dose escalation of SOMV can be implemented to prevent septic shock and overactive immune response.
Analyzing the immunologic profile in the TME, we observed a significant infiltration and activation of E7-specific CTLs in SOMV-9RE7 treated mice. The same trends in T cell proliferation were reflected in splenocytes. On the other hand, control treatments 9RE7 and SOMV yielded no significant amounts of E7-specific TILs (Fig.
3H) or splenocytes (Fig.
3F). Therefore, we can confidently conclude that 9RE7 antigen-display contributed to the differential E7+ T cell expansion difference between SOMV and SOMV-9RE7, which led to tumor-specific control. Interestingly, 9RE7 treated tumors showed a slight increase in CD8+ TILs (Fig.
3F), while splenocytes of SOMV treatment display an increase in CD8+ subpopulation (Fig.
4D) despite overall CD45+ CD3+ splenocyte decreased (Fig.
4B). These discrepancies in the observed local and systemic immunological profiles, however, cannot convey a comprehensive understanding of tumor-associated immune responses that correlate to tumor control. This further highlights the consistent cytotoxic lymphocyte profiles in SOMV-9RE7 immunotherapy that drive tumor-associated immunity.
Thus, we have demonstrated the successful synthesis of an MHC class I tumor-associated epitope displaying OMV vaccine with the polyarginine CPP method. Furthermore, SOMV-9RE7 exhibited immunogenic properties that activate E7-specific T cells via APC cross-presentation in vitro. Finally, administration of SOMV-9RE7 showed superior anti-tumor effects on TC-1 tumors by increasing E7-specific T cell infiltration and boosting systemic adaptive immunity. Future investigation will focus on the differences in therapeutic efficacy of 9RE7-coated
Salmonella and SOMV to determine the more efficacious and safer platform for bacterial immunotherapy in HPV-associated and other cancer models. Previous bacteria-based combination therapy results showed highly synergistic benefits of pairing cytokine or immune checkpoint inhibitors to enhance the efficacy of bacteria therapy [
8,
9,
28]. Our future experiments will dive into augmenting the CD8+ T cell-mediated tumor-specific immune cascade to determine synergistic combination therapy targets. In a phase II clincal trial of treating metastatic melanoma with modified HLA-A2*0201 bound gp100:209–217(210 M) peptide vaccine in combination with cytokine interlekin-2 (IL-2), the glycoprotein carrier can be replaced with our SOMV platform to introduce bacterial immunotherapy as a combination treatment of melanoma [
29]. Due to the simplicity and efficiency of polyarginine coating strategy, it can be broadly applied to personalized neoantigen targeted therapy that utilizes next-generation sequencing to identify highly immunogenic tumor-specific neoantigen [
30]. Our peptide-loaded SOMV can be seamlessly and effectively implemented in other neoantigen vaccines, such as the iNeo-Vac-P01 for pancreatic cancer[
31], and the HER2-derived MHC I peptide E75 vaccine used in clinical trial for ductal carcinoma in situ [
32]. Finally, small exogenous proteins and antibodies can also be displayed on OMVs using polyarginine CPPs as anchors. Engineered HPV L2 minor capsid targeting monoclonal antibodies can potentially be delivered through OMV surface presentation, which may lead to more efficient virus neutralization and offer new solutions to antibody penetration and targeting challenges [
33,
34].
By implementing our innovative 9RE7 antigen coating strategy to OMV and introducing the pioneering SOMV-9RE7, we have provided a feasible and economical approach for developing bacteria-based antigen-displaying vaccines. This strategy employs a straightforward production technique, eliminating the need for designing recombinant bacterial constructs, which allows the control over the peptide to OMV ratio. Furthermore, recent studies involving OMV vaccines in phase II clinical trials have demonstrated promising results [
35], indicating the translational potential of SOMV vaccines. While our SOMV-9RE7 work represents a groundbreaking advancement in bacteria-immunotherapy and vaccines for HPV-associated cancer, future preclinical and clinical research endeavors will continue to expand upon this strategy.
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