Nanocarriers based on bacterial membrane materials for cancer vaccine delivery

Here we present a protocol for the construction and use of two types of nanocarrier based on bacterial membrane materials for cancer vaccine delivery. Cancer vaccines induce tumor regression through triggering the specific T-cell responses against tumor neoantigens, a process that can be enhanced by nanocarrier delivery. Inspired by the body’s natural immune defenses against bacterial invasion, we have developed two different types of nanocarrier based on bacterial membrane materials, which employ genetically engineered outer-membrane vesicles (OMVs), or hybrid membrane vesicles containing bacterial cytoplasmic membrane, respectively. The OMV-based nanocarriers can rapidly display different tumor antigens through the surface modified Plug-and-Display system, suitable for customized cancer vaccines when the tumor neoantigens can be identified. The hybrid membrane-based nanocarriers are prepared through fusion of the bacterial cytoplasmic membrane and the primary tumor cell membrane from surgically removed tumor tissues, possessing unique advantages as personalized cancer vaccines when the neoantigens are not readily available. Compared with chemically synthesized nanocarriers such as liposomes and polymer without intrinsic adjuvant properties, owing to the large amounts of pathogen-associated molecular patterns, the two nanocarriers can activate the antigen-presenting cells while delivering multiple antigens, thus inducing effective antigen presentation and robust adaptive immune activation. Excluding bacterial culture and tumor tissue collection, the preparation of OMV- and hybrid membrane-based nanocarriers takes ~8 h and 10 h for tumor vaccine construction, respectively. We also detail how to use these nanocarriers to create cancer nanovaccines and evaluate their immunostimulatory and antitumor effects. This protocol details the construction of two types of nanocarrier based on bacterial membrane materials and their use in vaccine delivery to create cancer nanovaccines.


Introduction
As an emerging class of immune-oncology therapy, therapeutic cancer vaccines have demonstrated robust tumor-specific immunogenicity and antitumor activity in patients with melanoma, glioblastoma and other cancers in the past decade [1][2][3][4][5] . Different from the previously developed cancer vaccines targeting tumor-associated antigens that overexpress in tumor cells, the recent cancer vaccines trigger specific immune responses against neoantigens that are produced by genetic mutations in tumor cells, thus potentially avoiding off-target effects 6,7 . However, the immunogenicity of tumor neoantigens alone is often disappointing, and the use of immune adjuvants and/or delivery vehicles is an effective approach to improve the immunogenicity 8,9 . Immune adjuvants can stimulate the antigen-presenting cells (APCs) to provide the necessary costimulatory signals for successful antigen presentation, while delivery vehicles can enhance the uptake and processing efficiency of neoantigens by APCs 10,11 .
Currently, some promising nanocarriers with intrinsic immune adjuvant properties, such as polymeric and lipid nanoparticles with stimulator of interferon genes (STING) pathway activation ability, have been developed 12,13 . These nanocarriers can ensure that immune activation and antigen delivery occur in the same APCs, which is necessary for effective antigen presentation. Inspired by the body's natural immune defenses against bacterial invasion, our group has developed two different types of nanocarrier based on bacterial membrane materials for cancer vaccine delivery 14,15 . Owing to the large amounts of pathogen-associated molecular patterns (PAMPs), the bacterial membrane materials can act as excellent nanocarriers with intrinsic immune adjuvant properties [16][17][18] . To make these nanocarriers more suitable for use in cancer vaccines, two different loading methods for known and unknown tumor neoantigens have been applied for different application purposes, respectively (Table 1).
In the first study, the bacteria-derived outer-membrane vesicles (OMVs) were extracted through ultracentrifugation, which are the natural nanosized vesicles secreted by Gram-negative bacteria ( Fig. 1a) 19,20 . To simplify the display of tumor neoantigens on OMVs, we employed a Plug-and-Display system to decorate OMVs through genetic engineering (Fig. 1a) 14 . The Plug-and-Display system comprises the tag/catcher protein pairs, and the protein catchers were fused with the surface protein ClyA on OMVs 21,22 . Then, the OMVs decorated with different protein catchers can simultaneously display multiple tumor neoantigens labeled with the corresponding tags, thereby rapidly constructing the tumor nanovaccines (Fig. 1a). These OMV-based nanovaccines have been used to inhibit tumor growth and metastasis, which are more suitable for customized design when the tumor neoantigens have been identified and/or can be identified. According to the identified antigen information, the individualized matched neoantigens were synthesized and displayed on the OMV-based nanocarriers to create antigen-suited nanovaccines for patients with cancer.
In the second study, we developed hybrid membrane-based nanocarriers (HM-NPs), containing the bacterial cytoplasmic membrane from Escherichia coli (EM) and tumor cell membrane (TM) from the resected autologous tumor tissues (Fig. 1b) 15 . Briefly, the poly(lactic-co-glycolic acid) (PLGA) nanoparticles were constructed as a core, and the EM and TM were simultaneously coated onto the PLGA nanoparticles to form the final HM-NPs (Fig. 1b). The EM acts as the immune adjuvant to activate the innate immune response, and the autologous tumor neoantigens in the TM are the key messengers for transmitting the tumor-specific identification to the adaptive immune system [23][24][25][26] . These HM-NPs possess unique advantages as personalized cancer vaccines when the tumor neoantigens are not readily available and are more suitable for prevention of postoperative recurrence.
Here we provide protocols for the construction of these two types of vaccine based on bacterial membrane materials and the matched tumor antigens from different sources, and the potential applications and limitations of the nanovaccines are discussed. The characterization methods are validated and presented in detail, and the antigen delivery effect and immune stimulation mechanisms were validated both in vitro and in vivo.

Development of OMV-based nanocarriers and vaccines
Owing to their excellent immunostimulatory ability, OMVs are attractive candidates as vaccine carriers in the field of prophylactic vaccines against pathogenic microorganisms 20 . For example, the OMV-based vaccines against Group B meningococcus, namely MeNZB, have effectively decreased the incidence and mortality of meningitis in New Zealand 27 . In addition to preventing the invasion of their parental microorganisms, OMVs can be employed as a display platform of heterologous antigens to form vaccines against other pathogens through genetic engineering, chemical bonding and physical mixing 28 . To exploit OMVs as nanocarriers for cancer vaccines, the loading method of tumor neoantigens is the key issue. First, we adopted fusion expression to display a pattern antigenic Table 1 | Properties and applications of two types of nanocarrier based on bacterial membrane materials for cancer vaccines epitope of ovalbumin (OVA), OVA 257-264 (SIINFEKL) on the OMVs' surface, in which OVA [257][258][259][260][261][262][263][264] was fused with the C terminal of the surface protein ClyA (ClyA-OVA) on OMVs 29 . The strong antigen-specific immune response and antitumor effect induced by ClyA-OVA OMVs indicated that OMVs are outstanding vaccine nanocarriers for displaying tumor neoantigens to elicit antitumor immunity.
However, the tumor neoantigens generated from the gene mutations have high heterogeneity and variability 30 , making it impractical and time-consuming to produce OMV-based nanovaccines for every patient via fusion expression with neoantigens. The antigen display of tumor vaccine carriers should be rapid and flexible to meet the clinical requirement. Therefore, we employed the Plug-and-Display system to decorate OMVs, including a SpyTag (SpT)/SpyCatcher (SpC) pair and a SnoopTag (SnT)/SnoopCatcher (SnC) pair 21,22 . The SpC and SnC catchers were fused with the ClyA protein (ClyA-catchers, CC) on OMVs' surface and can spontaneously bind to the neoantigens labeled with SpT and SnT tags through isopeptide bond formation, respectively. These bioengineered CC OMVs were able to simultaneously and rapidly display multiple tumor neoantigens, thereby being competent for rapid preparation of the customized cancer vaccines when tumor antigens can be predicted and identified.

Development of HM-NPs and vaccines
Surgical resection is the primary treatment option for most solid tumors, and the recurrence and metastasis after surgery are still unmet clinical challenges 31,32 . Vaccination after surgery to induce the antigen-specific immune responses to eliminate residual tumor cells may have wide application 33 . TMs contain a high proportion of antigenic motifs and have similar antigen patterns as are displayed on the surface of tumor cells in vivo 34,35 . However, autologous tumor antigens can be recognized as 'self', and they are more likely to induce antigen-specific tolerance rather than antitumor immunity 36 . The body's immune defenses against bacterial invasion are rather sensitive; utilizing bacterial

Fig. 1 | Preparation of OMV-and HM-NPs for cancer vaccines. a, Preparation of OMV-based nanocarriers. The catchers (SpC and SnC) in the
Plug-and-Display system are fused with the ClyA protein (ClyA-catchers) on OMVs' surface, through E. coli genetic engineering. Then, the engineered bacteria-derived OMVs are extracted through ultracentrifugation. The OMVs decorated with two different catchers can simultaneously display multiple tumor neoantigens (Ag) labeled with corresponding tags (SpT and/or SnT), thereby rapidly constructing the antitumor nanovaccines. b, Preparation of HM-NPs. Tumors are surgically removed from tumor-bearing mice to obtain TM. E. coli are treated with lysozyme to remove the cell wall to generate protoplasts, and the cytomembrane is extracted with an extraction buffer to prepare E. coli cytomembrane (EM). HM vesicles are generated by mixing EM and TM, followed by extruding through an extruder. PLGA nanoparticles (NPs) as polymeric cores are then added to generate HM-NPs. Figure adapted from ref. 14 under a Creative Commons licence CC BY 4.0 (a) and ref. 15 , AAAS (b).
constituents that act as adjuvants to enhance immunogenicity is a promising strategy to overcome this limitation. However, lipopolysaccharide (LPS) and other cell wall components of bacteria as the first line of contact with immune cells could cause an undesirable immunopathological state 23 . The bacterial cytoplasmic membrane, which is spatially separated from the organism's cell wall, may be used as a potential adjuvant to reduce the magnitude of 'danger signals' 17,18 . Membrane fusion is a technology that can confer a hybrid membrane (HM) with properties inherited from two source cell membranes [24][25][26] . Thus, we developed the immunotherapy strategy using bacterial cytoplasmic membranes from E. coli and autologous tumor membranes from resected tumor tissue to confer a HM with the properties inherited from two source cell membranes 15 . These HM-NPs were able to codeliver antigen and adjuvant to APCs.

Overview of the procedures
First, we describe the preparation of the two types of nanocarrier based on bacterial membrane materials and the related nanovaccine constructions (Procedure 1, Steps 1-18 for OMV-based nanocarriers; Procedure 2, Steps 1-38 for HM-NPs). Next, the methods and processes for characterization of physicochemical properties and biological components are described in detail (Procedure 1, Steps 19-30 for OMV-based nanocarriers; Procedure 2, Step 39 for HM-NPs). Then, the antigen delivery efficiency and the immune response analysis are performed (Procedure 1, Steps 31-48 for OMV-based nanocarriers; Procedure 2, Step 40 for HM-NPs). Finally, we discuss the strategies to evaluate the in vivo antitumor effect in different mouse models using multiple antigens (Procedure 1, Step 49 for OMV-based nanocarriers; Procedure 2, Step 41 for HM-NPs). More detailed information on the two procedures is provided below.

Procedure 1: OMV-based nanocarriers and vaccines
Through genetic engineering, we splice the gene fragments fusing SpC or SnC and ClyA (ClyA-SpC and ClyA-SnC) respectively, and insert them into the co-expression plasmid pETDuet-1. Then, the co-expression plasmid is transformed into the engineered bacteria E. coli Rosetta (DE3). Finally, CC OMVs are extracted from the culture medium in massive quantities by bacterial fermentation and ultracentrifugation, followed by antigen display, vaccine preparation and functional characterization. In this protocol, the size and morphology of CC OMVs are characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The antigen display ability is evaluated by western blot. Finally, CC OMVs are used to display multiple tumor antigens including OVA 257-264 (termed 'OTI', an epitope that can stimulate the production of MHC class I-restricted OVA-specific CD8 + T cells in mice), OVA 223-339 (ISQAVHAAHAEINEAGR; 'OTII', an epitope that can stimulate the production of MHC class II-restricted OVA-specific CD4 + T cells in mice), an antigenic epitope of tyrosinase-related protein 2 (TRP2), TRP2 180-188 (SVYDFFVWL) and an antigenic epitope of Adpgk (CGIPVHLELASMTNMELMSSIVHQQVFPT) 37,38 . The in vitro and in vivo immune response and antitumor effect are determined in the pulmonary metastatic melanoma model and subcutaneous colon cancer model. Procedure 2: HM-NPs and vaccines HM-NPs are synthesized by preparation of the HM of EM and TM and coating the HM onto the synthesized PLGA nanoparticles. In this protocol, the size and morphology of HM-NPs are characterized using DLS and TEM, respectively. Immunogold staining for specific markers to identify EM and TM on HM-NPs is also observed by TEM. The enhanced tumor antigen uptake and maturation of bone marrow-derived dendritic cells (BMDCs) by HM-NPs are evaluated by flow cytometry and enzyme-linked immunosorbent assay (ELISA). The mouse tumor models of 4T1, CT26 and B16-F10 cells are then used to investigate the antitumor immune effects of HM-NPs.

Applications of the method
Potential applications of OMV-based strategy for vaccine development In this protocol, we used the OMV-based nanocarriers to rapidly display multiple antigens, including OTI, OTII, TRP2 180-188 and Adpgk. Of course, this platform can be also employed to deliver any other antigenic peptide, including human and murine antigens. Because of the high compatibility, this platform is suitable for the preparation of customized cancer vaccines for each patient with cancer. In the future, if a tumor antigen library is established, we can quickly identify the antigen NATURE PROTOCOLS PROTOCOL NATURE PROTOCOLS | VOL 17 | OCTOBER 2022 | 2240-2274 | www.nature.com/nprot information of patients with tumors by gene sequencing, so as to make use of the rapid antigen display capability of this platform for the preparation of customized cancer vaccines. In addition, OMV-based nanocarriers can also be used for rapid screening and identification of tumor antigens owing to their excellent immune stimulation, antigen transport and rapid antigen display capabilities.
As mentioned earlier, previous studies have focused on using OMVs to build vaccines against microorganisms. Therefore, in addition to cancer vaccines, prophylactic vaccines against infectious diseases can also use our OMV-based nanocarriers, especially for pathogenic microorganisms with highly variable antigens, such as influenza virus.
Potential applications of HM-based strategy for vaccine development The HM-NPs are integrated into a vaccine delivery nanoplatform with both bacterial cytoplasmic membranes and surgically derived TM to enhance both innate and adaptive immune responses. This HM-derived vaccine strategy is beneficial to multiple tumor models by codelivery of individualized tumor antigens and adjuvants into dendritic cells (DCs). In addition, these HM-NPs can be genetically or chemically modified to further expand their multifunctionality. The core of the nanocarriers can also load various cargos for integration of other treatment modalities to enhance the effects of immunotherapy.

Comparison with other methods
Several materials have been used to develop nanocarriers for tumor vaccine delivery, such as lipid, polymer, synthetic high-density lipoprotein and DNA origami [39][40][41][42][43][44] . Compared with these materials, the bacterial membrane material-based nanocarriers have a unique immune stimulation function and play the dual roles of immune adjuvants and antigen carriers. In addition, compared with the complexity of chemical synthesis, bacterial membrane material-based nanocarriers can be obtained in relatively large quantities through bacterial fermentation. This feature may ensure compatibility with industrial production in the future.
In the first Procedure, the Plug-and-Display system is employed to achieve the rapid display of peptide antigens on OMVs. The current clinical trials of tumor vaccines based on peptide antigens are mainly conducted by using a mixture of peptides and adjuvants 1,[3][4][5] . Nanocarriers can improve stability and immune system targeting of the antigens and realize the codelivery of antigens and adjuvants 9,10 . Therefore, the tumor vaccines based on nanocarriers have received more and more attention 9,10 . As mentioned above, the bacteria-derived OMVs have the dual functions of both carriers and adjuvants, while the chemical synthetic nanocarriers are mainly delivery vehicles. Therefore, the nanovaccines based on OMVs do not require additional adjuvants. In addition, our study demonstrated that the OMV-based nanovaccines can activate an antigen-specific T-cell response, as distinct from the previously reported B-cell-mediated antibody responses in the OMVbased prophylactic vaccines against pathogenic microorganisms 20,27 .
More importantly, we employed the Plug-and-Display system to achieve the rapid display of multiple peptide antigens. Compared with the fusion expression, physical encapsulation or chemical conjugation, this flexible approach of antigen display makes our OMV-based nanocarriers more suitable for rapid preparation of customized cancer vaccines. The Plug-and-Display system has been used in the construction of vaccines against microorganisms based on virus-like particle [45][46][47] . We firstly demonstrate the feasibility of this system for constructions of tumor vaccines. Compared with virus-like particle, OMVs have the advantage of immune adjuvant function, despite the disadvantage of complex composition. Furthermore, the current practice for antigen encapsulation into the nanoparticles during the production process lacks flexibility to load multiple antigens under different scenarios 41,43 . The vehicle and antigen(s) in our nanovaccine can be separately synthesized and used to rapidly prepare the vaccine through a simple combination procedure before immunization. This modular design allows one to establish a neoantigen library in advance and rapidly select appropriate antigens for individual patients, which may reduce the production time and realize the bedside preparation of tumor vaccines for individual patients in the future 14 .
In the second Procedure, the cancer vaccines are constructed through hybrid fusion of the surgically removed TM and the bacterial cytoplasmic membranes. Traditional methods of autologous tumor cell-based vaccines use whole-cell tumor vaccines or tumor lysate vaccines to elicit immunity against the entire collection of antigens expressed by the tumor 33,48,49 . Although whole-cell tumor or tumor lysate vaccines open a promising area for cancer immunotherapy, therapeutic efficacy may be severely limited as the immunogenicity of antigens in these vaccine formulations may be diluted or PROTOCOL NATURE PROTOCOLS 2244 inhibited by the unrelated proteins and nucleic acids. The current HM tumor vaccines are constructed through the fusion of the surgically removed TM and the bacterial cytoplasmic membranes, possessing unique advantages as personalized cancer vaccines with highly enriched antigens when the identified ones are not sufficient to trigger antitumor immunity or the neoantigens are not readily available.
Although TM are naturally enriched with a specific tumor antigen pool, these membrane antigens usually have low immunogenicity since tumorigenesis implies adaptation of tumor cells to the host immune system. Compared with the other tumor membrane-based nanocarriers, the introduction of the bacterial cytoplasmic membranes enhances the recognition of tumor membrane antigens by immune system. Meanwhile, bacterial cytoplasmic membranes also take advantage of the antibodydependent, cell-mediated cytotoxicity of NK cells, which lyse malignant cells even before the stimulation of specific T cells in an antigen-independent manner. Bacteria-derived substances (inactivated or attenuated bacteria, lipids, proteins and/or nucleic acids) provide 'danger signals' that alert the immune system to the potential infection and invasion 17 . However, bacteria-derived formulations, especially LPS, can lead to severe side effects, such as cytokine storm and sepsis 18 . Using bacterial cytoplasmic membranes can reduce the magnitude of 'danger signals' since most of the bacterial LPS is removed. Meanwhile, the bacterial cytoplasmic membranes can be rapidly and manageably prepared from inexpensive cultures of bacteria. This vaccine formulation protocol provides an appropriate balance among accessibility, safety and effectiveness.

Limitations
Some limitations of OMV-based nanocarriers for cancer vaccines should be considered. First, the OMVs contain large amounts of proteins, polysaccharides, lipids and trace amounts of nucleic acids from bacteria, and the complicated components may cause difficulty for the quality control of vaccine production. In this Procedure, we use DLS to detect the particle size and TEM to observe the morphology for quality control of OMVs. The ideal OMVs should have a low polydispersity index (PDI, <0.2). Another quality control from the production aspect of OMV-based products may be the presence of some characteristic proteins in the OMVs, such as outer-membrane protein A/C/F, which can be used as the fingerprint for OMV identification 20 . Second, the amount of LPS in OMVs is the main component of endotoxin, and the elimination of LPS in OMVs by genetic engineering is a feasible option for further improvement 50 . Third, the antigens used in this protocol include model antigens and murine tumor antigens, and the adaptability of this platform to human tumor antigens needs further study. Fourth, ultracentrifugation is used to isolate and purify OMVs in this protocol, but this method may not be suitable for mass production. Appropriate purification methods for industrial practices are in urgent need, and chromatographic separation according to the size of OMVs is an optional solution. Finally, the Plug-and-Display system used to create CC OMVs may be further optimized. In addition to ClyA protein, there are some other surface proteins on OMVs that can be employed as scaffold for the Plug-and-Display system, such as hemoglobin protease and outermembrane protein A/C/F 20 . In addition, updated versions of the Plug-and-Display system have been described, such as spycatcher003, with a stronger integration efficiency 51 . These novel scaffolds and Plug-and-Display systems should be further evaluated to determine whether they will be able to enhance the antigen display capability of the OMV-based nanocarriers.
One limitation of HM nanovaccines is that the immunosuppressive proteins of the TM have not been investigated. As tumor cells interact with the infiltration of immune cells and form the immunesuppressive environment, there is a high portion of immunosuppressive proteins. For example, the well-known immune checkpoint inhibitor, programmed cell death 1 ligand (PD-L1), is located in the TM 52 . Enhanced antitumor efficacy may be achieved by regulating these immunosuppressive factors. In addition, the contribution of other cell membranes to the antitumor immunity should be considered, since immune cells and stromal cells in the resected tumor tissue are not eliminated during HM nanovaccine preparation. Tumor cells could firstly be isolated and purified by the negative selection-based tumor cell isolation kit or culture plate adherent passaging, then the effectiveness of HM vaccines prepared from purified TM could be assessed in comparison. The fabricating methods could also be improved by the use of microfluidic methods instead of manual preparation and procedures. Last but not least, specific antigens on the TM recognized by the T cells are not yet identified. All these factors could either limit or benefit translation to the clinic.
In addition, the sterile production of the OMV-based nanocarriers and HM-NPs requires special attention. In the Procedure for the OMV-based nanocarriers, almost all the bacteria were removed by NATURE PROTOCOLS PROTOCOL NATURE PROTOCOLS | VOL 17 | OCTOBER 2022 | 2240-2274 | www.nature.com/nprot centrifugation at 5,000g for 10 min. In the following steps to separate OMVs from the supernatant, two filtration processes were carried out through 0.45 μm and 0.22 μm filters to ensure that the final OMVs did not contain bacteria. In the Procedure for the HM-NPs, the lysozyme and extraction buffer can lyse almost all the bacteria to obtain the cytoplasmic membranes of E. coli. In addition, in the following steps to prepare the HM-coated nanoparticles, the HM were extruded at least 13 times through the 400 and 200 nm cutoff sterile extruders, respectively. Therefore, the final HM-NPs do not contain bacteria. For future industrial-scale preparation, radiation sterilization may be suitable for large-scale production of the OMV-based nanocarriers and HM-NPs.

Experimental design
Selecting tumor antigens In our first Procedure, we adopt several model antigens (OTI and OTII) and murine tumor antigens (TRP2 180-188 and Adpgk) to make tumor vaccines against different cancer cells. Other tumor antigens can be selected according to the cancer cell type. For example, the gp100 antigen can be used to establish a cancer vaccine against B16-F10 melanoma cells, and the gp70 antigen is suitable for the treatment of CT26 colon cancer cells 53,54 . In addition, the combination of mass spectrometry and exome sequencing can effectively predict and identify neoantigens in tumor cells, which lays the foundation for customized cancer vaccines 38 . These identified tumor antigens can also be used in the experiments according to our protocol. Once the tumor antigens are selected, the SpT or SnT tag needs to be added at the N terminal, and the tag-labeled antigen peptides are synthesized via the Fmoc solid-phase peptide synthesis method. When there is only one type of tumor antigen, either SpC-SpT or SnC-SnT pair can be used for rapid display, and there is no notable difference between them. If there are two types of tumor antigen, it is recommended to use different pairs to display, so as to increase the efficiency of display. If there are more than two types of tumor antigen, we recommend that each antigen can be displayed separately using either SpC-SpT or SnC-SnT pair to construct different nanovaccines, and eventually mix together for immunization.
Selecting murine cancer models and mouse strains In our two Procedures, several murine cancer models are used, including B16-F10 and OVAexpressing B16-F10 (B16-OVA) melanoma, MC38 and CT26 colon cancer and 4T1 breast cancer. According to the origin of the tumor cells, different mice were used to make tumor models, including BALB/c mice for CT26 and 4T1 and C57BL/6 mice for B16-F10, B16-OVA and MC38. This protocol is also suitable for use with other murine tumor models. If the tumor vaccine is to be built against human tumor cells, mice with a humanized immune system should be used.

Design of controls
In the first Procedure, for OMV-based nanocarriers, the critical control group is a mixture of CN OMVs (from the bacteria expressing ClyA without the fused Catchers, ClyA-none) and tumor antigens, thus proving the importance of antigen display on CC OMVs for efficient immune stimulation. The control groups of separate antigens or separate empty CC OMVs are also necessary to illustrate the advantages of the system. If multiple antigens are displayed on the surface of CC OMVs to prepare tumor vaccines, control vaccines containing individual antigens should also be evaluated to determine synergistic effect between different antigens.
In the second Procedure, for HM-NPs, the critical control groups include the EM-coated nanoparticles (EM-NPs) and TM-coated nanoparticles (TM-NPs), and the physical mixture of two types of NPs (Mix NPs). The codelivery ones (HM-NPs) should result in a synergistic stimulation of an immune response, compared with the individual antigen and adjuvant nanoparticles or mixed ones.  (Lucigen,, in which LPS is genetically modified to not trigger the endotoxic response in human cells.

Plasmid
• pETDuet-ClyA-Catchers (synthesized by Suzhou Genewiz). The DNA fragments encoding the ClyA-SpC and ClyA-SnC (Table 2) are inserted into the backbone plasmid pETDuet-1 simultaneously to construct the co-expression plasmid pETDuet-ClyA-Catchers. The restriction enzymes NcoI and SalI are used for the ClyA-SpC insertion, and the restriction enzymes NdeI and KpnI are used for the ClyA-SnC insertion

Mice
• C57BL/6 mice (6-8 weeks old; Vital River Laboratory Animal Technology). Mice are housed in a room at 20-22°C with a 12-h light/dark cycle and a humidity of 30-70%, and provided food and water ad libitum c CRITICAL All animal protocols were approved by the Institutional Animal Care and Use Committee of the National Center for Nanoscience and Technology. ! CAUTION The mouse strain should be determined according to the source of tumor antigens and tumor cells in your experiments, such as using the C57BL/6 and BALB/c mice for the B16-F10 and CT26 cells, respectively. The sex and sex chromosomes do not affect the tumor growth or immune activation of vaccines. However, some sex-hormone-dependent tumors should mainly be used in mice of the corresponding sex, such as female mice for breast cancer and male mice for prostate cancer. In addition, we recommend the use of female mice because their body weight is relatively stable, which make it easy to control the dose-weight ratio.

Procedure 1
Solid LB medium. Prepare solid LB medium by dissolving 1 g NaCl, 0.5 g yeast extract, 1 g tryptone and 1.5 g agar in 100 ml deionized water. After sterilization under high temperature and high pressure, antibiotics (such as ampicillin sodium, kanamycin, etc.) were added when the temperature reduced to 60°C. After fully mixing, 10 ml sterilized solution was added to 10 cm dish and sealed with sealing film after solidification, which could be stored at 4°C for 1 month. ! CAUTION Temperature will seriously affect the activity of antibiotics, so antibiotics should be added when the liquid temperature drops to~50-60°C.
Liquid LB medium. Prepare liquid LB medium by dissolving 10 g NaCl, 5 g yeast extract and 10 g tryptone in 1 L deionized water. After sterilization under high temperature and high pressure, it can be stored at 4°C for 1 month.
Fekete's buffer. Prepare Fekete's buffer by mixing 70 ml ethanol, 10 ml formalin and 5 ml glacial acetic acid. The solution can be stored at 4°C for 1 week. ! CAUTION Ethanol, formalin and glacial acetic acid are highly volatile. Wear acid-resistant gloves, lab coat and goggles, and handle with caution in a fume hood.
PMA and ionomycin (PI) solution. Prepare PI solution by dissolving 5 μg PMA and 100 μg ionomycin in 1 ml sterilized PBS. The solution can be stored at 4°C for 1 week.
Tissue digestion solution. Prepare tissue digestion solution by dissolving 5 mg trypsin, 500 mg collagenase IV and 1 mg DNase-I in 100 ml RPMI 1640. The solution can be stored at 4°C for 3 d.
Procedure 2 c CRITICAL For preparation of plasma membranes from tumor tissue, all glassware should be washed with deionized water to avoid Ca 2+ contamination. Calcium contamination can cause swelling of mitochondria and rupture of the outer mitochondrial membrane. Special attention should be paid to use deionized water instead of ice and tap water in all preparations.

LB medium
Dissolve 10 g tryptone, 5 g yeast extract and 10 g sodium chloride in 1,000 ml deionized water, and the mixture sterilized at 121°C for 30 min. The solution can be stored at 4°C for 1 month. Extraction buffer Dissolve 1 mg DNAse-I and 95 mg MgCl 2 in 100 ml deionized water, and add 5 ml Tris-HCl (1 M, pH 8.0). The solution can be stored at 4°C for 1 week.

Tissue dissociation solution
Add 5 mg collagenase IV, 0.5 mg DNAse-I and 5 mg hyaluronidase to 5 ml GBSS buffer. The solution can be stored at 4°C for 3 d.
100 mM EGTA, pH 7.4 Dissolve 38.04 mg EGTA in 1 ml deionized water, and adjust the pH to 7.4 with KOH. The solution can be stored at 4°C for 1 week.
Isolation buffer (IB) Dissolve 4.1 g mannitol and 2.6 g sucrose in 100 ml deionized water, and add 3 ml Tris-HCl (1 M, pH 7.4). Place the buffer at 4°C for~30 min to allow it to cool. Check the pH of the buffer, and adjust it (if necessary) with KOH (1 M, if too low) or HCl (1 M, if too high). Dissolve 0.5 g of albumin in 100 ml solution above mentioned, add 500 μl 100 mM EGTA (pH 7.4). Protease inhibitor cocktail (20 μl/4 ml) and 1% (vol/vol) phosphatase inhibitor cocktail (final concentration) should be supplemented in IB buffer to avoid sample degradation. The solution can be stored at 4°C for 3 d. ! CAUTION EGTA is recommended for removing traces of Ca 2+ . It can be replaced by EDTA; however, a lower concentration (not greater than 0.1 mM) should be used. ! CAUTION IB buffer must be made fresh, not more than 1 d before the experiment.
ACK lysing buffer Dissolve 8.02 g NH 4 Cl, 1 g KHCO 3 and 37.2 mg Na 2 EDTA in 850 ml deionized water. Adjust the pH to 7.2-7.4, and add deionized water to 1,000 ml. The solution can be stored at 4°C for 1 week.
1% (wt/vol) sodium cholate Dissolve 0.2 g sodium cholate into 20 ml deionized water. The solution can be stored at 4°C for 1 week.
1% (wt/vol) BSA solution Dissolve 0.25 g BSA into 25 ml deionized water. The solution can be stored at 4°C for 1 week.
1% (wt/vol) glutaraldehyde Dissolve 0.025g glutaraldehyde into 2.5 ml deionized water. The solution can be stored at 4°C for 1 week.
Procedure c CRITICAL The methods for the preparation of OMV-based nanocarriers and HM-NPs for cancer vaccines, are described in Procedures 1 and 2, respectively. Procedure 1: plasmid transformation and bacterial culture • Timing~2 d 1 Place the centrifuge tube containing 100 μl competent cells (E. coli Rosetta (DE3)) in an ice bath until completely thawed. c CRITICAL STEP The competent cells should be stored at −80°C, and repeated freezing and thawing should be avoided to ensure the transformation efficiency. 2 Add 1 ng co-expression plasmid pETDuet-ClyA-Catchers into the competent cells. Mix gently using a 10 μl pipette ten times, and let stand in an ice bath for 30 min. 3 Place the centrifuge tube in a 42°C water bath for 60-90 s, and then transfer the tube quickly to an ice bath to cool the cells for 2-3 min without shaking during this process. 4 Add 900 μl sterile LB medium to the centrifuge tube, and incubate in a 150 rpm shaker at 37°C for 45 min.

NATURE PROTOCOLS
c CRITICAL STEP The LB medium in this step must be antibiotic-free because the resistance gene has not yet been expressed. 5 Add 100 μl transformed cells to a 10 cm culture plate of solid LB medium containing 50 μg/ml ampicillin, and gently spread the cells evenly with a sterile glass stick. c CRITICAL STEP The 10 cm culture plate of solid LB medium should be prepared in advance and stored in a sterile environment of 4°C (see 'Reagent setup'). 6 Place the culture plate at room temperature (20-25°C) until the liquid is absorbed, and culture at 37°C for 12-16 h. c CRITICAL STEP The amount of bacteria in the plate can be adjusted, ideally to obtain several dozens of colonies in a 10 cm plate. j PAUSE POINT The bacteria in the plate can be stored at 4°C for 1 month. ? TROUBLESHOOTING 7 Pick one bacteria colony on the plate carefully, and add it into 200 ml LB medium containing 50 μg/ml ampicillin in a conical flask. c CRITICAL STEP The conical flask is sealed using the sterile breathable sealing film to ensure oxygen supply for bacterial growth. 8 Incubate at 37°C with shaking at 180 rpm. Monitor the optical density at 600 nm (OD 600 ) of bacterial culture medium using a spectrophotometer. When the OD 600 reaches 0.6, add 0.1 mM IPTG into the medium and incubate at 16°C with shaking at 160 rpm for another 14 h. c CRITICAL STEP The IPTG-induced expression can be performed at 37°C for 2 h, but the expression efficiency is not as good as induction at 16°C.
OMV extraction • Timing~7 h 9 Divide the 200 ml bacteria medium into four 50 ml centrifuge tubes, and collect the supernatant after centrifugation at 5,000g and 4°C for 10 min. c CRITICAL STEP The weight of the centrifugal tubes must be balanced. 10 Filter the resulting 200 ml supernatant through a 0.45 μm filter, and then concentrate to 50-60 ml using a 50 kDa ultrafiltration tube through centrifugation at 3,000g and 4°C for~5-10 min. 11 Filter the concentrated solution through a 0.22 μm filter. 12 Put the concentrated solution into two ultracentrifugation tubes. c CRITICAL STEP The ultracentrifugation tubes should be cleaned and dried in advance. 13 Separate the CC OMVs through an ultracentrifugation at 150,000g and 4°C for 3 h. c CRITICAL STEP Bubbles are strictly prohibited in the liquid in the ultracentrifugation tubes. The weight of ultracentrifugation tubes must be balanced! All ultracentrifugation tubes have a weight error of less than 50 mg. The tube cover must be tightened. 14 Discard the supernatant, and resuspend the deposited CC OMVs at the bottom of the tubes using PBS to fill up the ultracentrifugation tubes. 18 Name the final OMV-based nanovaccines according to the display antigens, e.g., CC-SnT-TRP2 OMVs, CC-SpT-OTI OMVs, CC-SnT-OTII OMVs, CC-SpT-OTI/SnT-OTII OMVs and CC-SpT-Adpgk OMVs (Table 3). c CRITICAL STEP To accurately control the amount of antigens in the nanovaccines and ensure the same amount of antigens in the different groups, the SpT-or SnT-labeled antigens unbound to CC-OMVs are intended not to be removed. The 50 μg CC-SpT (or SnT)-antigen OMVs mean that there are 50 μg SpT (or SnT)-antigen and 50 μg CC-OMVs in the final nanovaccine. The 50 μg CC-SpT-OTI/SnT-OTII OMVs mean that there are 25 μg SpT-OTI, 25 μg SnT-OTII and 50 μg CC-OMVs in the final nanovaccine. c CRITICAL STEP To evaluate the loading rate of SpT-or SnT-labeled antigens, the total proteins of the final OMV-based nanovaccines (containing 3 μg antigens and 3 μg CC OMVs) are extracted according to Procedure 1, Step 16. The proteins are analyzed by electrophoresis and Coomassie blue staining 21,22 . According to the quantitative analysis of the bands in the gel using ImageJ software, the mass of the unbound antigens can be calculated, thereby evaluating the loading rate. The loading rates of SpT-labeled antigens and SnT-labeled antigens are~8.5% and 19.5%, respectively (Supplementary Fig. 1a,b). j PAUSE POINT The final OMV-based nanovaccines can be stored at −20°C or −80°C for 1 month.
Characterization of OMV-based nanovaccines • Timing~2 d 19 DLS analysis of size (~0.5 h). Dilute 100 μl of the final OMV-based nanovaccines from Procedure 1, Step 18 into 1 ml ultrapure water in a centrifuge tube. 20 Mix the solution by vortex, and transfer it to a DLS cuvette. 21 Test the size distribution using a Malvern Zetasizer 14 .
c CRITICAL STEP The salt in PBS can seriously affect the DLS measurement, so OMVs must be diluted in pure water. To reduce the effect of osmotic pressure, measurements should be taken within 2 min after dilution in Step The final OMV-based vaccines are named according to the display antigens.
PROTOCOL NATURE PROTOCOLS 2254 28 Validation of antigen display (~1 d). To confirm the successful antigen display on CC OMVs, add a peptide tag into the antigens during the synthesis process, such as HA tag, Flag tag or c-myc tag. c CRITICAL STEP The peptide tag should be placed between the antigen and SpT or SnT to avoid affecting the connection of tag/catcher protein pairs. 29 Then, prepare the OMV-based nanovaccines according to Procedure 1, Steps 17-18 and extract the total protein according to Procedure 1,Step 16. 30 Use western blot analysis to detect the displayed antigens 14 . Incubate the membranes first with primary antibodies against these peptide tags (1:1,000) and then HRP-conjugated secondary antibodies (1:10,000) before visualization. The expected band is at~45 kDa, the molecular weight of ClyA-SpC and ClyA-SnC. c CRITICAL STEP It must be noted that the addition of these tags has no effect on the immune stimulation of the final OMV-based nanovaccines.
Immune stimulation evaluation of OMV-based nanovaccines in vitro • Timing~8 d 31 Keep the C57BL/6 mice (6-8 weeks old) in a room at 20-22°C with a 12-h light/dark cycle and a humidity of 30-70%. Provide food and water ad libitum. 32 Kill the C57BL/6 mice by cervical dislocation. Dissect the mice, and obtain the femurs and tibias from the killed C57BL/6 mice. Flush the bone marrow cells using an injection syringe and RMPI 1640 medium containing 2% FBS, 100 U/ml penicillin G sodium and 100 μg/ml streptomycin. 40 ml medium is enough for femurs and tibias from one mouse.
! CAUTION The animal study should comply with the relevant ethical regulations for animal testing and research. Our animal protocols are approved by the Institutional Animal Care and Use Committee of the National Center for Nanoscience and Technology. 33 Collect the bone marrow cells through centrifugation at 800g and room temperature for 5 min. 34 Discard the supernatant, and resuspend the precipitated cells with 1 ml ACK lysis buffer to lysis the red blood cells. Incubate at room temperature for 90 s. 35 Add 3 ml PBS to stop the lysis, and filter the cells through a 70 μm cell strainer. 36 Collect the cells through centrifugation at 800g and room temperature for 5 min. 37 Discard the supernatant and resuspend the precipitated cells with 12 ml RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin G sodium, 100 μg/ml streptomycin, 1% HEPES, 0.05 mM β-ME, 20 ng/ml IL-4 and 20 ng/ml GM-CSF, and divide into six wells in a six-well plate. c CRITICAL STEP The medium should be prepared in advance. 38 Replace half of the medium every 2-3 d. 39 Collect non-adherent cells on day 6 for further investigation, which are known as BMDCs, through centrifugation at 800g and room temperature for 5 min. 40 Discard the supernatant, and resuspend the precipitated BMDCs with 10 ml RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin G sodium and 100 μg/ml streptomycin. Quantify the cell density using the Automated Cell Counter. 41 Place 100,000 BMDCs into a 1.5 ml centrifuge tube with 500 μl RPMI-1640 medium supplemented with 100 U/ml penicillin G sodium and 100 μg/ml streptomycin. 42 Add 50 μg OMV-based nanovaccine from Procedure 1 Step 18 into the BMDCs, and incubate in the cell incubator for 24 h. c CRITICAL STEP Herein, we use the nanovaccine containing the OTI antigen (SIINFEKL), CC-SpT-OTI OMVs as an example. Meanwhile, two necessary control groups should be evaluated, including the only antigen group (SpT-OTI, 50 μg) and the mixture group (SpT-OTI + CN OMVs, 50 μg + 50 μg). c CRITICAL STEP The centrifuge tube should be sealed with sealing film, but the air hole should be preserved to ensure oxygen supply. 43 Collect the stimulated BMDCs through centrifugation at 800g and room temperature for 5 min.   (ix) Collect the splenocytes through centrifugation at 800g and room temperature for 5 min. (x) Discard the supernatant, and resuspend the precipitated cells with 1 ml ACK Lysis Buffer to lyse the red blood cells. Incubate at room temperature for 90 s. (xi) Collect the splenocytes through centrifugation at 800g and room temperature for 5 min. (xii) Discard the supernatant, and resuspend the splenocytes with 5 ml RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin G sodium and 100 μg/ml streptomycin, then quantify the cell density using the Automated Cell Counter. (xiii) To analyze the antigen-specific T cells in the splenocytes using flow cytometry, plate the splenocytes from Procedure 1, Step 49A(xii) into the 48-well plate. There are 5,000,000 cells and 500 μl culture medium in one well. (xiv) Add 10 μg/ml TRP2 peptide to restimulate the splenocytes overnight. c CRITICAL STEP Set up the negative control group without adding antigen peptide and the positive control group in which the antigen peptide is replaced by the 1:1,000 PI solution. SpT-OTI + SnT-OTII + CN OMVs, 25 μg + 25 μg + 50 μg) and the single-antigen groups (CC-SpT-OTI OMVs, 50 μg SpT-OTI + 50 μg CC OMVs; CC-SnT-OTII OMVs, 50 μg SnT-OTII + 50 μg CC OMVs). There are 25 μg SpT-OTI, 25 μg SnT-OTII and 50 μg CC OMVs in the final CC-SpT-OTI/SnT-OTII OMVs. There are at least four mice in each group, and all the mice receive immunization with one of the different formulations. The two antigens are linked to CC OMVs by the two different molecular pairs, so they do not affect each other. According to the load rates calculated previously in Procedure 1, Step 18, there are 2.1 μg SpT-OTI and 4.9 μg SnT-OTII bound to the CC OMVs in the final CC-SpT-OTI/SnT-OTII OMVs. c CRITICAL STEP When the OMVs display two antigens that have a synergistic immune activation effect, such as the combination of antigens that stimulates CD4 + and CD8 + T cells, respectively, we recommend a two-dose immunization regimen to reflect the differences between different control groups. Preliminary experiments should be conducted to determine the final experimental protocol. However, at least two immunizations are required.
(iii) Kill the C57BL/6 mice by cervical dislocation on day 17. Dissect the mice, and collect the lungs and spleens. (iv) Evaluate the antitumor effect using the similar approach as in Procedure 1, Steps 49A(iv-v). (v) Prepare the single-cell suspension of splenocytes using the similar approach as in Procedure 1, Step 49A(vi-xii). (vi) Analyze the antigen-specific T cells in the splenocytes by flow cytometry using a similar approach as in Procedure 1, Steps 49A(xiii-xx). The only differences are restimulating cells using 5 μg/ml OTI and 5 μg/ml OTII peptides and staining cells using the FITC-antimouse CD3 (1:50, 4 μl), PE-anti-mouse CD4 (1:160, 1.25 μl), APC-anti-mouse CD8 (1:80, 2.5 μl) and PE/Cy7-anti-mouse IFNγ (1:20, 10 μl). c CRITICAL STEP The IFNγ + cells in CD3 + CD8 + or CD3 + CD4 + T lymphocytes in splenocytes are considered as the antigen-specific T cells. (vii) Analyze the antigen-specific T cells in the splenocytes by ELISPOT assay using the similar approach as in Procedure 1, Steps 49A(xxi-xxiv). The only difference is using 5 μg/ml OTI and 5 μg/ml OTII peptides to restimulate the cells. Step 49D(iii) into a 48-well plate. There are 5,000,000 cells and 500 μl culture medium in one well. (vi) Restimulate these splenocytes with 10 μg/ml OTI peptide overnight. (vii) Quantify the cell density using the Automated Cell Counter, and culture 5,000 B16-OVA or MC38 cells and 50,000 restimulated splenocytes from the previous step in a well of 96-well plate for 24 h. (viii) Remove the non-adherent cells, and wash the adherent cells with 100 μl PBS two times.
(ix) Perform the CCK8 assay following the protocol provided by the manufacturer, and calculate the percent of specific killing. (x) Challenge the at least ten vaccinated mice from Procedure 1, Step 49D(i) (ten mice in each group) through injecting 100 μl PBS containing 200,000 B16-OVA cells into mice via tail vein on day 60. (xi) Kill the mice by cervical dislocation on day 80, and dissect the mice and collect the lungs. (xii) Fix the lungs and count the melanoma nodules using a similar approach as in Procedure 1, Step 49A(iv-v). . c CRITICAL STEP The number of mice in this step depends on the experiment purpose. For characterization the physicochemical property of nanovaccine, a few mice is preferred. For tumor therapy, the total number of mice depends on the group setting (usually 10-12 mice per group for survival statistics). 12 Measure the tumor length and width with electronic calipers every other day. Calculate the tumor volume on the basis of the following formula: volume = 0.5 × length × width 2 . 13 Allow the average tumor volume to reach~300 mm 3 for each group, then anesthetize the mice and resect tumor tissue. j PAUSE POINT Resected tumor tissue can be stored at 4°C for 12 h. 14 Transfer the whole-tumor tissue (200-300 mg) to one well of a six-well plate containing 1.5 ml tissue dissociation solution. Use scissors to cut the tumor tissue into 1-2 mm 3 pieces, and incubate them at 37°C for 10 min. c CRITICAL STEP Cut the tumor tissue as small as possible. Incubation time can be extended to 20 min. 15 Repeat the dissociate Step 14 twice to prepare a single-cell suspension.
c CRITICAL STEP For the B16-F10 melanoma tissue that has less extracellular matrix than CT26 tumor, the 10 min incubation in Step 14 can be skipped, otherwise the excessive digestion of the tissue may lead to a loss of tumor membranes. 16 Filter the mixture with a 70 μm cell strainer to obtain the cell suspension, and collect cells by centrifuging at 300g and 4°C for 5 min. 17 Discard supernatant, and resuspend cells with 1 ml ACK lysing buffer for 1 min.
c CRITICAL STEP Lysis time should not be >1 min to avoid any unwanted degradation of protein on TM. 18 Add 10 ml PBS to stop lysis. 19 Collect tumor cells by centrifuging at 300g and 4°C for 5 min. Discard supernatant, and resuspend cells with 10 ml IB buffer. c CRITICAL STEP Resuspension and subsequent steps must be performed at 4°C to minimize the activation of proteases and phospholipases. 20 Break cells by sonication using a probe ultrasonicator at 35% power for 5 min.
c CRITICAL STEP Place the mixture on the ice bath, and the ultrasonicator should pulse on and off cycle every 1-2 s to avoid overheating. 21 Centrifuge the suspension at 3,000g and 4°C for 5 min. Transfer the supernatant into a new tube, and discard the pellet. 22 Centrifuge the collected supernatant at 10,000g and 4°C for 10 min. Transfer the supernatant into a new tube, and discard the pellet.

NATURE PROTOCOLS
2260 c CRITICAL STEP The supernatant of Steps 21 and 22 contains microsomes, and cytosolic proteins cannot be frozen before further centrifugation because freezing will cause damage to microsomal integrity and contaminate the plasma membrane fraction. 23 Centrifuge the collected supernatant at 100,000g and 4°C for 2 h. Discard the supernatant, and collect membrane pellet. c CRITICAL STEP Complete filling of the tubes and balancing the weights of tubes placed on opposite sides are important to maintain safety during centrifugation. 24 Resuspend cytoplasmic membrane pellet with 0.5 ml sterile PBS. Evaluate the total protein concentration using the BCA. c CRITICAL STEP Repeated freeze-thaw cycles degrade proteins. Aliquot the tumor membrane, and store at −80°C. j PAUSE POINT The obtained tumor membrane can be store for 1 year at −80°C.
Preparation of PLGA nanoparticle core • Timing~1.5 h 25 Dissolve 10 mg PLGA in 1 ml DCM. j PAUSE POINT PLGA in DCM can be stored for 2 weeks at 4°C. 26 Add 0.2 ml deionized water into PLGA solution.
c CRITICAL Replace 0.2 ml deionized water with 0.2 ml rhodamine B (0.5 mg/ml) for preparation of the rhodamine B-loaded HM-NPs in Step 40A(i). 27 Emulsify the mixture by sonication using a probe ultrasonicator at 25% power for 3 min. c CRITICAL STEP Place the mixture on the ice bath, and the ultrasonicator should pulse on and off cycle every 1-2 s to avoid overheating. 28 Add 2 ml 1% (wt/vol) sodium cholate solution to the mixture. 29 Emulsify the solution again by sonicating at 35% power for another 5 min. c CRITICAL STEP Place the mixture on the ice bath, and the ultrasonicator should pulse on and off cycle every 1-2 s to avoid overheating. 30 Add the mixture into 10 ml 0.5% (wt/vol) sodium cholate solution, and stir for 10 min. 31 Remove the organic solvent in the mixture by rotary evaporation under reduced pressure. 32 Collect the nanoparticles by centrifugation at 11,000g and 4°C for 15 min, and resuspend them in 500 μl deionized water. This solution can be stored for 1 week at 4°C. 33 Measure the size and PDI of PLGA nanoparticles by DLS following the manufacturer's specifications. c CRITICAL STEP Avoid using a phosphate buffer system to resuspend PLGA nanoparticles; otherwise, PLGA nanoparticles would easily precipitate. The PDI of PLGA nanoparticles should be <0.3.

Preparation of HM • Timing~0.5 h
34 Quantify the total protein concentration of the EM from Step 10 and TM from Step 24 using a BCA Protein Quantitation Kit according to the instructions. 35 Add 1.5 mg EM and 0.5 mg TM into the tube. Use PBS to adjust the total volume to 500 μl.
Incubate it at 37°C for 15 min of gentle oscillation at 50 rpm. 36 Extrude the mixture 13 times through the 400 nm cutoff sterile extruder. c CRITICAL STEP Prewash the extruder with deionized water. Do not overtighten the extruder, as this can cause the polycarbonate film to tear, leading to film leakage and ineffective sizing. The odd number of passes is necessary to obtain size-selected vesicles without too many unextruded vesicles or other contaminants. Use the HM immediately after preparation.
Preparation of HM-coated PLGA nanoparticles (HM-NPs) • Timing~0.5 h 37 To prepare HM-NPs, mix 2 mg HM from Step 36 (500 μl) and 10 mg PLGA nanoparticles (500 μl) from Step 32 in a tube. Sonicate the mixture for 10 min by a probe ultrasonicator at 30% power on the ice bath. Similarly, mix 2 mg TM (use PBS to adjust the volume to 500 μl) from Step 24 or 2 mg EM (use PBS to adjust the volume to 500 μl) from Step 10 with 10 mg PLGA nanoparticles (500 μl) from Step 32, and sonicate these mixtures for 10 min to prepare TM-coated PLGA nanoparticles (TM-NPs) and EM-coated PLGA nanoparticles (EM-NPs), respectively. c CRITICAL STEP Place the mixture on the ice bath, and the ultrasonicator should pulse on and off cycle every 1-2 s to avoid overheating. c CRITICAL STEP For the Mix-NPs, directly mix 1.5 mg TM-generated TM-NPs (750 μl) and 0.5 mg EM-generated EM-NPs (250 μl) from Step 38 without any other extrude procedures. c CRITICAL STEP Prewash the extruder with deionized water. Do not overtighten the extruder, as this can cause the polycarbonate film to tear, leading to film leakage and ineffective sizing. The odd number of passes is necessary to obtain size-selected vesicles without too many unextruded vesicles or other contaminants. Use the vaccine formulations immediately after preparation. Step 40A(i) (total membrane protein amount is 2 mg/ml) to the tube, and incubate for 8 h at 37°C in 5% CO 2 . c CRITICAL STEP Do not add BMDCs into a plate or dish for incubation with nanoparticles in this step. BMDCs, especially the matured BMDCs, easily adhere to the surface-modified plate, but they are less adhering on tube surface.

NATURE PROTOCOLS
(iv) Collect the cells by centrifuging at 300g and 4°C for 5 min. Aspirate the supernatant, and wash the cells once with 1 ml PBS with 2% FBS. (v) Collect the cells by centrifuging at 300g and 4°C for 5 min, discard the supernatant and resuspend the cell pellet using 500 μL PBS with 2% FBS. (vi) Evaluate the fluorescence intensity of rhodamine B in the collected cells using flow cytometry to detect the amount of EM-NPs, TM-NPs, Mix NPs or HM-NPs taken up by BMDCs. (B) TLR activation • Timing~3 d (i) Collect BMDCs from C57BL/6 mice (6-8 weeks old) using the similar approach as in Procedure 1, Steps 31-40. (ii) Seed BMDCs in a six-well plate at a density of 1 × 10 6 cells per well with 2 ml RPMI-1640 medium supplemented with 100 U/ml penicillin G sodium and 100 μg/ml streptomycin. (iii) Add 500 μl EM-NPs, TM-NPs, Mix NPs and HM-NPs from Procedure 2, Steps 37-38 (total membrane protein amount is 2 mg/ml) to the wells, and incubate for 24 h at 37°C in 5% CO 2 . (iv) Aspirate the medium, and add 3 ml PBS. Gently shake the plate for 3 min to wash the cells and aspirate the PBS. RPMI-1640 medium supplemented with 100 U/ml penicillin G sodium and 100 μg/ml streptomycin. c CRITICAL STEP Do not add BMDCs into plate or dish for incubation with nanoparticles in this step. BMDCs, especially the matured BMDCs, are easily adhere to the surfacemodified plate, but they are less adhering on tube surface. (iii) Add 100 μl EM-NPs, TM-NPs and HM-NPs from Procedure 2, Steps 37-38 (total membrane protein amount is 2 mg/ml) to the tube, and incubate for 24 h at 37°C in 5% CO 2 . (iv) Centrifuge the cells at 300g and 4°C for 5 min. Collect the supernatant, and use corresponding ELISA kits to analyze the concentrations of IL-6, TNF-α and IL-1β.

Troubleshooting
Troubleshooting advice can be found in Table 4. Step Problem Possible reason Solution

6
There are very few bacterial colonies on the plate (Supplementary Fig. 9a-c) The antibiotics are not being used in the right way Choose the right antibiotics according to the resistance gene in the backbone plasmid; use the antibiotic-free LB medium in Step 4 during competent cell resuscitation The activity of competent cells is insufficient Store the competent cells at −80°C until use, and avoid repeated freezing and thawing 15 The amount of extracted OMV is very low ( Supplementary Fig. 10a,b) The lack of bacterial activity leads to a decrease in OMV secretion Pick the single bacteria colony on the antibiotic plate as the culture source, not from the cryopreserved bacteria in glycerin 49A(i) Pulmonary metastases are rare ( Supplementary Fig. 11) There is something wrong with the tumor cellular viability Collect the tumor cells at a culture density of 70%, and keep on ice until injection 49A(xx) The number of antigen-specific T cells is very low There are problems with the antibody using in the detection by flow cytometry Store the antibody in a 4°C and dark environment, and set up the positive control group using a PI solution for quality control Procedure 2 Step Problem Possible reason Solution 6 The protoplasts pellet is much smaller than the original E. coli pellet in Step 4 or not visible The time of digest is too long, or the container is contaminated with denaturant or detergent Shorten the treatment time for cell wall digestion, or remove the denaturant or detergent contaminated from the container 9 The volume of the resident pellet is too high, or volume is similar to that of the original E. coli pellet in Step 4 The time of digest time is too short, or lysozyme is inactive

Procedure 1: OMV-based nanocarriers
The CC OMVs prepared in Procedure 1 exhibit a typical bilayer structure and uniform spherical morphology with a diameter of~30 nm (Fig. 2a). The antigen display of SpT-OTI has no obvious effect on the morphology and size of CC-SpT-OTI OMVs (Fig. 2b). After repeated freezing and thawing, the morphology and size of CC OMVs remain unchanged, indicating that CC OMVs can be stored at −80°C (Fig. 2c). The morphology and size are also unaffected by incubation in 10% FBS for 24 h, suggesting that CC OMVs are likely to remain stable in the body long enough for vaccination to be effective (Fig. 2c).

NATURE PROTOCOLS
To verify the antigen display on the CC OMVs, the HA-tagged SpyTag (SpT-HA) and SnoopTag (SnT-HA) are synthesized, respectively. The linkage between SpT-HA and SpC on CC OMVs, or SnT-HA and SnC on CC OMVs, is verified by western blot analysis using an anti-HA antibody, indicated by a concentration-dependent increase in the appearance of the ClyA-SpC-SpT-HA or ClyA-SnC-SnT-HA conjugate (45 kDa) (Fig. 2d). The connection between SpC and SpT or SnC and SnT is stable and is unaffected by storage at different temperatures or treatment with 10% FBS for 24 h (Fig. 2e).
In the immune stimulation evaluation in BMDCs in Procedure 1, Steps 31-48, the CC-SpT-OTI OMVs induce notable maturation and antigen presentation, indicated by the upregulation of the surface expression of CD80, CD86 and MHCI-OVA (Fig. 3a-c and Supplementary Fig. 2a-c). In the evaluation of CC OMVs displaying one tumor antigen (TRP2) in the pulmonary metastatic melanoma model, Procedure 1 Step 49A, immunization with CC-SnT-TRP2 OMVs almost eliminates B16-F10 tumor metastasis in the pulmonary metastatic melanoma model (Fig. 4a,b). All other formulations including the SnT-TRP2, CN OMVs and SnT-TRP2 + CN OMVs are less effective. Flow cytometry and ELISPOT analyses indicate that the CC-SnT-TRP2 OMVs elicit a strong increase in the numbers of IFNγ + cytotoxic T lymphocytes ( Fig. 4c and Supplementary Fig. 3a,b) and IFNγ production (Fig. 4d,e) in the splenocytes after restimulation with TRP2 antigen peptide. These results indicate that the CC OMVs displaying a specific tumor antigen can induce a strong antigen-specific immune response.
In the evaluation of CC OMVs displaying two model antigens (OTI and OTII) in the pulmonary metastatic melanoma model, Procedure 1 Step 49B, the strongest antitumor effect is found in the mice immunized with CC-SpT-OTI/SnT-OTII OMVs (Fig. 5a,b). Splenocytes from animals in the CC-SpT-OTI/SnT-OTII OMV group also secrete the most IFNγ when restimulated with the OTI and OTII antigen peptides (Fig. 5c,d). Interestingly, the increased proportions of IFNγ + in the CD3 + CD8 + and CD3 + CD4 + cells are detected in the mice immunized with the formulations containing OTI and OTII, respectively (Fig. 5e,. These data demonstrate that the OMV-based nanocarriers can simultaneously display different types of antigens to trigger a multiple T-cell-mediated, synthetic antitumor immunity. In the immune stimulation evaluation in the subcutaneous tumor model, Procedure 1, Step 49C, CC-SpT-Adpgk OMVs exhibit the strongest inhibition effects on tumor growth, even stronger than the mixture of SpT-Adpgk antigen with the clinically approved adjuvant Poly (I:C) (Fig. 6a). On day 50, 70% of mice in the CC-SpT-Adpgk group still survive, which is much more than the survivors in the Poly (I:C) + SpT-Adpgk group (30%) (Fig. 6b). Meanwhile, all mice die before day 43 in the saline and SpT-Adpgk + CN OMV groups. The results of tumor immune microenvironment analysis on day 29 show that the infiltration of CD3 + T cells, CD3 + CD8 + T cells, CD3 + CD4 + T cells, CD11b + Ly6G + activated neutrophils and CD11c + DCs are all significantly elevated in MC38 tumor tissues after immunization with the CC-SpT-Adpgk OMVs ( Fig. 6c and Supplementary Figs. 5a-d,  6a,b and 7a,b), and the immunosuppressive microenvironment mediated by CD3 + CD4 + Foxp3 + Tregs is alleviated effectively by CC-SpT-Adpgk OMV treatment (Fig. 6c).

PROTOCOL
NATURE PROTOCOLS (Fig. 7b). This effect disappears in the experiments using MC38 cells without OTI antigen (Fig. 7b). On day 60, the immunized mice are challenged with i.v. injection of B16-OVA cells. Compared with the obvious lung metastasis in the mice immunized with other formulations, there is almost no lung metastasis in mice in CC-SpT-OTI OMV group (Fig. 7c,d).

Procedure 2: HM-NPs
HM-NPs display a typical spherical structure at the nanoscale, the diameter of HM-NPs is~180 nm and the average surface charge is~20 mV (Fig. 8a,b). The immunogold staining followed by TEM imaging provides evidence that FtsZ and Na + /K + -ATPase, specific markers of EM and TM, respectively, are both present on the HM-NPs (Fig. 8c). Flow cytometry results reveal the cellular uptake of HM-NPs by BMDCs (Fig. 9a), and all bacterial cytoplasmic membrane-derived formulations (EM-NPs, Mix NPs and HM-NPs) exhibit~20-fold greater cell-associated fluorescence than the TM-NP group. After entering BMDCs, the HM-NPs induce the greatest proinflammatory cytokine concentration (Fig. 9c-e) and DC maturation (Fig. 9f,g).
Finally, we evaluated the ability of the HM-NP vaccine for postoperative immunotherapy in the CT26 and B16-F10 tumor models. In total, 100% and 90% of the mice vaccinated with HM-NPs after surgical treatment exhibit no tumor relapse within 60 d in CT26 and B16-F10 tumor models, respectively, whereas 100% tumor relapse is observed in the control groups (Fig. 10a,b). Meanwhile, . c, Tumor growth curves after tumor rechallenge. Postoperative mice with CT26 tumors are immunized with HM-NPs. On day 60, the mice treated with HM-NPs are randomized into different groups and inoculated with saline, CT26 or 4T1 cells (2 × 10 5 tumor cells per mouse; n = 12). Statistical significance is analyzed by two-way ANOVA with Bonferroni's multiple comparisons test for growth curves and log-rank (Mantel-Cox) test for comparing survival curves. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. NS denotes no significant difference (P > 0.05). Figure adapted with permission from ref. 15  the protection provided by HM-NPs is a specific and long-term immune response. The previously described HM-NPs-vaccinated mice in the CT26 tumor model are randomized into three groups and inoculated with saline, CT26 cells or 4T1 cells after 60 d. As shown in Fig. 10c, the mice inoculated with CT26 tumor cells show complete tumor elimination and a tumor inhibition rate of 100%. The tumor volumes in the mice inoculated with 4T1 cells reach up to 1,000 cm 3 after 3 weeks.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
The main data discussed in this protocol are available in the supporting primary research papers (refs. 14,15 ). Source data for Figs. 2-10 and Supplementary Fig. 1 are provided as Supplementary information. Source data are provided with this paper.

Corresponding author(s): Guangjun Nie
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Statistics
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A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The main data discussed in this protocol are available in the supporting primary research papers (ref. 14,15). Source data for Figures 2-10 and Supp. Figure 1 is provided as Supplementary information.
The B16-OVA cell is generously provided by Prof. Wang Hao at the National Center for Nanoscience and Technology, which was constructed from B16-F10 that was originally purchased from American Type Culture Collection (Manassas, VA, USA) through stable transfection of the ovalbumin gene (Gene ID: 396058)..

Authentication
All cell lines were validated with cell line authentication by the short tandem-repeat DNA profiling.

Mycoplasma contamination
All cell lines were carried out with mycoplasma detection and were negative for mycoplasma contamination.
Commonly misidentified lines (See ICLAC register) All cell lines are not listed in the database.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals
All animal studies were performed in accordance with ARRIVE guidelines. The mice were fed in a room at 20-22 °C with a 12-h light/ dark cycle and a humidity of 30-70%. Provide food and water ad libitum. Procedure 1: C57BL/6 mice (6-8 week old), Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). Procedure 2: C57BL/6 and BALB/c Mice (6)(7)(8) week old), Vital River Laboratory Animal Technology Co. Ltd (Beijing, China).

Wild animals
This study did not involve wild animals.
Field-collected samples The study did not involve samples collected from the field.

Ethics oversight
All animal studies were approved by the Institutional Animal Care and Use Committee of National Center for Nanoscience and Technology.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Sample preparation
The sample preparation method was described in the Protocol.

Instrument
BD Accuri C6 (BD Biosciences, USA) was used to analyze samples.

Cell population abundance
Over 10000 cells were analyzed for fluorescent intensity in the defined gate.

Gating strategy
A gate is drawn around the cells. Single cells are determined with the area and the height of the side scatter (SSC). The analysis was carried out in this gate.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.