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DOI: 10.1039/D4NR02323D (Paper) Nanoscale, 2024, Advance Article

A promising strategy to improve the stability and immunogenicity of killed but metabolically active vaccines: low-temperature preparation and coating of nanoparticles†

Ning Zhaoa, Jia-Xv Liab, Yong-Jiao Hanab, Li-Ping Lva, Jiang Deng*a and Yan-Yu Zhang*a
aAcademy of Military Medical Sciences, Beijing 100850, China. E-mail: ammsdjxm@163.com
bCollege of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China

Received 4th June 2024 , Accepted 7th August 2024

First published on 14th August 2024

Abstract

Bacteria are becoming an increasingly serious threat to human health. The emergence of super bacteria makes clinical treatment more difficult. Vaccines are one of the most effective means of preventing and treating bacterial infections. As a new class of vaccines, killed but metabolically active (KBMA) vaccines provide the immunogenicity of live vaccines and the safety of inactivated vaccines. Herein, a promising strategy is proposed to improve the stability and immunogenicity of KBMA vaccines. KBMA vaccines were produced at low temperature (4 °C), and the bacterial surface was engineered using mesoporous silica nanoparticle (MSN) coating. Compared to vaccines prepared at room temperature, the metabolic activity of KBMA vaccines prepared at 4 °C remarkably improved. Benefiting from the induction of MSNs, the stability of KBMA vaccines was increased and the preservation time was prolonged at 4 °C. Meanwhile, metabolomics analysis showed that the metabolite spectrum of live bacteria changed after photochemical treatment and MSN coating, which interfered with organic acid metabolism pathways, lipid metabolism and biosynthesis of secondary metabolites. Furthermore, the immune response in the mice treated with KBMA/MSN vaccines was similar to that in those treated with live vaccines and stronger than that in those treated with inactivated vaccines. In comparison with the control group, bacteria tissue burdens of KBMA/MSN group were significantly reduced. CD4+ T cells dominated immune responses for the protection of mice. Thus, the current work promotes the application of KBMA vaccines, providing an alternative choice for treating bacterial infections.

Introduction

Intracellular pathogenic infections remain a challenge in the international biomedical community as they can cause many serious human diseases, such as malaria, AIDS, hepatitis and tuberculosis. Different types of broad and narrow-spectrum antibiotics are common treatment for bacterial infections. However, according to the World Health Organization (WHO), the emerging increasingly multidrug-resistant bacteria are a major threat to public health.1

Vaccines are essential to control the spread of intracellular pathogens by inducing broad cellular immunity. Generally, three types of vaccines are commonly used, i.e., killed whole pathogens, live attenuated vaccines and subunit vaccines.2 However, various side effects have been reported in practice. While safe, killed whole pathogens or subunit vaccines induce weak cellular immunity. Conversely, live attenuated vaccines could elicit high immune response, but in practice, there are safety risks.

To address the above problem, a killed but metabolically active (KBMA) vaccine platform was proposed.3–5 Bacteria are sensitive to long-wavelength UV light after deletion of any one of the three uvr genes (ABC), which are necessary for nucleotide excision repair. Moreover, micromolar amounts of psoralens (S-59 or 8-mop) and DNA/RNA form covalent monoadducts and crosslinks when exposed to ultraviolet A (UVA) light. After treatment, bacteria cannot replicate but remain metabolically active, which perfectly combines the safety of inactivated vaccines and efficiency of live vaccines. In principle, KBMA vaccines provide a way out of vaccinologists’ dilemma. However, the metabolic activity of conventionally prepared KBMA vaccines decreases over time, seriously affecting their immunogenicity and practical application. To overcome this deficiency, engineered KBMA vaccines have been reported.6,7 However, such a strategy may also be associated with some unpredicted risks and preservation problems. As a consequence, there is an urgent need for an improvement strategy for KBMA vaccines to promote their clinical application.

We focus on the process of preparation of KBMA vaccines. First, on account of the great heat under UVA illumination, bacterial activity will suffer.8,9 Temperature control becomes very critical to maintain bacterial metabolic activity. In addition, the surface engineering of live organisms shows extraordinary promise in biotechnology, and it has been extensively reported for bacteria protection.10–13 Bacteria coated with synthetic nanoparticles are able to effectively escape phagosomes and significantly improve the tolerance of bacteria in stomach and intestines.14–16 Some nanomaterials can be used as immune adjuvants.17–19 More recently, mesoporous silica nanoparticles (MSNs) have shown the ability to deliver antigens and elicit obvious immune response.20,21 Thus, MSNs may be developed as an ideal cover material to overcome the weakness of KBMA vaccines.

Inspired by previous works, we hypothesized that low temperature preparation and nanoparticles coating may improve the property of KBMA vaccines. In this study, a promising strategy to improve the stability and immunogenicity of KBMA vaccines was established. Intracellular pathogen Listeria monocytogenes (L. monocytogenes) was applied as a model strain to test the above ideas. KBMA vaccines were produced at 4 °C, and vaccines surface were engineered using MSNs coating. Metabolic activity and different metabolites were analyzed in detail. Moreover, the immune protection of the novel vaccines against bacteria challenge was evaluated. A schematic protocol for the preparation and characterization of KBMA/MSN vaccines in vitro and in vivo is displayed Fig. 1.


image file: d4nr02323d-f1.tif
Fig. 1 Schematics of the (A) preparation, (B) metabolomics analysis and (C) protective effect of vaccines.

Materials and methods

Bacterial strains, media and reagents

All L. monocytogenes (Lm) strains were derived from L. monocytogenes ATCC10403S. uvrA gene in Lm was deleted via homologous recombination with reference to other reports.3,5,22 All bacterial strains were cultured in Brian Heart Infusion (BHI) broth at 37 °C with vigorous shaking at 200 rpm. Vaccine stocks were prepared in a mixture of 30% glycerol and PBS and stored at −80 °C. MSNs functionalized with polyethyleneimine (PEI, M.W. 1800) was provided by Nanjing NanoEast Biotech Co., Ltd, China. Simulated body fluid (SPF) was purchased from Solarbio Science & Technology Co., Ltd (Beijing, China). Antibodies were purchased from Invitrogen, namely, CD3 conjugated to APC (17-0031-82, eBioscience), CD4 conjugated to FITC (11-0041-82, eBioscience), CD8 conjugated to PE (12-0081-82, eBioscience), and TNF-α conjugated to AF700 (56-7349-42, eBioscience).

Preparation of KBMA vaccines and KBMA/MSN vaccines

KBMA vaccines. Lm ΔuvrA was grown in BHI medium to an optical density at 600 nm (OD600) of 0.5. 8-Methoxypsoralen (8-mop, CAS no. 298-81-7) was added directly to the cultures for 1 h. Then, the bacterial cultures were transferred to 6-well plates (2 mL per well) and UVA irradiated at a dose of 25 J cm−2. The illumination over the samples was 125 mW cm−2. It was measured by an ultraviolet illuminometer (Linshang Technology Co., Ltd Shenzhen, China). The inactivated bacteria, known as KBMA vaccines, were stored as mentioned above.
KBMA/MSN vaccines. KBMA vaccines (2 mL) were harvested by centrifugation at 8000 g for 10 min and then incubated in 100 μg mL−1 PEI-MSNs (500 μL) aqueous solution at 37 °C on a rotary shaker (150 rpm) for 40 min. The bacterial pellets were collected by centrifugation and washed twice with physiological saline. The treated bacteria, known as KBMA/MSN vaccines, were stored as mentioned above.

Metabolic-activity assay

MTS analysis. The metabolic activity of the prepared vaccines was assessed by the Cell Proliferation Assay Kit (Promega, G3580) according to the manufacturer's instructions. The optical density (OD) of formazan was monitored at 490 nm (Molecular Devices).
Non-targeted metabolomics analysis. Bacterial pellets or supernatant (prepared vaccines resuspended in physiological saline, and the supernatant of vaccines was collected after 24 h at 4 °C) were mixed with 100 μL of extraction solution (MeOH[thin space (1/6-em)]:[thin space (1/6-em)]ACN[thin space (1/6-em)]:[thin space (1/6-em)]H2O, 2[thin space (1/6-em)]:[thin space (1/6-em)]2[thin space (1/6-em)]:[thin space (1/6-em)]1 (v/v)). The extraction solution contained deuterated internal standards, and the mixed solution was vortexed for 30 s. After homogenization and ultrasonic extraction, the supernatant was analyzed by Vanquish (Thermo Fisher Scientific), and the column was ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) (Waters). The raw data were converted to the mzXML format using ProteoWizard and processed with an in-house program, which was developed using R and based on XCMS, for peak detection, extraction, alignment, and integration. The R package and the BiotreeDB (V3.0) were applied for metabolite identification.23

Characterization of prepared vaccines

The morphology of the samples was observed by TEM (JEM1200EX) equipped with an energy-dispersive X-ray (EDX) spectrometer and SEM. X-ray diffraction (XRD) patterns were obtained on a Bruker D8-Advanced X-ray instrument. Fourier transform infrared (FTIR) spectra of all the samples were acquired using a Nicolet IS10 FTIR spectrometer.

The release behavior of KBMA/MSN in SPF was analyzed by ICP-MS. Samples were digested after treatment with SPF for 24 hours, 36 hours, and 48 hours for the measurement.

Animal experiment

BALB/C mice (seven-weeks-old male, 18–20 g) were used in our study. The mice conditions were strictly kept at 22 °C–25 °C and 12/12 h light/dark cycle, and the mice were free to access food and water during the experiment. At least one week was necessary for mice to adapt to the new environment. Mice were randomly divided into 6 groups (6 mice per cage): Control, Heat Kill (HK) (cultured Lm incubated at 70 °C in a water bath for 1 h), Live, KBMA, KBMA/MSN, MSN.

Protection studies of the vaccine

Immunization was performed by the intramuscular injection of all the groups using the following doses: Control, 200 μL physiological saline; HK, 107 CFU; Live, 104 CFU; KBMA, 107 CFU; KBMA/MSN, 107 CFU; MSN, 0.1 mg. All the injection doses were referred to from previous reports.24

Mice were vaccinated on day 0, 14 and 28. Three weeks after the third dose, mice were challenged intraperitoneally (i.p.) with a sublethal dose of L. monocytogenes (8 × 105 CFU per mouse) and sacrificed 3 days after the challenge. At the end of the experiment, liver and spleen were collected for colony counting and histopathology. In brief, liver and spleen homogenates were serially diluted with physiological saline, and then 100 μL samples were plated on BHI agar medium.

Statistical analysis

The statistical data were evaluated by GraphPad Prism 8.0 software. In order to compare the data among multiple groups, one-way analysis of variance (ANOVA) with Tukey's multiple range test and Student's test was used. Results are shown as the mean ± SEM. At least three repetitions were needed for each experiment. P < 0.05 was considered statistically significant, *P < 0.05, **P < 0.01, *P < 0.001.

Results and discussion

Preparation of KBMA vaccines at low temperature

To prepare the KBMA vaccines, nucleotide excision repair mutants were first constructed by removing the related gene uvrA (Fig. S1†). Also, it was determined whether the deletion of the uvrA gene leads to growth defect. As observed in Fig. S2,† there were no obvious alterations in the growth curves in BHI between strain Lm with uvrA deletions and its sister strain.

Then, the potential toxicity of the photosensitizer (8-mop) was measured before the study. It could be found from Fig. 2A that Lm viability was not affected after co-incubation with the indicated 8-MOP concentration for 0.5 h, 1 h or 2 h. Apparently, the metabolic activity of L. monocytogenes was not affected either (Fig. S3†). Therefore, 8-MOP is non-toxic to L. monocytogenes and can be used for KBMA vaccines preparation.


image file: d4nr02323d-f2.tif
Fig. 2 (A) The effect of 8-MOP on bacterial viability. Co-incubation of 8-MOP with bacteria for 0.5 h, 1 h or 2 h. (B) Viability of Lm and LmΔuvrA after treatment with the indicated 8-MOP concentration; UVA intensity was 25 J cm−2. (C) Metabolic activity of Lm and LmΔuvrA before and after 8-MOP/UVA treatment. (D) Types and percentages of metabolites in bacteria. (E) Metabolic activity of KBMA vaccines produced at room temperature and 4 °C.

After that, the relative sensitivity of Lm and LmΔuvrA toward photochemical inactivation was analyzed over a range of 8-mop concentration. UVA intensity (25 J cm−2) used in the experiment referred to our previous study.25 As illustrated in Fig. 2B, ≥8 logs were inactivated at 320 ng mL−1 8-MOP for LmΔuvrA, but 1280 ng mL−1 8-MOP was required to get the same effect for Lm. The results suggested that LmΔuvrA was more sensitive to photochemical inactivation and easier to be inactivated. It is generally known that safety is one of the key problems for live bacterial vaccine, and the inhibition of bacterial proliferation is necessary for vaccine preparation. Therefore, compared to Lm, LmΔuvrA was preferred to prepare vaccines. 320 ng mL−1 8-MOP was used in the following preparation experiment.

To prove another characteristic of the prepared vaccine as KBMA bacteria, we determined the metabolic activity. As shown in Fig. 2C, the metabolic activity of 8-MOP/UVA Lm ΔuvrA (KBMA) was significantly higher than that of 8-MOP/UVA Lm, but no difference was found compared with live Lm, which was same as the observation in a previous study done before on another KBMA bacteria.5 This is likely due to the different responses to photochemical treatment stress.

To know well the response of bacteria to photochemical inactivation in detail, bacterial samples were analyzed using metabolomics. A total of 2084 compounds were detected and quantified, mainly in the categories of organoheterocyclic compounds (21.977%), organic acids and derivatives (13.58%), benzenoids (13.532%) and lipids and lipid-like molecules (6.286%) (Fig. 2D). Detailed information on some major metabolites are displayed in Table 1, demonstrating the capability of KBMA bacteria to synthesize macromolecules, including proteins and cell-wall constituents. The difference in some metabolites (e.g., benzenoids, organic acids and derivatives) between the KBMA bacteria and the live bacteria may be due to the undecomposed chromosomes.26

Table 1 Detailed information on some major metabolites
Metabolites Number of metabolites
KBMA Live
Amino acids and peptides 84 89
Benzenoids 224 257
Carbohydrates 32 34
Fatty acids 47 49
Lipids and lipid-like molecules 93 104
Organic acids and derivatives 225 251
Organic nitrogen compounds 30 29
Organic oxygen compounds 51 57
Organoheterocyclic compounds 369 414

In addition to photochemical effects, it has also been found that temperature has a great influence on bacterial metabolic activity.8,27 To solve this problem, a temperature control system was installed in a UVA-LED irradiation setup. In this way, the temperature around the bacterial samples was successfully reduced to 4 °C during illumination (Fig. S4†). As expected, the metabolic activity of KBMA vaccines prepared at 4 °C was obviously higher than that of the vaccines prepared at room temperature (Fig. 2E). It is easy to understand that most proteins exhibit better activity at a relatively low temperature.

On the whole, these results indicated that KBMA vaccines, which lack bacterial cell proliferation ability but have metabolic activity, were successfully prepared. Besides, vaccines prepared at 4 °C exhibit higher metabolic activity.

Preparation and characteristics of KBMA/MSN vaccines

To improve the immunogenicity and stability of KBMA vaccines, the bacterial surface was modified using MSNs coating (Fig. 3A).
image file: d4nr02323d-f3.tif
Fig. 3 (A) Schematic of the preparation process of KBMA/MSN vaccines. (B) Zeta potential of Lm and PEI-MSN. (C) FT-IR spectra of the samples; SEM images of (D and E) KBMA/MSN and (F and G) KBMA. EDX elemental mapping images of (H) KBMA/MSN and (I) KBMA.

Firstly, MSNs were functionalized with a positively charged functional group (PEI). Then, the zeta potential of Lm and PEI-MSNs was measured at physiological pH, and the corresponding results are displayed in Fig. 3B. Obviously, the zeta potential of Lm was negative and positive for PEI-MSNs. Opposite zeta potential promotes the combination of Lm and PEI-MSNs. Furthermore, the zeta potential of KBMA/MSN proved the correctness of our conjecture that MSNs were enriched in the bacterial surface.

The characteristics of KBMA/MSN vaccines were also investigated. As shown in Fig. 3C, in comparison with KBMA, there were some new characteristic peaks in the Fourier transform infrared (FT-IR) spectrum of KBMA/MSN, which might be related to the –OH bonds of MSNs. The wide angle XRD patterns of different samples are presented in Fig. S5.† In contrast with KBMA vaccines, diffraction peaks were clearly observed in KBMA/MSN vaccines.

Furthermore, TEM and SEM were utilized to observe the morphology of the samples. As displayed in Fig. 3D–G, MSNs were well distributed in the KBMA surface. TEM and the corresponding EDS mapping images in Fig. 3H and I further supported the results.

In brief, the above results demonstrated that MSNs were well enriched in the surface of KBMA bacteria, and KBMA/MSN vaccines was prepared successfully.

Metabolic activity of KBMA/MSN vaccines

Clearly, metabolic activity is one of the important features that distinguishes KBMA vaccines from inactivated vaccines. Hence, the effect of coating MSNs on the metabolic activity was measured in our study.

First, the influence of MSNs and KBMA co-incubation on the metabolic activity was determined. As illustrated in Fig. 4A, MSNs made no difference to the metabolic activity of KBMA vaccines within a certain range of concentrations. However, the metabolic activity was seriously affected at high concentration of MSNs (2500 μg mL−1), demonstrating the toxic effects of high concentration MSNs on KBMA/MSN vaccines. Moreover, the co-incubation times on the metabolic activity were assessed. No obvious change was found in the metabolic activity after 160 min of co-incubation (Fig. 4B), suggesting that the metabolic activity was not affected by the co-incubation time. Therefore, 100 μg mL−1 MSNs-PEI and 40 min co-incubation time were applied throughout the whole preparation experiment of KBMA/MSN vaccines. Conclusively, KBMA bacteria still cannot replicate but remain metabolically active after coating nanomaterials MSNs. Thus, this work confirms that KBMA/MSN vaccines’ preparation is feasible.


image file: d4nr02323d-f4.tif
Fig. 4 (A) Metabolic activity of KBMA/MSN after treatment with the indicated MSN concentration (control group: OD490 = 1.32 ± 0.054). (B) Metabolic activity of KBMA vaccines after incubation with MSNs (100 μg mL−1) for different durations. Metabolic activity of KBMA/MSN and KBMA after storage at (C) 4 °C, (D) 25 °C and (E) 40 °C for different times. (F) Concentration of Si in the supernatant after treatment of SPF for 24 h, 36 h or 48 h.

Then, the effect of storage temperature on the metabolic activity of KBMA vaccines after MSNs coating was evaluated. Surprisingly, the metabolic activity of KBMA/MSN vaccines remained over 80% after 30 hours of storage at 4 °C. After being stored for 100 hours at 4 °C, the metabolic activity remained 27.4%, while the metabolic activity of KBMA vaccines rapidly decreased with increasing storage time and was less than 50% after 30 hours of storage at 4 °C (Fig. 4C). However, it was disappointing that the metabolic activity of KBMA vaccines and KBMA/MSN vaccines at 25 °C (Fig. 4D) and 40 °C (Fig. 4E) decreased with increasing storage time. After being stored for 30 hours at 25 °C and 40 °C, the metabolic activity remained only 34.3% and 5.7%, respectively. MSNs coating does not seem to have a protective effect on KBMA vaccines when stored at 25 °C and 40 °C, suggesting the negative effects of high temperature (25 °C and 40 °C) on bacterial protein activity. Anyway, MSNs formed a transient barrier on the surface of Lm, providing protection for KBMA vaccines during storage at 4 °C. The stability of KBMA vaccines was improved and the preservation time at 4 °C was prolonged by coating MSNs materials. MSNs nanoparticles coating overcame a critical issue in the application of KBMA vaccines, presenting new opportunities for disease treatment and bioremediation.

In addition, we investigated the controlled release behavior of KBMA/MSN in SPF, which is a prerequisite for application. After treatment with SPF for 48 hours, the Si concentration in the supernatant significantly increased compared to that after 24 h treatment (Fig. 4F). Similar results were obtained by comparing the morphology change. The rule shape of MSNs was completely destroyed after the treatment with SPF (Fig. S6A–D†). Moreover, the breakup of the MSNs shell structure ensured the release of enclosed Lm so as to play its role as a vaccine.

Metabolomics analysis

To study the metabolism secretions of KBMA/MSN vaccines, the supernatant of vaccines was collected after 24 h at 4 °C for metabolomics analysis. First, metabolites were analyzed using the supervised discriminant analysis statistical method OPLS-DA model. Fig. 5A presents a separation between live vaccines and KBMA/MSN vaccines, indicating the different metabolite spectrum of the two vaccines.
image file: d4nr02323d-f5.tif
Fig. 5 (A) PCA score plot of metabolites in the live bacteria group and KBMA/MSN bacteria group. (B) The expression volcano plot of different up and downregulated metabolites. (C) Donut plot of different metabolites.

The changes in the metabolite spectrum directly determined the potency of vaccines and is an important indication of the novel antigenic screening.28 Therefore, the different metabolites were further screened to explore which metabolite changes caused the separation between live vaccines and KBMA/MSN vaccines. Compared with live vaccines, the photochemical treatment and MSNs coating significantly changed the content of 1529 metabolites: 627 metabolites were up-regulated and 902 metabolites were down-regulated (Fig. 5B). Among the different metabolites, organoheterocyclic compounds occupied the largest proportion, accounting for 19.48% of all metabolites, followed by benzenoids (14.72%), organic acids and derivatives (13.42%), and lipids and lipid-like molecules (6.93%) (Fig. 5C). Additionally, the different metabolites interfered with organic acid metabolism pathways, lipids metabolism and biosynthesis of secondary metabolites,29–31 which contribute to explaining the mechanism by which KBMA/MSN stimulate the immune response.

Protective effect of KBMA/MSN vaccines against bacterial challenge

To test the possibility of using KBMA/MSN vaccines against bacterial challenge, the protective immunity induced in mice was assessed in our study. The overall experimental procedure is displayed in Fig. 6A. The protective efficacy was evaluated by observing changes in the body weight, bacterial load in mice organs, histological analysis, and inflammatory cytokines.
image file: d4nr02323d-f6.tif
Fig. 6 (A) Schematic of the animal experiment. (B) Mouse body weight change (n = 6). Bacterial load in the mice (C) spleens and (D) livers (n = 6). (E) Histological sections of the mouse livers (H&E magnifications, 200×) (n = 3). All data are represented as mean ± SEM.

Body weight exhibits a significant decrease in the control group after 3 days of L. monocytogenes infection, while the weight loss was prevented after vaccination with heat kill vaccines, live vaccines, KBMA vaccines or KBMA/MSN vaccines, which indirectly suggested the protection of vaccines (Fig. 6B). As is known to all, the tissue burdens in organs can visually demonstrate the role of vaccines. We determined the bacterial content in the spleen and liver 3 days after the L. monocytogenes challenge. As depicted in Fig. 6C and D, regardless of whether live or KBMA/MSN vaccines, it was an approximate 4-log reduction in spleen colonization and 3-log reduction in liver colonization, suggesting the comparable protective immunity of the two vaccines (live and KBMA/MSN vaccines). As expected, the bacterial load in organs was also significantly lower in mice receiving KBMA/MSN vaccines than those receiving HK vaccines. These results further confirmed that KBMA/MSN vaccines have both the advantages of the potency of live vaccines and the safety of heat-killed vaccines.

Moreover, similar change tendency was observed in the histological analysis of mice livers. Compared with the control group, the massive infiltration of immune cells was significantly alleviated in the vaccines’ treatment group (Fig. 6E). But the histological analysis of mice spleens in different groups changed a little (Fig. S7†), perhaps due to the powerful function of spleen cells in controlling bacterial infections.32–34 Besides, several literature reports show that attenuated Listeria strains offered protection and were applied as antitumor vaccine vectors35–38 due to the fact that L. monocytogenes could induce robust cellular immunity. It is well known that T cells play a critical role in the protective immunity. Among them, the development and maintenance of CD8+ cytotoxic T lymphocytes cannot happen without CD4+ helper T cells.39,40 Accordingly, we then characterized the immune response of different vaccines. As shown in Fig. 7A and B, there was no difference in the percentage of the activated CD8+ T cells between the groups, but the percentage of the activated CD4+ T cells in live and KBMA/MSN vaccines was significant higher than that in control, indicating that vaccines induced CD4+ T cells-dominated immune responses.


image file: d4nr02323d-f7.tif
Fig. 7 Induction of (A) CD4+ and (B) CD8+ T cells in mice (n = 3).

Conclusions

In the current work, novel KBMA vaccines with good stability and immunogenicity were prepared by low temperature and nanoparticles (MSNs) coating. Intracellular pathogen Listeria monocytogenes was selected as a model platform. Interestingly, the metabolic activity of KBMA vaccines was increased significantly after preparation at 4 °C. Meanwhile, benefiting from the induction of MSNs, KBMA/MSN vaccines were more stable at 4 °C, resulting in a longer storage time. Metabolomics analysis showed the different metabolite spectrum of live vaccines and KBMA/MSN vaccines interfered with the organic acid metabolism pathways, lipids metabolism and biosynthesis of secondary metabolites. Furthermore, the protective effect of KBMA/MSN vaccines against bacterial challenge in vivo was evaluated. As predicted, the immune efficacy of KBMA/MSN vaccines is similar to that of live vaccines and stronger than that of inactivated vaccines. CD4+ T cells dominated the immune responses for mice protection.

Ethical statement

Committee on Animal Use and Care of the Academy of Military Medical Sciences was performed strictly during the animal experiment. All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Author contributions

Ning Zhao: investigation, formal analysis, data curation. Jia-Xv Li: formal analysis, writing-original draft, software. Yong-Jiao Han: investigation, data curation, writing-original draft. Li-Ping Lv: methodology, formal analysis. Jiang Deng: project administration, data curation, data curation. Yan-Yu Zhang: project administration, investigation, data curation, data curation.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4nr02323d
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