key: cord-1031367-jk04u17k authors: Zhao, Zhangting; Ma, Xingyuan; Zhang, Ruihuan; Hu, Fabiao; Zhang, Tong; Liu, Yuping; Han, Myong Hun; You, Fang; Yang, Yi; Zheng, Wenyun title: A novel liposome-polymer hybrid nanoparticles delivering a multi-epitope self-replication DNA vaccine and its preliminary immune evaluation in experimental animals()() date: 2020-11-13 journal: Nanomedicine DOI: 10.1016/j.nano.2020.102338 sha: 1b500daef057d370c3cb1d3953580ae2cc1bb10c doc_id: 1031367 cord_uid: jk04u17k DNA vaccine is an attractive immune platform for the prevention and treatment of infectious diseases, but existing disadvantages limit its use in preclinical and clinical assays, such as weak immunogenicity and short half-life. Here, we reported a novel liposome-polymer hybrid nanoparticles (pSFV-MEG/LNPs) consisting of a biodegradable core (mPEG-PLGA) and a hydrophilic shell (lecithin/PEG-DSPE-Mal 2000) for delivering a multi-epitope self-replication DNA vaccine (pSFV-MEG). The pSFV-MEG/LNPs with optimal particle size (161.61 ± 15.63 nm) and high encapsulation efficiency (87.60 ± 8.73%) induced a strong humoral (3.22-fold) and cellular immune responses (1.60-fold) compared to PBS. Besides, the humoral and cellular immune responses of pSFV-MEG/LNPs were 1.58- and 1.05-fold than that of pSFV-MEG. All results confirmed that LNPs were a very promising tool to enhance the humoral and cellular immune responses of pSFV-MEG. In addition, the rational design and delivery platform can be used for the development of DNA vaccines for other infectious diseases. Infectious diseases are caused by living pathogenic microorganisms including viruses, bacteria, and fungi. These diseases can be transmitted, directly or indirectly, from person to person by contact. According to the World Health Organization (WHO), 3 infectious diseases are ranked in the top 10 causes of death worldwide in 2016, including lower respiratory infections (3.0 million deaths), diarrheal diseases (1.4 million deaths), and tuberculosis (1.3 million deaths). 1 Thus far, more than 10 millions confirmed cases and almost half million deaths worldwide have been reported due to the coronavirus disease pandemic. 2 Given that infectious diseases are a significant threat to public health and the global economy, significant progress has been made in infectious disease management. Since the 1940s, antibiotics have saved millions of lives and play a critical role in the treatment and prevention of various infectious diseases. 3 However, though powerful, antibiotics only work for the bad bacterial infections but not for viral infections. What's worse, after decades of misuse and overuse of antibiotic, they are becoming less effective and has led to the emergence of "superbugs", which also know as antimicrobial resistance bacteria (ARB). 4 In recent years, the rise and spread of ARB is building up to become an epic global public health crisis. 7 reagents used in this study were of analytical grade. Normal liver L-02 cells were acquired from the Type Culture Collection Committee of Chinese Academy of Sciences (Shanghai, China). Fetal bovine serum (FBS), Dulbecco's Modified Eagle Medium (DMEM), and penicillin-streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin) were obtained from Thermo Fisher Scientific (Waltham, USA). Heat-labile-enterotoxin B (LTB), colonization factor antigen-I (CFA/I), CFA/IV surface antigen 6 (CS6), and invasive plasmid antigen B (IpaB) are the main antigens in ETEC and Shigella that cause traveler's diarrhea (TD). [18] [19] [20] [21] The protein sequences of LTB, CFA/I, CS6, and IpaB were obtained from the PDB database. The linear B-cell epitopes were analyzed by ABCpred and Bcepred, while the conformational B-cell epitopes were predicted by DiscoTope 2.0 and ElliPro. T-cell epitopes were restricted by major histocompatibility complex (MHC) and classified into 2 types, MHCI and MHCII. Helper T lymphocytes (HTL) and cytotoxic lymphocyte (CTL) belongs to MHCI and MHCII, respectively 22 , which were respectively analyzed by NetMHCpan 4.0 and IEDB. Besides, both T-cell epitopes were evaluated by RANKPEP. Similarly, the modest CTL epitopes candidates were predicted by CTLPred and PAComplex. The selected 16 B-and T-cell of linker peptides ((GGGGS) 2 , GPGPG, KAAA, AAY, and EAAAK) were used to connect different types epitopes to ensure the independence and integrity of each epitope and improve the efficiency of epitope recognition ( Figure S3A ). Besides, the universal Th epitopes and MHCI trafficking domain (MITD) sequences were introduced at the N-and C-terminal of T-cell epitopes respectively to reduce the MHC polymorphism and improve the efficiency of epitopes presentation and recognition ( Figure S3A ). 23 The quality of the designed MEG was accessed by I-TASSER, DNAStar, and PROCHECK, respectively. Besides, the immunogenicity and proteasome cleavage site of the MEG were analyzed by PAProC and the overall antigenicity of MEG was evaluated by VaxiJen 2.0 (with a 0.4 threshold). All the online bioinformatic softwares used in this study were listed in Table 1. In addition, Kozak sequence (GCCGCC) and His tags were respectively added before and after MEG sequences. Codon optimization and gene synthesis were performed by Reidi Biotechnology co., LTD (Shanghai, China) to improve the expression level of MEG antigen in mammalian cell. Two high-level expression vectors (pcDNA3.1 and pSFV) were selected to construct 5 recombinant plasmids (pSFV, pcDNA3.1-MEG, pSFV-MEG, pcDNA3.1-MEG-EGFP (green fluorescent protein), and pSFV-MEG-EGFP) ( Figure S3B ). All primers used in this study were listed in Table S1 . lecithin and 0.36 mg of PEG-DSPE-Mal 2000 were dissolved in 9 mL of 4% ethanol solution (molar ratio of 9 : 1) (water phase). The aqueous phase was preliminarily heating to 65-70 °C; then, the organic phase solution was added dropwise (1 mL/min) to the preheated water phase under slow stirring. After that, the mixture was vortexed vigorously for 3 min, and slowly stirred at 25 °C for 2 h to evaporate the organic phase. The obtained nanoemulsion was concentrated with a 100 KDa ultrafiltration tube (Millipore, USA) and washed twice with ultrapure water to remove unencapsulated DNA. Finally, the DNA-loaded LNPs was frozen and saved at 4 °C. DNA-loaded mPEG-PLGA NPs and PLGA NPs were prepared as the same method. L-02 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C with 5% CO 2 . When the confluence reached 80-90%, cells were trypsinized and seeded into 24-well plates for each experiment. The concentration of DNA in the supernatant of LNPs was determined by an ultra-micro nucleic acid detector (Quawell, USA). All samples were performed in triplicate. Encapsulation efficiency (EE) was calculated using the equation (1) shown below. Initial amount of DNA added − Amount of DNA in supernatant Initial amount of DNA added × 100% (1) Japan). The zeta potential and particle size of LNPs were measured and calculated by dynamic light scattering (DLS) (Malvern, UK) at 25 °C. Each sample was measured in triplicate. The protective effect of LNPs on pSFV-MEG were evaluated by agarose gel electrophoresis. Naked pSFV-MEG and pSFV-MEG/LNPs incubated with DNase at 25 °C for 30 min, and then all samples were analyzed by agarose gel. The gray value of the electrophoretic band was quantified by Image J software (National Institutes of Health, USA). The release profile of pSFV-MEG from LNPs, mPEG-PLGA NPs, and PLGA NPs The stability of dried pSFV-MEG/LNPs was evaluated by particle size and encapsulation efficiency after 3-and 7-week storage at 4 °C and 25 °C, respectively. MTT assay was performed in vitro to determine the cytotoxicity of DNA-loaded LNPs. L-02 cells (6 × 10 4 cells/well) were seeded in 96-well plates and cultured overnight, and µL of MTT (5 mg/mL) was added and continue incubated for another 4 h. The culture supernatant was discarded and 150 µL of DMSO was added to dissolve the precipitated crystals. The OD 490nm value was measured to calculate the cell viability. The red blood cells (RBC) were collected from BALB/C mice by centrifugation and diluted L-02 cells (2 × 10 5 cells/well) were seeded in 24-well plates and fixed with 4% paraformaldehyde after washing twice with PBS. After that, cells were incubated with 0.1% Triton X-100 for 10 min and 3% BSA for 1 h. Cells were then sequentially incubated with His-tagged mouse monoclonal antibody, goat anti-mouse AlexaFluor 488 ® , and Hoechst 33342 in the dark. Finally, the intracellular fluorescence was observed by invert fluorescence microscopy (Olympus, Japan). Western blotting was used to test the expression of MEG antigen in L-02 cells. Proteins IgG antibodies were measured in serum samples by ELISA with the mouse IgG total ready-set-go kit. Briefly, 100 µL of capture antibody solution was added to Corning™ The major organs (heart, liver, spleen, lung, and kidney) of mice treated with PBS, pcDNA3.1-MEG/LNPs, and pSFV-MEG/LNPs were collected and fixed with 4% paraformaldehyde for paraffin slicing, followed by hematoxylin-eosin (HE) staining for histological examination. All experiments were performed at least 3 times and results were expressed as mean ± J o u r n a l P r e -p r o o f Journal Pre-proof standard deviation (SD). Statistical analysis of the data was performed by SPSS 22.0 software (IBM, USA). P < 0.05 was considered to be statistically significant. Several critical factors were taken into consideration in the rational design of the MEG sequence. Firstly, the antigen sequences (LTB, CFA/I, CS6, and IpaB) were retrieved from the PDB protein database (Scheme S1) to screen the optimal epitopes. According to the comprehensive evaluation of the online tools, the optimal epitopes (HTL, CTL, linear B-cell, and conformational B-cell epitopes) of each antigen with the highest score were chosen for the further studies (Table 1 and Table S1 ). Secondly, to enable efficient and stable expression of fusion proteins and also ensure the independence of functional epitopes, several linkers included HTL (GPGPG linker), CTL (KAAA linker), linear B-cell (AAY linker), and conformational B-cell (EAAAK linker) were selected to unite the same epitopes. Moreover, different types of epitopes was jointed by (GGGGS) 2 linker to ensure the independence and integrity. [25] [26] [27] [28] Thirdly, T-cell epitopes were connected in series in front of linear B-cell epitopes, which could improve the expression efficiency of antigens. 29 Fourthly, since it was important to avoid the MHC polymorphism restriction and improve the efficiency of epitope presentation and recognition, the universal Th epitope and MITD sequence were introduced at the N-and C-terminal of T-cell epitopes, respectively. 30 Finally, the corresponding tertiary structures were obtained by simulating the arranged MEG sequence with the three-dimensional (3D) simulation software I-TASSER, which J o u r n a l P r e -p r o o f Journal Pre-proof could automatically generate high-quality model predictions of 3D structure and biological function of protein molecules from their amino acid sequences. Five different tertiary structures were successfully obtained (C-score within -5 to 2), and their corresponding C-scores were -1.08, -1.69, -2.66, -2.57, and -4.33 ( Figure S2 ), respectively. Due to the first 3D structure has the highest C-score value indicating high confidence, its corresponding amino sequence ( Figure S3A ) was selected as the optimal MEG sequence. Besides, additional analysis showed that only 28.2% amino residues of the first structure had a positive normalized B factor (NBF) value, while the NFB value of the rest four structures were all positive ( Figure S2 ). Since residues with higher NBF value than 0 indicated that the protein was structurally unstable, this result further confirmed that the first structure was the most suitable and could be used as a DNA vaccine candidate ( Figure 1 ). To verify the general and local quality of MEG antigen, Prosa and PROCHECK were used to analyze the predicted MEG tertiary structure. As shown in Figure 2A , the Prosa Z-score of MEG antigen was -4.05, which was perfectly within the range of scores typically found for native proteins of similar size. The Ramachandran plot showed that 76.1% and 16.2% of the residues located in the favored and allowed areas, respectively, whereas only 7.7% residues were in disallowed regions ( Figure 2B ). The results above demonstrated that the structure of MEG antigen had a very high resolution and good structural quality, and the steric atomic clashes among the residues were minimal. Besides, the instability index (II) of MEG obtained from the ProtParam tool was 38.85, which classified MEG antigen as J o u r n a l P r e -p r o o f Journal Pre-proof stable. Furthermore, the overall quality score of MEG antigen from ERRAT was 90.68% ( Figure 2C ). All the above results revealed that the selected MEG antigen had a stable tertiary structure. The secondary structural properties of MEG antigen were evaluated by PAProC tool and DNAstar. There were several proteasome cleavage sites in the sequence, but most of them appeared at the junction, making the designed MEG antigen not only retained the integrity of the cell epitopes but avoided unnecessary immune response caused by the emergence of new epitopes ( Figure 3A) . Besides, the B-cell epitope consisted of 43.7% helix and 56.3% random coil, which was consistent with the fact that B-cell epitopes most likely existed in the β-turns and random curls regions to be easily recognized by B cell receptor ( Figure 3B ). What's more, the B-cell epitopes were found to be composed of Table 3) . To MTT and hemolysis assay were performed to evaluate the cytotoxicity of LNPs. Figure 6A showed that after 24 h of incubation with blank LNPs, pcDNA3.1-MEG/LNPs, and pSFV-MEG/LNPs, the cell viability could still reach more than 90% even at the highest DNA concentration (100 μg/mL), and the high cell viability could be maintained for at least another 24 h without any significant difference ( Figure 6B ). These results indicated that blank LNPs or DNA-loaded LNPs had little cytotoxicity towards normal cells. Moreover, the highest concentrations of blank LNPs, pcDNA3.1-MEG/LNPs, or pSFV-MEG/LNPs hardly resulted in hemolysis of mice erythrocyte (less than 5%) after incubation at 37 °C for 3 h ( Figure 6C ), suggesting that the blank LNPs and DNA-loaded LNPs rarely caused cellular hemolysis. All the above results further reflected that LNPs were safe as a DNA vaccine carrier. To explore the expression pattern of DNA-loaded LNPs, both naked DNA and DNA-loaded LNPs were transfected into L-02 cells, respectively. As shown in Figure 7A and To evaluate the humoral immune response of pSFV-MEG/LNPs, the total amount of serum IgG antibodies of mice was detected by ELISA. 32 The fitting of the ELISA standard curve of IgG antibodies was shown in Figure S7 . As presented in Figure 8A , CD4 + T cells are helper T lymphocytes, whose main function is to enhance the humoral immune response mediated by B cells. While CD8 + T cells are inhibitory/lethal T lymphocytes, whose primary function is to specifically kill target cells directly. 33, 34 The distribution of CD4 + T and CD8 + T cells in the spleen of mice was analyzed to examine the MEG-induced cellular response. As can be seen from Figure 8B and S9, there was no statistical difference of CD8 + T cells in each group, but the CD4 + T cells increased significantly after the third time immunization. Compared to the PBS group, the CD4 + T cells in blank LNPs group increased 1.20-fold, demonstrating that LNPs alone enhanced the CD4 + T cell immune response. Besides, the CD4 + T cells in pSFV-MEG/LNPs group increased significantly among all groups, which was 1.60-fold higher than PBS group. The pSFV-MEG/LNPs induced a strong CD4 + T cell immune responses (1.05-fold) compared to pSFV-MEG. Moreover, the CD4 + T cell levels in pSFV-MEG and pSFV-MEG/LNPs groups were also higher than that in the pcDNA3.1-MEG and pcDNA3.1-MEG/LNPs groups (1.14-and 1.02-fold), which reconfirmed that the self-replication vector pSFV could enhance CD4 + T cell immune response. J o u r n a l P r e -p r o o f To evaluate the biosafety of different LNPs in vivo, the body weight of mice was monitored during the whole immunization experiment and the main organs (heart, liver, spleen, lung, and kidney) were collected for health evaluation. During the immunization period, the mice did not show any signs of discomfort and the body weight of mice treated with different LNPs increased regularly ( Figure 9A ). Besides, main organs were identified to be healthy after immunization regardless of types of reagents (LNPs or PBS) ( Figure 9B ). These results further demonstrated that the novel developed LNPs were safe enough for DNA vaccine delivery. As one of the most prevalent disease in low-income countries worldwide, TD infects 300 million people every year. 35 According to Intestinal Center Study, TD is mainly caused by two major pathogens, including Enterotoxigenic Escherichia coli and Shigella. 36 Although current antibiotics are critical treatment and prevention drugs for TD, they have been gradually denied because of the increasing number of ARB. 37 To the best of our knowledge, DNA vaccines have potential advantages compared to antibiotics and other traditional vaccines, which is easy to adapt to new and fast-emerging diseases by changing the antigen sequence. 6 Meanwhile, the advantages of multi-epitope DNA vaccines have been gradually discovered, especially with immunodominant B-and T-cell epitopes. Until now, the safety and effectiveness of multi-epitope DNA vaccines in human have been tested by many clinical trials, such as NCT02348320 and NCT02157051 for breast cancer, NCT02172911 for cervical cancer. 8 However, low antigenicity and lack of J o u r n a l P r e -p r o o f Journal Pre-proof stable delivery system limit the use of DNA vaccines. Currently, several studies have reported that the design of multi-epitope DNA vaccines based on online softwares. 22, 23 Partial protection in BALB/C mice had been seen, and the multi-epitope vaccines displayed suboptimal immunogenicity or weaker immune responses. Thus, we designed the multi-epitope DNA vaccine reasonably, and the design principles as follows : 1) active B-and T-cell epitopes to enhance immunogenicity; 2) leader sequence to enhance the stability of mRNA; 3) Kozak sequence and strong promoter to improve transfection efficiency in mammalian cells; 4) appropriate linker peptides to improve the immunogenicity; 5) codon optimization to improve expression level. 17 Study results showed that pSFV-MEG improved both humoral (1.19-fold) and cellular immunity (1.14-fold) compared to pcDNA3.1-MEG. Previous studies have shown that self-replication plasmid pSFV can enhance the immune response without integration into the genome. 38 Besides, the selected 16 epitopes based on online tools were consistent with clinically attenuated multivalent vaccines for TD. [39] [40] [41] The Z-score (-4.05) of MEG antigen was simlilar to the other reports. 12, 42 Furthermore, the developed vaccine had an antigenicity probability of 1.13, which was higher than that in Gummow et al. 12 (0.48) and Pourseif et al. 35 Splenocytes from three mice in each group after the third immunization were stained with mouse anti-CD4 and anti-CD8 monoclonal antibodies. Data were expressed as mean ± SD (n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001. J o u r n a l P r e -p r o o f Data were expressed as mean ± SD (n = 3). J o u r n a l P r e -p r o o f Antibiotic resistance is ancient The Science of Antibiotic Discovery DNA Vaccines-How Far From Clinical Use? 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