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/lp/springer-journals/novel-adjuvant-nano-vaccine-induced-immune-response-against-r0nSYwXDcC
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- Springer Journals
- Copyright
- Copyright © The Author(s) 2023
- eISSN
- 2191-0855
- DOI
- 10.1186/s13568-023-01531-0
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- See Article on Publisher Site
Abstract
Developing adjuvant vaccines to combat rising multidrug‑resistant (MDR) Acinetobacter baumannii (A. bauman- nii) infections is a promising and cost‑ effective approach. The aim of this analysis was to construct a pDNA‑ CPG C274‑adjuvant nano ‑ vaccine and investigate its immunogenicity and protection in BALB/c mice. The CPG ODN C274 adjuvant was chemically synthesized and cloned into pcDNA3.1( +), and the cloning was verified using PCR and BamHI/EcoRV restriction enzyme digestion. Then, utilizing a complex coacervation approach, pDNA‑ CPG C274 was encapsulated by chitosan (CS) nanoparticles (NPs). TEM and DLS are used to explore the properties of the pDNA/CSNP complex. TLR-9 pathway activation was investigated in human HEK‑293 and RAW 264.7 mouse cells. The vaccine’s immunogenicity and immune‑protective effectiveness were investigated in BALB/c mice. The pDNA‑ CPG C274/CSNPs were small (mean size 79.21 ± 0.23 nm), positively charged (+ 38.87 mV ), and appeared to be spherical. A continu‑ ous slow release pattern was achieved. TLR-9 activation was greatest in the mouse model with CpG ODN (C274) at concentrations of 5 and 10 μg/ml with 56% and 55%, respectively (**P < 0.01). However, in HEK‑293 human cells, by increasing the concentration of CpG ODN (C274) from 1 to 50 μg/ml, the activation rate of TLR-9 also increased, so that the highest activation rate (81%) was obtained at the concentration of 50 μg/ml (***P < 0.001). pDNA‑ CPG C274/ CSNPs immunized BALB/c mice produced increased amounts of total‑IgG, as well as IFN‑γ and IL ‑1B in serum samples, compared to non‑ encapsulated pDNA‑ CPG C274. Furthermore, liver and lung injuries, as well as bacterial loads in the liver, lung, and blood, were reduced, and BALB/c mice immunized with pDNA‑ CPG C274/CSNPs showed potent protection (50–75%) against acute fatal Intraperitoneal A. baumannii challenge. pDNA‑ CPG C274/CSNPs evoked total‑ IgG antibodies, Th1 cellular immunity, and the TLR-9 pathway, as well as protection against an acute fatal A. baumannii challenge. Our findings suggest that this nano ‑ vaccine is a promising approach for avoiding A. baumannii infection when used as a powerful adjuvant. Key points • Novel CPG-DNA adjuvant nano-vaccine against A. baumannii was constructed. • The bacterial load in the serum of mice infected with pDNA-CPG C274/ CSNP post-challenge was substantially lower than in control groups. • In BALB/c mice, pDNA-CPG C274/ CSNP induced specific total-IgG antibodies, Th1 cellular immunity, the TLR-9 pathway, and protection against a fatal intraperitoneal A. baumannii challenge. *Correspondence: Abbas Doosti abbasdoosti@yahoo.com Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Piri‑Gharaghie et al. AMB Express (2023) 13:31 Page 2 of 16 Keywords A. baumannii, CPG‑adjuvant vaccine, Polymeric nanoparticles, Chitosan Introduction activation (Lai et al. 2019). Class A ODNs are powerful ESKAPE pathogens are a major cause of nosoco- inducers of IFN-alpha, which leads to the development mial infections and often contain antibiotic resistance of plasmacytoid dendritic cells (pDCs) (Bencze et al. mechanisms (Benkő et al. 2020). Enterococcus faecium, 2021). Type I IFNs are only moderately induced by class Staphylococcus aureus, Klebsiella pneumoniae, A. bau- B ODNs, but they are powerful activators of human B mannii, Pseudomonas aeruginosa, and Enterobacter cells and monocyte maturity (Martinson et al. 2007). spp. are all members of the ESKAPE type of microor- Class C ODNs contain components from both Classes ganisms. This collection of microbes is resistant to A and B ODNs, causing IFN-alpha production in pDC numerous antimicrobial treatments and is presently the and B cell stimulation (Martinson et al. 2007; Shirota most common cause of nosocomial infections (Mulani et al. 2017). Class C ODNs is a novel form of immu- et al. 2019a, b). In clinical research, ESKAPE isolates nological adjuvant that includes dinucleotides with resistant to all current antibiotics are becoming more unmethylated CpG patterns and has been examined common, making therapy difficult, if not impossible, in a variety of animal systems for protective immune for these illnesses (Santajit et al. 2016). A. baumannii response versus viral, bacterial, and parasite diseases is one of the most significant members of this group, (Shirota et al. 2017). Many conducted clinical investi- as it possesses an escape strategy against the human gations have shown that Class C CpG ODN like C274 immune response in addition to conventional resist- might be used as an adjuvant because of its influence ance mechanisms (Sarshar et al. 2021). A. baumannii on immune stimulation (Keshavarzian et al. 2020; Bode has developed a method of evading the immune sys- et al. 2011). The bulk of research on CpG ODN have tem (Morris et al. 2019). A. baumannii avoids phago- been conducted on animals of various species that pro- cytosis by sticking to the membrane of neutrophils and tect them from bacterial, viral, and protozoan diseases taking advantage of their migratory capacity to spread (Kuo et al. 2020; Lin et al. 2020; Goonewardene et al. throughout the host. Natural killer (NK) cells identify 2021). A plasmid carrying CpG ODN has also been bacteria that have passed through the epithelial and found to be effective as an adjuvant for DNA vaccina - respond by secreting CXCL1 and attracting more neu- tions (Klinmana et al. 1999). The combination of CpG- trophils (García-Patiño et al. 2017). Toll-like receptor enriched plasmids and a DNA vaccination encoding (TLR) detect bacteria in other innate immune cells, the HIV envelope glycoprotein dramatically enhanced such as macrophages and dendritic cells (DCs) (García- HIV-specific cell mediated and humoral immunity in Patiño et al. 2017). Toll-like receptors (TLRs) and BALB/c mice (Kojima et al. 2002). The presence of CpG NOD-like receptors (NLRs) recognize infectious agents patterns in plasmid DNA was shown to interact with by detecting common pathogen-associated molecular toll-like receptor 9 (TLR-9) and effectively trigger the patterns (PAMPs) by host specific receptors (Li et al. synthesis of IL-12 and IFN (interferon)- in dendritic 2021). TLRs are a class of cell specific receptors which cells, giving it adjuvant properties (Tudor et al. 2005). interact to a range of infectious like bacterial, fungal The receptor of CpG ODN has been identified as and viral molecular patterns (Li et al. 2021). One of the TLR-9, a pattern recognition receptor (PRR). TLR-9 is most significant aims of researchers in the last decade an internal receptor whose active form is only found against infections, which is triggered by CPGs, has been in end lysosomes and detects unmethylated CpG pat- to stimulate TLRs, particularly TLR-9 (Zheng et al. terns in bacterial or viral DNA (Behzadi et al. 2021). 2020). Synthetic oligodeoxynucleotides (ODN) with an In Klebsiella pneumoniae, Legionella pneumophila, unmethylated deoxycytosine-deoxyguanosine (CpG) and Streptococcus pneumoniae variants of pneumo- pattern have immunomodulatory activity comparable nia, as well as a Neisseria meningitides type of sepsis, to bacterial DNA and can trigger immune response in mice missing TLR-9 had higher mortality and bacterial animals via Toll-like receptors 9 (TLR-9) and hence can loads (Noto et al. 2015). TLR-9 may detect CpG ODN, be used as adjuvants (Nigar et al. 2019). An adjuvant is promoting the development of antigen presentation a substance that boosts the capacity of co-inoculated cells (APCs) and the production of cytokines such as antigens to induce early, long-lasting, and increased interferon (IFN), interleukin (IL), tumor necrosis fac- immune systems (Pulendran et al. 2021). CpG ODN tor (TNF), and others. In mouse models of acute bac- adjuvants are divided into three groups, each of which terial pathogens, TLR-9 signaling has varying impacts has a distinct modulatory impact on innate immune on illness consequences (Krogmann et al. 2016). When P iri‑Gharaghie et al. AMB Express (2023) 13:31 Page 3 of 16 CpG enters the physiological milieu, it diffuses and the Institutional Animal Care and Use Committee guide- degrades quickly. Since a result, using nanocarriers lines of Islamic Azad University, Shahrekord, Iran (14th for adjuvant administration has a number of benefits, 2020 with ethics code: IR.IAU.SHK.REC.1400.046) and as these nanoscale vehicles can preserve the delicate all methods were carried out in accordance with relevant adjuvant payload throughout transit (Pautu et al. 2021). guidelines and regulations. All methods are reported in Nanomaterials can be more efficiently accumulated to accordance with ARRIVE guidelines (https:// arriv eguid lymphoid regions by migrating dendritic cells engulfing elines. org). Eight-week-old female BABL/c mice were and transporting them, or by free, diffusional migration purchased from Isfahan Royan Laboratory Animal Sci- through lymphatic channels (Jia et al. 2018). Surface ence Center (Isfahan, Iran). The mice were maintained morphology, shape, particle size, and adjuvant contents under specific-pathogen-free conditions (20–25 °C, may also be intricately tailored to adapt to particu- 40–45% humidity, 12 h light/dark cycle; food and water lar innate immune activation and targeting to specific access). After the experiments were completed, the immune cells, due to nanomaterials’ synthetic flexibil - remaining limbs were autoclaved, and all efforts were ity (Pati et al. 2018). TLR agonists such as CpG have made to minimize suffering. been tried to improve immunomodulatory capacity in mice via nanomaterials entrapment or surface conjuga- CpG ODN and plasmids construction tion (Rajput et al. 2018). Polymeric systems are one of Synthetic oligodeoxynucleotides (ODN) containing the most widely utilized nanoparticles for drug deliv- unmethylated deoxycytosine-deoxyguanosine (CpG) ery due to their biodegradability and biocompatibility. motif are equivalent to bacterial DNA in the immu- Chitosan nanoparticles (NPs) are potential polymeric nostimulatory activity, which can induce innate immu- and organic NPs that have gotten a lot of controversy nity via Toll-like receptor 9 (TLR-9) in mammals. 20 in recent decades (Gharaghie et al. 2020; Beiranvand repetitions of CpG ODN C274 (480 bp) were syn- et al. 2021). They have a lot of promise as nanostruc - thesized and inserted into pcDNA3.1 ( +), by Beijing tures for encapsulating compounds like drugs or active company (Shenzhen, China). The following oligodeoxy - chemicals, delivering them to a specified location, and nucleotide sequences were used, Uppercase letters in releasing them in a regulated manner. Our objective the following sequences represent PS linkages, and low- was to examine the effect of C274 adjuvant against A. ercase letters represent PO linkages: CpG ODN C274, baumannii by stimulating TLR-9 and chitosan nano- 5’-tCGtCGaaCGttCGagatgat-3’. Oligodeoxynucleotides particles, taking into account the bacterium’s primary were confirmed by Bam HI (GGA TCC ) (1400 bp) and PstI difficulties and escape strategy. (CTG CAG ) (4000 bp) endonuclease digestion assay and DNA sequencing. Recombinant plasmids were provided Materials and methods by puncture bacteria and powder and stored at −20 °C. Materials The plasmids were all transformed into E. coli TOP10F Chitosan powder (Sigma-Aldrich, USA), Endo-free and purified with the Endo-free Plasmid DNA Extraction Plasmid DNA Extraction mini kit (Favorgen, Taiwan), mini kit (Favorgen, Taiwan). CpG ODN C274 and pcDNA3.1 ( +), by BGI Genomics Company (Shenzhen, China), SYBR Safe (Invitrogen), Preparation of CpG‑loaded polymeric chitosan TLR-9 activation was performed as follows by abeom- nanoparticles (pDNA‑CPG C274/CSNPs) ics, Inc (San Diego, CA 92121, United States). Quant-iT CpG encapsulating chitosan nanoparticles (CpG-CSNP) RiboGreen RNA Kit (Invitrogen), karmania pars gene were produced as previously described, using a water in ELISA kits (KPG, IRAN), LB medium, fetal bovine serum oil in water (w/o/w) double emulsion method (Lin et al. (FBS) and antibiotic- ampicillin were all purchased from 2019; Rasmussen et al. 2020; Instruments et al. 2011). Invitrogen (Carlsbad, CA). Briefly, a polymer solution was prepared by dissolving 50 mg of chitosan nanoparticles (purchased from Sigma- Experimental animals, bacterial strains and ethics Aldrich, USA) in hydrochloric acid at 0.5% (w/v) using a A. baumannii strain (ATCC: 19606) and E. coli (TOP10F) 1:2 chitosan: HCl ratio (50 mg chitosan in 100 mL HCL were purchased from the Iranian Biological Resource 0.5%) in a rotary mixer. The diluted chitosan solutions Center (IBRC). The strain was cultivated on Luria–Ber - were sterile filtered using a 0.2 µm syringe filter before tani (LB) agar (Difco, USA) at 37 °C overnight. All ani- being mixed with pDNA-CPG. The inner aqueous phase mal protocols were performed in accordance with the (pDNA solution) was prepared by dissolving 100 µg Ethical Committee and Research Deputy of the Islamic of CpG-C274 in the 100 µl phosphate buffer (pH = 7). Azad University of Shahrekord Branch, Iran for the Care 500 μL of sterile diluted chitosan solution was added to and Use of Laboratory Animals and were approved by 250 μL of pDNA, pipetting up and down, heated at 40 °C, Piri‑Gharaghie et al. AMB Express (2023) 13:31 Page 4 of 16 and gently tapping the tubes to make pDNA-CPG /CSNP HeK-293 cell lines were placed at a density of 1 × 10 (1:2). cells per well in 96-well plates with 100 μL of the form of media for the cell viability experiment. Cell adhesion Transmission Electron Microscopy (TEM) and Dynamic was then promoted by overnight incubation at 37 °C in Light Scattering (DLS) a humid environment with 5% CO2. The cell lines were A variety of analytical methods, such as dynamic light then exposed to various nanovaccine concentration levels scattering (DLS), transmission electron microscopy (0.125–1024 μg/mL) for 24 h. The media was then mixed (TEM), and Fe-SEM, were used to analyze nanoparticles with 10 μL of CCK-8 solvent and incubated for an extra for their physicochemical characteristics by Beam Gostar 2 h. Using a spectrophotometric method, optical density Taban Material Analysis Laboratory (IRAN). DLS was (OD) values were calculated at 450 nm wavelength (Bio- used to determine the size of nanoparticles and their zeta Rad iMark). potential (Brookhaven Instruments Corp., USA). Field scanning electron microscopy (FESEM) and TEM model Vaccine and immunization MIRA3 were used to examine NP surface morphology In this investigation, 70 female Balb/C mice aged 6 to (TESCAN, Czech Republic). 8 weeks and weighing 15–20 g were employed. Animals have injected pDNA-CPG C274-CSNPs through IM injection in both hind legs (100 μL of total volume per Release kinetics of pDNA‑CPG C274‑ CSNPs leg, the total dose of 100 µg of DNA). After initial vac- The release kinetics was determined in 10 mM sodium cination, the mice were boosted on days 0, 7, 14 and 30, phosphate buffer (pH 7.0) and 10 mM acetate buffer using the identical injection techniques. The mice were (pH 5.0). The Slide-A-Laser MINI Dialysis units (10 kDa separated into 6 groups of 10, depending on whether the MWCO, Thermo Fisher Scientific) were loaded with injection was done intramuscularly (n = 60), and 10 addi- 100 μL of CpG encapsulated CSNPs and placed in tanks tional mice were used as negative controls, receiving PBS containing aforementioned buffers at 37 °C. After 20, 40, therapy intramuscularly (n = 10). Table 1 shows the num- 60, 80, and 100 h, the nanoparticles remaining in dialy- ber of mice utilized in each group. sis units were collected and disrupted by 100% acetone. After acetone was evaporated, remaining CpG was ana- ™ ® Analysis of IgG total antibody in serum lyzed by the reagent in Quant-iT RiboGreen RNA Kit Serum samples were collected from all groups before vac- (Invitrogen). cination, 7, 14 and 30-days post immunization(dpi). The blood was taken and kept at 37 °C for 1 h before being In‑Vitro analysis of TLR‑9 activation centrifuged to extract the serum. By using an indirect Previous studies using models of A. baumannii mouse ELISA test, the levels of total IgG were measured to eval- pneumonia and systemic infection have shown that TLR- uate the humoral immune response. First, 10 µl of each 9 Actively contributes to the innate immune response sample was added to the particular coated wells, which against A. baumannii (Noto et al. 2015). Analysis of were then washed and incubated with goat anti-mouse TLR-9 activation was generally performed as follows secondary antibody conjugated with horse-radish peroxi- by abeomics, Inc (San Diego, CA 92121, United States). dase (HRP). Plates were then cleaned and optical density Briefly, Harvest NF-kB Leeporter —RAW 264. 7 mouse was measured between 460 and 630 nm. All experiments cells and TLR-9/NF-kB Leeporter —HEK293 human were performed in triplicate. cells seed cells into a white solid-bottom 96-well micro- plate in 100 ul of growth medium at 5 × 10 cells/well. Incubate cells at 37 °C in a CO2 incubator for overnight. The next day, stimulate cells with various amounts of Table 1 The number of mice used in this experiment CpG ODN C274 incubate at 37 °C in a CO2 incubator for Group Injection composition Number Average Type of 6–16 h. Add 30–50 ul of luciferase assay reagent (Pierce number of mice weight of injection Firefly Luciferase Glow Assay Kit, Catalog number: mice 16176, Thermo Fisher Scientific, Netherlands) per well. 1 pDNA‑ CPG C274/CSNP 10 18.7 IM Incubate at room temperature for 1–5 min and meas- 2 pDNA‑ CPG C274 10 18.2 IM ure luminescence using a microplate luminometer (Haas 3 Free‑ CPG C274 10 18.9 IM et al., 2014). 4 Free‑pDNA/CSNP 10 18.4 IM 5 Free‑pDNA 10 18.3 IM Cytotoxicity research 6 CSNP 10 18.6 IM CCK-8 experiment was used to measure the cytotoxic 7 PBS 10 18.9 IM effects of nanovaccines on RAW/HeK-293 cells. RAW/ P iri‑Gharaghie et al. AMB Express (2023) 13:31 Page 5 of 16 Detection of cellular immune response Tukey– Kramer post hoc test with a 95 percent confi - The levels of IFN-γ, and IL1β were measured to evalu - dence interval. A Chi square test with Yates’ correction ate the cellular immune response caused by the plas- was used to compare the survival rates between immu- mids. The amounts of functional and, proinflammatory nized mice and the control group. Differences were cytokines were determined in serum sample using kar- considered significant at p < 0.05 and very significant at mania pars gene ELISA kits (KPG, IRAN) according with p < 0.01. owner’s manual. The coating antibody was coated over - night at 4 °C onto a 96-well plate (0.2 μg/well). PBS with Results 0.5% BSA and 0.1%Tween 20 was then used to block the Construction and identification of pDNA‑CPG C274 plate. Following that, 100 μL of sample were distributed CpG-C274 adjuvant was cloned into the pcDNA3.1( +) into two replicated wells, and 50 μL of 1 μg/mL detecting eukaryotic expression vector as shown in Fig. 1A. The antibody were added. The plate was sealed and shaken CPG sequencing from the recombinant plasmids was for 2 h at room temperature (700 rpm). Following washes done (Fig. 1C). The plasmids were digested with Bam HI with PBS containing 0.1% Tween 20, 100 μL of streptavi- and PstI. The digestion products electrophoretically din- HRP (1:1024) was added and incubated for 30 min isolated at 1400 bp and 4000 bp, respectively, showing on a shaker at room temperature (700 rpm). that the recombinant plasmid was effectively produced (Fig. 1B). The existence of a 480-bp band following diges - Bacterial load assessment in the blood, liver and lung tion with BamHI (GGA TCC ) and EcoRV (GAT ATC ) Blood samples were obtained from mice in each group enzymes further suggests that the cloning and synthesis at 24 h post-challenge. To determine the bacterial loads of the recombinant plasmid pcDNA3.1 ( +)/CPG. in the blood, samples were serially diluted and plated on blood agar plates. After blood collection, the mice were Characterization of CpG‑Loaded polymeric nanoparticles killed, and the liver and lungs were removed aseptically, The nanoparticles were studied using DLS and TEM weighed, and homogenized. Serial dilutions of tissue imaging. The results revealed unimodal round form and homogenates were plated onto blood agar plates. Bacte- thin-shell empty nanoparticles (without the CpG load- rial CFUs were enumerated after 24 h of incubation at ing = CSNP) with an average diameter of 25.93 ± 0.98 nm, 37 °C. being comparable in size to the internally and externally CpG loaded nanoparticle (pDNA-CPG C274-CSNPs), Challenge experiments which had a diameter of 79.21 ± 0.23 nm (Fig. 2A, B). To investigate the protective efficacy of different DNA The zeta-potentials of CSNP and pDNA-CPG C274- vaccine, all groups were challenged with A. bauman- CSNPs were determined to be + 73.2 mV and + 38.87 mV, nii ATCC19606 by intraperitoneal injection 3 weeks respectively (Fig. 2C, D). Nanoparticles with zeta poten- after the last immunization. Briefly, A. baumannii strain tials between −10 and + 10 mV are roughly neutral, but ATCC19606 was raised to the late-logarithmic phase in nanoparticles with zeta potentials larger than + 30 mV or LB broth, at 37 °C/150 rpm. The cells were collected by less than −30 mV are strongly cationic and strongly ani- centrifugation at 4000 × g for 10 min, then washed and onic, respectively (Rasmussen et al. 2020; Instruments resuspended in PBS. Mice were given lethal dosages of et al. 2011). As a result, the nanoparticles synthesized ATCC19606 (2 × 10 CFU) in a total volume of 100 µL in this study (CSNP and pDNA-CPG C274/CSNPs) are Intraperitoneally. The mice were tracked for seven days, strongly cationic nanoparticles. The cationic nature of with each group’s body weight, clinical score, and sur- chitosan is unique, as most polysaccharides are usually vival rate recorded every day. Each mouse’s overall clini- either neutral or negatively charged in an acidic envi- cal sign was rated on a range of 0 to −5 on a sliding scale. ronment. This property allows it to form electrostatic Individual clinical ratings ranged from 0 (normal, active, complexes or multilayer structures with other negatively healthy), −1 (slightly sick, slightly ruffled fur, otherwise charged synthetic or natural polymers and CpG ODNs. normal), −2 (ill, ruffled fur, sluggish movement, hunch - Encapsulation of CpG ODNs are negative in charge ing), −3 (extremely sick, ruffled hair, very slow move - causes CpG ODNs placed inside a layer of chitosan par- ment, stooped, eyes shut), −4 (moribund), and −5 (dead) ticles with a positive charge. Therefore, the resulting (Du et al. 2021). nanoparticles show a positive charge (Additional file 2) (Tamara et al. 2018). Statistical analysis The size of the nanoparticles was measured before and GraphPad Prism 5.0 was used to examine the data and after a month of freezing at −20 °C. The nanoparticle size perform statistical tests. A one-way analysis of variance increased during storage, which might be attributed to (ANOVA) was used to compare means, followed by a the presence of negatively charged pDNA-CPG C274 at Piri‑Gharaghie et al. AMB Express (2023) 13:31 Page 6 of 16 Fig. 1 Construction and identification of pDNA‑ CPG C274. A Cutting sites and map of plasmid recombinant. B To separate the DNA plasmids, electrophoresis was utilized. Lane 1: pDNA‑ CPG C274; Lane 2: Expected size after digestion; Lane M: DNA markerIII C The CPG sequencing from the recombinant plasmids full‑length gels is included in an Additional file 1: Figure S1 the CSNP Surface, causing aggregation with nearby nano- pDNA-CpG C274 release kinetics of pDNA-CpG C274/ particles due to gravitation between negative and positive CSNP analysis shows that Chitosan-based CpG-NP was charge and treatment of nanoparticles with DNase enzyme more stable in pH 7.0 than pH 5.0 (Fig. 2F), indicating that did not increase the size of nanoparticles (Fig. 2E). The early interruption of pDNA-CpG C274/ CSNP and lack P iri‑Gharaghie et al. AMB Express (2023) 13:31 Page 7 of 16 of adjuvant in the normal biological environment may be were nontoxic to RAW/HeK-293 cells and supporting their prevented. Furthermore, the sensitivity to low pH pro- safety and biocompatibility for in vivo research. The growth motes the unloading of pDNA-CpG C274/CSNP in acidic rate of cells treated with various formulations is also shown endosomes after absorption by cells. in Fig. 3D. CpG ODN (C274) activated TLR‑9 in human and mouse cells pDNA‑ CPG C274/CSNP enhanced IgG total generation (In‑Vitro) The mice were subsequently vaccinated intramuscu - Analysis of TLR-9 activation in mouse and human cells larly with the vaccines listed in Table 1. The total IgG was performed by abeomics, Inc. TLR-9 actively contrib- titer was greatest after vaccination with a combination utes to the innate immune response against A. baumannii, of pDNA- CPG C274 plasmids and CSNP nanoparti- according to previous research employing A. baumannii cles (P ≤ 0.0001). Recombinant CPG plasmid appears mouse pneumonia and systemic infection models (Noto to be related with high immunogenicity (Fig. 4A), indi- et al. 2015). According to the results of Fig. 3, CpG ODN cating that the pDNA-CPG adjuvant elicited a humoral (C274) was able to activate TLR-9 in human and mouse immune response, as seen by an increase in total IgG cell lines. TLR-9 activation was greatest in the mouse in serum (P ≤ 0.01). The Free-C274 group additionally model with CpG ODN (C274) at concentrations of 5 and enhanced total IgG (P ≤ 0.05), however the Free-pDNA/ 10 μg/ml with 56% and 55%, respectively (**P < 0.01). How- CSNP, Free-pDNA and CSNP groups showed identical ever, at a concentration of 50 μg/ml, the activation rate of effects to the PBS group and did not increase total IgG. TLR-9 was 43% (*P < 0.05) (Fig. 3A). However, in HEK-293 In fact, there were no or low antibody titers in this groups human cells, by increasing the concentration of CpG ODN (Fig. 4A). As shown in Fig. 4B, the average serum anti- (C274) from 1 to 50 μg/ml, the activation rate of TLR- body titers IgG Total in the pDNA-CPG C274/CSNPs 9 also increased, so that the highest activation rate (81%) group were stronger than other groups, suggesting that was obtained at the concentration of 50 μg/ml (***P < 0.001) these types of plasmids (carrying CPG) in conjunction (Fig. 3B). However, when the concentration of CpG ODN with CSNPs were primarily priming Th1 type immune (C274) was raised from 1 to 50 μg/ml in HEK-293 human response. Furthermore, as compared to days 0, 7, and 14, cells, the activation rate of TLR-9 increased as well, with immunization at 30 dpi resulted in the highest blood IgG the maximum activation rate (81%) achieved at the 50 μg/ total levels (Fig. 4C). ml concentration (***P < 0.001). In mouse and human cells, the maximum activation of TLR-9 and hence the innate immune response was shown at concentrations of 10 and pDNA‑ CPG C274/CSNP induces high levels of functional 50 μg/ml, respectively (Mendez et al. 2020). and, proinflammatory cytokines in mice To investigate the effect of CpG on cellular immune Nanovaccines’ cytotoxic effect on RAW/HeK‑293 cells response expression in vivo, serum of each group was In biological applications, toxicity is a significant problem sampled and the changes of functional and proinflamma - when employing nanoparticles, particularly biodegrad- tory cytokines (IFN-γ, IL-1β) were investigated. The con - able polymeric materials. We used RAW/HeK-293 cells centrations of functional and proinflammatory cytokines to assess the toxicity of various nanovaccine formula- in serum significantly increased following immunization tions. Additional file 3: Figure S3 shows the growth of with pDNA- CPG C274/CSNP (p ≤ 0.0001), pDNA- CPG RAW/HeK-293 cells in a culture medium used for CSNP, C274 (p ≤ 0.001), and CPG C274 (p ≤ 0.05), compared pDNA-CSNP, and pDNA-CpG-CSNP groups. Research on to the control (groups 4, 5, 6, 7). Furthermore, groups toxicity that depends on time and dosage was carried out administered chitosan nanoparticles (pDNA- CPG C274/ across 24 periods in RAW/HeK-293 cells exposed to vari- CSNP) exhibited a greater cellular immune response than ous nanovaccine formulations at concentrations ranging the control group, with higher levels of functional and from 0.125 μg/mL to 1024 μg/mL. No concentration of proinflammatory (IFN-γ, IL-1β) cytokines. Furthermore, Nanovaccines altered the viability of RAW/HeK-293 cells the maximum concentrations of cytokines are observed at any of the studied periods (Fig. 3C), proving that they (See figure on next page.) Fig. 2 Characterization of CpG C274‑Loaded Polymeric Nanoparticles. Transmission electronic microscopy was used to visualize CSNPs A and pDNA‑ CPG C274/CSNPs B with an average diameter of 25.93 0.98 nm and 79.21 ± 0.23 nm rspectively. CSNP nanoparticles C and pDNA‑ CPG C274/ CSNPs D zeta sizer load diagrams. E Nanoparticle size measurement following storage by freezing at −20 °C for 1 month. F In vitro release kinetic of CpG from CpG‑NP at pH 5 and pH 7. *p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.001. G Oligreen fluorescent test to determine the degradation of DNA at 16, 12, and 72 h. The results show no DNA degradation after 72 h. The highest amount of DNA was obtained after 72 h, which indicates the slow release of DNA from the nanoparticle Piri‑Gharaghie et al. AMB Express (2023) 13:31 Page 8 of 16 Fig. 2 (See legend on previous page.) P iri‑Gharaghie et al. AMB Express (2023) 13:31 Page 9 of 16 Fig. 3 TLR-9 activation in human and mouse cells. A CpG ODN C274‑mediated mouse TLR-9 activation in NF‑kB Leeporter —RAW 264.7 cells. B CpG ODN C274‑mediated human TLR-9 activation in TLR-9/NF‑kB Leeporter —HEK293 cells. *p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.001. C The cytotoxicity of different formulations of nanovaccines after 24 h on RAW/HeK ‑293 cells. D Cell colonies of RAW/HeK ‑293 trated with CSNP, Free ‑pDNA‑ CSNP and pDNA‑ CpG‑ CSNP were stained with Giemsa. Experiments were performed in triplicate Piri‑Gharaghie et al. AMB Express (2023) 13:31 Page 10 of 16 Fig. 4 Humoral immune responses to plasmids containing CpG ODNs and other groups in mouse. A Total IgG in different groups on 0, 7, 14 and 30 dpi. B Average concentration (pg/ml) of Total IgG in different groups. C Average concentration (pg/ml) of Total IgG on 0, 7, 14 and 30 dpi. Data in the plot present the mean ± SEM, n = 10 (ns, nonsignificant, *p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001) 30-days post immunization, suggesting that the cellular to be evaluated for bacterial load in the blood, liver, and immune response is at its peak after that time (Fig. 5). lung tissue (Fig. 6). Mice immunized with pDNA- CPG C274/CSNP had lower bacterial loads in the blood than Bacterial loads were reduced as a result of immunization mice from the other groups (Fig. 6A). After blood col- We tested the efficiency of the pDNA- CPG C274/CSNP lection, mice in each group were sacrificed, and the liver vaccine using an i.p injection model in BALB/c mice and lungs were collected. The right lobes of the lungs to see if it provided protection. All groups were chal- were prepared for CFU analysis. The liver and lungs of lenged with fatal dosages of A. baumannii ATCC19606 the pDNA- CPG C274/CSNP groups had a significantly (2102 × 108 CFU/mouse) 3 weeks after the last boost vac- reduced bacterial load than the control groups, as illus- cination. Mice were selected at random from each group trated in Fig. 6B–C. Furthermore, when comparing the P iri‑Gharaghie et al. AMB Express (2023) 13:31 Page 11 of 16 Fig. 5 Cytokine levels in the sera. Groups of female Balb/c mice (n = 10) were immunized subcutaneously with 100 μg CPG C274 formulated with pDNA and CSNP on day 0, 7, 14, and 30. pDNA‑ CPG C274/CSNP increased concentrations of IL ‑1β (A) and IFN‑γ (C) in serum. The highest levels of IL‑1β (B) and IFN‑γ (D) were observed on day 30. Data in the plot present the mean ± SEM, n = 10 (ns nonsignificant, *p ≤ 0.05; **p ≤ 0.01, ****p ≤ 0.0001) pDNA-CPG C274/CSNP vaccinated group to the pDNA- CSNP, pDNA- CPG C274, and CPG C274 had 7-day CPG C274, and CPG C274 groups, the bacterial load was survival rates of 75%, 50%, and 25%, respectively, after considerably lower in the pDNA-CPG C274/CSNP vac- the fatal dosage of ATCC19606, strains challenged, cinated group. These findings suggested that immunizing which were considerably greater than mice immunized mice with pDNA-CPG C274/CSNP could decrease A. with PBS (0%). After the A. baumannii challenge, the baumannii colonization in the liver and lungs. body weight and clinical symptom scores (Table 2) of each group decreased to their lowest levels three days post-infection. Then, the symptoms of mice in Survival rate, body weight changes and clinical scores the pDNA- CPG C274/CSNP, pDNA- CPG C274, and of mice post‑challenge CPG C274 gradually improved. The body-weight of Six mice from each group were chosen to record sur- the mice vaccinated with pDNA- CPG C274/CSNP vival, body weight changes, and clinical scores every returned to that before the challenge, and the symp- day for 7 days after being infected with A. baumannii. toms disappeared seven days post-challenge but in the All mice in the Free-pDNA/CSNP, Free-pDNA, CSNP, other 2 groups, the symptoms of the disease were mild and PBS groups died 24 h after the challenge, as shown to severe. These findings showed that mice immunized in Table 2. Mice vaccinated with pDNA-CPG C274/ Piri‑Gharaghie et al. AMB Express (2023) 13:31 Page 12 of 16 Fig. 6 Bacterial burdens in BALB/c mice’s blood, liver, and lung tissues. The procedure of vaccination and A. baumannii challenge. BALB/c mice were infected with fatal concentrations of A. baumannii ATCC19606 (2108 CFU/mouse) 3 weeks after their last vaccination. A Bacterial burdens in mice’s blood 24 h after the challenge. Bacterial burdens in the liver and lungs B–C. After a 24‑h challenge with A. baumannii, the liver and lungs were removed. The mean SD (n = 6) is indicated by the bars. (ns nonsignificant, *p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001) with the pDNA- CPG C274/CSNP nano-vaccine were treat such infections are urgently required (Doosti et al. more resistant to fatal dosages of A. baumannii infec- 2015). Despite years of effort in the development of A. tion than mice inoculated with the pDNA- CPG C274 baumannii vaccines, Unfortunately, no vaccine against and CPG C274 groups. this pathogen is currently available. There has recently been an increase in attention in developing vaccinations Discussion that include only the minimum necessary of pathogen Multidrug-resistant A. baumannii is a rapidly develop- components (Parvizpour et al. 2020; Skwarczynski et al. ing disease that causes diseases with high fatality rates 2014). The development of vaccines that employ CpG- due to insufficient medical treatment (Vazquez et al. DNA as an adjuvant has recently sparked renewed atten- 2020; Ghorbani-Dalini et al. 2015; Chuang et al. 2019; tion. CpG-DNA boosts the host immune system’s power Mulani et al. 2019a, b). New methods to prevent and to defend both intracellular and extracellular pathogens P iri‑Gharaghie et al. AMB Express (2023) 13:31 Page 13 of 16 Table 2 Survival rates, body weight changes and clinical scores each day after A. baumannii challenge Group number After challenge Day post‑ challenge Body weight Clinical score Survival rate (%) pDNA‑ CPG C274/CSNP Before challenge 18.7 0 100 1 Day post‑ challenge 17.5 −1 100 3 Day post‑ challenge 15.8 −2 100 7 Day post‑ challenge 18.1 −1 75 pDNA‑ CPG C274 Before challenge 18.2 0 100 1 Day post‑ challenge 17.1 −2 75 3 Day post‑ challenge 14.1 −3 75 7 Day post‑ challenge 17.8 −4 50 Free‑ CPG C274 Before challenge 18.9 0 100 1 Day post‑ challenge 17.9 −2 75 3 Day post‑ challenge 14.8 −3 75 7 Day post‑ challenge 18.2 −5 25 Free‑pDNA/CSNP Before challenge 18.4 0 100 1 Day post‑ challenge 17.8 −5 0 3 Day post‑ challenge 15.1 −5 0 7 Day post‑ challenge 18 −5 0 Free‑pDNA Before challenge 18.3 0 100 1 Day post‑ challenge 18.1 −5 0 3 Day post‑ challenge 15.3 −5 0 7 Day post‑ challenge 17.9 −5 0 CSNP Before challenge 18.6 0 100 1 Day post‑ challenge 18.1 −5 0 3 Day post‑ challenge 16.2 −5 0 7 Day post‑ challenge 18.2 −5 0 PBS Before challenge 18.9 0 100 1 Day post‑ challenge 18.0 −5 0 3 Day post‑ challenge 16.1 −5 0 7 Day post‑ challenge 18.3 −5 0 0 (normal, active, healthy), −1 (slightly sick, slightly ruffled fur, otherwise normal), −2 (ill, ruffled fur, sluggish movement, hunching), −3 (extremely sick, ruffled hair, very slow movement, stooped, eyes shut), −4 (moribund), and −5 (dead) (Kim et al. 2019). In vitro and in vivo delivery of CpG- concentration, suggesting a widespread transcription- DNA protects the host from Gram-positive and Gram- based mechanism of CpG-mediated gene regulation. negative pathogenic bacteria, respectively. The protective TLR-9 induction and increased high CpG transcription effects of the CpG-C274 generated TLR-9 pathway in are linked. TLR9 activation is boosted by pDNA-CpG, mice models against A. baumannii infection were the strengthening adaptive immune responses and signifi - subject of this study. Several studies have demonstrated cantly increasing cellular infiltration. that administering CpG-DNA three days before a bac- To preserve CPG against fast degradation and terial infection boosts the production of cytokines IL-6, enhance immune responses, nontoxic and effective IL-12, IFN-γ, and TNF-α, which all assist the host defend administration mechanisms are required. CSNP, a (Judy et al. 2012). CpG-DNA treatment substantially new form of polymeric nanoparticle, has been shown boosted the level of TLR-9 pathway (Noto et al. 2015) to improve the stability of encapsulated CPG-based and improved mouse survival and bacterial clearance in vaccines while also boosting their release (Wibowo the evaluated primary organs after A. baumannii infec- et al. 2020; Azim et al. 2019). We cloned 20 repeti- tion, according to this study. CPG-based vaccines have a tions of CPG C274 into pcDNA3.1( +) and encapsu- great deal of promise as safer and more effective alterna - lated it in CSNPs with a high entrapment efficiency tives to traditional immunization procedures. De novo rate and a gradual release pattern in this study. To our transcriptional activity was impacted by the pDNA-CpG information, this is the first research to encapsulate Piri‑Gharaghie et al. AMB Express (2023) 13:31 Page 14 of 16 the pDNA-CPG C274 adjuvant in CSNPs, character- in mouse and human cells, respectively. TLR-9 appears ize its physical-structural properties, and determine to have a function in A. baumannii infection host its immunological and protective effectiveness. Prior defense, which is consistent with TLR-9’s participa- to this investigation, no report of the CSNP delivery tion in other bacterial pneumonia models. Mice lack- system being employed in the construction of bacte- ing TLR-9 had higher mortality and bacterial loads in rial pDNA-CPG adjuvant nano-vaccines was found. the lungs as compared to wild type mice in K. pneu- Although there has been research on free CPG encap- moniae, L. pneumophila, and S. pneumoniae, models sulation, none have been done on pDNA-CPG encap- (Arafa et al. 2020; Bhan et al. 2010, 2008, 2007; Albiger sulation and from this point of view the present study et al. 2007). These findings showed that humoral and is novel (Lin et al. 2020). The release of biomaterials cellular immune responses, particularly Th1 immune from nanoparticles can be influenced by the mor- responses and TLR-9 pathway, were produced and phological properties of nanoparticles. Dispersion of provided complete protection. The challenge stud- biomolecules is enhanced by a leaky or porous struc- ies revealed that animals inoculated with pDNA-CPG ture, but burst release is reduced by a smooth surface C274/CSNP had effective resistance against A. bau- (Makadia et al. 2011). The morphological and sub- mannii ATCC 19606 infection. The bacterial load in stance properties of pDNA-CPG C274/CSNP indi- the serum of mice infected with pDNA-CPG C274/ cated that it had a uniform spherical structure with CSNP post-challenge was substantially lower than in a homogenous morphology. The particle size dis- the non-encapsulated pDNA-CPG C274 group and tribution of pDNA-CPG C274/ CSNP was similarly the control groups. The high titer of antigen-specific homogeneous, and it was adequately diffused without antibody led to robust protection in mice inoculated aggregation. Because it may impact both nanoparticle with pDNA-CPG C274/ CSNP, according to these integrity and nanoparticle adhesion, the zeta potential findings. In a study, Sun et al. showed that adding CpG value is one of the most essential particle characteri- based on an aluminum adjuvant might further boost zation factors (Liang et al. 2018). CSNP had a posi- the immune response, particularly cellular immunity. tive charge, as seen in Fig. 2C and D, which allowed These results imply that an A. baumannii infection it to adsorb more molecules, enhance entrapment vaccination might be effective against the A. bauman- efficiency, and enhance stability. The immune system nii Ata (Sun et al. 2022). response and the vaccination regimen, such as the pro- In conclusion, a novel CPG-DNA adjuvant nano-vac- tein concentrations and the frequency of vaccination, cine against A. baumannii was described in this study. are influenced by the release profile of a molecule in In BALB/c mice, pDNA-CPG C274/ CSNP induced nanoparticles, which is critical for the formulation of specific total-IgG antibodies, Th1 cellular immunity, a nano-vaccine. The pDNA-CPG C274/CSNP release the TLR-9 pathway, and protection against a fatal intra- patterns in this investigation were biphasic, with a peritoneal A. baumannii challenge. According to our burst of molecule followed by a prolonged release. The findings, this nano-vaccine is a promising option for first day’s burst release might trigger a powerful immu- preventing A. baumannii infection. nological response. The molecule’s long-term slow release was a desirable feature for a DNA-vaccine since Supplementary Information it may minimize the number of vaccinations while also The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13568‑ 023‑ 01531‑0. improving the peptide’s presentation to APCs (Duran et al. 2019; Arafa et al. 2020). The intra-muscular (I.M) Additional file 1: Cloning confirmation result of recombinant pcDNA3.1‑ delivery of the pDNA-CPG C274/CSNP nano-vaccine CPG C274 adjuvant. generated systemic antibody responses, according Additional file 2: General flowchart of preparation and synthesis of to the findings. In the serum of mice vaccinated with chitosan adjuvant nano‑ vaccine. pDNA-CPG C274/CSNP, significant titers of total-IgG Additional file 3: The growth of RAW/HeK ‑293 cells in a culture medium used for CSNP, pDNA‑ CSNP, and pDNA‑ CpG‑ CSNP groups. antibodies were obtained, as shown in Fig. 5. Further- more, compared to control mice, pDNA-CPG C274/ CSNP immunization resulted in considerably greater Acknowledgements The authors would like to thank the staff members of the Biotechnology levels of antigen-specific IFN-γ and IL-1β production Research Center of the Islamic Azad University of Shahrekord Branch in Iran for in the serum in the pDNA-CPG C274/CSNP and non- their help and support. This research did not receive any specific grant from encapsulated pDNA-CPG C274 groups. The present funding agencies in the public, commercial, or not‑for ‑profit sectors. study also showed that CPG C24 at concentrations of 10 and 50 μg/ml showed the highest TLR-9 activation P iri‑Gharaghie et al. AMB Express (2023) 13:31 Page 15 of 16 Author contributions Bhan U, Ballinger MN, Zeng X, Newstead MJ, Cornicelli MD, Standiford TJ (2010) TPG conducted research and drafted the manuscript, AD conceived and Cooperative interactions between TLR4 and TLR-9 regulate interleukin designed research and made manuscript revision, SAM designed research and 23 and 17 production in a murine model of gram‑negative bacterial analyzed data, TPG provided funding support. All authors read and approved pneumonia. PLoS ONE 5(3):e9896 the final manuscript. Bode C, Zhao G, Steinhagen F, Kinjo T, Klinman DM (2011) CpG DNA as a vaccine adjuvant. Expert Rev Vaccines 10(4):499–511. https:// doi. org/ 10. Funding1586/ erv. 10. 174 Not applicable. Chuang YC, Cheng A, Sun HY (2019) Microbiological and clinical characteristics of Acinetobacter baumannii bacteremia: implications of sequence type Availability of data and materials for prognosis. J Infect 78(2):106–112. https:// doi. org/ 10. 1016/j. jinf. 2018. 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Journal
AMB Express – Springer Journals
Published: Mar 11, 2023
Keywords: A. baumannii; CPG-adjuvant vaccine; Polymeric nanoparticles; Chitosan