Aluminum (oxy) Hydroxide Nanorods Activate an Early Immune

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Biological and Medical Applications of Materials and Interfaces

Aluminum (oxy) Hydroxide Nanorods Activate an Early Immune Response in Pseudomonas aeruginosa Vaccine Yingli Chen, Feng Yang, Jun Yang, Yali Hou, Leilei He, Houxiang Hu, and FengLin Lv ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18164 • Publication Date (Web): 27 Nov 2018 Downloaded from http://pubs.acs.org on November 29, 2018

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ACS Applied Materials & Interfaces

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Aluminum (oxy) Hydroxide Nanorods Activate an Early

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Immune Response in Pseudomonas aeruginosa Vaccine

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Yingli Chen1+, Feng Yang1+, Jun Yang1, Yali Hou1, Leilei He1, Houxiang Hu2,* & Fenglin Lv1,*

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1College

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Repair Engineering, Key Laboratory of Biorheological Science and Technology,

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Chongqing University, Chongqing, 400030, PR China.

of Bioengineering, “111 Project” Laboratory of Biomechanics & Tissue

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2Department

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Nanchong 637000, Sichuan, PR China.

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of Cardiology, Affiliated Hospital of North Sichuan Medical College,

These authors contribute equally to this work.

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*Corresponding

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Fenglin Lv, Chongqing Shapingba District 174 Shazheng Street, Chongqing, 400030,

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PR China; Fax: 86-23-65102507, E-mail: [email protected]

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ORCID:

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Fenglin Lv: 0000-0001-6321-4936

author:

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Running Head: Aluminum (oxy) Hydroxide Nanorods

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Word counts for the abstract: 199 words.

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Word counts for the text: 5986 words.

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The number of figures: 9 figures. 1

ACS Paragon Plus Environment

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Abstract

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Bacterial vaccines have been widely used to prevent infectious diseases, especially in veterinary medicine. Although there are many reports on bacterin adjuvants, only a few contain innovations in bacterin adjuvants. Taking this into consideration, in this study, we designed and synthesized a new aluminum (oxy) hydroxide (AlOOH) nanorod (Al-NR) with a diameter of 200 ± 80 nm and a length of 1.1 ± 0.6 μm. Using whole-Pseudomonas aeruginosa PAO1 as antigens, we showed that the bacterial antigens of P. aeruginosa PAO1 adsorbed on the Al-NRs induced a quick and stronger antigen-specific antibody response than those of the other control groups, especially in the early stage of immunization. Furthermore, the level of antigen-specific IgG was approximately 4-fold higher than that of the no adjuvant group and 2.5-fold higher than those of other adjuvant groups in the first week after the initial immunization. The potent adjuvant activity of the Al-NRs was attributed to the rapid presentation of antigen adsorbed on them by APCs. Additionally, Al-NRs induced a milder local inflammation than the other adjuvants. In short, we confirmed that Al-NRs, enhancing both humoral and cellular immune responses, are a potentially promising vaccine adjuvant delivery system for inhibiting the whole-pseudomonas aeruginosa infection.

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Keywords: bacterial vaccine, aluminum (oxy) hydroxide whole-Pseudomonas aeruginosa, rapid stimulation, immune responses

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nanorods,

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1. Introduction

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Bacteria are a major hazard in the infection of human and animal diseases.1 Pseudomonas aeruginosa induces hemorrhagic pneumonia and is one of the most serious bacterial infectious diseases affecting mink farming. In the densely bred regions such as Zhucheng and Weihai in Shandong, the incidence of the disease is approximately 20% and the mortality rate ranges from 10% to 50%, causing large economic losses for most farmers.2 In addition, P. aeruginosa also causes ventilation-associated pneumonia in humans with a mortality rate as high as 13.5%.3 Actinobacillus Pleuropeumoniae (APP) is a highly contagious, fatal respiratory infection Gram-negative pathogen in pigs. A variety of pigs are susceptible to this pathogen and infection with it can reach 82.2%.4 Currently, many bacterial infections are still not preventable with the available vaccines. To combat the spread of bacterial infection, an effective vaccine or adjuvant is urgently needed.

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The whole-bacterial vaccines are chosen as a vaccine candidate. One great advantage for the whole-bacterial development of vaccines is the simple fabrication and high efficiency against pathogen invasion. In addition, the inactivated whole-bacteria vaccines present a complex array of antigenic determinants to the immune system, avoiding the frequent alterations of some bacteria-associated virulence factors.5-7 Moreover, their cost is quite low, especially for veterinary vaccines, compared to the component vaccines, recombinant vaccine or DNA vaccines.

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To develop effective bacterial vaccines, vaccine adjuvants were investigated in addition to choosing the antigen. One research direction was to screen promising vaccine adjuvants. To date, many vaccine adjuvants have been studied, including aluminum salt minerals, oil emulsions, cytokines, liposomes,8-10 and various polymeric nanoparticles.11,12 All of these adjuvants have provided a big breakthrough, especially for nano-adjuvants. Compared to traditional adjuvants, nano-adjuvants can increase their half-life, minimize inflammation reaction at injection sites, and promote the delivery of immunomodulatory and immunostimulatory substances to antigen presenting cells (APCs).13 Another method is to change the physicochemical properties of the adjuvant. It has been demonstrated that the size,14 shape, crystallinity, hydroxyl content,15 and surface chemistry16,17 determine the activity of immunostimulation, of which, shape has already been considered a critical factor contributing to efficient antigen uptake. A previous study discovered that the morphologies of nano-aluminum adjuvants strongly affected the NLRP3 inflammasome activation and antigen-specific immune responses.15 Moreover, aluminum oxyhydroxide nanorods are more effective in activating NLRP3 3



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inflammasomes than nanoplate and nanopolyhedron. Another study reported that the stick-like aluminum (oxy) hydroxide nanoparticle had a high adjuvant activity in adsorbing and delivering antigens (OVA) into antigen-presenting cells, activated inflammasomes and stimulated OVA-specific antibody responses.18 However, the application is limited to the model antigen OVA, and no studies have yet been reported on the immune expression of rod-shaped aluminum nanoparticles combined with bacterial antigens,15,18 which has a realistic application value, especially in veterinary vaccines.

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Although the advantage of using rod-shaped nano-adjuvants is obvious, reverse adsorption occurs when they are combined with bacterial antigens due to the difference in size between the nanoparticles and the antigen. Based on the above considerations, herein, we report an improved rod-shaped nano-aluminum adjuvant that achieves a good combination of rod-shaped nanoparticles and bacterial antigens. We synthesized aluminum (oxy) hydroxide (AlOOH) nanorods (Al-NRs) using a bicontinuous reverse microemulsion (RM) technique containing NH4OH microemulsion and AlCl3 microemulsion. Next, the Al-NRs were used to adsorb P. aeruginosa PAO1 (the entire inactivated bacterial) to examine whether it has adjuvant activity against the whole bacterin of P. aeruginosa PAO1. Then, we comparatively investigated whether Al-NRs are more potent than other adjuvants (Freund’s, Al(OH)3 and AlPO4 gels) in boosting the immune response. Moreover, we studied the relationship of antigen-presenting cells to whole P. aeruginosa PAO1-adsorbed Al-NRs in boosting the immune response. In addition, we examined the antibody titers at different stages after immunization, the levels of IgG subgroups, and the potential of antigen-specific INF-γ and IL-4 secreting lymphocytes and inflammation. We demonstrated that the Al-NRs could rapidly stimulate an immune response and generate numerous antibodies of IgG and cytokines (IL-4, IFN-γ). More importantly, they could be more readily internalized and presented by antigen presenting cells. All these results demonstrate that the Al-NRs are a promising aluminum adjuvant against whole P. aeruginosa PAO1 infection via fully activated cellular and humoral immune responses. In addition, this provides a scientific and theoretical basis for the design of optimal alum-based adjuvants for bacterin vaccination.

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Materials and Methods

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Ethics statement

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All animal care and use protocols in this study were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of the People’s Republic of China. All animal experiments in this study were approved by the Animal Ethical and Experimental Committee of Chongqing University in accordance with their rules and regulations. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.

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Materials

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Ammonium hydroxide solution, glycerol, benzyldodecyldimethylammonium bromide and cyclohexane were purchased from MACKLIN®. Mouse 1 × lymphocyte separation medium, serum-free medium for ELISPOT, nylon net filters, a mouse IL-4 precoated ELISPOT kit and a mouse IFN-γ precoated ELISPOT kit were purchased from Dakewe Biotech Co. Ltd. RPMI medium 1640, sodium chloride (NaCl) and aluminum chloride (AlCl3) hexahydrate, ethanol anhydrous, phosphate-buffered saline (PBS) were purchased from Solarbio, and Pierce™. A BCA protein assay kit was purchased from Beyotime. Fetal bovine serum (FBS), horseradish peroxidase-labeled goat anti-mouse immunoglobulins (IgG, IgG1, IgG2a and IgG2b) and adjuvant of Freund’s were purchased from Sigma-Aldrich (St. Louis, MO, USA). Adjuvants of Al(OH)3 and AlPO4 were purchased from Pierce.

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Preparation of P. aeruginosa PAO1 antigens

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P. Aeruginosa international standard strain PAO1 was purchased from ATCC (Manassas, VA, USA). Bacterial PAO1 were cultured in LB medium, then washed three times in sterile PBS. After the last wash, bacterial were resuspended in PBS containing 4% (vol/vol) formalin and incubated overnight at 4 °C for 24 hours. Excess formalin was removed by three washes with PBS.19 Then bacterial were diluted to an appropriate cell concentration, as determined by spectrophotometry at 600 nm (OD600). The bacterial count was defined by determining the CFU from a gradient dilution count on LB solid plate. To ensure that there is no viable bacteria, 200 μL of the inactivated suspensions were added on LB solid plate and cultured at 37 °C. After one week of cultivation without bacterial growth, the formalin-inactivated bacterial were used as an antigen for the P. aeruginosa vaccines.

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Mice and cell lines

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Female BALB/c mice, 8 to 12 weeks of age, were purchased from the Animal Experiment Center of Chongqing Medical University (Chongqing, People’s Republic of China). Mice were matched for age and sex, and kept under specific pathogen-free (SPF) conditions.

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Mouse J774A.1 macrophage cells were from the American Type and Culture Collection (Manassas, VA) and grown in DMEM media supplemented with 10% fetal bovine serum (FBS), 100 U/mL - 100 µg/mL penicillin-streptomycin. Lymphocytes were prepared from the spleen of female BALB/c mice and cultured in RPMI-1640 containing 10% fetal bovine serum (FBS), 100 U/mL - 100 μg/mL penicillin-streptomycin.

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Synthesis of aluminum (oxy) hydroxide nanorods

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The synthesis of aluminum (oxy) hydroxide (AlOOH) nanorods (Al-NRs) was performed using previously described methods (the RM technique), with some modifications.18, 20-21 First, the emulsifier benzyldodecyldimethylammonium bromide and glycerol was dissolved in cyclohexane via sonication to obtain a homogeneous mixture. Thereafter, an aqueous solution of AlCl3 (1 M, 1 mL) and ammonium hydroxide (25-28%, 3 mL) was added into each mixture solution with magnetic stirring, followed by the addition of water and sonication in a water bath until the system became transparent. Two types of RM were prepared, one containing AlCl3 microemulsion and the other containing ammonium hydroxide microemulsion. Then, the growth process was prepared by mixing the two systems together using a syringe pump to add ammonium hydroxide solution at a rate of 1 mL/min to AlCl3 solutions to initiate the reaction between Al3+ and OH-. The reaction mixture was kept at 50°C under magnetic stirring for 2 h. Once the reaction was completed, the mixed solution was transferred into two volumes of 95% anhydrous ethanol and the resultant mixture was centrifuged at 12000 × g for 30 min. The precipitate was washed twice using 95% anhydrous ethanol, resuspended in distilled water, and stored at 4 °C for further use.

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Characterization of aluminum (oxy) hydroxide nanorods 6


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The morphology of the Al-NRs was measured via scanning electron microscopy (SEM, FEI, Nova 400, USA). Specimens were prepared by placing a small volume (5 µL) of the aqueous suspension of Al-NRs onto a titanium plate, and after drying, spraying gold (ten seconds, three times) for observation. Atomic force microscopy (AFM. IPC-208B), specimens were prepared by placing a small volume (10 µL) of the ethanol suspension of Al-NRs onto a conductive glass.

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X-ray powder diffraction (XRD) was used to characterize the particle component of Al-NRs. Specimens were prepared by grinding after drying. Energy dispersive spectrometer (EDS) was performed to determine the particle element types of the Al-NRs.

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Characterization of P. aeruginosa PAO1-adsorbed aluminum (oxy) hydroxide nanorods

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The adsorption of P. aeruginosa PAO1 on aluminum (oxy) hydroxide nanorods was carried out by mixing the Al-NRs with an equal volume of the inactivated P. aeruginosa PAO1 suspensions, followed by gently rotating at 4 °C for 12 h to assist conjugation. The morphology of the PAO1-adsorbed Al-NRs was evaluated using scanning electron microscope (SEM, FEI, Nova 400, USA). The hydrodynamic sizes and zeta potentials of the PAO1-adsorbed Al-NRs were also measured using Zetasizer Nano (Malvern Instruments, Co. Ltd,UK ) at 25 °C.

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Uptake of the P. aeruginosa PAO1-adsorbed aluminum (oxy) hydroxide nanorods by macrophages in culture

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Murine J774.1A macrophages were seeded into 24-well plates containing cover glass with a density of 2 × 105 per well, followed by overnight incubation at 37°C in 5% CO2. Then, the culture medium was replaced with a fresh one containing free CFSE-labeled P. aeruginosa PAO1, P. aeruginosa PAO1-adsorbed Al-NRs or Al(OH)3. After further incubation for 6 h, the cells were washed with PBS and the cell membranes were stained with DiO for 10 min. The cells were washed with PBS three times and then fixed with 4% paraformaldehyde for 15 min. The cells were washed with PBS. Subsequently, 0.2% Triton X-100 was added to enhance cell membrane 7



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permeability for 5 min, and the cells were washed with PBS twice more. Finally, the cell nuclei were stained with DAPI for 10 min, washed with PBS and observed with a CLSM (Leica, SD AF, Germany).

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Immunization of the mice

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Inactivated whole P. aeruginosa PAO1 was used as the antigen, and then antigen was emulsified 1:1 (volume ratio) in Al-NRs, Al(OH)3, AlPO4 and Freund’s. Al-NRs, Al(OH)3, AlPO4 were mixed and gently rotated at 4 °C for 12 h to assist conjugation, Freund’s adjuvant was emulsified by grinding on the ice. Female BALB/c mice (18-20 g) were immunized with 200 μL of the emulsion on days 0, 14, and 21. As controls, mice were injected with sterile PBS and inactivated P. aeruginosa PAO1 alone (Fig. 1). The dose of the PAO1 and aluminum was 6 × 108 CFU per mouse and 0.3 mg per mouse, respectively.

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Figure 1. Schematic illustration of the immune process. BALB/c mice were intramuscularly injected with vaccine on days 0, 14 and 21. In addition, mice were infected with a lethal dose of P. aeruginosa PAO1 on day 7 or 28. Blood was collected on days 7, 14, 21, 28, 35 and 56 from their tail veins for serum IgG and subtype investigation. On day 28, the mice of each group were exsanguinated for histological examination at the injection site and the mice were exsanguinated for cytokine detection on day 56.

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Infectious challenge with P. aeruginosa PAO1

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The P. aeruginosa PAO1 was grown in LB medium at 37°C for 6 h. Then, the bacteria were washed with PBS and resuspended to the appropriate number of CFU/mL. One week after the first and last immunization, the mice in all groups were challenged with a lethal dose (2 LD50) of P. aeruginosa PAO1 suspension through the tail vein, respectively. The survival rate in each group was monitored every 12 hours in a 7-day observation period postchallenge (Fig. 1). 8


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Enzyme-Linked Immunosorbent Assay (ELISA)

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To determine the serum antibody responses to P. aeruginosa PAO1 in immune mice, mice were bled on days 7, 14, 21, 28, 35 and 56 during immunization. Enzyme-linked immunosorbent assay (ELISA) was performed with plates coated with PAO1 bacteria lysed using ultrasonic treatment (400 ng per well) in 0.05 M carbonate buffer (pH = 9.5) overnight at 4°C as described previously.22,23 Diluted serum samples were used as the primary antibodies, and the secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG. Then, the optical density at 450 nm was measured. By comparison of serum subtypes among antigens in the immunized mice, the levels of the IgG1, IgG2a and IgG2b subtypes were also evaluated.

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ELISPOT assays

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For cytokines detection, the splenic cells of mice were enumerated via ELISPOT assays.24,25 Splenic cells were seeded with 1 × 105 cells/well in an IL-4 or IFN-γ ELISPOT plate and stimulated with P. aeruginosa PAO1 outer protein of the final concentration of 10 μg/mL. After incubation for 24 h, the cells were discarded and the plates were incubated with biotinylated anti-mouse IL-4 or IFN-γ for 1 h, followed by streptavidin-HRP for 1 h, AEC dyeing for 20 min and then spots were detected with a counting board (Dakewe Biotech Co., Ltd). Data are presented as the mean spot-forming units (SFU) per million cells ± SE.

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Histological analysis

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Seven days after the last injection, mice from each group were sacrificed for histological examination. The skin tissues at the injection sites were removed, fixed in 4% paraformaldehyde and then stained with hematoxylin-eosin (H&E) for observation of histological changes of inflammation and damage.14,21

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Statistical analysis 9



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Data are shown as the mean ± standard deviation (SD) or the mean ± standard error of the mean (SEM). Statistical analyses were performed using the two-tailed Student's t-test for two-group analysis or one-way ANOVA for multiple comparisons among different groups. SPSS 13.0 (SPSS Inc., USA) and GraphPad Prism 6.0 (GraphPad Software, Inc., USA) were used to perform the statistical analyses. P ≤ 0.05 was considered statistically significant.

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Results and discussion

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Synthesis and physicochemical characterization of aluminum (oxy) hydroxide nanorods

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The Al-NRs were synthesized via a well-established RM method with minor changes.18, 20-21 The RM system used for the synthesis of Al-NRs consisted of H2O, benzyldodecyldimethylammonium bromide and glycerol as the emulsifier, and cyclohexane as the oil phase; one containing NH4OH microemulsion and the other containing AlCl3 microemulsion. The reaction was initiated when these were mixed with each other. Al-NRs were synthesized in the aqueous phase by stirring during the preparation process.

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The ultimate morphologies and size of the as-synthesized Al-NRs were characterized with microscopic techniques. As shown in Figs. 2A and 2B, all SEM and AFM images show that the obtained products have a uniform rod morphology with a mean diameter of 200 ± 80 nm and a length of 1.1 ± 0.6 μm. The mean aspect ratio of Al-NRs was approximately 6.55. The surface topography observed via SEM was rough, which was consistently validated via AFM images showing slightly high or low regions of the Al-NRs. The rough surface of the Al-NRs was expected to improve the adsorption properties for bacteria as a rougher surface of substrates can have a higher bacterial attachment.26 In addition, an X-ray powder diffraction (XRD) analysis was conducted to further investigate the composition and crystal structure of the Al-NRs. As shown in Fig. 2C, the XRD patterns of the Al-NRs exhibit a typical boehmite structure and a small amount of bayerite, the typical peaks located at 2θ = 14, 28, 38, 49, 65 and 72.21 The elemental analysis via energy dispersive spectrometry (EDS) further demonstrates that the composition of the material was Al and O, the mass ratio of Al : O in the Al-NRs was approximately 5:6 and the atomic ratio of Al : 10


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O in the Al-NRs was approximately 1: 2 (Fig. 2D). Taken together, the XRD and EDS results confirmed that the Al-NRs are primarily amorphous AlOOH. These results demonstrated that the Al-NRs were successfully prepared.

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Figure 2. Characterization of the Al-NRs. (A) Representative SEM images of AlOOH nanorods (mag: 10000x). (B) AFM images of the AlOOH nanorods. (C) XRD patterns of the AlOOH nanorods. (D) EDS analyses of the AlOOH nanorods.

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Adsorption of P. aeruginosa PAO1 antigens on aluminum (oxy) hydroxide nanorods

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In order to evaluate the adsorption capacity of whole P. aeruginosa PAO1 onto Al-NRs, inactivated whole P. aeruginosa PAO1 was emulsified 1:1 (volume ratio) in Al-NRs, followed by gently rotating at 4 °C for 12 h to assist conjugation. Shown in Fig. 3A are the size distribution and zeta potentials of the Al-NRs and PAO1-adsorbed Al-NRs. The mean diameters of the Al-NRs and PAO1-adsorbed Al-NRs were 97 ± 50 nm and 220 ± 80 nm, respectively; The mean lengths were 1.0 ± 0.3 μm and 3.2 ± 0.8 μm, respectively; And their zeta potentials were 38 ± 1.5 and 12 ± 1.1, respectively. The size of Al-NRs increased after the adsorption of PAO1. In addition, after the adsorption of PAO1, the zeta potentials of the Al-NRs became less positive. Moreover, a representative SEM images clearly show the adsorption effect of whole P. aeruginosa PAO1 onto Al-NRs (Fig. 3B).

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The size distribution of the Al-NRs and PAO1-adsorbed Al-NRs had two distinguish peaks, which represent the transverse plane and longitudinal plane, respectively.27 Although this method may not accurately reflect the hydrodynamic sizes of the non-spherical materials, it can still demonstrate the degree of dispersion and the adsorption capacity.21 In addition, the changed zeta potentials still confirmed the the adsorption capacity of whole P. aeruginosa PAO1 onto Al-NRs. The zeta potentials of Al-NRs were positive, but it became less positive after the adsorption of PAO1, which may be due to the combination of PAO1 that covered the surface charge of the Al-NRs. Besides, the Al-NRs had a relatively larger surface area, and the effect of the surface area also have contributed to the adsorption capacity.14 Therefore, the Al-NRs may have a good adsorption capacity for whole P. aeruginosa PAO1.

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Figure 3. Physical characterization of Al-NRs after adsorption of P. aeruginosa PAO1 . (A) Particle sizes and zeta potentials of Al-NRs and PAO1-adsorbed Al-NRs. Data shown are mean ± SD (n = 3). (B) A representative SEM images of PAO1-adsorbed Al-NRs (mag: 30000x). Scale bar: 2 µm.

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Uptake of P. aeruginosa PAO1 antigens adsorbed aluminum (oxy) hydroxide nanorods by J774A.1 macrophages in culture

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The uptake, allocation and presentation of the P. aeruginosa PAO1 antigens-adsorbed Al-NRs at the cellular level were first investigated using CLSM on J774A.1 macrophages. It is well known that macrophages are one of the major antigen presenting cells (APCs) currently used in vaccine research. Herein, the macrophages were incubated with free PAO1 (no adjuvant) or PAO1-adsorbed Al-NRs and Al(OH)3 for six hours. The PAO1 internalization ratio is shown in Fig. 4A. Free PAO1 produced a minimum cell uptake at 6 h, whereas the PAO1-adsorbed Al-NRs group displayed a significantly higher uptake percentage than that of the no adjuvant control group (P = 0.0008) and the Al(OH)3 adjuvant control group (P = 0.0034), indicating that Al-NRs were more easily internalized by APCs. This view was confirmed by comparing the intracellular green fluorescence signal in cells 12


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incubated with adjuvant (Al-NRs and Al(OH)3) to that of the free PAO1 control group (Fig. 4B). A significantly strong green fluorescence signal was produced in cells with PAO1-adsorbed Al-NRs, whereas a low green fluorescence was observed in the PAO1-adsorbed Al(OH)3 group. Most of the fluorescence signals were extracellular, which could be explained by the well-established phagocytosis mechanism of nanoparticles.28 Meanwhile, the result is consistent with the previous finding that when the size of antigen-loaded nanoparticles is equivalent to that of the pathogen, they are more easily recognized and ingested by APCs.29,30 The Al-NRs that we prepared were comparable with the P. aeruginosa PAO1 antigen in length, therefore, the Al-NRs were more easily internalized and presented by antigen presenting cells.

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Figure 4. Cells uptake of P. aeruginosa PAO1 antigens-adsorbed aluminum (oxy) hydroxide nanorods and Al(OH)3. (A) The internalized percentage of free P. aeruginosa PAO1 (no adjuvant), P. aeruginosa PAO1-adsorbed Al-NRs (Al-NRs) and P. aeruginosa PAO1-adsorbed Al(OH)3 (Al(OH)3) by J774A.1 cells in culture (***, P < 0.001, and **, P < 0.01). Data shown are mean ± SD (n = 3). (B) 13



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Representative confocal laser scanning microscopy (CLSM) images of J774A.1 cells treated with free P. aeruginosa PAO1 (a), P. aeruginosa PAO1-adsorbed Al-NRs (b) and P. aeruginosa PAO1-adsorbed Al(OH)3 (c) for 6 h. Blue: cell nuclei; green: CFSE-labeled P. aeruginosa; red: cell membrane. Scale bar: 25 µm.

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Vaccination with aluminum (oxy) hydroxide nanorods induce earlier immune responses

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To determine the protective efficacy of the Al-NRs in vaccines against P. aeruginosa PAO1 strains, PAO1 adsorbed Al-NRs (v/v = 1:1) were s.c. injected into mice on days 0, 14 and 21. Free PAO1 (no adjuvant) and PAO1-adsorbed adjuvant (Al(OH)3, AlPO4 and Freund’s) were used as controls. Serum samples were collected on days 7, 14, 21, 28, 35 and 56 to measure antigen-specific antibody levels. As shown in Figs. 5A and 5B, the Al-NRs group induced significantly higher levels of antigen-specific IgG than the other adjuvants control groups in the first week after the initial immunization (P Al-NRs vs no adjuvant = 0.0013, P Al-NRs vs Al(OH)3 = 0.0170, P Al-NRs vs AlPO4 = 0.0054, P Al-NRs vs Freund’s = 0.0170); in the second week, the Al-NRs group still maintained a high level of antibody titer (P Al-NRs vs no adjuvant = 0.0005, P Al-NRs vs Al(OH)3 = 0.0321, P Al-NRs vs AlPO4 = 0.0054, P Al-NRs vs Freund’s = 0.0038); in the fifth week after immunization, the antibody titer of the Al-NRs group was higher than that of the no adjuvant control group (P = 0.0004), but there was no significant difference among the four adjuvant groups. Notably, the antibody titer in the Al-NRs group underwent almost no increase in the third week compared with the second week. During the remainder of the observation time, there was an overall upward trend, but there was no significant difference between each group.

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All these results suggested that Al-NRs can induce the generation of antigen-specific antibodies in the early stages of immunity and sustain a high antibody titer for a long time. The underlying mechanism may be due to that the antigen is a T cell-independent antigen, so it could directly be presented by B cells without the need of T cell presentation process. It may be also due to the properties of the Al-NRs, which have a diameter in nanometers and a length in microns, and therefore have not only the small size effect of the nanometer but are also consistent with the size of the antigen in length, and a rod shape is more easily taken up by APC, that is: the burst effect after the initial immunization. This effect was also observed by Raghuvanshi,31 who found that immunization of mice with tetanus toxoid co-adsorption of nano-PLGA adjuvant and aluminum adjuvant can produce higher antibody titers on 14


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

the 15th day of immunization and speculated that this result is due to the “synergistic effect” of small size nanoparticles and aluminum adjuvant. The burst effect is the rapid release of the “antigen depot”. AndrzejMyc et al. dissolved influenza A virus in a nanoemulsion and detected INF-γ in the serum and spleen lymphocytes on the 4th day after nasal immunization of mice, which may be attributed to the rapid presentation of antigen by APCs.32 Evidence of mucosal macrophage internalization of nanoemulsion has been observed in lung research.33 Nano-adjuvants are more effective in rapidly releasing soluble mediators, such as cytokines, chemokines and immunomodulatory molecules.34 However, the reaction time was limited and the “immune stimulating effect” that followed was not immediately effective. There is a time difference where the burst effect of the nano-adjuvant quick release occurs and disappears within a short time, while the latter still do not immediately take effect, resulting in the fact that the increase in antibody titer was not significant in the third week. Conventional adjuvants do not have the burst effect and the reaction is mild and slow, so the antibody titer is gradually increased.

16 17 18 19 20 21 22 23 24 25

Figure 5. Total IgG levels in the serum samples of mice immunized with the free P. aeruginosa PAO1 (no adjuvant) and P. aeruginosa PAO1-adsorbed adjuvants (Al-NRs, Al(OH)3, AlPO4 and Freund's). (A) Comparison of the total IgG level trend at different times in the immunized mice (n = 5). Serum was obtained on days 7, 14, 21, 28, 35 and 56. The levels of total IgG were expressed as the means of log2 titers. (B) Serum IgG titer on days 7, 14 and 35. The levels of total IgG were expressed as the means of log2 titers. Multiple comparisons among different groups were analyzed using one-way ANOVA (***, P < 0.001, **, P < 0.01 *, P < 0.05, ns = no significance). Data are shown as the mean ± SEM.

26 27 28

Vaccination with aluminum (oxy) hydroxide nanorods elicits a comprehensive Th1 and Th2 responses 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

To further investigate the Al-NRs adsorbed PAO1 vaccination-mediated protection, the type of antibody response (IgG1, IgG2a and IgG2b) in the immunized mice serum was evaluated. As shown in Fig. 6, Al-NRs induced higher levels of antigen-specific IgG1, IgG2a and IgG2b subtypes than the no adjuvant control group (P IgG1 = 0.0006, P IgG2a < 0.0001 and P IgG2b < 0.0001). Furthermore, the IgG2a subtypes induced by each experimental group were slightly higher than that of IgG1 and IgG2b. Among them, Al-NRs-induced IgG2a antibody levels were higher than that of IgG1 (P = 0.0241). Moreover, The mice immunized with adjuvants (Al-NRs, Al(OH)3, AlPO4 and Freund’s) all induced significantly higher levels of IgG1, IgG2a and IgG2b than those of the free PAO1 antigen control group (IgG1, P Al-NRs and Freund’s < 0.01, P Al(OH)3 and AlPO4 < 0.05, P IgG2a < 0.01, P IgG2b < 0.001). IgG1 and IgG2a are markers of the Th2 and Th1 type immune response, respectively. These results suggested that Al-NRs, Al(OH)3, AlPO4 and Freund's adjuvant groups can induce the system Th1 and Th2 immune response. Notably, the IgG2b levels are relatively high in the adjuvant group, which should be attributed to the stronger cellular immunity produced. Th1-type immune responses are closely related to the production of subtypes of IgG2a, IgG2b and IgG3.35 In addition, previous data have shown that IgG2b, compared with IgG1, can led to different degrees of activation for dendritic cells (DC) through Fc receptor signaling, resulting in the enhancement of the immune response.36 In summary, cellular and humoral facets of the immune responses can be simultaneously activated.

23 24 25 26 27 28

Figure 6. Comparison of serum IgG1, IgG2a and IgG2b subtypes among antigens with different adjuvants in the immunized mice (n = 5). Serum was obtained at two weeks after the final immunization, and the levels of IgG1, IgG2a and IgG2b were expressed as the mean of log2 titers. Multiple comparisons among different groups were analyzed using one-way ANOVA (*, P < 0.05, ns = no significance). The results 16


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1

are expressed as the mean ± SEM.

2 3 4

Vaccination with aluminum (oxy) hydroxide nanorods primarily induces Th1 type immune response

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

The cellular and humoral immune response is an important indicator of the body's immune capacity. Consequently, ELISPOT technology was used to assess the capability for producing antigen-specific INF-γ and IL-4 secreting lymphocytes of mouse splenocytes after the last blood draw (8th week). As shown in Figs. 7A and B, the splenocytes from mice immunized with adjuvant (Al-NRs, Al(OH)3, AlPO4 and Freund’s) produced significantly more IFN-γ and IL-4 spot numbers than did the free PAO1 control group. Notably, there were more spots observed in the IFN-γ treated group than in the IL-4 group (P no adjuvant = 0.0438, P Al-NRs = 0.0112, P Al(OH)3 < 0.0001, P AlPO4 = 0.0082, P Freund’s = 0.0221). Because IFN-γ and IL-4 are secreted by Th1 and Th2 cells, respectively, and represent different antigen presentation pathways, Th1 cells mediate cellular immune responses primarily by secreting Th1 type cytokines (IFN-γ), whereas Th2 cells mediate the immune response primarily through the secretion of Th2 type cytokines (IL-4). Therefore, the significantly higher secretion of antigen-specific INF-γ than IL-4 suggests that a Th1-biased immune response was induced in mice immunized with Al-NRs, Al(OH)3, AlPO4 and Freund's adjuvant, which was consistent with the result of IgG subtypes by serum. In addition, the immune response elicited by these adjuvant formulations was Al(OH)3 > Al-NRs ≥ Freund's > AlPO4 (Fig. 7B), which was consistent with the trend of serum titer in the 8th week. These results suggest that the Al-NRs can elicit cellular and humoral immune responses and this ability is directly related to the time of immunization. This result may be partially attributed to the burst effect of nano-adjuvants, which leads to a lower immune effect than that of conventional aluminum adjuvants in the late stage of immunization, whereas it only led to a limited reduction compared to the other groups.32

17



1 2 3 4 5 6 7 8 9 10

Figure 7. The ELISOPT assay for cytokine secreted spleen cells. (A) The spot numbers produced by spleen cells in the mouse immunized with free P. aeruginosa PAO1 (no adjuvant) and P. aeruginosa PAO1-adsorbed adjuvant (Al-NRs, Al(OH)3, AlPO4 and Freund's, respectively). Multiple comparisons among different groups were analyzed using one-way ANOVA and each value represents the mean of three replicates (*, P < 0.05, **, P < 0.01). (B) The secretion of IFN-γ and IL-4 by spleen cells treated with free P. aeruginosa PAO1 (no adjuvant), P. aeruginosa PAO1-adsorbed adjuvant groups (Al-NRs, Al(OH)3, AlPO4 and Freund's, respectively). The number represents the spots number.

11 12 13

Vaccination with aluminum (oxy) hydroxide nanorods protects mice against P. aeruginosa PAO1 infection

14 15 16

To understand whether Al-NRs can provide an early and broad protection, immunized mice in each group were challenged with a lethal dose of PAO1 on day 7 18


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1 2 3 4 5 6 7 8 9 10 11 12

and 28. As shown in Fig. 8A, the survival rate in all experimental group mice was lower on day 7, whereas the Al-NRs group had a higher survival rate than the other control group (P Al-NRs vs no adjuvant = 0.0003, P Al-NRs vs Al(OH)3 = 0.0444, P Al-NRs vs AlPO4 = 0.0031, P Al-NRs vs Freund’s = 0.0460). Al-NRs could protect 50% of the mice from the P. aeruginosa PAO1 challenge when compared with the PBS and no adjuvant controls group. The common adjuvants of Al(OH)3, AlPO4 and Freund’s only provide 10% to 20% protection in the early stages of immunization. In addition, the four adjuvant groups showed a high protection efficacy on day 28 (Fig. 8B). Even though all the infected mice became weak 12 h after the infection, all vaccine groups had a significantly higher recover than the PBS group. Until the 7th day after immunization, the immunized with adjuvant groups still maintained an 80% to 100% survival rate. However, there were no significant differences among the four adjuvant groups.

13 14 15 16 17 18 19 20 21 22 23

In this study, the Al-NRs group had a higher survival rate (50%) than the other adjuvants control groups (10% - 20%) on day 7, suggesting that vaccination with Al-NRs could provide an earlier protection. This protection was primarily attributed to antibodies induced in the early stages of immunization. Furthermore, the survival rates in all adjuvant groups were significantly greater with the increase in antibodies on day 28. These results revealed that antibody is critical in protecting mice from PAO1 infections, which is consistent with the reports that antibodies are critical for protection against P. aeruginosa infection.37 Another study reported that passive immunization with pcAb or MAbs has shown potential in protecting against P. aeruginosa infection.38

24 25

Figure 8. Survival rates of immunized mice (n = 10) challenged with P. aeruginosa

26

strains PAO1. BALB/c mice (n = 10) were immunized with P. aeruginosa PAO1 plus

27

different adjuvants (Al-NRs, Al(OH)3, AlPO4 and Freund’s). The control groups

28

included the PBS groups and the no adjuvant group. (A) One week after the first

29

immunization, the immunized mice and control mice (n = 10) were challenged with a 19



1

lethal dose of PAO1. The survival rate in each group was monitored every 12 hours in

2

a 7-day observation period postchallenge. (B) One week after the final immunization,

3

all mice (n = 10) were challenged with PAO1 (6.0 × 108 CFU/mouse). Survival was

4

monitored for one week. The P-value was compared with that of the other adjuvant

5

control group using the Mantel-Cox log-rank test (ns = no significance).

6 7 8

Vaccination with aluminum (oxy) hydroxide nanorods induced milder local inflammation reactions

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

The use of adjuvants in human vaccines is always associated with adverse reactions,39-41 which are often as classified local reactions such as erythema, subcutaneous nodule, contact allergies and granulomatous inflammation,42 or under autoimmune (or autoinﬂammatory) syndrome induced by adjuvants.43 Therefore, to ensure the practical feasibility of Al-NRs for potential vaccine applications, the degree of inflammation and in vivo toxicity of the Al-NRs was assessed by collecting the skin samples at the last injection site for histological examination. As expected, there was almost no or little inflammation in the PBS and no adjuvant controls group (Fig. 9A-B). The common adjuvants of Al(OH)3, AlPO4 and Freund’s group had varying degrees of subcutaneous nodules, granulomas and abscesses, of which Freund's adjuvant group was much more severe and the inflammation of the leg injection site was visible to the eye (Fig. 9D-F). And Al-NRs group had a slight local inflammations (Fig. 9C). However, the inflammation induced by the Al-NRs was significantly lower than the common adjuvants group. Taken together, we confirmed that the Al-NRs adjuvant could efficiently reduce local inflammation of the injection site. Further meticulous studies are still needed to evaluate the long-term adverse reactions in vivo.

27 20


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1

Figure 9. Representative H&E histograms of the skin samples in the injection sites.

2

BALB/c mice were immunized with free P. aeruginosa PAO1 (no adjuvant), P.

3

aeruginosa PAO1-adsorbed adjuvants (Al-NRs, Al(OH)3, AlPO4 and Freund’s) on

4

day 0, 14 and 21. As controls, mice were injected with sterile PBS alone. On day 28,

5

mice were euthanized and the skin samples in the injection sites were collected for

6

H&E staining (scale bar, 500 µm, magnification = 200x).

7 8

Conclusions

9 10 11 12 13 14 15 16 17 18

In this study, we successfully designed and fabricated aluminum (oxy) hydroxide nanorods with a diameter of 200 ± 80 nm and a length of 1.1 ± 0.6 μm. For the first time, we applied Al-NRs adjuvant to adsorb whole P. aeruginosa to immune mice. Compared to the commercial Al(OH)3, AlPO4 and Freund’s adjuvants, Al-NRs adjuvants are more effective in adsorbing and presenting the whole bacterial antigens into antigen-presenting cells, reducing inflammation at the injection site and enhancing antigen-specific immune responses, especially in the early stage of immunization. Therefore, the rod-shaped (oxy) hydroxide nanorods in this study were proven to be a promising bacteria vaccine adjuvant.

19 20

Acknowledgments

21 22 23 24 25 26 27 28

The authors would like to thank College of Bioengineering, “111 Project” Laboratory of Biomechanics & Tissue Repair Engineering, Key Laboratory of Biorheological Science and Technology, Chongqing University in providing fund and support toward this work. The authors are thankful to Prof. Xueheng Yang, Physics College of Chongqing University for instrumental and technical support. The authors also thank Prof. Fenglin Lv and Dr. Feng Yang for valuable contributions to the revision of this paper.

29 30

Author Contributions Statement

31 32 33 34

FL, FY and YC designed the research; YC and JY conducted the experiments, analyzed the data, wrote the main manuscript text and prepared the figures; LH and YH helped to conduct the experiments and supervised the project. FL and FY helped 21



1 2

with the discussion of the results and manuscript refinement. All authors reviewed the manuscript.

3 4

Conflict of Interest Statement

5 6

The authors declare that the research has no conflict of interest.

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by Adjuvants. Reumatismo. 2011, 63, 63-66.

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Aluminum (oxy) Hydroxide Nanorods Activate an Early Immune

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