Intranasal Vaccination against HIV-1 with Adenoviral Vector-Based

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Intranasal vaccination against HIV-1 with adenoviral vector-based nanocomplex using synthetic TLR-4 agonist peptide as adjuvant Man Li, Yuhong Jiang, Tao Gong, Zhirong Zhang, and Xun Sun Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00802 • Publication Date (Web): 29 Jan 2016 Downloaded from http://pubs.acs.org on February 2, 2016

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Molecular Pharmaceutics

Intranasal vaccination against HIV-1 with adenoviral vector-based nanocomplex using synthetic TLR-4 agonist peptide as adjuvant Man Li#, Yuhong Jiang#, Tao Gong, Zhirong Zhang ,Xun Sun*

Key Laboratory of Drug Targeting, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, 610041, People’s Republic of China

#The authors contributed equally to this work. *Corresponding author: Xun Sun Key Laboratory of Drug Targeting, Ministry of Education, Sichuan University, No. 17. Section 3. Southern Renmin Road, Chengdu 610041, People’s Republic of China. Tel: 86-28-8550 2037; Fax:86-28-85501615 E-mail: [email protected] (X.Sun)

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Abstract

Recombinant type 5 adenovirus(rAd5)vaccines hold the promise to prevent HIV-1 infections. Intranasal vaccination not only stimulates systemic immunity, but also elicits mucosal immunity that provides first defense for mucosally transmitted diseases like HIV-1. Adjuvants such as TLR agonists are usually co-delivered with antigens to enhance the immunogenicity of vaccines. Here, we present a rAd5 vaccine delivery system using DEG-PEI as the carrier. Adenovirus encoding HIVgag was used as antigen, and was complexed with DEG-PEI polymer via electrostatic interaction. A novel synthetic TLR-4 agonist, RS09, was either chemically linked with DEG-PEI (DP-RS09) or physically mixed with it(DP/RS09) to enhance the immunogenticity of rAd5 vaccine. After intranasal immunization, the systemic antigen-specific immune responses and cytotoxicity T lymphocytes responses induced


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by DP-RS09-rAd5 and DP/RS09-rAd5were analyzed. The mucosal secretive IgA level was detected in both nasal and vaginal washes to determine the mucosal immunity. Furthermore, cytokine productions on RAW264.7 cells were tested after pre-incubation with TLR-4 pathway inhibitors. The results indicated that DEG-PEI could facilitate the intranasal delivery of rAd5 vaccine. Both chemically linked (DPRS09) and physically mixed RS09 (DP/RS09) could further enhance the mucosal immunity of rAd5 vaccine via TLR-4 pathway. This RS09 adjuvanted DEG-PEI polymer represents a potential intranasal vaccine delivery system and may have a wider application for other viral vectors.

Key words: adenovirus vaccine, TLR4, RS09, intranasal delivery



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1. Introduction

Human immunodeficiency virus (HIV) is perhaps the most dreadful pandemic since it was first discovered. Facing this threat, vaccination offers a promising and feasible approach to prevent the spread of this disease1-3. Great efforts have been made on the design and development of anti-HIV vaccines4. Since the first HIV vaccine clinical trial in 1987, different vaccination concepts have been explored including DNA, subunit protein and viral vector vaccines5-7. Among them, recombinant adenoviral vector has been the most immunogenic vaccine candidate8. During the past few years, two vaccine concepts based on recombinant adenovirus serotype-5 (rAd5) were evaluated in clinical trials. The Step study showed that rAd5gag/pol/nef failed to protect against infections, and pre-existing neutralizing antibodies may play a role for this failure5, 9. Alternative vaccination with adenovirus originated from other species (i.e. chimpanzee-derived vectors) may circumvent the neutralizing antibodies prevalent in humans10. Although currently no evidence of increased risk of HIV infection for other Ad vectors were shown, the Ad serotype cross reactivity may impair the specific immune response of other Ad vectors due to the fact that most human and primate Ad species share highly conserved hexon regions11-13. Therefore, the current study aimed to improver Ad5-based vaccination


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through pharmaceutical technologies, such as the addition of adjuvants or change of administration route for HIV vaccination.

Traditional vaccine delivery routes include intravenous14, subcutaneous15, or intradermal delivery16. Recently, non-invasive routes, such as intranasal route, has been successfully applied in adenovirus vaccine studies17-19, which induced both systemic and mucosal immune responses and provided the first defense against HIV-1 in the port of virus entry. Mucosal administered rAd5 vaccines could bypass the preexisting immunity against adenovirus thus resulting in protection against speciesadapted virus challenge18, 20. However, the immune responses induced by intranasal vaccines are relatively lower than intramuscular vaccines, partly owing to the mucociliary clearance and low antigen uptake efficiency. To enhance efficiency of intranasal vaccination, functional materials such as cationic polymers have been applied in the delivery of rAd5 vaccines. Low molecular weight chitosan and its derivatives showed excellent mucoadhesivity and gene transduction ability21-23. Polyethyleneimine (molecular weight 25k, PEI 25k) was capable of enhancing the immunogenicity of vaccines24, however the high toxicity hindered its application in mucosal routes. In our previous work, we synthesized a serious of PEI derivatives by crosslinking low molecular weight PEI to reduce the toxicity of cationic PEI. DEGPEI obtained by conjugating diethylene glycol (DEG) and PEI 2k proved an efficient



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carrier for rAd5. This cationic polymer complexed with rAd5 via electrostatic interaction, and facilitated the transduction of rAd5 in a coxsackievirus and adenovirus receptor (CAR) independent manner. Moreover, DEG and PEI 2k are crosslinked via ester/amide bonds and can be degraded under physiological conditions.

In addition to proper delivery systems, incorporation of appropriate adjuvants has been proven critical in enhancing the magnitude of immune response. The adjuvants provide the first signals to initiate the immune responses. In the innate immune system, antigen presenting cells express pattern recognition receptors such as Toll-like receptors (TLR)25. Triggering via TLR stimulates the production of proinflammatory cytokines/chemokines that increase the magnitude of immune responses and eliminate the pathogen. Moreover, this innate immune response also supports the subsequent development of adaptive immunity, and further enhances the induction of vaccine-specific responses26, 27. Thus, co-delivery of rAd5 vaccine and TLR ligands may lead to an improved immune response against the encoded antigen of rAd5 vaccine. To date, adjuvants which act as agonists to TLR-2, TLR-3, TLR-4, TLR-5, TLR7/8, and TLR-9 have been wells studied26, and one of the TLR-4 agonists, monophosphoryl lipid A (MPLA), has been approved by FDA. Although many of these agonists are quite effective, their bacterial nature may arise safety concerns. To address this issue, the use of synthetic peptides as TLR agonists seems a rational way.


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Different synthetic TLR-4 agonists have been developed with high specificity, and applied in different vaccination regimens including intranasal vaccination, exhibiting less side effects and better adjuvanticity28. Arulkumaran Shanmugam et al. have successfully developed a peptide named RS09 (Ala-Pro-Pro-His-Ala-Leu-Ser) showing great potential as vaccine adjuvant by interacting with TLR-4 and elevating the magnitude of immune responses29. Moreover, this synthetic adjuvant can be employed with a wide range of antigens, including proteins, peptides and viruses. Based on these, RS09 may exert potent adjuvanticity in a rAd5 vaccine delivery system.

Here, we synthesized a polymer containing blocks of DEG and PEI 2k and explored its efficacy in intranasal rAd5 vaccine delivery. Together, a TLR-4 agonist peptide RS09 was used as adjuvant to improve the immune response. The chemical conjugation form (DEG-PEI-RS09, DP-RS09) and mixture form of DEG-PEI and RS09 (DEG-PEI/RS09, DP/RS09) were complexed with rAd5. The morphology and physical characteristics of different complexes, in vitro cell uptake and transduction efficiency, as well as in vivo immune responses were analyzed and compared after intranasal administration in BALB/c mice.

2. Materials and methods



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2.1. Cell culture and animals

293T cells, Madin-Darby cane kidney (MDCK) cells, murine dendritic cells (DC2.4) and murine macrophage RAW264.7 cells were obtained form Chinese Academy of Science and maintained in Dulbecco’s modified Eagle medium (DMEM) or 1640 medium (Hyclone, NY, USA) supplemented with 10% fetal bovine serum (Hyclone, NY,USA), 100 units/ml penicillin and 100 µg/ ml streptomycin.

BALB/c mice (6 to 8 weeks old) were purchased from West China Experimental Animal Center of Sichuan University (Chengdu, China) and then housed in a specific pathogen-free and temperature-controlled facility. All experiments were conducted according to China’s Animal Welfare Legislation and the Institutional Animal Care and Use Guidelines of Sichuan University.

2.2. Synthesis of DEG-PEI and DEG-PEI-RS09

DEG-PEI polymer was synthesized by our group as previously reported30. Briefly, 0.053 g diethyleneglycol (DEG) was dried and dissolved in 25 mlanhydrous ethylene dichloride, then 302 mg nitrophenylchloroformate (NPC) was added, followed by adding 208 µl triethylamine dropwise under continuous stirring at room temperature. Six hours later, the reaction solution was extracted by saturated sodium chloride solution and the organic phase was collected. Subsequently, the product was


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dried and added to 20 ml ethylene dichloride solution with 2.0 g PEI 2k. After a further 16 h stirring at room temperature, the solvent was removed by rotary evaporation and the residue was dialyzed for 7 days in a dialysis bag (3500 Da cutoff) against ddH2O.

To synthesis DEG-PEI-RS09, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) was used as cross-linker. 40 mg DEG-PEI was dissolved in 3 ml PBS-EDTA. After adding 7.5 mg SPDP, the reaction was stirred for 45 min in room temperature, followed by adding 16 mg RS09-Cys under continuous stirring over night. Then the product was purified by using PD-10 desalting column. Finally, the product was obtained after lyophilization.

2.3. Recombinant adenovirus vectors

The E1/E3-deleted adenovirus serotype 5 vector encoding HIV-1 gag p24 protein (kindly provided by Prof. Hildegund C.J. Ertl, Wistar Institute of Anatomy and Biology, Philadelphia, PA, USA) was amplified in 293 T cells and purified by CsCl density gradient centrifugation. The virus particles number of purified adenoviral vector was determined by ultraviolet spectrophotometry.

2.4. Preparation of rAd5 nanocomplex



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Nanocomplexwas assembled by electrostatic interaction. The rAd5 particles were diluted in 5% glucose solution to a final dosage of 2 × 109 viral particles (vp, ~1.2 × 107 plague forming unit, pfu). Then, the polymers (DP, DP-RS09 or mixture of DP and RS09) diluted in 5% glucose solution were mixed with rAd5 (equal volume), followed by vortex for 30s and incubation for 30 min at room temperature for the complexes to form.

2.5. Characterization of rAd5 nanocomplexes

Photon correlation spectroscopy (Zetasizer Nano ZS90, Malvern, UK) were used to determine the mean particle size, zeta potential and polydispersity index were of at 25°C. Samples were diluted to a final volume of 1 ml in 5% glucose solution. Measurements were taken at a fixed angle of 90°and each sample was analyzed in triplicate.

To analyze the morphology of DP-rAd5, DP-RS09-rAd5 and DP/RS09-rAd5 nanocomplexes, samples were freshly prepared, loaded on a copper grid and incubated for 5 min. After staining with 1% (w/v) aqueous uranyl acetate for 2 min, the samples were air-dried after removing the dye and observed using transmission electron microscopy (TecnaiG2F-20, FEI, Holland).

2.6. In vitro transduction efficiency


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To compare whether the addition of RS09 peptide could enhance the transduction efficiency of DP-rAd5 complex, the rAd5 expressing lacZ was used as reporter system. DC 2.4cells or MDCK cells were seeded at a density of 1.5 × 105 cells/well in 24-well plates one day before experiments. Nanocomplexes were formed with rAd5-lacZ and DP, DP-RS09 or mixture of DP and RS09. In our preliminary experiments, the optimal dosage of DP and rAd5 were 2 × 106 pfu (a multiplicity of infection, MOI of 20, ~3 × 108 vp) rAd5 and 500 ng DP (at a concentration of 1.25 µg/ml) per well, respectively. To analyze the effects of RS09 on the transduction, the dosage of RS09 in DP/RS09-rAd5 complex and DP-RS09 in DP-RS09-rAd5 complex were set as 500 (1.25 µg/ml), 1000 (2.5 µg/ml), 1500 (3.75 µg/ml) or 2000 ng (5 µg/ml). After 4 h of infection, adenovirus complexes were replaced with complete medium and cells incubated a further 48 h. The target gene expression of rAd5-lacZ was quantified using the β-galactosidase (β-gal) enzyme assay system according to the manufacturer’s instructions (Beyotime, China). The total protein content per well was determined using the BCA assay (Pierce, Thermo Fisher, USA). The transduction efficiency was defined as the units of β-gal activity per mg total protein. Each sample was tested in triplicate.

2.7. In vitro uptake assay



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To detect the cell uptake efficiency after addition of RS09 peptide, rAd5-HIVgag was labeled with Alexa Fluor®488 dye and purified by dialysis. DC 2.4 cells were seeded in 6-well plates at a density of 6 × 105/well. Nanocomplexes were formed with Alexa Fluor®488-labeled rAd5-HIVgag with DP, DP-RS09 or DP/RS09 as described in transduction experiment. Cells were incubated 1 h at 37°C. Naked Alexa Fluro®488 labeled rAd5-HIVgag was used as negative control. Cells were then digested and analyzed by flow cytometry using a FC500 flow cytometry (Beckman Coulter, CA, USA).

2.8. Cytotoxicity

The cytotoxicity of RS09 and DP-RS09 were analyzed on DC 2.4 cells and MDCK cells using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method. The dosage of DP and rAd5 were1.25 µg/ml and 7.5 × 107 vp (5 × 105 pfu), respectively, as determined in the tranduction assay. The dosage of RS09 and DP-RS09 were 1.25, 2.5, 3.75, 7.5 µg/ml. One day before experiment, DC 2.4 cells or MDCK cells were seeded in 96-well plate at a density of 1 × 105 cells/well. After addition of complexes and incubation for 24 hours, MTT (5 mg/ml, 20 µl) was added and incubated for 4 h at 37°C, followed by replacing the media with 150 µl dimethysulfoxide (DMSO) to dissolve the formazan crystals. The absorbance at 570


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nm was measured using a Varioskan Flash reader (Thermo Fisher Scientific, Waltham, MA). OD570 readings were normalized to untreated cells for comparison.

2.9. Animal immunization

Female BALB/c mice were randomized into 6 groups (n = 5). Mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg) and inoculated intranasally with 2 × 109 viral particles (~1.2 × 107 pfu) of rAd5-HIVgag, either in naked form or complexed with DP, DP/RS09 and DP-RS09 in 20 µl volume. The dose of DP. RS09 and DP-RS09 were 5 µg, 15 µg and 15 µg, respectively. Mice were maintained in an upright position for at least 30 min to ensure uniform dosing and avoid swallowing. Mice intramuscularly injected with rAd5 were set as the positive control. Two weeks later, mice were sacrificed by neck dislocation under anesthesia for immune responses analysis.

2.10. Cytotoxic T lymphocyte response (CTL) assay

The cytotoxicity of antigen-specific CD8+ T cells was examined by in vivo CTL assay. Briefly, splenocytes were isolated from naïve mice, red blood cells were lysed, and the cell suspension was divided equally into two aliquots. One aliquot was incubated with 2 µM synthetic HIVgag peptide (H-2Kd-restricted peptide, AMQMLKETI, p24 197-205), and the other was incubated with medium only. After



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incubation for 2h at 37°C, these two aliquots were stained with high Carboxyfluoresceinsuccinimidyl ester (CFSE, Invitrogen, CA, USA) concentration (4 µM) and low CFSE concentration (0.4 µM) for 5 min at 37°C, respectively. The staining process was then quenched by addition of FBS to a final concentration of 20% (v/v). The two aliquots (1 × 107 cells/100 µl) were mixed equally, and injected to naïve (control) or immunized mice via tail vein (2 × 107 cells/mouse). After 18 h, spleen mononuclear cells from recipient mice were prepared and analyzed by flow cytometry. The percentage of specific lysis was calculated as follows:   ratio of CFSE low / CFSE high cells re cov ered from naive mice   Specific Lysis ( % ) = 100 × 1 −   low high   ratio of CFSE / CFSE cells re cov ered from immunized mice  

2.11. Intracellular cytokine staining

Splenocytes were isolated and prepared as described in CTL assay. After washing, the splenocytes suspension was cultured in RPMI 1640 medium supplemented with 10% FBS and 2-mercaptoethanol with 2 µg/ml HIV gag peptide. One hour later, each sample was added with brefeldin A (eBioscience, San Diego, CA, USA) and incubated for another 5 hours at 37°C. After washing, cells were incubated with 1:100 dilution of anti-mouse CD8a-flourescein isothiocyanate (FITC) or anti-mouse CD4-FITC (eBioscience, CA, USA) at 4°C for 30 minutes. Then cells were washed, fixed, and permeabilized with Fixation and Permeabilization buffers


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(eBioscience). After centrifugation, cells were incubated in permeabilization buffer with a 1:100 dilution of anti-mouse IFN-γ-PE or anti-mouse IL-4-polyethyleneimine (PE) in the dark for 20 min, respectively (eBioscience). After washing, cells were diluted in PBS with 1% BSA and were analyzed by two-color flow cytometry (Beckman Coulter, CA, USA). CD8+/IFN-γ+ and CD4+/IL-4+ double positive percent were analyzed using the Kaluza software (Beckman Coulter, CA, USA).

2.12. Serum and mucosal antibody response

ELISA was used to detect the HIV gag-specific serum antibodies (IgG total, IgG1 and IgG2a) and mucosal wash (nasal and vaginal) sIgA antibodies. Polystyrene 96-well plates (Corning 3590, USA) were coated overnight at 4°C with 100 µl HIV gag p24 protein (1 µg/ml) per well. After washing with PBS plus 0.2% Tween-20 (PBST), the plates were incubated with 100µltwo-fold serially diluted serum samples (1:8 to 1:2048 for IgG, IgG1 and IgG2a) for 2 h. Then plates were washed and incubated with 100 µl HRP-conjugated anti-mouse IgG (1:10000 dilution for IgG total, 1:5000 for IgG1 and IgG2a) and IgA (1:5000 dilution) antibodies for 1 h. After washing, TMB substrate (BD PharMingen, CA, USA) was added and the reaction was stopped with 2M H2SO4 after incubating for 30 min in dark. The optical density was measured at 450 nm using a Microplate Reader (VarioSkan; Thermo Fisher Scientific, MA, USA).



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2.13. The TLR-4 agonist activity

To analyze the TLR agonist property of DP-RS09 and mixture of DP and RS09, a TLR-4 inhibitor TAK-242 was used to block the TLR-4 signaling. Murine macrophage RAW264.7, which expresses TLR-4 was seeded in 96-well plate at a density of 1 × 105 cells/well. The stock solution of TAK-242 was dissolved in N,Ndimethylformamide at a concentration of 138 mM. Then TAK-242 was diluted with cell culture media to a working concentration of 100 nM. Half of the cells were incubated with TAK-242 for 1h, after which cells were washed with PBS and incubated with DP-RS09-rAd5, DP/RS09-rAd5, RS09-rAd5 complexes or rAd5 alone. Polymers without rAd5 (DP-RS09, DP/RS09 or RS09) were used as controls. The other half cells were incubated with complexes or rAd5 without pre-incubation of TAK-242. The dosage of rAd5 and RS09 were 1×107 vp (~1.5× 109 pfu)and 1 ng/well. For DP-RS09, the dosage was calculated as the ratio of conjugated RS09 in the polymer. After incubation, the cell culture media were collected and subjected to TNF-α (6 h) and NO (20 h) measurements.

2.14. Statistic analysis

Experiments were performed at least in triplicate unless otherwise noted. Quantitative data were depicted in graphs as mean ± SD. Statistical significance was


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checked using one-way analysis of variance (ANOVA) with Bonferroni post hoc test and Student’s t-test. p

Intranasal Vaccination against HIV-1 with Adenoviral Vector-Based

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