Combination Nanovaccine Demonstrates Synergistic Enhancement in

Article pubs.acs.org/journal/abseba

Combination Nanovaccine Demonstrates Synergistic Enhancement in Efficacy against Influenza Kathleen Ross,†,‡ Justin Adams,†,‡ Hyelee Loyd,§ Shaheen Ahmed,⊥ Anthony Sambol,∥ Scott Broderick,# Krishna Rajan,# Marian Kohut,△ Tatiana Bronich,⊥ Michael J. Wannemuehler,○ Susan Carpenter,§ Surya Mallapragada,*,‡ and Balaji Narasimhan*,‡ ‡

Chemical and Biological Engineering, §Animal Science, △Kinesiology, and ○Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, Iowa 50011, United States ⊥ Pharmaceutical Sciences and ∥Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198, United States # Materials Design and Innovation, University at BuffaloThe State University of New York, Buffalo, New York 14260, United States ABSTRACT: H5N1 influenza virus has the potential to become a significant global health threat, and next generation vaccine technologies are needed. In this work, the combined efficacy of two nanoadjuvant platforms (polyanhydride nanoparticles and pentablock copolymer-based hydrogels) to induce protective immunity against H5N1 influenza virus was examined. Mice received two subcutaneous vaccinations (day 0 and 21) containing 10 μg of H5 hemagglutinin trimer alone or in combination with the nanovaccine platforms. Nanovaccine immunization induced high neutralizing antibody titers that were sustained through 70 days postimmunization. Finally, mice were intranasally challenged with A/H5N1 VNH5N1-PR8CDC-RG virus and monitored for 14 days. Animals receiving the combination nanovaccine had lower viral loads in the lung and weight loss after challenge in comparison to animals vaccinated with each platform alone. These data demonstrate the synergy between polyanhydride nanoparticles and pentablock copolymer-based hydrogels as adjuvants in the design of a more efficacious influenza vaccine. KEYWORDS: H5 hemagglutinin, influenza, nanoparticle, PDEAEM, polyanhydride, hydrogel, combination nanovaccine

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INTRODUCTION Highly pathogenic avian influenza A (HPAI) H5N1 represents a significant global threat, with documented cases in at least 15 countries resulting in up to 60% mortality.1 Although the virus is usually transmitted to humans through contact with infected poultry, the possibility of mutation or reassortment with more human tropic influenza virus strains would provide the virus the ability to transmit between human hosts.2 Although there are currently two FDA-approved vaccines against H5N1, both require multiple doses (90 μg hemagglutinin per dose) and their efficacy varies considerably among vaccine recipients.3,4 Because of these limitations, next-generation vaccine adjuvants that promote robust neutralizing antibody titers with broad epitope recognition are necessary to prevent and control HPAI H5N1 influenza. Previously, polyanhydride nanoparticles encapsulating H5 hemagglutinin trimer (H53) were shown to induce protection against influenza viral challenge.5 Polyanhydride nanoparticles represent a dual-functional platform capable of enabling both sustained antigen release and adjuvanting subunit proteins such as H53.6,7 Further, the chemistry of polyanhydride copolymers can be tailored to provide sustained release kinetics of © XXXX American Chemical Society

encapsulated antigen, as well as protect antigen from exposure to degradative, aqueous environments before release.8,9 In addition, the use of polyanhydride nanoparticles has been shown to induce long-lived antibody titers with high avidity that recognize a broad range of antigen-specific epitopes.6,7,10 The breadth of immune responses elicited by these nanovaccines may be helpful in inducing cross-protection against highly mutating pathogens such as the influenza virus. Although the immune responses induced by polyanhydride nanovaccines against influenza are promising, recent literature suggests that novel combinations of adjuvants and/or delivery vehicles may enhance immune responses by targeting different immune pathways.11−13 We previously demonstrated that hydrogels consisting of Pluronic F127 (PF127) and a pentablock copolymer based on PF127 with cationic polydiethylaminoethyl methacrylate (PDEAEM) outer blocks were effective gene, drug and protein delivery vectors.14−16 The finding that hydrogels promoted the rapid development of high Received: November 10, 2015 Accepted: January 27, 2016

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DOI: 10.1021/acsbiomaterials.5b00477 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering Table 1. Vaccine Formulations group saline soluble alone nanoparticles hydrogel nanoparticles + hydrogel

nanoparticle- encapsulated H53 (μg)

20:80 CPTEG:CPH nanoparticles (μg)

hydrogel- entrapped H53 (μg)

PDEAEM: PF127:PVA hydrogel (mg)

soluble H53 (μg)

0 0 2 0 2

0 0 200 0 200

0 0 0 10 8

0 0 0 25 25

0 10 8 0 0

titer antibody responses against ovalbumin17 suggests this platform will be effective as a vaccine adjuvant and delivery vehicle for protein antigens. Because of the temperaturedependent gelation properties of PF127, these block copolymer hydrogels can be injected as liquid solutions and form gels at physiological temperature to create a depot at the injection site that typically lasts for 3−4 days followed by persistent low amounts of the hydrogel for up to one month postinjection.17 Additionally, these pentablock copolymers have demonstrated minimal cytotoxicity in vitro18,19 and a favorable safety profile in mouse studies.20 In this work, we present the ability of novel combination nanovaccine based on the pentablock copolymer and polyanhydride nanoparticles to induce protective immunity following a prime/boost immunization regimen. Neutralizing antibody titers were evaluated in serum collected 35 and 70 days after the primary immunization and before an intranasal viral challenge was performed. As measures of vaccine efficacy, the viral load in the lungs and changes in body weight of the animals were measured following the challenge. Altogether, the data demonstrated that animals immunized with the combination nanovaccine presented less severe clinical disease following influenza virus infection compared to animals immunized with the other vaccine regimens.

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copolymer with cuprous oxide nanoparticles from Pluronic macroinitiator has been previously described.18 Briefly, Pluronic macroinitiator (10 g, 0.78 mmol) was added to a round-bottom flask containing 50 mL of cuprous oxide nanoparticles (0.24 g, 1.68 mmol) in toluene. The reaction flask was degassed by vacuum-argon three times to remove air after fitting with a rubber stopper and cable tie. The N-propylpyridynyl methanimine (NPPM) ligand (0.5 mL, 3.40 mmol) and DEAEM monomer (4 mL, 2.53 mmol) were carefully added into the reaction flask. Several freeze−pump−thaw cycles with liquid nitrogen were used to further remove oxygen. The reaction was carried out inside of a water bath at 70 °C and 300 rpm for 20 h. The reacted product was passed through a column of basic alumina with a 1:1 dichloromethane:toluene solution. The eluate from the column was evaporated with a rotary evaporator and the resulting pentablock copolymer was precipitated in chilled n-heptane. The precipitant was collected using a Büchner funnel and dried inside a vacuum oven. The molecular weight was determined using 1H NMR (VXR 300 MHz, Varian). Hydrogels were created with a composition of 15 wt % poly(vinyl alcohol) (PVA), 5.9 wt % Pluronic F127 and 4.1 wt % PDEAEM pentablock copolymer in aqueous solutions, based on in vivo safety studies.20 First, 30 wt % PVA was heated in PBS to 80 °C. After dissolution and formation of a viscous solution, the PVA was pipetted into a chilled solution of block copolymer. In addition, PBS containing H53 protein and/or nanoparticles was pipetted into the solution and vortexed to form a temperature-dependent hydrogel. Mice. Female BALB/c mice were purchased from Charles River Laboratories (Wilmington, MA). All mice were housed under specific pathogen-free conditions where all bedding, caging, water, and feed were sterilized prior to use. Animal procedures were conducted with the approval of the Iowa State University and University of Nebraska Medical Center Institutional Animal Care and Use Committees. Immunizations. Each formulation was suspended in 100 μL of sterile PBS and administered subcutaneously to the nape of the neck of the mice. As outlined in Table 1, each animal received a total of 10 μg of H53 at each immunization. In particular, nanoparticle formulations were composed of 2 μg of H53 encapsulated in 200 μg of 20:80 CPTEG:CPH nanoparticles delivered with 8 μg of soluble H53 (sH53), which has previously been described as an optimal formulation to both prime (sH53) and sustain (encapsulated H53) the immune response.5,6 Nanoparticle formulations were prepared by suspending the particles in sterile PBS and sonicating to disperse aggregates before immunization. Hydrogel formulations were chilled on ice for ∼5 min to obtain a viscous solution that facilitated the uniform distribution of the added nanoparticles and/or sH53 within the hydrogel when sonicated. The formulation was then allowed to reach room temperature to begin gelation and then immediately administered to mice. Control formulations included 10 μg of sH53 alone and saline. All mice received two immunizations: a primary (day 0) and booster immunization (day 21). Secondary (i.e., booster) immunizations were prepared and administered using the same methods as the primary immunizations. Evaluation of Neutralizing Antibody Titers. Blood was withdrawn at 35 and 70 days after primary immunization via the saphenous vein and serum was prepared and stored frozen until used. Neutralizing antibody titers were determined using a HA-pseudotyped luciferase reporter virus (which expresses the A/Whooper Swan/ Mongolia/244/05 H5 antigen) as previously described.5 Briefly, sera

MATERIALS AND METHODS

Expression and Purification of H5 3 Protein. Plasmids containing the full length HA gene from HPAI H5N1 influenza virus A/Whooper Swan/Mongolia/244/05 were cloned and the H53 protein expressed as previously described.5,9 Briefly, Sf9 cells (Invitrogen, Grand Island, NY) were infected with recombinant baculoviruses generated using the Bac-to-Bac Baculovirus Expression System (Invitrogen). Supernatants were collected after 96 h, clarified by centrifugation, and incubated overnight with Ni-NTA beads at 4 °C. The H53 proteins were then eluted from the Ni-NTA beads and stored at −80 °C until use. H5 3 -Nanoparticle Synthesis. Monomers of 1,6-bis(pcarboxyphenoxy)hexane (CPH) and 1,8-bis(p-carboxyphenoxy)-3,6dioxaoctane (CPTEG) were synthesized as previously described.21,22 The 20:80 CPTEG:CPH polymer was then synthesized via melt polycondensation.22 The final composition, molecular weight, and purity were verified using 1H NMR (VXR 300 MHz, Varian, Palo Alto, CA). Polyanhydride nanoparticles encapsulating the H53 protein were synthesized using the water−oil−oil double emulsion process previously described.5,9 Briefly, H53 in nanopure water was homogenized for 90 s with 20 mg/mL 20:80 CPTEG:CPH in methylene chloride. The particles were precipitated in chilled pentane and collected via vacuum filtration. The size and morphology of the H53-nanoparticles were characterized using scanning electron microscopy (FEI Quanta 250, FEI, Hillsboro, OR) and found to be consistent with previous work.5,9 The encapsulation efficiency was determined by degrading the nanoparticles in 40 mM sodium hydroxide and quantifying the total H53 released using a microbicinchoninic acid (BCA) (Pierce, Rockford, IL) assay. Synthesis of PDEAEM Pentablock Copolymer and Formation of Hydrogels. The synthesis of PDEAEM pentablock B


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ACS Biomaterials Science & Engineering samples were serially diluted 3-fold in culture medium and incubated for 1 h at 37 °C with equal volumes of diluted pseudovirus stock. Next, the serum and pseudovirus mixtures were added to HEK293T cells and luceriferase activity in cell lysates assayed after 48 h using a Oneglo Luciferase assay system (Promega). Neutralizing titers were reported as ID50, or the reciprocal of the serum dilution that neutralized 50% of the HA-pseudotyped reporter virus. Neutralizing antibody titers of ID50 ≤ 50 were considered non-detectable (i.e., the limit of detection of the assay is ID50 = 50). Viral Challenge and Clinical Evaluation. Mice were challenged 77 days after primary immunization with the influenza A/H5N1 VNH5N1-PR8CDC-RG obtained from Influenza Reagent Resource (Manassas, VA). This virus is a PR8-based reassortant virus that contains the HA and NA genes of the H5N1 A/Vietnam/1203/04 clade 1 virus. The virus stock (2.1 × 108 CEID50/mL) was diluted 1:1000 with PBS before inoculation. Mice were anesthetized with 100 μL of 20 mg/mL xylazine and 100 mg/mL ketamine before being intranasally inoculated with 30 μL of the diluted virus stock. A control group of mice receiving no vaccine or challenge was housed under similar conditions. Lung tissue was collected from half of the mice 3 days postchallenge as previously described,5 while the remaining mice were monitored daily for weight loss over the 14 days postchallenge. Analysis of Viral Load. Homogenized lung tissue collected 3 days postchallenge was used to quantify viral load via PCR as previously described.5 Briefly, the total RNA concentration for each sample was normalized to 40 μg/μL. The PCR reaction used a template of 5 μL and was performed on an Applied Biosystems 7500 Fast Real-Time PCR System, on the standard mode, using the AgPath-ID One-Step RT-PCR Reagents (Life Technologies, Grand Island, NY, USA) in conjunction with the Fast-Track Diagnostics FTD-21−96/12 Kit (Junglinster, Luxembourg), which contains Brome Mosaic Virus (BMV) internal PCR extraction control, positive control, and primer/probes for universal influenza A antigen. Principal Component Analysis. Principal component analysis (PCA) was applied to project the data onto a dimensionally reduced principal component space, where the axes are a linear combination of the input descriptors including viral load, neutralizing antibody titer, and weight loss. PCA operates by identifying the covariance between descriptors in the data, and then defining the combination of descriptors and ordering them in terms of information captured.23,24 In this way, a reduced number of dimensions are sufficient for capturing all the input information. In this work, the challenge is in combining spectral data (percent original body weight measured every day for 14 days) with point data (viral load and/or neutralizing antibody titer) because given the dimensionality difference the spectral data is expected to dominate the analysis. The spectral data was therefore first parametrized using PCA. Previously, PCA has been used to characterize spectral data with the ability to extract key signatures and to perform parametrization based on physically interpretable conditions.25−28 By applying PCA, instead of curve fitting, the parametrization carries physically meaningful information. Using the first principal component as the primary parameter, with this PC capturing greater than 75% of the variance in the data, a single point value was used to represent the spectra with minimal loss of information. A second PCA was then performed on all the point data, including this new parameter. Thus, the dimensionality of this data was reduced from 17 (including 14 dimensions in the spectra) to two, while capturing 87% of the variance in the data. Statistics. Statistical differences among vaccine formulations were determined using the nonparametric Kruskal−Wallis and Mann− Whitney tests using GraphPad (Prism 6, GraphPad Software, La Jolla, CA). A repeated measures ANOVA was used to determine statistical differences of weight loss among treatment groups postchallenge. The denoted statistical significance indicates a p-value ≤0.05.

nanoparticles (Figure 1A) were found to be consistent with previous work (approximately 200 nm in diameter).5,9 In

Figure 1. Characterization of vaccine formulations. Scanning electron photomicrographs of (A) 1% H53-loaded 20:80 CPTEG:CPH nanoparticles and (B) pentablock copolymer hydrogel. Scale bar = 1 and 5 μm, respectively.

addition, hydrogels (Figure 1B) were prepared and mice were immunized subcutaneously at days 0 and 21 with the H53-based formulations as outlined in Table 1. Serum samples were collected at 35 and 70 days after primary immunization and analyzed for anti-H53 neutralizing antibody titers. Serum samples from most mice immunized with any of the H53 vaccine formulations demonstrated measurable neutralizing antibody titers at both 35 and 70 days postimmunization (Figure 2). These titers were sustained between days 35 and 70 and no significant differences were observed when comparing the titers at the two time points for the same formulation. Although mice receiving H53-containing nanoparticles were not found to have neutralizing antibody titers that were significantly different from animals receiving sH53, the mice receiving the H53-containing hydrogel formulation showed enhanced neutralizing antibody titers at both time points. In addition, mice immunized with the combination nanovaccine demonstrated enhanced antibody titers in comparison to sH53 at day 70. Finally, it was also observed that greater numbers of animals (7−8/8) receiving the hydrogel formulations (with or without nanoparticles) responded to the vaccine in comparison to animals (4−6/8) that received sH53 or H53-containing nanoparticles. Combination Nanovaccine Enhanced Protection in Mice Postchallenge. Mice were challenged with live A/ H5N1 VNH5N1-PR8CDC-RG virus 77 days after the primary immunization and the ability of the vaccine formulations to provide protection (based on weight loss and virus load) from clinical disease was assessed. Following infection, most mice receiving immunizations experienced weight loss; however, all these mice began to regain body weight between six and eight days postchallenge (Figure 3). In contrast, mice administered saline (i.e., no vaccine, but infected) experienced prolonged and more severe weight loss than the vaccinated mice, with only modest recovery observed at 14 days postchallenge. On the basis of the absence of clinical signs of disease, only the mice immunized with the combination nanovaccine formulation demonstrated enhanced protection as evidenced by their ability to maintain their body weight over the course of the ̈ noninfected mice (Figure 3). experiment similar to naive, Viral load was measured in the lungs of mice 3 days postchallenge. Although all the vaccine formulations reduced viral load when compared to that of the saline-administered mice, the median viral load of mice receiving the combination nanovaccine was lowest (Figure 4). Mice immunized with the combination nanovaccine had nearly 9-fold less viral particles

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RESULTS Nanovaccines Induced Sustained Neutralizing Antibody Titers. Polyanhydride nanoparticles encapsulating H53 were synthesized and the size and morphology of the C


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ACS Biomaterials Science & Engineering

Figure 4. Reduced viral load in mice administered polyanhydride nanoparticles and hydrogel. Viral load was measured in the lungs of mice 3 days postchallenge. Statistical differences among groups were determined using Kruskal−Wallis and Mann−Whitney tests. Formulations with different letters are significantly different. p ≤ 0.05. Error bars represent interquartile range of the median.

adjuvanted formulations had similar viral loads postinfection, only formulations containing the hydrogel (either alone or in combination with nanoparticles) demonstrated a significant decrease in viral load when compared to mice receiving sH53. The viral load observed in the vaccinated mice was inversely correlated with the weight of the animals 7 days postinfection (Figure 5A). Although less apparent, an inverse correlation (as calculated by Pearson’s correlation coefficient, r) was also observed between viral load and neutralizing antibody titer (Figure 5B). In addition, no correlation was observed between weight loss at 7 days and neutralizing antibody titer (Figure 5C). This analysis suggests that while neutralizing antibody titer is important, there are perhaps other host immune parameters (e.g., T cells) that may be involved in conferring protection against clinical sequelae. The combined relationship among the various vaccine formulations and clinical/virological/immunological outcome was more easily discerned using PCA, with the degree of correlation being defined by the geometric proximity between data points. PCA provides a visualization of the same data but in spatial context that permits the variance among the measurements. This approach linearly combines original data weighted based on the information contained, and thus, definitive conclusions not previously observed become clear when visualized in this manner. Of particular note, the change in conclusions with the iterative removal of individual measurements ensures that the final visualization is not overly sensitive to outlier measurements, providing a high level of statistical robustness. Mice that received the combination nanovaccine (closed triangle) were the furthest Euclidean ̈ mice (closed distance away from the immunologically naive diamond), suggesting that this formulation was the most efficacious (Figure 5D). Similarly, mice administered sH53 were closest to saline, and therefore, most similar to salineadministered mice. The property that had the largest influence on this result was the viral load, with the shift in -PC1/-PC2 direction corresponding with decreased viral load, thereby providing a quantitative measure of the difference in formulations, such as the decreased viral load in the combination vaccine group as opposed to the hydrogel only formulation. Although separate administration of both nanoparticles and hydrogel improved responses compared to

Figure 2. Combination of adjuvants results in enhanced neutralizing antibody titers. Serum collected at 35 and 70 days postinitial immunization was analyzed for neutralizing antibody titers. Statistical differences among groups were determined using Kruskal−Wallis and Mann−Whitney tests. Formulations with different letters are significantly different from one another at one time point (i.e., day 35 or day 70). p ≤ 0.05. No statistical differences were found comparing days 35 and 70 of a single formulation. Error bars represent interquartile range of the median. Limit of detection: ID50 = 50.

Figure 3. Mice immunized with combination platform maintained body weight upon infection. Mice were intranasally challenged 77 days after the primary immunization with a live A/H5N1 VNH5N1PR8CDC-RG virus. The body weight of each mouse was monitored daily for 2 weeks and presented as a percentage of their initial body weight. Statistical differences among groups were determined using repeated measures ANOVA. Formulations with different letters are significantly different. p ≤ 0.05. Error bars represent standard error of the mean.

per mL of lung homogenate when compared to animals that received sH53. Although mice immunized with any of the D


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Figure 5. Combination nanovaccine correlates with reduced viral load and weight loss. The Pearson’s correlation coefficient, r, was calculated to determine correlations among the data. Correlations between (A) viral load vs body weight (r = −0.600), and (B) viral load vs neutralizing antibody titer (r = −0.342) were noticed. However, no correlation (r = 0.079) was observed for (C) neutralizing antibody titer vs body weight. (D) Principal component analysis was performed with the degree of correlation begin defined by the geometric proximity of points.

administration of sH53 (as measured by their Euclidean distances from the saline control), using a combination of the two nanoadjuvant/delivery platforms resulted in enhanced efficacy.

that utilized three immunizations. Thus, the ability to combine two nanovaccine platforms allowed for the use of 1/3rd less antigen compared to that used previously and one less immunization.5 This ability of the combination nanovaccine to induce protective immunity following the administration of few immunizations is significant because their use may lead to enhanced patient compliance and the use of less antigen will improve the cost-effectiveness of the vaccine. Although the ability to generate high and protective neutralizing antibody titers in response to influenza vaccination is important, the longevity of protection must also be considered. Vaccines against pandemic influenza viruses that would induce long-term protection would be beneficial in highrisk individuals, such as the elderly or the very young, where protection declines faster than in low-risk individuals.30 In this work, mice immunized with the combination nanovaccine formulation maintained high, neutralizing antibody titers for at least 50 days after the second immunization (Figure 2). Whereas each separate nanoadjuvant formulation induced antibody titers that were maintained between days 35 and 70, the combination nanovaccine generated the greatest titer and efficacy (i.e., lower morbidity) postinfection. The longevity of the neutralizing antibody response induced by the combination

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DISCUSSION There is an urgent need to generate vaccine technologies that can prevent and control HPAI H5N1 influenza. Despite the development of two FDA-approved H5N1 vaccines, their large antigen dose and variability in efficacy leave room for improvement.3,4 Previously, polyanhydride nanoparticles were demonstrated as a viable platform to adjuvant and release stable H53 antigen.5,9 In this work, we describe a new adjuvant platform based on pentablock copolymer hydrogels and demonstrate that combining the polyanhydride nanovaccine with the H53-containing pentablock copolymer hydrogel synergistically enhanced vaccine efficacy while reducing antigen dose and variability in response. In previous work, nanovaccines containing polyanhydride nanoparticles or pentablock copolymer-based hydrogels were shown to enhance antibody titers.5,17,29 In this work, mice receiving two immunizations of the combination nanovaccine (nanoparticles + hydrogel) demonstrated neutralizing antibody titers (Figure 2) similar to those reported in previous studies5 E


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system provided immunological synergy that results in the enhancement in protection against influenza virus challenge and sets the stage for future development of combination nanovaccines to prevent influenza.

nanovaccine may be beneficial in the development of pandemic influenza vaccines. The variability in vaccine efficacy among immunized individuals is a major challenge in the development of influenza vaccines. For example, the FDA-approved H5N1 influenza vaccine has been shown to be only 45% effective.4 Host factors, such as age and obesity, have also been shown to influence the ability to generate an efficacious immune response to the vaccine, and lead to increased variability among individuals.31,32 Although no statistically significant differences in neutralizing antibody titer were observed at day 70 between the different adjuvanted H53 formulations in this work, it is important to note the reduced variability in neutralizing antibody titer, weight loss, and viral load (Figures 2−4, respectively) in the mice that received the pentablock copolymer-based hydrogel formulation, suggesting that the kinetics of antigen availability may influence the development of neutralizing antibody responses. We hypothesize that the hydrogel creates a homogeneous depot of available antigen, which could reduce the variability of the responses among animals. However, the addition of the hydrogel to the polyanhydride nanoparticles did not reduce the variability in titer attributed to the nanoparticles. Polyanhydride nanoparticles, in contrast to the pentablock copolymer hydrogel, have been shown to release antigen over several months.8,9 Although this sustained antigen release has been shown to promote the development of long-lived plasma cells,33,34 the antigen available to immune cells at any given time point would presumably be less than the antigen associated with the hydrogel depot. In addition, the nanoparticles are polydisperse in size, and therefore, erode at different rates. These characteristics may also contribute to a more heterogeneous availability of antigen, and therefore, increased variability among responses in animals receiving nanoparticle formulations. Although neutralizing antibodies are known to be important against influenza, recent literature suggests that other host response mechanisms (e.g., T cells, mucosal immunity, innate immunity, and cross-reactivity) may be necessary for optimal protection.35−37 For example, only weak correlations were observed between neutralizing antibody titer and viral load (r = −0.342) or between viral load and weight loss (r = −0.600), suggesting that additional mechanisms are involved in the resulting protection from virus challenge. On the basis of these results, we posit that the assessment of vaccine efficacy is complex and needs to encompass a combination of physiological, immunological, and virological parameters (e.g., neutralizing antibody titer, weight loss, viral load in the lungs, etc.) and a holistic methodology (such as PCA) is required to identify lead candidate vaccine formulations. As demonstrated in Figure 5, mice immunized with the combination formulation were observed to have the least clinical responses after vaccination and infection when compared to saline-administered mice. We hypothesize that the combination nanovaccine formulation composed of both polyanhydride nanoparticles and the pentablock copolymer hydrogel activated multiple components of the immune response to confer optimal protection. It is possible that the polyanhydride nanoparticles and pentablock copolymer hydrogel induce differential inflammatory responses and cytokine profiles, stimulate different pattern recognition receptors on antigen presenting cells, and differentially activate B and T cells that better approximate the host response following natural infection. Thus, combining the benefits of each nanoadjuvant/delivery

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CONCLUSIONS There is an urgent need to develop effective vaccine strategies against HPAI H5N1 influenza. The studies presented here demonstrate the successful development of a H5N1 influenza vaccine formulation using a combination of polyanhydride nanoparticles and a pentablock copolymer-based hydrogel. Mice immunized with the combination nanovaccine formulation developed the highest neutralizing antibody titers. These enhanced neutralizing titers were maintained for at least 70 days postinitial immunization and effectively protected mice against challenge. Mice administered the combination nanovaccine maintained their body weight upon infection, similar to healthy, noninfected mice. Finally, the mice receiving the combination nanovaccine also showed a significant reduction in viral load, which was highly correlated with the enhanced antibody titers. Collectively, all these data provide evidence that combining nanoadjuvants/delivery systems based on polyanhydride nanoparticles and pentablock copolymer hydrogels provides a synergistic enhancement in the efficacy of HPAI H5N1 influenza vaccines.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: 515-294-7407. Fax: 515294-2689. *E-mail: [email protected]. Phone: 515-294-8019. Fax: 515294-2689. Author Contributions †

K.R. and J.A. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge financial support from the U.S. Army Medical Research and Materiel Command (Grants W81XWH09-1-0386 and W81XWH-10-1-0806) and from the Iowa State University Nanovaccine Initiative. B.N. gratefully acknowledges the Vlasta Klima Balloun Professorship and S.K.M. is grateful for support from the Stanley Chair. The authors acknowledge valuable discussions with Dr. Phillip Dixon of the Department of Statistics at Iowa State University on the appropriateness of the statistical methods used in this work. In addition, the authors also acknowledge Dr. Metin Uz of the Department of Biological and Chemical Engineering at Iowa State University for his assistance in preparing scanning electron photomicrographs of the pentablock copolymer hydrogel.

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REFERENCES

(1) Van Kerkhove, M. D.; Mumford, E.; Mounts, A. W.; Bresee, J.; Ly, S.; Bridges, C. B.; Otte, J. Highly pathogenic avian influenza (H5N1): pathways of exposure at the animal-human interface, a systematic review. PLoS One 2011, 6 (1), e14582. (2) Jackson, S.; Van Hoeven, N.; Chen, L. M.; Maines, T. R.; Cox, N. J.; Katz, J. M.; Donis, R. O. Reassortment between avian H5N1 and human H3N2 influenza viruses in ferrets: a public health risk assessment. J. Virol. 2009, 83 (16), 8131−40.

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