M2e conjugated gold nanoparticle influenza vaccine displays thermal stability at elevated temperatures and confers protection to ferrets

Abstract

Currently approved influenza vaccines are not only limited in breadth of protection but also have a limited shelf-life of 12–18 months when stored under appropriate conditions (2–8 °C). Inadvertent alteration in storage temperatures during manufacturing, transportation, distribution until delivery to patient, can damage the vaccine thus reducing its efficacy. A thermally stable vaccine can decrease the economic burden by reducing reliance on refrigeration system and can also enhance outreach of the vaccination program by allowing transportation to remote areas of the world where refrigerated conditions are scarce. We have previously developed a broadly protective influenza A vaccine by coupling the highly conserved extracellular region of the matrix 2 protein (M2e) of influenza A virus to gold nanoparticles (AuNPs) and upon subsequent addition of toll-like receptor 9 agonist – CpG, as an adjuvant, have shown its breadth of protection in a mouse model. In this study, we show that the vaccine is thermally stable when stored at 4 °C for 3 months, 37 °C for 3 months and 50 °C for 2 weeks in its lyophilized form, and later it was possible to readily reconstitute it in water without aggregation. Intranasal vaccination of mice using reconstituted vaccine induced M2e-specific IgG and IgG subtypes in serum similar to the freshly formulated vaccine, and fully protected mice against lethal influenza A challenge. Immunization of ferrets intranasally or intramuscularly with the vaccine induced M2e-specific IgG and there was reduced virus level in nasal wash of ferrets immunized through intranasal route.

1. Introduction

Influenza virus has caused the deadliest pandemics known to humankind. The first recorded pandemic was in 1918 (Spanish flu), which was followed by three more, one in 1957 (Asian flu), another in 1968 (Hong Kong flu) [1] and the most recent in 2009 (H1N1pdm09). The first pandemic of 1918 claimed an estimated 20–50 million lives [1–3] whereas the recent pandemic of 2009 led to 150,000–575,000 mortalities worldwide [4]. Influenza virus continues to cause seasonal epidemics each year, which annually result in 3–5 million cases of severe illnesses and around 290,000–650,000 deaths worldwide [5]. Current vaccines licensed for influenza are based on highly variable epitopes of surface glycoproteins namely, hemagglutinin (HA) and neuraminidase (NA) [6]. These epitopes are strain-specific and can undergo antigenic shifts and drifts reducing vaccine effectiveness, prompting for continuous annual redesign of the seasonal vaccine to incorporate these new variants in the vaccine formulation. For example, the 2009 pandemic strain was caused by antigenic shift due to triple reassortment of genes from human, swine and avian influenza viruses [7]. This process of yearly amending the vaccine formulation limits the validity of seasonal influenza vaccine for one year. The 2009 pandemic revealed possible shortcomings of current vaccination programs and urges improvements in our abilities to respond quickly and effectively and to develop an influenza vaccine that has more universal effectiveness.

Additionally, current licensed influenza vaccines are thermally labile and require storage at temperatures between 2 °C to 8 °C (known as ‘cold-chain’- an uninterrupted refrigeration chain) during handling, transportation and routine immunizations [8, 9]. Exposure of the vaccine to temperatures outside this narrow range can affect their biological property and can result in reduced potency of the vaccine making the process of storage and distribution a delicate as well as an expensive one. Proper storage and handling of the vaccine is the most important step for distribution of the vaccine from manufacturer to healthcare professionals until it has been finally administered to the patients.

M2e, the extracellular domain of transmembrane protein M2 of the influenza virus, is a suitable candidate for making a vaccine of universal nature because M2e has remained fairly conserved in the human influenza strains [10]. However, M2e is just 23 amino acids long and as such has poor immunogenicity [10]. In one strategy aimed to enhance its immunogenicity, M2e has been attached to carrier molecules [11]. Recently our group has shown that attachment of the consensus M2e peptide to gold nanoparticles (AuNPs) can significantly enhance the immunogenicity of M2e [12], and that the intranasal (IN) delivery of M2e-conjugated AuNPs (AuNP-M2e) with soluble CpG (sCpG, ‘s’ stands for soluble) as an adjuvant (AuNP-M2e+sCpG) protects mice against lethal challenge of influenza A subtypes [13].

Building upon the previous encouraging results, in this study our objective was to characterize the AuNP-M2e+sCpG vaccine by determining its thermal stability and its effectiveness in a ferret animal model. Ferrets are one of the most attractive species for influenza vaccine development [14] because they are highly susceptible to infection by human influenza strains and show similar clinical symptoms as humans [15, 16]. In order to determine stability and potency of the lyophilized vaccine formulation under different temperatures, AuNP-M2e+sCpG1826 (AuNP-M2e supplemented with CpG ODN 1826) vaccine was freeze-dried to a solid dry powder and was stored either at 4 °C for three months, or at 37 °C for three months or at 50 °C for two weeks. Temperatures of 4 °C and 37 °C were selected to mimic potential real-life storage temperatures corresponding to refrigerated cold-chain and ambient temperature, respectively. For practicality, the storage time was selected as three months, however, to predict longer term effects we performed an accelerated degradation study by storing the vaccine at 50 °C for two weeks. At significantly elevated temperatures, the degradation kinetics of vaccines accelerates, and such accelerated degradation studies are often used in lieu of studies where the vaccine is stored for many years but at lower temperatures [17]. After storage, the vaccine was reconstituted and used to vaccinate mice through the IN route and the immunogenicity was evaluated through measurement of antibody response and protection against lethal influenza A virus challenge. Immunogenicity of the AuNP-M2e+sCpG2006 (AuNP-M2e supplemented with CpG ODN 2006) vaccine in ferrets was evaluated by administering the freshly made vaccine through the IN and intramuscular (IM) routes, and its efficacy was evaluated by subsequently challenging the ferrets with the 2009 pandemic influenza strain and measuring different clinical symptoms and viral titers in the nasal passages. Experiments showed that the vaccine is thermally stable and is effective in ferrets. The broadly protective nature of the AuNP-M2e+sCpG vaccine along with its thermal stability could provide an attractive alternative to seasonal vaccines. It could negate the need to manufacture a new vaccine annually and reduce the cost associated with the cold-chain.

2. Materials and methods

2.1. Chemicals, peptide, oligonucleotides and antibodies

Gold (III) chloride trihydrate (520918–5G), trisodium citrate dihydrate (S1804–500G), O-phenylenediamine (OPD) powder (P9029–50G), tween 20 (P1379–1L) and phosphate-citrate buffer tablet (P4809–50TAB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Consensus M2e peptide (acetylated-SLLTEVETPIRNEWGSRSNDSSDC-amidated, MW: 2736 Da) was chemically synthesized at 95% purity by AAPPTec (Louisville, KY, USA). Mouse CpG oligodeoxynucleotide (ODN) 1826 (5’-TCCATGACGTTCCTGACGTT-3’) and the ferret CpG ODN 2006 (5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’), recognized by toll-like receptor 9 (TLR9), were synthesized by Integrated DNA Technologies (Coralville, IA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibodies were bought from Southern Biotech (Birmingham, AL, USA). HRP-conjugated goat anti-ferret IgG was purchased from KPL Inc. (Gaithersburg, MD, USA). ELISA plate blocking buffer concentrate (80061) was obtained from Alpha Diagnostic International (San Antonio, TX, USA). 10X Tris/Boric acid/EDTA (1610733) was purchased from BioRad. 10X Phosphate buffered saline (PBS) (46013CM) was purchased from Fisher Scientific. Milli-Q water with a resistance of 18.2 MΩ cm was used in all the experiments.

2.2. Vaccine preparation and storage at different conditions

AuNP-M2e+sCpG vaccine formulation was prepared as described in our previous work [12]. Briefly, 1 ml of synthesized AuNPs (0.0225 nmol/ml equal to about 224 μg of AuNPs supplemented with 0.1 % tween 20) were centrifuged at 17000 g for 25 minutes at 4 °C in a 1.5 ml eppendorf tube (022363212, Eppendorf, Hauppauge, NY, USA). Excess liquid (932 μl) was aspirated and AuNPs (collected as a pellet at the bottom of the tube) were resuspended in the remaining 68 μl volume. Next, 12 μl of 1 mM M2e solution (equivalent to 32.8 μg of M2e) was added in dropwise manner to the 68 μl volume. The mixture was incubated overnight at 4 °C to complete the AuNP functionalization process. AuNP-M2e was further supplemented with 80 μg CpG ODN 1826, in a volume of 20 μl, to bring the final volume of the formulation to 100 μl. This recipe enables immunization of four mice where each mouse received 25 μl volume containing 56 μg of AuNPs, 8.2 μg of M2e and 20 μg of CpG ODN 1826. Eppendorf tube (1.5 ml) containing liquid form of AuNP-M2e+sCpG1826 vaccine formulation was lyophilized overnight to obtain the dry powder form. Tubes containing lyophilized AuNP-M2e+sCpG1826 dry powder were stored either at a) 4 °C for three months, or at b) 37 °C for three months, or at c) 50 °C for two weeks. To avoid moisture ingress from the refrigerator environment, the tube stored at 4 °C was placed in a vacuum chamber. Another tube, containing dry powder of AuNP-M2e+sCpG1826, was placed in a 37 °C incubator (Percival, IA, US) without any additional control over humidity. To store the vaccine at 50 °C, tube containing lyophilized vaccine was placed in a heating block (Thermo Fisher Scientific, Inc., MA, US) heated to 50 °C. Post-storage, the lyophilized dry powder was reconstituted by pipetting 100 μl of water into the tubes. Contents were gently vortexed until a homogeneous solution was formed. The recipe in each eppendorf tube allowed immunization of four mice. Freshly made vaccine formulation without any lyophilization step or storage was prepared as a control.

For immunization of ferrets, the AuNP-M2e+sCpG vaccine was prepared as described above, with the exception that CpG ODN 1826 was replaced with CpG ODN 2006 (AuNP-M2e+sCpG2006).

2.3. UV-vis spectral analysis

Absorption spectra of different vaccine formulations were obtained at excitation wavelengths from 700 nm to 400 nm in 1 nm increments using Cary 300 UV-vis spectrophotometer.

2.4. Transmission electron microscopy

Vaccine formulation after reconstitution was analyzed using a transmission election microscope (TEM) (Hitachi H-8100, MI, USA).

2.5. Dynamic light scattering (DLS)

Particle size and size distribution of the vaccine formulation was investigated using a light scattering instrument (Mobius, Wyatt Technology, CA, USA).

2.6. Measurement of free M2e in vaccine formulation

AuNP-M2e+sCpG1826 formulation (100 μl: a dose for four animals) was freeze dried and the resulting lyophilized powder was stored at 50 °C for 2 weeks. Post-storage, lyophilized powder was resuspended in 100 μl of either water or 1X PBS. Out of this, 25 μl (volume used to immunize one animal) was then transferred to a new Eppendorf tube. Water or 1X PBS (475 μl) was added and the tube was centrifuged at 17,000 g for 25 minutes. Supernatant was collected. Standard solutions were prepared by dissolving known amounts of M2e into either water or 1X PBS. The amount of free M2e in the supernatant was quantified using Micro BCA assay following manufacturer’s protocol (ThermoFisher Scientific, Rockford, IL, USA). To find M2e attached to AuNPs, the amount of M2e in supernatant (free M2e) was subtracted from 8.2 μg, the amount of M2e that was originally added to make the formulation.

2.7. Agarose gel electrophoresis

AuNP-M2e+sCpG1826 formulation (100 μl: a dose for four animals) was freeze dried and the resulting lyophilized powder was stored at 50 °C for 2 weeks. Post-storage, lyophilized powder was resuspended in 100 μl of either water or 1X PBS. Out of this, 25 μl (volume used to immunize one animal) was then transferred to a new Eppendorf tube. TriTrack loading dye (6X) (R1161, ThermoFisher Scientific) was then further added to the tube. To analyze stability of CpG ODN 1826, 10 μl of this mixture was loaded in a well of agarose gel (3 % agarose in 1X Tris/Boric acid/EDTA buffer). Gel was run at 90 V for 90 minutes. GeneRuler 1kb Plus DNA ladder (SM1333, ThermoFisher Scientific), was also loaded on the gel.

2.8. Mice and ferret immunizations

The protocol was approved by Institutional Animal Care and Use Committee (IACUC) at Texas Tech University (TTU). Mice (BALB/c, female, 6–8-week-old, n= 5–8 mice per group) obtained from Charles River Laboratories (Wilmington, MA, USA) were primed on day 0 and boosted on day 21 with different reconstituted formulations. A group of mice immunized with a freshly made vaccine served as a positive control. All formulations were administered in a volume of 25 μl given dropwise to the nares. Blood was collected from mouse submandibular (facial) vein at day 0 (before vaccination) and then on day 21 and day 42. Collected blood was centrifuged at 12000 g for 15 min and the serum was stored at −80 °C until analysis.

Female fitch ferrets, approximately 8–10 months of age (Triple F Farms, Sayre, PA, USA), that were confirmed to be seronegative by hemagglutination inhibition (HI) assay for the prevailing human influenza viruses, were used in this study. Experimental protocols were approved by the IACUC at TTU. Isoflurane-anesthetized ferrets (n = 3/group) were immunized with freshly prepared AuNP-M2e+sCpG2006 on day 0 followed by a booster on day 21 and day 42. Ferrets were immunized with a total volume of 250 μl vaccine, either injected through IM route or through IN route. Each vaccinated ferret received a total of 1120 μg of AuNP, 164 μg of M2e and 400 μg of CpG ODN 2006. A third group of ferrets, which did not receive any vaccine, was kept as a negative control (unvaccinated). To assess the antibody response in ferrets, blood was collected prior to vaccination and on days 21, 42 and 63 post first vaccine. Blood was processed, and serum was stored at −80 °C until analysis.

2.9. Measurement of M2e-specific immune response

M2e-specific antibodies in serum of the immunized mice were measured by ELISA as described before [18]. To determine M2e-specific serum antibody responses in a ferret, ninety-six well plates (Maxisorp, Nunc) were coated with 50 μl of 5 μg/ml M2e peptide in phosphate buffered saline (PBS) and stored overnight at 4 °C. Plates were blocked with 200 μl of 2x ELISA plate blocking buffer concentrate in distilled water for 2 h at room temperature. Serum from individual ferrets was diluted to 1:8, added to wells, and incubated for 1.5 h at room temperature. Next, plates were incubated with 50 μl of 1:500 dilution of HRP-labeled anti-IgG antibody for 1.5 h at room temperature. Plates were washed three times with PBST (0.05 % tween 20 in PBS) between each step using ELx405 microplate washer (BioTek, VT). Color was developed with OPD dissolved in phosphate-citrate buffer (pH 5.0) containing 0.03 % H₂O₂, as substrate. After 10 minutes, 50 μl of 3 M phosphoric acid was added to terminate the reaction. Absorbance at 492 nm was recorded using SpectraMax Plus384 microplate reader (Molecular Devices LLC., CA)

2.10. Virus challenge

On day 42, mice were challenged intranasally with 5 × LD₅₀ of influenza A virus - A/PR/8/34 (H1N1). Mice were weighed prior to virus challenge and the weight was recorded on a daily basis for 14 days. If mice experienced weight loss of over 25 %, they were euthanized and included as an experimental end point.

On day 63 after the first vaccination dose, ferrets were infected with 1 × 10⁷ TCID₅₀ of A/California/07/2009 (H1N1pdm09) influenza virus, diluted in 0.5 ml of sterile PBS and delivered to the nostrils (0.25 mL per nostril). Post-infection, nasal washes were collected on day 3 and day 6 in 1 ml of PBS. Nasal wash titers were determined by standard TCID₅₀ assay on MDCK cells and measured as log₁₀ TCID₅₀/ml.

2.11. Statistical analysis

All statistical analyses were performed using GraphPad Prism for windows, version 6.05 (GraphPad Software, Inc., La Jolla, CA). Comparison of antibody titers between groups of mice was performed with two-way analysis of variance (ANOVA) and a Bonferroni test at a value of p<0.05 for statistical significance. Kaplan-Meier survival curves were generated and compared by the Log-rank (Mantel-Cox) test followed by pairwise comparison using the Gehan-Breslow-Wilcoxon test. Comparison of antibody titers and virus titers was performed with two-way analysis of variance (ANOVA).

3. Results

3.1. Effect of storage at different conditions

To assess thermal stability of the AuNP-M2e+sCpG1826 vaccine, the formulation was lyophilized, and the resulting powder was stored at 4 °C for three months or 37 °C for three months or 50 °C for two weeks (Fig 1). Post storage, the formulations were reconstituted in water and their absorbance spectra were measured. All reconstituted formulations showed similar absorption spectra (Fig 2A, 2B), which were similar to the absorption spectra of a freshly prepared vaccine formulation. The wavelengths corresponding to the maximum absorption did exhibit a shift from 522 nm for the freshly prepared formulation to 526, 528 and 530 nm for formulations stored at 4 °C, 37 °C, and 50 °C, respectively. To determine whether the AuNPs had aggregated, we visually inspected them under a TEM, and the corresponding images (Fig 2C) showed that the particles were well separated.

Figure 1. Vaccine formulation and storage conditions.

Storage scheme for lyophilized AuNP-M2e+sCpG1826 vaccine formulation. Post-storage, the lyophilized vaccine formulations were reconstituted in water and were used to immunize BALB/c mice to confirm its potency.

Figure 2. Stability of lyophilized vaccine formulation.

AuNP-M2e+sCpG1826 formulation was freeze dried and stored at 4 °C for 3 months, 37 °C for 3 months or 50 °C for 2 weeks. Post-storage, the lyophilized vaccine formulation was reconstituted in DI water and examined using UV-vis spectroscopy and transmission electron microscopy (TEM). (A) Absorption spectra, (B) a close-up of peaks for absorption spectra in ‘A’, and (C) TEMs.

3.2. Effect of accelerated stability test on vaccine formulation

Since storage of the vaccine formulation at 50 °C resulted in greater deviation as compared to storage at 4 °C and 37 °C, we performed a more detailed analysis on the formulation stored at 50 °C. First, to better evaluate changes in properties we reconstituted the stored vaccine in water or 1X PBS. PBS was selected to mimic in vivo physiological pH and salt concentrations. The UV absorption spectra for water-resuspended and PBS-resuspended formulations were similar and exhibited a peak at 530 nm and 529 nm respectively compared to a peak at 522 nm for the freshly prepared formulation (Fig 3A, 3B). To better characterize the peak shift, we measured particle size using DLS. As compared to an average particle diameter of 15.2 ± 0.1 nm for bare AuNPs and 20.6 ± 0.2 nm in a freshly prepared vaccine, the average diameter of particles in vaccine resuspended in water and 1X PBS was found to be 41 ± 0.6 nm and 36.6 ± 0.2 nm, respectively (Fig 3C). Similar to water-resuspended formulation (Fig 2C), the PBS-resuspended formulation also showed no aggregation as observed under TEM (Fig 3D). To determine the state of M2e in the formulation we quantified the free and immobilized M2e in PBS-resuspended and water-resuspended formulations. About 49% and 44% less M2e was obtained in the supernatant of water-resuspended and PBS-resuspended formulations, respectively (Table 1). Finally, to determine the stability of CpG during accelerated degradation study, we ran the formulations through an agarose gel. In none of the formulations degraded oligonucleotide bands were seen (Fig 3E).

Figure 3. Stability of lyophilized vaccine formulation at accelerated storage condition.

AuNP-M2e+sCpG1826 formulation was freeze dried and stored at 50 °C for 2 weeks. Post-storage, the lyophilized vaccine formulation was reconstituted in either DI water or 1X PBS and examined using UV-vis spectroscopy, dynamic light scattering (DLS) and transmission electron microscopy (TEM). (A) Absorption spectra, (B) a close-up of peaks for absorption spectra, (C) dynamic light scattering data showing size distribution and (D) TEMs. (E) Stability of CpG ODN 1826 was examined using agarose gel electrophoresis. Lane 1: ladder, lane 2: empty, lane 3: CpG ODN 1826 (6.66 μg loaded on the gel), lane 4: freshly prepared AuNP-M2e (no CpG1826), lane 5: freshly prepared AuNP-M2e+sCpG1826, lane 6: lyophilized AuNP-M2e+sCpG1826 (@ 50 °C for 2 weeks) resuspended in water, lane 7: lyophilized AuNP-M2e+sCpG1826 (@ 50 °C for 2 weeks) resuspended in 1X PBS and lane 8: empty.

Table 1. Amount of free M2e in the vaccine formulation.

AuNP-M2e+sCpG1826 formulation was freeze dried and stored at 50 °C for 2 weeks. Post-storage, the lyophilized vaccine formulation was reconstituted in either DI water or in PBS and the amount of free M2e was examined using a Micro BCA assay. Data represented as mean ± standard deviation. n = 2 to 4 samples. Samples read as triplicates.

Sample ID	Amount of free M2e in supernatant (μg) ‘A’	Amount of M2e in immobilized on surface of AuNPs (μg) ‘B’= 8.2-A
AuNP-M2e+sCpG1826, Fresh	6.5 ± 0.4	1.7 ± 0.4
AuNP-M2e+sCpG1826, 50 °C @ 2 weeks, resuspended in water	3.3 ± 0.1	4.9 ± 0.2
AuNP-M2e+sCpG1826, 50 °C @ 2 weeks, resuspended in PBS	3.6 ± 0.2	4.5 ± 0.2

3.3. Immunogenicity of lyophilized AuNP-M2e+sCpG1826 vaccine formulations and their protective potential

We next investigated immunogenicity of formulations that were stored at different temperatures. For comparison, a control group of mice received a freshly made vaccine formulation without lyophilization. The mice were primed on day 0 and received a booster on day 21 (Fig 4A). Mice immunized with the freshly prepared vaccine formulation and reconstituted formulations exhibited a significant increase in M2e-specific IgG antibody response (Fig 4B). There was no significant difference in anti-M2e IgG (Fig 4C), IgG1 (Fig 4D), and IgG2a (Fig 4E) antibody responses in mice receiving reconstituted formulations that had been stored at 4 °C, 37 °C, or 50 °C in comparison to the fresh vaccine. This shows that lyophilization and storage of the formulation at 4 °C, 37 °C, or 50 °C did not adversely affect the vaccine immunogenicity.

Figure 4. Immune response in mice from reconstituted AuNP-M2e+sCpG1826 formulations.

(A) BALB/c mice (n= 5–8) were vaccinated on days 0 and 21 with reconstituted vaccine formulations that had been stored under different temperature conditions. Each animal received 8.2 μg M2e and 20 μg sCpG, and serum was collected on days 0, 21 and 42 for analysis. (B) M2e-specific IgG antibody in serum of individual mice at days 0, 21 and 42 (@ 1:1600 dilution). (C) M2e-specific IgG on day 42 (@ 1:1600 dilution). (D) M2e-specific IgG1 on day 42 (@ 1:1600 dilution). (E) M2e-specific IgG2 on day 42 (@ 1:1600 dilution). (F) Survival of mice challenged on day 42 with 5 x LD₅₀ of influenza A/PR/8/34 (H1N1) virus. (G) Percent change in body weight of challenged mice. Data represented as mean±SD. ****p<0.0001 and ns: not significant.

We next tested if the storage conditions affected the ability of the formulation to protect mice against lethal influenza virus challenge. All vaccinated mice were challenged using a lethal dose of influenza A/PR/8/34 (H1N1) virus (~5 × LD₅₀) and all of them showed 100% survival irrespective of the vaccine used (stored or fresh) (Fig 4F) with minimal weight loss (Fig 4G). Thus, the vaccine storage temperature had no effect on either the ability of the vaccine to induce a strong anti-M2e antibody response or its ability to protect the mice against lethal virus challenge.

3.4. Formulation and characterization of the vaccine in ferrets

Ferrets are the preferred large animal model to study influenza vaccine efficacy. Therefore, we next sought to evaluate the effectiveness of our vaccine in ferrets. Because our vaccine comprised CpG, a TLR 9 agonist as an adjuvant, we had to reformulate the vaccine for use in ferrets. This reformulation was necessary because the same CpG nucleotide sequence does not result in effective stimulation of TLR 9 in different mammalian species, and although ODN 1826 is an effective stimulant of TLR 9 in mice it does not adequately stimulate TLR 9 in other mammals [19]. We reformulated the vaccine for ferrets by swapping ODN 1826 with ODN 2006. Previously we have shown that the presence of ODN 1826 in the vaccine formulation does not destabilize the colloidal AuNP and M2e mixture [12]. To establish that ODN 2006 also does not cause vaccine colloidal instability we analyzed the absorption spectra of the AuNP-M2e solution supplemented with ODN 2006 (AuNP-M2e+sCpG2006) using UV-vis spectroscopy. Addition of CpG ODN 2006 to the AuNP-M2e solution did not alter the wavelength corresponding to the peak maxima (Fig 5 A, B, C). To assess the stability further, we added saline to the vaccine until 1 % NaCl concentration was achieved to mimic the in vivo physiological salt content. Even at 1 % NaCl the AuNP-M2e+sCpG2006 remained stable (Fig 5 D and E). The drop in peak in Figs 4D and 4E are due to dilution effect caused by addition of NaCl solution. Absence of any aggregates in the formulation was further verified by visual observation under TEM (Fig 5 F and G). Together these data show that the vaccine containing ODN 2006 is stable.

Figure 5. Stability characterization of gold nanoparticles formulated with CpG2006 for ferrets.

(A) Absorption spectrum of AuNPs. (B) Absorption spectra of AuNPs after conjugation of M2e and subsequent addition of soluble CpG ODN 2006. (C) A close-up of peaks for absorption spectra in ‘B’. (D) Absorption spectra in the presence of 1% of NaCl. (E) A close-up of peaks for absorption spectra in ‘D’. (F, G) Transmission electron micrographs of AuNP-M2e+sCpG2006 formulation.

3.5. Immunogenicity and efficacy of AuNP-M2e+sCpG2006 in ferrets

Ferrets (n= 3/group) were immunized on days 0, 21 and 42 (Fig 6A) either through IM or IN routes with a freshly prepared AuNP-M2e+sCpG2006 formulation. Detectable levels of anti-M2e IgG antibodies were seen, on day 21, after single immunization for both the vaccination routes (Fig 6B). The two booster doses of AuNP-M2e+sCpG2006 given on days 21 and 42 further enhanced the antibody response in ferrets as compared to the unvaccinated (UnVacc.) control group (Fig 6B). On day 63, ferrets immunized through the IN route showed a slightly better anti-M2e IgG antibody response as compared to the IM route (Fig 6B); however, the difference was not statistically significant.

Figure 6. Immunogenicity and protection in ferrets.

(A) Ferrets were vaccinated with AuNP-M2e+sCpG2006 (160 μg M2e and 400 μg sCpG2006) on days 0, 21, and 42 through intramuscular (IM) or intranasal (IN) routes. Ferrets were challenged IN with A/California/07/2009 virus on day 63. (B) M2e-specific IgG antibody in serum of ferrets at days 0, 21, 42 and 63 (@ 1:8 dilution). (C) Virus titer in nasal wash post infection. Data represented as mean±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns: not significant. n= 3 ferrets per group.

To evaluate vaccine efficacy the ferrets were challenged intranasally with 1 × 10⁷ TCID₅₀ dose of A/California/07/2009 (H1N1pdm09) virus on day 63. Virus replication in the upper respiratory tract was determined by titrating nasal washes in MDCK cells (Fig 6C). As compared to unvaccinated ferrets, ferrets immunized through the IN route presented a 32 fold and 355 fold reduction in virus titer on days 3 and 6 post infection, respectively. In contrast, ferrets immunized through the IM route did not show any significant reduction in virus titers. All ferrets eventually recovered from infection.

Discussion

The objectives of this study were to evaluate the thermal stability of the vaccine formulation consisting of the conserved M2e sequence attached to AuNPs with CpG as an adjuvant, and to assess its immunogenicity in ferrets.

Thermal stability of a vaccine has several implications in terms of both cost and accessibility of the vaccine to the public. Current seasonal influenza vaccine’s main immunogenic component, HA, is prone to degradation at higher temperatures [20] and this vulnerability can affect potency of the marketed vaccine. To counter this problem the vaccine formulation, which is provided in a liquid form [21], is kept refrigerated at 2–8 °C (‘cold-chain’). Unfortunately, the requirement of a cold-chain puts additional burden on the economics of vaccine distribution, and disruption in cold-chain is one of the major cause of vaccine wastage [22]. To overcome this limitation, the vaccine can be converted from a liquid state to a dry solid form, since the solid form exhibits superior thermal stability [9].

In the current study, even after storage of the dry form of AuNP-M2e+sCpG1826 vaccine for three months at 4 °C (simulates refrigerated storage), or for three months at 37 °C (simulates ambient temperature storage), or for two weeks at 50 °C (accelerated stability test), the vaccine was found to readily disperse in water and formed a transparent colloidal solution. UV-vis spectroscopy is a technique often used to study AuNP colloidal solutions. The wavelength at which the maximum absorbance for a AuNP colloidal solution occurs has been shown to increase when the size of AuNPs increases [23]. We observed that for all formulations stored at 4 °C, 37 °C, or 50 °C, the UV-vis spectra were similar after resuspension in water, although their peak wavelengths were higher as compared to a freshly prepared formulation. To check for aggregation, we performed TEM however, none of the formulations revealed presence of any AuNP aggregates. Since increase in peak wavelength was highest for the formulation stored at 50 °C, we analyzed this formulation in greater detail. Upon resuspending the formulation that had been stored at 50 °C in PBS, the increase in peak wavelength was found to be lower as compared to the water-resuspended formulation, indicating that presence of salts may contribute to improved dispersion of the vaccine. Consistent with lower peak wavelength for the PBS-resuspended formulation, the particle diameter measured with DLS was also lower for the PBS-resuspended formulation as compared to water-resuspended formulation. CpG was found to remain intact under the accelerated stability test condition at 50 °C. Quantification of free M2e in the supernatant of the PBS- and water-resuspended formulations showed a decrease. This decrease could happen due to M2e aggregation; however, the TEM images did not reveal formation of new aggregates. Another plausible explanation could be that upon drying, M2e molecules get attached to AuNPs forming multiple layers, which would also explain the increase in size of particles after resuspension. Additional studies are needed to verify this postulate.

Besides colloidal stability, elevated temperatures can also cleave the critical conjugation between the AuNP and M2e peptide even when the formulation is in its dry solid form [24]. In our previous study we have shown that only ~1.2–1.3 μg of the total 8.2 μg M2e added is immobilized on the AuNP through thiol linkage whereas the remaining ~7 μg remains unconjugated [18]. In this study, we again found that 1.7 μg M2e was attached to AuNPs while 6.5 μg was free. We postulate that this presence of excess free M2e could compensate for any disruption of the AuNP-M2e thiol linkage that might occur during storage. The excess M2e in the formulation could reestablish the thiol linkage to AuNPs upon reconstitution. Our data agrees with previous findings where it was shown that mercaptoacetic acid capped AuNPs resisted aggregation upon lyophilization [25]. Storage conditions did not alter IgG2a immune response across all the immunized groups. Since IgG2a response is primarily caused by inclusion of CpG [12], this suggests that CpG was also not degraded during storage.

Additionally, we sought to determine efficacy of the vaccine in an animal model that closely resembles human physiology especially in case of influenza infections. Ferrets exhibit high susceptibility to influenza infection, carry similar cellular receptors as humans and show similar clinical signs of infection [26]. Ferrets also display similar signs of clinical symptoms post infection as the humans, such as lethargy, fever, weight loss, sneezing and nasal discharge [14]. This has made ferrets the gold standard model for studying influenza vaccine efficacy [27]. Thus, we evaluated efficacy of a freshly made AuNP-M2e+sCpG2006 vaccine by either the IM or IN administration in ferrets. We observed that immunization of ferrets with AuNP-M2e+sCpG2006 was able to induce a significant anti-M2e antibody response as compared to unvaccinated ferrets. Although this humoral immune response was independent of the route of immunization, we found that ferrets immunized through the IN route had faster recovery characterized by lower virus shedding in upper respiratory tract. Our findings suggest that the vaccine formulation based on the conserved sequence of M2e peptide conjugated to AuNP and supplemented with CpG ODN 2006 as an adjuvant can induce a strong antibody response and protect the ferrets against lethal challenge of A/California/07/2009 (H1N1pdm09).

Similar to our results, other studies using M2e as an antigen have shown protection against virus infection in ferrets. For example Music et al. demonstrated that supplementing M2e to the split vaccine (A/California/07/2009 virus) enhanced immunogenicity of the split vaccine in ferrets (delivered through IM route), and also conferred cross-protection to ferrets against seasonal A(H1N1) strain BR/59 virus challenge [28]. In another study by Rosendahl et al., it was shown that immunization of ferrets with M2e (along-with other conserved B and T cell epitope) reduced the virus replication in lungs of ferrets [29]. The importance of including CpG as an adjuvant in the vaccine formulation has also been demonstrated previously. Fang et al. showed that inclusion of CpG (500 μg) in the vaccine formulation enhanced virus specific-antibody levels in serum of ferrets immunized with Fluviral® through the IM route [30].

In this study, besides showing protection of immunized mice against influenza A/PR/8/34 (H1N1) and that of ferrets against influenza A/California/07/2009 (H1N1pdm09), we did not emphasize the breadth of protection conferred by the AuNP-M2e+sCpG vaccine. However, in our earlier studies we have demonstrated that in mice the vaccine provide protection against A/Victoria/3/75 (H3N2) and the highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1) [13].

The AuNP-M2e+sCpG vaccine formulation possesses several positive attributes. The broad protection conferred by the AuNP-M2e+sCpG vaccine could provide immunogenicity against an emergent pandemic strain. The thermally stable dry-powder formulation of the AuNP-M2e+sCpG could simplify stockpiling of vaccines even in remote areas with less-favorable storage and transportation conditions. The convenience and ease of self-administration of the vaccine through the IN route could be another potential benefit of the vaccine. It is possible to speculate that since the vaccine is thermally stable, in the event of a pandemic with restrictions of a lockdown that keeps individuals inside their homes, the vaccine could be delivered to their mailboxes through the postal services or other delivery services, enabling safe mass vaccinations.

In conclusion, our findings suggest that an influenza vaccine based on the conserved epitope of M2e conjugated to AuNP and supplemented with CpG, a TLR 9 agonist, should be further evaluated as a potential broadly protective influenza vaccine candidate.

Highlights.

• Freeze dried M2e-conjugated gold nanoparticle formulation is thermally stable

• Thermal stability is exhibited even at 50 °C when stored for two weeks

• Stored vaccine upon reconstitution protects mice from lethal influenza A challenge

• Vaccine is immunogenic in ferrets and reduces lung viral titers in a challenge model