compared to subunit, split or virosomal vaccines.Heat stability of liquid and lyophilized influenza vaccines
The heat stability of lyophilized influenza vaccines was determined by storing liquid and lyophilized influenza vaccines in glass vials at 4°C, 24°C, 37°C and 60°C for one month. For both liquid and lyophilized vaccines, the recovery of HA1 and antigenicity was determined by RP-HPLC and SPR respectively (Table 2). Additionally, the residual moisture content was determined for lyophilized vaccines and used for calculation of the predicted glass transition temperature (Tg) with the Gordon-Taylor equation for a binary water-trehalose system as described in literature [18].
The residual moisture content may affect the mobility of biopharmaceuticals entrapped in glassy matrixes such as lyophilized (glassy) trehalose. In general, a higher moisture content indicates a lower Tg and consequently may increase molecular movement at lower storage temperatures giving risk to destabilization of the entrapped biopharmaceutical. The residual moisture content was found to be 2.1% or less for all four lyophilized influenza vaccines. The predicted Tg at a residual moisture content of 2.1% was calculated to be approximately 95°C, which is well above normal or elevated storage conditions.
Both liquid and lyophilized influenza vaccine formulations were not affected by storage at 4°C and 24°C for a month. All vaccines showed a recovery of both HA1 and antigenicity that ranged between 90 to 110% (data not shown). After one-month storage at 37°C, freeze-dried vaccines show HA1 recoveries of approximately 100%, whereas antigenicity slightly decreased to approximately 80%. In contrast, antigenicity of liquid subunit, split and WIV vaccines was lowered below 70% after one month at 37°C, while liquid virosomal vaccines retained 81% of their original antigenicity. HA1 recoveries for liquid vaccines after one-month storage at 37°C ranged from 84 to 103%. After
Vaccine Formulation RMC (%) Tg (°C) HA1 recovery (%) Antigenic recovery (%)
37°C 60°C 37°C 60°C
WIV Liquid n.a.a n.a.a 99 ± 5 n.d.b 69 ± 5 n.d.b
Lyophilized 1.3 ± 0.1 104 96 ± 20 91 ± 12 82 ± 7 83 ± 25
Virosome Liquid n.a. n.a. 103 ± 6 n.d. 81 ± 6 n.d.
Lyophilized 1.8 ± 0.2 99 104 ± 7 84 ± 6 87 ± 13 93 ± 11
Split Liquid n.a. n.a. 89 ± 3 n.d. 64 ± 16 n.d.
Lyophilized 1.4 ± 0.2 103 105 ± 8 83 ± 7 85 ± 8 103 ± 9
Subunit Liquid n.a. n.a. 84 ± 1 n.d. 69 ± 3 n.d.
Lyophilized 2.1 ± 0.4 95 109 ± 5 82 ± 10 74 ± 12 96 ± 3
Table 2. Heat stability of lyophilized influenza vaccine formulations. Residual moisture content (RMC) of lyophilized vaccine formulations was determined directly after lyophilization. Glass transition temperatures (Tg) were calculated from RMCs.Lyophilized vaccines were stored for one month at 37°C and 60°C. Recovery of HA1 and antigenicity was determined after reconstitution and is shown as a percentage of initial recovery after lyophilization. Data represent mean ± SD (n = 3).
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one-month storage at 60°C, lyophilized influenza vaccines were minimally affected, with HA1 and antigenicity recoveries being higher than 83%. Strikingly, liquid influenza vaccines showed no detectable recoveries of either HA1 or antigenicity after one-month storage at 60°C, indicating that the liquid vaccines were completely deteriorated.Biodistribution of vaccine-filled Bioneedles To investigate the vaccine release profile of influenza vaccine-filled Bioneedles, an infrared dye was either filled in Bioneedles or bound to influenza WIV vaccine before filling and subsequent freeze-drying. Nude mice received either IRdye or IRdye-labeled WIV s.c. or by Bioneedle. The release of IRdye was tracked over time with a bioimaging system. The images are presented in
0 hr 6 hr 1 hr 2 hr 24 hr 48 hr 72 hr s.c. WIV
s.c. IRdye Bioneedle IRdye Bioneedle WIV
Time 2.0 1.5 1.0 0.5 Radiant Efficiency ([p/s/cm²/sr] / [µW/cm²]) x 108
Figure 7.In vivo imaging of vaccine-filled Bioneedles. IRdye and labeled WIV were administered either s.c. or by Bioneedles in mice, after which the fluorescence was tracked over time.
bioneedle-delivered influenza vaccines
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Figure 7. Subcutaneous administered IRdye rapidly disappears from the site of injection (SOI), and is completely undetectable after 6 hours at the SOI. In contrast, Bioneedle-delivered IRdye remained visible at the SOI up to 24 hours post administration, indicating that the IRdye was slowly released from the Bioneedle (Figure 8A). Surprisingly, s.c. administered WIV remained at the SOI for a prolonged period of time, similar to Bioneedle-delivered WIV (Figure 8B), although Bioneedle-delivered WIV maintained a steady antigen presence at the SOI even after 72 hours. Total release of WIV over time did not differ between s.c. and Bioneedle groups (Figure 8C).
0 2 4 6 0 24 48 72 168 Time (hr) s.c. IRdye Bioneedle IRdye 1.0x109 8.0x108 6.0x108 4.0x108 2.0x108
A
Radiant Efficiency ([p/s/cm²/sr] / [µW/cm²]) x 10 8 0 2 4 6 0 24 48 72 Time (hr) Bioneedle WIV s.c. WIV 2.0x108 1.5x108 1.0x108 5.0x107B
Radiant Efficiency ([p/s/cm²/sr] / [µW/cm²]) x 10 8C
s.c. Bioneedle s.c. Bioneedle 0 IRdye WIV 8.0x109 6.0x109 4.0x109 2.0x109AUC of Radiant Efficiency
([p/s/cm²/sr] / [µW/cm²]) x 10
8
Figure 8. Quantitative fluorescence at the site of injection over time for both IRdye (A) and WIV (B). Area under the curve (AUC) was calculated for each mice (C). Data represents mean ± SD (n= 5).
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This study demonstrates that influenza vaccines delivered by Bioneedles elicit equal or improved immune responses in C57BL/6 mice compared to conventional liquid vaccines. Furthermore, lyophilization of the different influenza vaccine types formulated with trehalose and HBS greatly improved the heat stability of the influenza vaccines.
The four influenza vaccine types were selected for their differences in compositions and particulate organization. All vaccines were produced from a single virus batch, enabling a fair comparison between the vaccines. Comparative immunogenicity studies remain few [8, 19], and studies on subcutaneous influenza vaccines are mostly limited to a single vaccine type. This knowledge gap makes it difficult to preselect the most suitable influenza vaccine candidate for delivery with Bioneedles. Therefore, a head-to-head comparison between these four vaccines was performed in this study. Characterization of the produced influenza vaccines confirmed that WIV and split vaccines contained all viral components, whereas virosome and subunit vaccines only contained the membrane proteins HA and NA. Determination of the particle size and polydispersity of the vaccines showed that WIV and virosome vaccines do have a relatively monodisperse particle size. In contrast, the produced split and subunit vaccines showed heterodisperse particle sizes, indicating that the vaccines contained viral components in a less organized fashion. WIV and virosome vaccine retained their particle size after freeze-drying, while split and subunit vaccines displayed still a heterodisperse particle size. These differences however did not influence the survival of the trehalose/HBS stabilized vaccines after freeze-drying in vials; a complete recovery of HA1 protein (HPLC) and vaccine antigenicity (SPR) was observed after freeze-drying. This indicates that influenza vaccine containing Bioneedles can be produced without the loss of vaccine structure and antigenicity.
Immunization of C57BL/6 mice with all influenza vaccine-filled Bioneedles induced strong systemic humoral responses. Serum HI titers induced by vaccines delivered by Bioneedles were higher than the HI titers induced by intramuscular administered subunit vaccine and comparable to HI titers induced by intramuscular administered WIV vaccine. Overall, Bioneedle-delivered influenza vaccines induced similar HI titers as their liquid counterparts, indicating that this alternative method of administration could be used for influenza vaccines. The HI titers induced after i.m. immunization with WIV or subunit vaccine were comparable to those found in the study by Hagenaars et al[8]. This might indicate that in terms of HI titers, subcutaneous and intramuscular administered liquid influenza vaccines elicit comparable responses.
The current correlate of protection (CoP) for human influenza vaccines is indicated by an HI titer higher than 40 in humans after vaccination [20]. However, previous studies have shown that cellular immune responses are important as well, especially for pandemic vaccine candidates [21]. Additionally, it is believed that cellular responses may play an important role in the elderly, in which vaccines fail to elicit adequate antibody responses [22]. Analysis of the cellular immune responses