Stabilized single-injection inactivated polio vaccine elicits a strong neutralizing immune response

Figures

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  Fig. 1.. Eudragit E and bPEI have buffering capacity and affect PLGA degradation. The buffering capacity of Eudragit E, PLL, and bPEI was measured (A). After they were blended with PLGA and formulated into microspheres, Eudragit E and bPEI accelerated PLGA degradation over the course of a release study (B), observed as a difference in the rate at which the peak molecular weight (Mp) of the PLGA decreased. The release of acid from the microspheres into the external release medium correlated with PLGA degradation and total release of type 1 IPV, all of which are affected by incorporation of Eudragit E or bPEI (C–F). Release of Eudragit E or bPEI from the microspheres (plotted as a percentage of the total loading) was also pulsatile and matched the timing of IPV release. Data are reported as mean ± SD.

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  Fig. 2.. Acid-induced aggregation of IPV is prevented by complexation with polycations. IPV forms a broad distribution of large aggregates at low pH (A) when diluted in PBS without excipients. Cationic polymers, such as PLL, Eudragit E, and bPEI, prevent acid-induced aggregation at low pH (B). After 24 h of incubation at low pH, PLL is the only cation to preserve a large population of virus particles with diameters close to 30 nm at low concentration (25 μg/mL), while Eudragit E prevented some but not most of the aggregation (C). Serotypes 1 and 3 act differently in the presence of polycations, with type 3 showing the least aggregation at low pH in the presence of polycations (D–F).

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  Fig. 3.. The polycation PLL mitigates acid-induced aggregation of IPV. IPV can be observed mostly as individual 30-nm particles at pH 7.4 (A) but forms massive aggregates at low pH 4.5 (B). Addition of PLL causes only a few minor aggregates at neutral pH (C) and prevents the formation of large aggregates upon acidification to pH 4.5 (D).

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  Fig. 4.. PLL and polyethylenimine promote stable IPV release from microspheres. All microspheres shown above contained in the same number of doses of tIPV and the same aqueous sugar- and salt-based excipients. Microspheres with Eudragit E cationic polymer in the organic phase released more stable IPV initially and in the second burst after addition of small amounts of PLL in the internal aqueous phase during emulsification, listed as molar ratio of PLL to IPV (A–C). Microspheres included PLL in the hydrophilic compartment and varying amounts of bPEI or Eudragit E in the organic polymer compartment. Microspheres containing 3–6% bPEI tended to release more stable IPV initially and in the second burst than those containing 3% Eudragit E (D–F). Groups that performed statistically significantly better than the control (“No PLL” in A–C; “3% Eudragit E” in D–F) are marked in the figure (*P < 0.05, **P < 0.01).

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  Fig. 5.. F1 and F2 microspheres release stable IPV over >3 wk. F1 and F2 microspheres contain Eudragit E, PLL, and/or bPEI as excipients, as depicted in the schematic (A). As depicted in B, the initial burst of release results from diffusion of IPV on or near the surface of the particles, followed by a lag phase as PLGA begins to degrade by chain scission and finally a second burst as degradation proceeds enough for the particle to lose mass and release internally encapsulated cargo. F1 and F2 microspheres have smooth morphology by SEM (C). (Scale bar, 20 µm.) Release (D–F) and cumulative release (G–I) graphs show that all three IPV serotypes are released in D-antigen conformation in two distinct bursts and that F2 microspheres deliver more stable IPV than F1.

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  Fig. 6.. F1 microspheres elicit a strong neutralizing response against type 1 and type 2 poliovirus. The neutralizing antibodies in the serum of immunized rats is shown as the geometric mean absolute titer (A–C) for types 1, 2, and 3, respectively. A single bolus injection of liquid IPV is represented by the gray X; a single bolus injection of liquid IPV along with empty microspheres is represented by closed gray circles; two bolus injections of liquid IPV at t = 0 and 4 wk is represented by open black diamonds; and a single injection of F1 microspheres is represented by closed black squares. Data represent geometric mean ± geometric SE.

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  Fig. 7.. Formulations F1 and F2 combined elicit a strong neutralizing antibody response to all three IPV serotypes. The total IgG binding titers against IPV type 1 (A), 2 (B), and 3 (C) are shown 4 wk after injection. The absolute neutralizing antibody titers against poliovirus types 1 (D and G), 2 (E and H), and 3 (F and I) elicited by the bolus controls and formulations F1 and F2 are shown before (D–F) and after (G–I) the bolus control group received its second injection. Seroconversion (the percent of animals with detectable neutralizing antibody titers) was comparable in rats immunized with a single injection of F1 or F2 microspheres as in rats administered two standard injections of the liquid bolus (J–L). Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01) compared with the control (bolus injected at t = 0 and t = 4 wk) at the time point shown.