Injectable Slow-Release Hydrogel Formulation of a Plant Virus-Based COVID-19 Vaccine Candidate

Abstract

Cowpea mosaic virus (CPMV) is a potent immunogenic adjuvant and epitope display platform for the development of vaccines against cancers and infectious diseases, including coronavirus disease 2019. However, the proteinaceous CPMV nanoparticles are rapidly degraded in vivo. Multiple doses are therefore required to ensure long-lasting immunity, which is not ideal for global mass vaccination campaigns. Therefore, we formulated CPMV nanoparticles in injectable hydrogels to achieve slow particle release and prolonged immunostimulation. Liquid formulations were prepared from chitosan and glycerophosphate (GP) before homogenization with CPMV particles at room temperature. The formulations containing high-molecular-weight chitosan and 0–4.5 mg mL –1 CPMV gelled rapidly at 37 °C (5–8 min) and slowly released cyanine 5-CPMV particles in vitro and in vivo. Importantly, when a hydrogel containing CPMV displaying severe acute respiratory syndrome coronavirus 2 spike protein epitope 826 (amino acid 809–826) was administered to mice as a single subcutaneous injection, it elicited an antibody response that was sustained over 20 weeks, with an associated shift from Th1 to Th2 bias. Antibody titers were improved at later time points (weeks 16 and 20) comparing the hydrogel versus soluble vaccine candidates; furthermore, the soluble vaccine candidates retained Th1 bias. We conclude that CPMV nanoparticles can be formulated effectively in chitosan/GP hydrogels and are released as intact particles for several months with conserved immunotherapeutic efficacy. The injectable hydrogel containing epitope-labeled CPMV offers a promising single-dose vaccine platform for the prevention of future pandemics as well as a strategy to develop long-lasting plant virus-based nanomedicines.

Injectable Hydrogel Containing Cowpea Mosaic Virus Nanoparticles Prevents Colon Cancer Growth

“…This ranking is consistent with the viscosity of these liquid formulations we previously recorded at 25 °C (0.099 Pa•s for F1; 0.202 Pa•s for F2; and 0.482 Pa•s for F3). 31 The formulation F3 was found to be the most viscous, but the recorded viscosity value (<1 Pa•s) and passage through a 26 G needle suggest that the F3 formulation has huge potential for good flow and spreading within the TME; 34 other authors established the injectability of a chitosan/GP-based formulation by passing through a 23 G needle. 35 We used Cy5-CPMV particles to characterize hydrogel degradation, swelling, and release profiles; these data are reported in Nkanga et al 31 Although no obvious gel degradation or swelling was observed, the UV spectrometric analysis of the release medium revealed continuous release of CPMV particles from the formulated hydrogels.…”

“…This is consistent with previous observations from scanning electron microscopy of freeze-dried CPMV hydrogels pre-and post-CPMV release. 31 The release rate of CPMV from all the hydrogels was much slower (only 20% released by day 28) compared to that of the controls where CPMV was sampled from PBS (up to 95% was depleted by day 10 due to constant sampling and medium renewal). The fast diffusion of CPMV particles in solution is essentially governed by the Brownian motion, whereas the slow release of CPMV from the hydrogels may reflect the existence of physical or/and chemical hindrances within the polymeric matrix; 36,37 thus, we characterized CPMV particles from hydrogels to verify whether their properties remained unchanged (Figure S2).…”

“…Key formulation properties, such as gelation time, gel swelling, degradation, and release profiles, were determined as previously reported. 31 The particulate characteristics of in vitro released CPMV particles were investigated by SEC, DLS, and TEM to confirm particles' integrity. Detailed procedures of hydrogel preparation and characterization are described in the Supporting Information file.…”

Gelatin-methacryloyl hydrogels containing turnip mosaic virus for fabrication of nanostructured materials for tissue engineering

“…Plant viruses are, in principle, incapable of infecting mammalian cells (Lauria et al, 2017;Balke and Zeltins, 2020). Due to their protein nature, they are expected to eventually undergo enzymatic degradation under physiological conditions (Nkanga et al, 2022). However, to our knowledge, no experiments have assessed the fate of implanted hydrogels containing TuMV.…”

“…Overall, the results suggest that plant virus-based vaccines could be introduced into clinical and veterinary practice in the near future (Folia Biotech Inc, 2000;Chichester et al, 2018). More research is needed before plant viruses can be declared safe for use in hydrogels destined for medical implantation, particularly as several plant viruses have proven to be potent adjuvants (Folia Biotech Inc, 2000;Sánchez et al, 2013;Chichester et al, 2018;Nkanga et al, 2022) and may trigger clinically relevant immune responses if implanted.…”