Stability of enveloped and non-enveloped viruses in a liquid formulation of hydrolyzed gelatin | Journal of Virology

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The experiments discussed above revealed the stability of enveloped and non-enveloped viruses in liquid gelatin formulations. Gelatin liquid formulations with high molecular weights of 60,000 and 160,000 showed a drastic loss of infectivity, which was greater than 2 LRV, compared to that of HG at 25°C. The same experiment did not was performed at 4°C due to the high gelation factor of high molecular weight gelatin. Higher molecular weight had a negative effect on the stability of the virus, which can probably be caused by the high viscosity factor disadvantaging the stability of the virus particles in the formulation. Another possibility is the greater loss of viral infectivity when thawing the gelled samples at 37°C, which required a longer melting time due to the higher gel strength than HG in which it does not. There was no gel strength and no thawing process was required after storage at 4°C or 25°C prior to CPE testing. Among the HG samples, the 4000 MW sample appeared to improve the thermal stability of the virus at 25°C or 4°C compared to HG samples of lower MW including 650 and 1980, and the 5% concentration improved. proven to be the optimal concentration to maintain virus stability. Hydrolyzed gelatin acts as an antioxidant and electron donor to produce stable products that can react with free radicals [21]. Hydrolyzed gelatin is commonly present in some vaccines at different concentrations as a stabilizer [22]and the optimal concentration may depend on the buffer or other ingredients in the formulation.

All viruses studied showed stability at 4°C for at least 8 weeks, while BHV-1 and AdV remained stable for more than 30 weeks in storage. In this report, BHV-1 was also stable for 3 weeks at 25°C, showing an improved result compared to previous formulations. In another report, stability of HSV lasted for a period of 39 weeks at 4°C in a 0.5% partially HG or rHSA liquid formulation [23]. The observed difference could be explained by some factors, including the heterogeneous nature of HG, the difference in buffer compositions used in each study, the virus species used for the stability test or, most likely, the initial stock of virus. Both BHV and HSV belong to the same Herpesviridae subfamily; HSV belongs to the genus Simplexvirus and BHV belongs to the genus Varicellovirus [24]. However, the BHV-1 progeny appear to comprise an abundance of capsid-less, non-infectious light particles, unlike HSV, and both show variations in tegument content. [25]. BHV-1 in gelatin-free GTS buffer was stable at −80°C (0.2 log10 reduction) after 8 weeks of storage, but not at ambient temperatures such as 4°C or 25°C (Supplementary File 1 : Table S1). Moreover, its stability was reduced (0.7 log10) when it underwent a series of three freeze-thaw cycles and was significantly different from that of GTS with HG. The only difference between the two formulations in the freeze-thaw cycle test was the presence of HG; therefore, we speculate that the potential cryoprotective characteristics of HG used in this study in the freeze-thaw process had a positive impact on viral stability. The cryoprotective characteristics of HG have previously been reported with immune biomarkers [26] and food products [27] or probiotics [28, 29]and further study is needed to understand these features in the freezing process and their impact on virus stability.

The LRV of AdV-5 was 0.6 log10 after 30 weeks of storage at 4°C. Previous studies have reported stability of recombinant adenovirus at 4°C with optimal formulation for 12 weeks or 24 months [30, 31]. There are possible factors contributing to the difference in long-term stability over 24 months (104 weeks) in the previous study [31] compared to that at 30 weeks in this study. These factors include the use of glass vials for virus storage and surfactants such as polysorbate 80 to further prevent virus adsorption to the glass surface or additional ingredients in the other study formulation, which are most likely needed for longer storage times. However, wild-type adenovirus is known to be stable as a non-enveloped virus for several years at -80°C, but its stability profile at ambient temperatures, such as 4°C and 25°C, is poor. known. In fact, the wild-type adenovirus used in this study is reported by the manufacturer to have a significant drop in infectious titer after 12 months of storage at -20°C. Another study in which the stability of an adenovirus strain in a freeze-dried formulation lasted 24 weeks at 4°C [32] did not show greater stability than the 30-week stability in the liquid formulation in this study. In addition, the adenovirus remained stable at 25°C after 8 weeks of storage compared to 1 month (4 weeks) at 30°C in the previous study. Improving virus stability, including for vaccine protection and viral oncolytic therapy, would alleviate the cold chain burden and cost required.

Neither the PIV nor RV models remained stable over 3 weeks of storage at 25°C, unlike the DNA virus models used in this study. Components of the outer capsid of RV play a critical role in virus stability, and the inner core appears to be more thermostable than the outer capsid. The σ3 outer capsid protein preserves infectivity by stabilizing the μ1 protein, which functions to penetrate the host cell membrane [33, 34]and its alteration by chemical or physical agents probably influences the infectivity of the virus [35, 36]. However, a higher infectious stock virus titer may have improved the stability results of PIV and RV because one of the factors of virus storage stability is the stock virus titer. [23, 37]. In fact, the stock titers of PIV and RV were a thousand times lower than the stock titers of BHV and AdV, indicating that they were stable in the HG liquid formulation. Therefore, the poor stability profile of PIV and RV in HG is most likely the result of low virus stock titers rather than the developed HG liquid formulation. To confirm this claim, another new virus stock was prepared to achieve a higher titer for RV. Evaluation of stability with higher titer in the same formulation of HG revealed better stability compared to stability with lower viral titer (Supplementary File 1: Table S2).

Interestingly, among the HG samples, those that resulted from the basic extraction (alkaline treated) showed better results than the sample obtained by the acid extraction method (acid treated) with the BHV-1 model. , and an interpretation of this result would require further investigation. In general, the pi (isoelectric point) values ​​of alkaline-treated and acid-treated gelatin are low (acidic) and high (basic), respectively. These HG samples with pI around 5 or 8 were produced by alkaline and acid extraction methods respectively. The pi of herpesvirus would be around 9.6 [38]. There is a possible electrostatic interaction between HG with a pI of 5 and the herpes virus to maintain the stability of the surface proteins of the virus during storage at physiological pH. These results should be interpreted with caution. It is too preliminary to conclude that pi is a factor affecting virus stability. Further studies are needed to explore the potential effect of gelatin pi on virus stability.

Nevertheless, this research has some limitations. The stability profiles of other viruses, including PIV, RV and AdV, with known gelatin parameters, such as MW, concentration or method of extraction, have not been evaluated. In addition, only the stability of BHV was assessed in gelatin-free buffer or in freeze-thaw stability tests. Additionally, the effect of the current formulation on a vaccine model or genetically modified virus such as oncolytic virus for potential retention of immunogenicity or thermal stability has not been evaluated and would be investigated in a future study.

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