In this study, a cell culture-based production process for purely clonal DI244 particles without STV contamination was established. The production process was scaled up from shake flask to a STR with a working volume of 500 mL. The produced material showed high interfering efficacies in an in vitro interference assay, which was further improved after membrane-based SXC purification. Animal trials performed with unclarified shake flask harvests passed toxicity testing. Finally, mice infected with a lethal dose of IAV could be rescued by co-treatment with this DI244 material, demonstrating its antiviral potential.
Advantages of DIPs over currently used small molecule antivirals
The administration of DIPs to prevent IAV infection might have several benefits. Specifically, DIPs show a fast mode of action, as their protective ability does not depend on the adaptive immune system, which can take up to 2–3 weeks to establish full protection in the case of vaccine administration. Because of their mode of action, DIPs could be used either prophylactically or even therapeutically [10]. It was shown by Dimmock and Easton that egg-derived and UV-inactivated DI244 material administered 7 days before infection still protected mice from a lethal dose of IAV [25]. Moreover, mice infected with a lethal dose of IAV and treated with DI244 24 h after the challenge survived [10]. Partial protection was observed, when DIPs were administered 48 h after the infection [10]. Furthermore, an antiviral effect of DI244 against a variety of IAV strains was shown [10, 23], including pandemic and highly pathogenic avian strains [30]. This suggests that DIPs, in contrast to currently used vaccines, might have the potential to act universally against IAV [25]. Additionally, an antiviral effect against influenza B and pneumovirus was demonstrated [11, 31]. This might be explained by an unspecific protection due to an induction of the innate immune response. Currently, IAV vaccines require annual adaptation to seasonal circulating strains, including accurate prediction, time-consuming generation of a seed virus, and egg or cell culture-based production. In contrast, DIP production in suspension cells would allow manufacturing a high number of doses of an antiviral drug to be better prepared for the next IAV pandemics.
Mice infected with a lethal dose of STV and treated with DI244 did not show symptoms of disease, but still developed an immunity to the pathogenic STV [10]. Here, it was speculated that DIP co-treatment results in the release of non-infectious particles carrying the surface proteins of the pathogenic virus and therefore acts like a live attenuated vaccine [25]. In line with this, it was shown that compared to an untreated control group, DI244 co-treatment did not influence the amount of specific IAV antibodies produced in ferrets. In contrast, reduced antibody titers were detected in oseltamivir treated ferrets [23], emphasizing potential advantages of DIPs over conventional antivirals. Furthermore, resistance against the small molecule antivirals oseltamivir and zanamivir has already been reported [4,5,6], whereas it is highly unlikely that resistance arises against DIPs: the RNA-dependent RNA polymerase complex (comprising the polymerase subunits PB2, PB1, and PA proteins) would need to mutate to not recognize and replicate the DI vRNA anymore [25]. However, the same polymerase complex replicates all eight vRNA segments [32,33,34]. Thus, in addition to mutation of the viral polymerase complex, it would be necessary that mutations of the polymerase recognition sequences of all eight STV vRNA segments arise simultaneously. Only under these circumstances could STV replication without DIP replication take place [25]. The probability that this happens is extremely low and was previously estimated to be around 1E−45 [25]. In conclusion, DIPs with their unique antiviral mechanism are very interesting candidates for prophylactic and therapeutic treatments showing advantages over currently used small molecule antivirals.
Advantages and opportunities of a cell culture-based production processes
The cell culture-based production process established has several advantages over the previously reported egg-based process [10]. First, it has improved sterility, scalability, and flexibility. Second, it allows for comprehensive monitoring, and tight process control enables a reproducible product quality [19, 20, 35, 36]. Additionally, genetically modified cells can be used to allow production of purely clonal DIP preparations [26, 27], which completely overcomes the necessity of STV inactivation. Previously, UV light was used to disrupt the STV vRNA [10, 36] by introducing photodimeric lesions [37] or unspecific chain breaks [38, 39]. Here, it was speculated that larger STV vRNA (~ 2.0 kb) should be faster inactivated than the rather small DIP vRNA (~ 0.4 kb), as the probability of damaging photoreactions is higher for the STV vRNA [10]. However, it was shown recently that also the DIP vRNA is damaged by UV light, resulting in a decreasing interfering efficacy over UV inactivation time [40]. Moreover, UV inactivation was also used in the current study to generate a negative control, which did not show any interfering efficacy in the interference assay (Fig. 6) or in animal experiments (Figs. 7 and 8). In contrast, the interfering efficacy of the purely produced DIP material was maintained at a very high level. Using our approach, concerns regarding biosafety, i.e., the risk of residual infectious STV due to incomplete inactivation after UV treatment can be avoided.
In principle, the genetically modified MDCK-PB2(sus) may be used universally for cell-culture based production of any IAV Seg1 DIP [26]. The interfering efficacy of a DIP seems to be affected by many factors including genome length, genome sequence, and breaking point [16, 41,42,43]. Therefore, other DIPs could have a higher interfering efficacy or offer additional advantages over DI244. The generation of genetically modified cell lines expressing another viral protein, e.g., the viral PB1 or PA protein, would also allow production of purely clonal Seg2 or Seg3 DIPs. These segments are of special interest, as deletions in Seg1–3 are most frequently observed [16, 44]. Here, it was hypothesized that DIPs originated from polymerase genes (Seg1–3) may have advantages over DIPs originating from structural genes (Seg4–8) [22]. For the generation of a purely clonal DIP seed virus, the reverse genetics approach reported earlier represents a universal platform [26].
Lastly, the separation principle of the SXC allows purification of any IAV strain using a single recipe [45]. Therefore, the established platform covering cell line generation, seed virus generation, DIP production, and DIP purification allows to quickly produce a wide range of DIP candidates for testing in an animal model for further use as an antiviral.
The MODIP affected the incorporation of DI244 vRNA in the produced virus particles
In the present study, Seg5 and Seg8 vRNA levels (considered representative for all STV vRNA segments) were approximately equimolar for each MODIP and each sampling time point. In contrast, DI244 vRNA levels were always lower. This might suggest that Seg5 and Seg8 vRNA were present in every virus particle, whereas the DI244 vRNA was absent in some virus particles. Usually, virus assembly and budding were considered a well-organized process, where each of the eight vRNAs is incorporated in the produced virus particle only once [46, 47]. This is facilitated by the packaging sequence, present at the 3′ and 5′ end of each vRNA segment [48, 49]. Nevertheless, depending on the strain, up to 20% of produced viruses do not package at least one vRNA segment [50]. This results in the generation of semi-infectious particles [51]. Naturally occurring virus mutants which completely miss several vRNA segments have also been observed [17].
The MDCK-PB2(sus) cell line used here expressed the viral PB2 protein, encoded by Seg1 vRNA. With the cell line providing the missing PB2 protein, the virus propagation theoretically does not require an intact Seg1 vRNA. Concurrently, also the deleted Seg1 vRNA from DI244 is not essential for replication. Therefore, the MDCK-PB2(sus) cell line might not only allow production of purely clonal Seg1 DIPs, but also propagation of viruses with only seven segments, completely missing the Seg1 vRNA. This might also explain the lower level of DI244 vRNA in the produced virus particles. Furthermore, DI244 vRNA levels decreased with lower MODIPs. A possible explanation may be that higher MODIPs result in overall more co-infections and therefore a higher probability that all eight segments are present in an infected cell. Here, most produced viruses would incorporate all eight segments. In contrast, in a low MODIP scenario, the likelihood for single-hit infections is increased drastically. Under this condition, cells may occasionally be infected by a virus without DI244 vRNA. Consequentially, those cells could only produce viruses also missing DI244 vRNAs. Additionally, the produced 7-segmented viruses will further accumulate in subsequent infection waves (which are characteristic for low MODIPs).
The lack of DI244 vRNA could also explain the observed differences between plaque and interference assay. More specifically, MODIPs of 1E−2 to 1E−4 resulted in very comparable DI244 titers. In contrast, large differences in the interfering efficacy were observed, where material produced with lower MODIPs induced a less pronounced titer reduction. The DI244 titer was evaluated in a plaque assay with MDCK-PB2(adh) cells. Here, virus particles without incorporated DI244 vRNA could still replicate and would therefore contribute to the DI244 titer. On the other hand, particles without DI244 vRNA would not interfere with STV replication. Therefore, these particles would not contribute to the interfering efficacy determined in the interference assay. Consequentially, material produced at a MODIP of 1E−2, showing the highest DI244 vRNA level (for MODIP 1E−2 to 1E−4) resulted in the most pronounced titer reduction. DIP material produced at a MODIP of 1E−1 showed comparable vRNA levels, but lower DI244 titers. A possible explanation could be that the interfering efficacy of this material is lower due to a faster onset of DI244 production, resulting in an earlier onset of degradation of biologically active virus particles and a reduced number of virions carrying DI244 vRNA to the cells. Another explanation for the observed reduction of DI244 and HA titers of material produced at a MODIP of 1E−1 might be a self-interference of DIPs at high MODIPs, as reported by other groups [52, 53].
The mechanism proposed here, where DI vRNAs might not be efficiently incorporated in the produced virus particles, when produced with a complementing cell line, would have implications for the DIP production process. The risk that high amounts of virus particles without any therapeutic effect might be produced, especially at lower MODIPs (usually used for cell culture-based viral vaccine production) is of particular importance. Therefore, optimization of the MODIP seems crucial for the establishment of a production process as it might drastically affect the quality of DIP harvests.
Purification of DI244 harvests
The product yield of the SXC purification step measured by hemagglutination assay was 92.3%. This is consistent with previous results for downstream processing of IAV [54], as was the concentration of the HA antigen in the SXC eluate of this work (16.0 μgHA/mL). The clearance of host cell DNA was 97.1%, and adding a DNA digestion step prior to SXC increased the DNA clearance to 99.95%. The total protein clearance was 97.2%, which is also consistent with previously reported data [54].
The dsDNA concentration of the clarified virus harvest in this work was 4495 ng/mL compared to around 4300 ng/mL from a similar process for IAV production using SMIF8 chemically defined medium [54]. With a dsDNA concentration of 192 ng/mL in the eluate, the estimated dsDNA concentration of this material was 0.05 ng/μL and therefore about 100-fold lower than that of the unpurified material (4.6 ng/μL) administered to mice for toxicity testing. A further reduction in the amount of DNA can be achieved, for instance with optimizations in cell culture that could reduce the total burden of DNA introduced into the purification train, a longer DNA enzymatic digestion, or with additional polishing steps, such as pseudo-affinity chromatography with sulfated membrane adsorbers [55] or ion exchange chromatography.
Another interesting topic would be the separation of active DI244 particles from 7-segmented viruses, discussed in the previous section. The difference in the nucleotide cargo between the two virus populations might result in an isoelectric point difference that could be exploited for their chromatographic separation by isoelectric focusing [56, 57] or even ion exchange. These options provide alternatives for future work.
Evaluation of DI244 interfering efficacy in animal and in vitro models
Administration of DI244 alone did not induce body weight loss nor result in a decreased survival rate and therefore did not appear to be highly toxic. To further elaborate potential toxicity of DI224 administration, histopathology or blood chemistry of mice could be performed [58]. Co-treatment with DI244 did not show a positive impact on body weight loss or survival rate in PR8 (H1N1) STV infected D2-Mx1−/− mice. In strong contrast, infected D2-Mx1r/r mice treated with DI244 showed a reduced body weight loss and all animals survived the infection.
MX1 represents an interferon-induced protein, which binds to the ribonucleo-protein particles of IAV and thereby inhibits viral replication [59, 60]. The protective activity of MX1 against IAV was originally discovered in A2G mice that carry a wild-type Mx1 allele [59, 61]. However, most laboratory strains, including the most commonly used strain, C57BL/6, do not carry a functional Mx1 allele [59, 62]. These common laboratory mice express Mx1 transcripts with a deletion or nonsense mutation in the open reading frame resulting in a non-functional protein [59, 62]. The wild type functional Mx1 allele has been transferred from A2G to C57BL/6 mice [63] to generate strain B6.A2G-Mx1r/r (B6-Mx1r/r). B6-Mx1r/r mice are per se highly resistant against infections with IAV [63, 64]. Since these mice scarcely show clinical symptoms and mortality, they do not allow testing of anti-viral treatments.
Humans carry a functional MX1 allele, but are still susceptible to IAV infections. Therefore, we generated a mouse model that better mimics the human situation. We introduced the wild type Mx1 allele into DBA/2 mice, resulting in mouse strain D2-Mx1r/r, which now expresses a fully functional MX1 protein. In contrast to B6-Mx1r/r mice, D2-Mx1r/r mice are still highly susceptible to IAV infections. However, after pretreatment with interferon, D2-Mx1r/r become resistant to IAV infections demonstrating that in these mice a fully functional and protective MX1 protein can be produced. We previously [29] have described the detailed characterization of the D2-Mx1r/r model. Thus, our D2-Mx1r/r IAV infection model which has been used here for the DI244 functional studies represents an ideal system that better reflects the human situation and allows testing of antiviral treatments in the context of a fully functional Mx1 allele.
The infection experiments with the different mouse models demonstrate the importance of a functional innate immune response for the antiviral effect of DIPs in vivo. Here, we hypothesize that DI244 in mice inhibits viral replication, as we have demonstrated in our in vitro studies, but in addition induces interferon, which subsequently activates the highly protective functional Mx1 gene in D2-Mx1r/r mice. Both effects will lead to lower viral loads in the lung, the rapid induction of a potent innate immune response, and protection from a lethal outcome. Virus-specific antibodies and cytotoxic T cells typically start to appear at 7 days post infection and are fully mounted after 14 days. Thus, an adaptive immune response would come too late in a primary infection to protect against a severe outcome during the first week. We thus conclude that in this model, DI244 does not primarily have a vaccination effect. However, mice treated with DIPs will survive the infection, and in these mice, an adaptive immune response will be mounted. Such an adaptive immune response will protect against a secondary infection [10]. Therefore, DIP treatment will not only be beneficial for protection against severe disease in the early phase of a primary IAV infection but also contribute to mounting a long-lasting protective immunity. Here, it would also be of interest to investigate the impact of the time of DIP application (e.g., a few days before or after challenge virus administration).
The in vitro interference assay used here was carried out with MDCK cells, which express a canine MX1 lacking activity against the human IAV strain PR8 [65]. Furthermore, trypsin added to the medium used in the interference assay degrades the secreted interferon [66]. Therefore, the interfering effects observed in the in vitro assay are most likely explained by DIPs interfering with the replication of the STV, rather than induction of the innate immune response. In order to understand better the contribution of the innate immune response to the interference of DIPs in vitro, additional experiments are necessary. For example, the interference assay could be carried out with a human cell line carrying a functional MX1 (e.g., A549 or HEK293 cells). Additionally, to avoid interferon degradation by trypsin, the virus strain A/WSN/33, which does not rely on trypsin addition for its propagation [67], could be used and is topic of ongoing research.
Finally, it would be desirable to conduct infection experiments in ferrets, as they are susceptible to human IAV and air-borne virus transmission [68,69,70]. In a next step, infection experiments in macaques could be carried out, as their clinical symptoms closely resemble those found in humans [71]. Trials in both animals would represent a significant step towards studies in humans to demonstrate the protective effect of DIPs and the use of DIP preparations as antiviral drugs. Here, it would also be of interest to investigate the impact of the time of DIP application (e.g., a few days before or after challenge virus administration).