MS2 VLPs are produced in E. coli through expression of the MS2 bacteriophage coat protein (CP). This self-assembles into a roughly spherical shell that mimics the structure of the virus. The resulting particles are non-infectious and can be produced in large quantities. Moreover, CP tolerates significant insertions (up to 29 amino acids) or substitutions, meaning that heterologous peptides can be incorporated into the particle (Mastico et al., 1993, Peabody, 1997a, Peabody, 1997b, Peabody, 2003, Peabody et al., 2008). These characteristics have been explored with the aim of developing MS2 as an antigen presentation platform (Heal et al., 1999, Tumban et al., 2012, Ord et al., 2014, Basu et al., 2018, Mogus et al., 2020). In addition to their use in vaccine development, MS2 VLPs have also been employed for encapsulation of chemical compounds (Wu et al., 1995, Ashley et al., 2011, Aanei et al., 2018), fluorophores (Capehart et al., 2013), contrast agents (Datta et al., 2008) or short DNA fragments (Wu et al., 2005, Zhang et al., 2015, Zilberzwige-Tal et al., 2021). Studies on how MS2 bacteriophage packages its own RNA genome revealed that during capsid assembly the CP specifically interacts with a stem-loop RNA structure called the “pac site”, which in turn triggers its encapsulation (Peabody, 1993, Stockley et al., 1995, Peabody, 1997a, Peabody, 1997b, Toropova et al., 2008). Importantly, this process is retained when the pac site sequence is fused to a foreign sequence of interest. This knowledge has subsequently been exploited to package heterologous RNA fragments (Pickett and Peabody 1993), which in turn has applications in siRNA delivery or “armored RNA” technology (probes for RT-PCR quantification of RNA viruses) (Pan et al., 2012, Zambenedetti et al., 2017, Naskalska and Heddle, 2024). Lastly, if MS2 VLPs are chemically disassembled and subsequently reassemble in the presence of negatively charged molecules, they can entrap them (Glasgow et al., 2012, Aanei et al., 2018).

However, although several applications of MS2 VLPs have been demonstrated, some aspects of their use remain challenging. For instance, installation of large antigens on the surface of MS2 VLP cannot be achieved by genetic insertion in the CP, as this is structurally and sterically too disruptive to VLP assembly. Further, some antigens require posttranslational modifications to confer immunogenicity – a requirement that E. Coli expression systems are unable to fulfill. One approach to overcome this issue is incorporation of a universal adapter molecule into the MS2 VLP, allowing attachment of an antigen of choice, which itself may be produced in a tailored (e.g. eukaryotic) expression system. SpyTag peptide or SpyCatcher protein can serve as such universal adapter molecule (Zakeri et al., 2012) and the utility of the SpyTag/SpyCatcher technology in displaying antigens on VLPs has been proven by numerous studies (Brune et al., 2016, Brune and Howarth, 2018).

The volume of the MS2 VLP lumen imposes restrictions on the size and quantity of potential cargoes. This is an important consideration given that potentially the most appealing application is packaging and delivery of functional, heterologous mRNA. Additionally, the packaging of short, single stranded oligodeoxynucleotides (ODNs), which can act as adjuvants, is of interest for vaccine development (Bode et al., 2011, Hanagata, 2017). To be maximally functional, these ODNs must be protected from nucleases and efficiently delivered to the intracellular receptors of antigen presenting cells (Zhang and Gao 2017).

We previously engineered MS2 VLPs to have an increased size (Biela et al., 2022), with the aim of creating a prototype universal vaccine platform consisting of a recombinant MS2 VLP, bearing multiple externally displayed and accessible SpyTag peptides and having a diameter increased by 4.4 nm (compared to wild type MS2 VLPs). Of note, our enlarged MS2 VLPs exhibit altered particle geometry, with a population having T = 4 symmetries instead of the T = 3 that is typically seen for wild type MS2 VLPs (Stockley et al., 2007) (Supplementary Fig. 1 and Supplementary Table 1). The resulting increase in size translates to an increased surface area and a larger number of attachment points for SpyCatcher-fused antigens (120 compared to 90 in the wild type VLP). The lumen volume is also increased (from 5188 nm3 to 8156 nm3), allowing for encapsulation of larger sized and/or greater numbers of cargo molecules (Supplementary Table 2).

In this work we examine whether these engineered MS2 VLPs can be externally decorated with a large, complex antigen such as the Receptor Binding Domain (RBD) of SARS-CoV2 spike protein, and if they can also carry fluorescently labeled ODNs or functional mRNA in their lumens. We tested two variants of MS2 VLPs with externally displayed SpyTag, named: MS2-SpyTag and MS2-SpyTag4. These two variants differ in peptide length and therefore the ratio of T = 3 and T = 4 particles (Supplementary Table 1 and (Biela et al., 2022)).