key: cord-0663106-d6npxzqz authors: Francis, Ziad; Incerti, Sebastien; Zein, Sara A.; Lampe, Nathanael; Guzman, Carlos A.; Durante, Marco title: Monte Carlo simulation of SARS-CoV-2 radiation-induced inactivation for vaccine development date: 2020-05-13 journal: nan DOI: nan sha: 5326515d4062391a68718f5e8c1c1d73b4143d88 doc_id: 663106 cord_uid: d6npxzqz Immunization with an inactivated virus is one of the strategies currently being tested towards developing a SARS-CoV-2 vaccine. One of the methods used to inactivate viruses is exposure to high doses of ionizing radiation to damage their nucleic acids. Although gamma-rays effectively induce lesions in the RNA, envelope proteins are also highly damaged in the process. This in turn may alter their antigenic properties, affecting their capacity to induce an adaptive immune response able to confer effective protection. Here, we modelled the impact of sparsely and densely ionizing radiation on SARS-CoV-2 using the Monte Carlo toolkit Geant4-DNA. With a realistic 3D target virus model, we calculated the expected number of lesions in the spike and membrane proteins, as well as in the viral RNA. We show that gamma-rays produce significant spike protein damage, but densely ionizing charged particles induce less membrane damage for the same level of RNA lesions, because a single ion traversal through the nuclear envelope is sufficient to inactivate the virus. We propose that accelerated charged particles produce inactivated viruses with little structural damage to envelope proteins, thereby representing a new and effective tool for developing vaccines against SARS-CoV-2 and other enveloped viruses. The threat from the new SARS-CoV-2 is omnipresent in all regions of the world. Several companies and research groups report possible success in the development of vaccines or drugs. However, the results of the clinical studies are still pending and it is not certain whether these candidates are safe and/or effective (1). Some of the SARS-CoV and MERS-CoV vaccines developed so far followed the most traditional approaches based on inactivated or live attenuated viruses (2) , as well as subunit vaccines. Traditional chemical inactivation methods can lead to the modification of critical epitopes in both inactivated and subunit vaccines, thereby affecting overall vaccine efficacy (3) . Although live attenuated virus-based vaccines generally induce potent cellular immune response, antibody production is often less efficient, and the use of attenuated viruses is often not indicated in individuals who are immunosuppressed as a result of disease or therapeutic interventions. In the current pandemics, RNA vaccines are emerging as very attractive candidates, due to their ease of manufacture. However, no such vaccine has been approved yet for humans and their true value remains to be proven in the field. In this regard, radiation technology is of interest to vaccine manufacturers, because it penetrates pathogens to damage the nucleic acid without residual chemical contaminants (4) . Nevertheless, the development of irradiated vaccines has not been pursued actively over the past 20 years for two main reasons. First, the development of new radiation techniques has been considered impractical or difficult due to issues accessing radiation equipment. Second, it has been thought that modern subunit vaccines would provide a solution, as they can be developed more easily (5) . However, recent successful development of irradiated vaccines for human malaria (6) and influenza (7) have not only demonstrated the feasibility and practicality of this technique, but also the efficacy of such vaccination approaches, prompting research in applying the method to other viruses (8, 9) . Nevertheless, the problem of epitope damage caused by high-dose g-ray exposure is well known and has been recently tackled using radioprotectors (10) . Formaldehyde and other chemicals commonly used for virus inactivation also induce severe damage to the protein structures involved in the elicitation of adaptive immune response post vaccination (11) . With conventional g-ray sterilization, many photons are needed to induce lesions in RNA. However, the track structure of charged particles, such as those used in cancer therapy (12) , suggests that a single traversal is sufficient to induce lethal lesions in RNA with minimal membrane damage. In this work we performed a comprehensive simulation of ionizing radiation damage to the SARS-CoV-2 using Geant4-DNA (13), a Monte Carlo simulation toolkit which is used for modeling biological damage induced by ionizing radiation at the DNA scale. We developed a Geant4-DNA application to simulate the SARS-CoV-2 structure, and calculated the damage to the virus' spike and membrane proteins, and the viral RNA following exposure to radiation of different quality, that is, low-or high-linear energy transfer (LET) radiation. High-LET (densely ionizing) radiation is in arXiv.org 13.5.2020 4 fact densely ionizing and may inactivate the virus with less membrane damage than low-LET (sparsely ionizing) radiation. The Geant4-DNA extension (13) (14) (15) (16) of the Geant4 Monte-Carlo toolkit (17) (18) (19) was used to simulate ionizing particle tracks and energy deposition inside the virus model. The virus membrane was represented by a spherical volume, the outer and inner diameters were set to 100 nm and 80 nm, respectively. These dimensions are based on experimental data from the literature. Figure 1 shows the configuration of the described virus structure. The approximate dimensions of the proteins of the SARS-CoV-2 were estimated based on the descriptions from the protein data bank (24, 25) . The nucleic acid contained in the envelope is a ss-RNA of approximately 30 kbp (26) . For a comparison of sparsely and densely ionizing radiation in the SARS-CoV-2 target, we show in Figure 2 the simulation of the damage produced in the virus by: 60 Co g-rays (LET=0.24 keV/µm), the standard radiation used for virus inactivation in vaccine development; 3 MeV a-particles, a common standard in high-LET radiation (LET=126 keV/µm) with a range of only 18 µm in water; and 56 Fe 450 MeV/amu (LET=178 keV/µm), a densely ionizing ion often used at accelerators in studies of space radiation protection (27) and with a range of approximately 12 cm in water. Probability distributions of the number of RNA damage sites were normalized to one incident particle for Fe-ions, and to ejected g-electron for g-ray photons. Photon cross sections are low in comparison with charged particles and a large number of photon tracks cross the virus volume without interaction, therefore considering the number of primary photons for normalization would be misleading in this comparison. The first bin of the distribution, showing the probability that no damage is induced, is highest for gelectrons, with a probability of ~90% (that is, the probability of at least one RNA damage event is only 10%). For Fe-ions the damage probability is almost 75%, and it becomes 99.8% if only ion traversals though the nuclear envelope are considered. The number of RNA lesions per incident particle reaches up to 30 damage points for Fe-ions, while the largest number observed for g-electrons is 5. arXiv.org 13.5.2020 6 Figure 2 (right column) also shows the distribution of the number of damaged spike proteins for particles and g-electrons, normalized per primary particle, showing a higher damage yield for densely ionizing charged particles. Ideally, we would be looking for a radiation type leading to a high RNA damage yield and a small number of damaged proteins. However, the apparently higher number of damaged proteins is simply due to the dense ionizing track that has a high probability to damage any protein when traversing it. In Figure 2 we focus on arXiv.org 13.5.2020 7 the damage in the nucleic acid (single-stranded RNA) and the spike proteins, which is considered a main antigen target for SARS-CoV-2 vaccine development (28) . The average number of damaged spike proteins normalized to single RNA break is shown in Figure 3 for different incident particles according to their LET. In addition to the particles shown in Figure 2 , we have included protons at 30 MeV and 80 MeV, accelerated at cyclotrons for radionuclide production or eye tumor therapy; 12 C-ions at 80 MeV/amu, available in several research and medical accelerators; and electrons, that are used for sterilization of surfaces. The electron energy of 200 keV was selected since that electron beam studied experimentally as an alternative to grays for vaccine production (29) . A second electron energy of 2 keV was also selected based on the results of the recent Monte Carlo study by Feng et al. (30) that shows that at this energy there is a maximum efficiency in virus sterilization by electrons. We found a decreasing damage ratio with increasing LET, and the minimum value, ~0.5, is obtained using a-particles and iron ions. The ratio for g-rays and 200 keV eis ~8-9. The high ratio obtained for conventional g-rays is due to a low average value of the RNA damage distribution. The estimated survival curves were calculated considering that a single RNA single-strand break is sufficient to inactivate the virus, as it cannot be repaired and will result in an interrupted amino acids chain. Thus, the probability of inducing at least one RNA damage was used in an exponential survival model. Figure 3 (right) shows the SARS-CoV-2 survival probability as a function of the dose for sparsely and densely ionizing particles. As expected, high-LET radiation requires a much arXiv.org 13.5.2020 8 higher dose to inactivate the virus because the dose is proportional to the LET and the particle fluence must remain high enough to hit all the targets. As explained in the Materials and Methods section, calculations presented in Figures 2 and 3 were obtained using a 10 eV threshold of the biological molecule damage. Even if this number is sound, variations in the range 5-20 eV have been used in the literature (31) (32) (33) , and it is therefore necessary to assess the robustness of our simulation to this parameter. Figure 4 shows the average The robustness analysis should consider whether the LET dependence of the ratio (Figure 3) is affected by the modified protein/RNA ratio. Figure 4 (B and D) shows the ratio of the results obtained for the different particles in the left panel, for spike and membrane proteins respectively, formalized to the reference radiation source (g-rays). Results for 5 eV and 10 eV thresholds are almost similar, since ionization is triggered at 10.73 eV but above this value the increase in the damage ratio becomes more significant. Notably, the ordering of the different particles is not influenced by the threshold except for electrons versus carbon ions above a threshold of 15 eV, which is seen in both parts related to spike proteins in Figure 4 (A and B) . Simulation of radiation damage in SARS-CoV-2 is interesting for many reasons. Here, we analyze the potential use of ionizing radiation for virus inactivation in vaccine manufacturing. As with other chemical agents, conventional inactivation with g-rays unavoidably damages the membrane proteins, whereas ideally an inactivated virus with intact membrane antigens would elicit the most effective vaccine response. Here, we show that using densely ionizing charged particles, such as accelerated heavy ions or a-particles, the ratio of protein/RNA damage is reduced by more than an order of magnitude (Figure 3) . Charged particles can, therefore, represent a powerful tool for developing SARS-CoV-2 (or other viruses) vaccines based on inactivated virus, which is still one of the strategies currently used for pandemics. While Geant4-DNA is validated as an accurate Monte Carlo code for microscopic calculations, every model is affected by uncertainty, and in particular the minimum energy required to induce damage. While the minimum energy for damage in nucleic acids, characterized by strand breaks, arXiv.org 13.5.2020 10 is well known, this uncertainty is even higher for proteins, where the energy required for a conformational change, leading to a failed recognition as an antigen, could be higher. However, we have shown that our conclusions are robust in relation to the threshold energy for damage ( Figure 4 ), meaning the advantage of densely ionizing radiation remains, or could even be bigger by an order of magnitude, with different simulation parameters. While the simulations clearly suggest that accelerated heavy ions can be a powerful tool for vaccine development, the practical implementation may be more complex. Energetic charged particles require large accelerators and high doses. As shown in Figure 3 , inactivation doses for heavy ions are higher than for g-rays. Survival curves similar to those in Figure 3 were measured in the past for many different viruses (34, 35) . The increase in lethal dose is simply due to the fact that D (Gy) = LET x fluence, and the fluence (in particles per cm 2 ) must be very high to hit all the viruses in the target samples, according to Poisson statistics. The best particle for virus inactivation with minimal membrane damage depends on the LET , as shown in Figure 3 , and also on the track structure, similar to biological effects in mammalian cells (36) . However, the choice of the best ion will be dominated by practical considerations. Large numbers of viruses have to be irradiated in closed cryovials in dry ice to keep the virus frozen (37) . Particles with very small penetration range (such as a-particles and low-energy electrons) cannot be used, as they are unable to penetrate the wall of a cryovial. Concerning electrons, our results support the experimental data showing that inactivation with 200 keV electron beam maintain the antigenic properties of g-rays (29) , and that in principle slow (2 keV) electrons would be more efficient (30) , but the short range of these electrons (in water, ~0.45 mm at 200 keV and ~0.2 µm at 2 keV) hampers their practical usage. High-energy heavy ions, such as Fe-ions simulated in this paper, are more suitable for vaccine production because they are both high-LET and long range, so whole cryoboxes can be irradiated in one session. High beam intensity is required to reach a kGy-level dose in a short time. In a preliminary safety test at the SIS18 synchrotron at GSI (Darmstadt, Germany), a high-intensity and -energy synchrotron (38) , we found that a few kGy of 1 GeV/n Fe-ions (~28 cm range in water) can be delivered to cryoboxes containing many frozen cryovials surrounded by dry ice in a few hours, without any damage to the container and with limited and quickly reduced sample activation. These tests show that such irradiations are feasible and can represent a completely new application of large-scale particle accelerators currently in operation and under construction worldwide (39) . In conclusion, our Monte Carlo simulations show that inactivation of SARS-CoV-2 can be achieved with accelerated heavy ions minimizing the damage to the epitopes in membrane proteins. Validation experiments at accelerators will be necessary to compare the capacity to induce antibody-and cell-mediated immune responses of viruses inactivated by either heavy ions or conventional methods. arXiv.org 13.5.2020 11 The incident radiation is a parallel beam with a circular cross-section 132 nm in diameter, uniformly covering the complete geometry of the virus including the spike proteins. Simulations were carried out for 3 MeV a-particles, 450 MeV/amu 56 Fe-ions, 80 MeV/amu 12 The Geant4-DNA processes (14) (15) (16) 40) were used to simulate step-by-step tracks of ions and electrons. The Livermore models (19, 41) were used to simulate gamma interactions. Processes included for protons and alpha particles were nuclear scattering, electronic excitation, ionization, and charge transfer. For electrons, elastic scattering, electronic excitation, ionization, vibrational and rotational excitations, and electron attachment were considered. Electrons were followed down to 2 eV, below this energy an electron is stopped and its remaining kinetic energy is locally deposited. Heavy ions interactions consisted only of ionization which was simulated using the relativistic Rudd model with an effective charge scaling as described in (42) and previously used for carbon ion fragments (43) . Since all the included processes of Geant4-DNA were validated for liquid water, we proceeded with a density scaling to account for the different virus materials. Therefore, the different protein densities were calculated based on the constitutions mentioned in (30) and the cross sections were scaled accordingly. The calculated densities are 1.4 g/cm 3 for the spike proteins, 1.38 g/cm 3 for the membrane protein, and 1.46 g/cm 3 for the inner sphere, equal to the RNA density. Energy deposition points were calculated in the spike proteins, membrane proteins, and RNA molecule. The RNA size was calculated based on the genome sequence published by the National Center for Biotechnology Information (44) . The viral ss-RNA sequence length is 29903 bases and its mass is 6,648 MDa. Considering the RNA density, we can estimate its approximate volume to be 7562 nm 3 . This value is equal to ~3% of the inner volume of the virus, the 80 nm sphere shown in Figure 1 . Therefore, for each energy deposition point inside the virus a random sampling was applied with a 3% probability that this point is positioned on the RNA. Studies from the literature on DNA damage reported breaks induced by low energy electrons. The incident energies ranged between 1 eV and 30 eV (45) (46) (47) (48) , and damage was induced by energies as low as 3-5 eV. Taking these values into account we determined that an energy deposition above a threshold of 10 eV, positioned on the RNA volume, is considered a damage. arXiv.org 13.5.2020 12 For spike proteins, if the sum of energy depositions inside one protein was higher than the 10 eV damage energy threshold, we consider the protein as damaged. The number of damaged proteins was counted and normalized to a single RNA damage. As we are looking for the ionizing radiation type that would yield the lowest amount of protein damage per RNA damage to inactivate the virus, this normalization is desirable. In a second stage, in order to verify the influence of the damage threshold on our results, calculations were evaluated for 4 different threshold values, 5 eV, 10 eV, 15 eV and 20 eV. 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The safety test on high-dose irradiation of cryovials were performed in Cave A in the frame of FAIR Phase-0 supported by the GSI Helmholtzzentrum für Schwerionenforschung in Darmstadt (Germany). We thank Dr.Uli Weber and his crew for support in those tests. We thank IN2P3/CNRS for the support to SZ and Geant4-DNA.