The present invention relates to a method for preparing an inactivated virus and an antiviral vaccine based on an inactivated virus.

In greater detail, the invention relates to a method for preparing an inactivated virus and an antiviral vaccine based on an inactivated virus, wherein the virus can be a coronavirus, to be used both in the medical field and in the veterinary field, wherein the process of inactivating the virus is capable of safeguarding the antigenic protein structure of the virus itself, such as, for example, SARS-CoV-2.

It is well known that vaccines can be prepared by means of various technologies.

Taking, for example, among coronaviruses, the SARS-CoV-2 virus, at the present state of knowledge the following types of anti SARS-CoV-2 vaccines can be listed: inactivated viral vaccines produced by growing the SARS-CoV-2 virus in cell cultures and inactivating it chemically or thermically, so that it is not capable of causing disease but is capable of inducing immune responses similar to those induced by natural infection; vaccines with live attenuated viruses produced by generating a genetically weakened version of the virus which replicates to a limited degree, not causing disease but inducing immune responses similar to those induced by natural infection; recombinant protein vaccines based on the spike protein, or the receptor binding domain (RBD) or virus-like particles (VLPs); viral vector vaccines typically based on an existing virus (generally a replication-incompetent adenovirus) which carries the genetic code sequence which codes for the spike protein;

DNA vaccines based on plasmids modified in such a way as to carry genes that code in general for the spike protein, which is then produced in the vaccinated individual;

RNA vaccines based on messenger RNA (mRNA) or a self- replicating RNA that provides the genetic information for the spike protein.

In general, the inactivation of a virus can be achieved by thermal treatment methods and/or chemical treatment methods. However, these known methods for inactivating a virus can cause alterations in the protein structure of the virus, in particular in the antigenic portions of the virus responsible for the immune response of the affected organism (i.e. the response on the part of the immune system, which is capable of lending protection against the virus and the disease caused by the virus). Therefore, the inactivated virus vaccines obtained with such methods could induce an immune response in an individual thus vaccinated which is not suitable for immune coverage against the virus, because of the alterations in the protein structure of the virus itself caused by the inactivation method.

On the other hand, it is essential that the inactivation methods are sufficiently effective to obtain virions that have absolutely no ability to infect or replicate. This may require inactivation conditions that are not compatible with maintaining the protein structure of the virus. Therefore, the need for safety often conflicts with the need to maintain the protein structure of the virus unaltered.

In the light of the foregoing, for the production of a vaccine based on an inactivated virus there clearly appears to be a need to have a method capable of overcoming the disadvantages of the known methods of preparation.

The solution according to the present invention is comprised in this context, which aims to provide a method for preparing an vaccine based on an inactivated virus which is capable of providing an effective, totally safe vaccine.

It is well known that, in the food & beverage industry, HPP (high pressure processing) technology is used to inactivate the microbial load (both pathogenic and spoilage causing) in packaged foods in order to guarantee the safety and prolong the shelf life thereof. This technology, which exploits the effects of high hydrostatic pressure on microorganisms, has shown to be effective in inactivating the infective or metabolically active forms of the microbial structures (bacteria, yeasts, moulds, viruses) present in food matrices, while preserving the product’s organoleptic and nutritional characteristics as much as possible. More in particular, this technology is effective for the inactivation of numerous microbial forms, also including viruses potentially present in foods (e.g. hepatitis, norovirus, poliovirus, HAV, HRV, FCV, MNV-1 , HuNoV, CAV9 etc.).

However, to date no one has evaluated the possibility of using HPP for the inactivation of viruses to be used as vaccines, wherein the inactivated virus must have characteristics suitable for the purpose of vaccination, a purpose that is not necessary in the treatment of foods. In this regard, it should be noted that foods can be subject to drastic treatment conditions, which could not be applied to virion structures for subsequent use for active immunisation purposes.

According to the present invention, a method has now been developed for the preparation of an inactivated virus, suitable for use in vaccine therapy, by means of a high pressure treatment. The virus inactivation method according to the present invention makes it possible to obtain an inactivated virus in which the antigen structure is maintained intact. Therefore, the inactivated virus according to the method of the present invention is suitable for vaccine prophylaxis purposes. The present invention thus also concerns a method for preparing a vaccine based on an inactivated virus, wherein the step of inactivating the virus is carried out by means of a high pressure treatment, and the vaccine obtainable with said method.

In particular, according to the present invention, a process for preparing a vaccine based on inactivated SARS-CoV-2 has been developed, wherein the step of inactivating the SARS-CoV-2 virus was conducted through the application of high hydrostatic pressures (or HHP or HPP or pascalization); subsequently, a verification was conducted firstly of the inactivation of the infectivity of the virions treated with HPP for permissive cell cultures in vitro, and secondly of the maintenance of the antigenicity thereof with the consequent possibility of using the preparation obtained as an anti-coronavirus vaccine. Therefore, the method according to the present invention is a nonthermal method for inactivating a virus, such as, for example, SARS-CoV- 2, for the purpose of producing vaccines from viral cells that have been completely inactivated without the use of thermal or chemical treatments typically adopted for that purpose.

According to the present invention it has been observed that the method makes it possible to obtain SARS-CoV-2 virions that are absolutely devoid of any infective capacity in vitro for cells susceptible to SARS-CoV-2 infection, without any evident antigenic alterations of the virion structure.

The inactivating method according to the present invention makes it possible to obtain a preparation with an anti-coronavirus vaccine function based on an inactivated virus, obtained by applying specific pressure values to the SARS-CoV-2 virus for certain exposure times and temperatures, treatments which ensure that the virions lose their ability to replicate, as shown by the experimental results described further below. Once the HPP treatment suitable for certain inactivation of the infectious agent was identified, a study was conducted into possible alterations of the virus structure induced by the process in order to confirm the suitability of the ‘cold’ method proposed for the preparation of the vaccine which, as stated above, entails the complete absence of any thermal or chemical treatments which could decisively modify the structure (in particular the protein structure) of the virus, thereby rendering the immune response of the vaccinated individual unsuitable as vaccine coverage against the virus.

As regards the medical and health sector, the HPP treatment would make mass production of the vaccine preparation easier and more effective, also vis-a-vis new emerging viral variants, since the proposed treatment is decidedly more respectful of the morphology and the molecular and antigenic structure of the virus compared to traditional thermal and chemical treatments, and it is thus plausible that it will enable a vaccine to be obtained which is more effective because substantially identical in its antigenic structure to the infective virion, obviously without possessing the capacity thereof to infect eukaryotic cells. Accordingly, the vaccine preparation would be much more capable of inducing an immunogenic stimulus that is very similar to what occurs in the course of a natural infection and consequently more recognisable by the immune system of the individual undergoing active prophylaxis.

Therefore, the method according to the present invention would be a simple, safe and extremely repeatable method. The inactivation method makes it possible, downstream of the inactivation process, to proceed with stabilisation of the preparation itself with different systems, such as, for example, freezing and/or simple refrigeration, thus guaranteeing the easy storage and transport of the vaccine preparation thus obtained.

More specifically, the method according to the present invention has the following advantages compared to the known inactivation methods for the preparation of vaccines: extremely reduced production times (about 5-10 minutes for the HPP treatment step, with viral suspension volumes of up to 400-450 litres per treatment batch); greatly reduced production costs (estimated at around 50-70 euro cents per kg of viral suspension); effectiveness independent of the genetic variants of the virus(es) (for example, in reference to coronavirus, the effectiveness would be independent of the type of coronavirus, e.g. native SARS-CoV-2, the Delta variant, the Omicron variant, and the other VoCs identified up to now), since the HPP treatment inhibits the infective capacity of the virus by acting as a physical process on the cellular structure of the virion, which is common to all variants, but not on the virus genome, which, by contrast, is the part that can mutate from variant to variant; therefore, it is plausible that the prophylaxis may obtain results independent of the VoC used to prepare the vaccine itself;

- storage at deep-freezing temperatures (-18 °C) and/or refrigeration temperatures (0 - 4 °C) for the same inactivated virus, without the need to resort to extreme cryogenic conditions (e.g. -80 °C); simplicity of the method for obtaining large amounts of viral suspension that is inactivated in terms of infectivity; finally, as the same inactivated virus is involved, the vaccine thus obtained might be more widely accepted by people who show little confidence in vaccines produced with other techniques (particularly the ones that entail genetic engineering operations and administration of RNA derivatives of the viral genome).

The method according to the present invention can advantageously be applied without the need for a chemical and/or heat treatment to inactivate the virus, or else it can be used in combination with said chemical and/or heat treatments, using the latter under milder conditions compared to the conditions used in the respective conventional methods.

The specific subject matter of the present invention thus relates to a method for preparing a virus that is inactivated and suitable for use in vaccine therapy, said method comprising inactivating a virus by means of a high pressure technique (HPP), wherein a suspension of a virus is subjected to a hydrostatic pressure of 200 to 1200 Mpa at a temperature of 1 to 60 °C.

The method according to the present invention does not relate to the inactivation of a virus contained in a food, but is rather carried out on an isolated virus in suspension, i.e. it is not carried out for example on food products for the purpose of inactivating the viruses contained therein.

According to the present invention, the term “suitable for use in vaccine therapy” means that the inactivated virus possesses the characteristics for being used in a vaccine; therefore, the inactivated virus must at least have lost its infective capacity and have maintained its protein structure (for example tertiary) intact in order to be able to induce an immune response against the virus in a human or animal body.

According to the present invention, a virus suspension means a suspension of a virus, or rather of virions, in a fluid such as, for example, water, a gel or an oil, provided that said fluid is not capable of chemically inactivating the virus. The virions can be obtained with cell cultures, for example in Minimal Essential Medium (MEM) supplemented with 5% foetal bovine serum.

According to the method of the present invention, the hydrostatic pressure can be comprised in a range of 200 to 1000 Mpa, preferably 400 to 600 Mpa.

The latter pressure range has shown to be particularly effective in inactivating SARS-CoV-2. Therefore, it is plausible that a pressure of 400 to 600 Mpa may be equally effective on other coronaviruses or on other viruses having morphological and/or physiological characteristics similar to those of SARS-CoV-2.

The pressure can be applied increasingly up to an established maximum pressure value, immediately followed by an abrupt decrease in pressure, or else it can be applied increasingly up to an established maximum pressure value, maintained for a certain period of time and subsequently followed by an abrupt decrease in pressure.

The hydrostatic pressure at the above-mentioned values will be applied for the time necessary to obtain inactivation of the virus. In general, the time of application of the hydrostatic pressure at the above- mentioned values may vary from 1 to 120 minutes, for example for a period of time of 4 to 10 min, more preferably 5 to 6 min.

When the established maximum hydrostatic pressure is maintained for a certain period of time, the time of application of pressure refers to the time of application of said established maximum hydrostatic pressure, whereas the time for reaching said maximum hydrostatic pressure and that of the decompression step are not included.

According to the method of the present invention, the temperature can be comprised in the range of 1 to 50 °C, preferably 1 to 40 °C, more preferably 4 to 20 °C. The temperature must be such as not to thermally degrade the tertiary protein structure of the virus.

According to the method of the present invention, the amount of virus in the suspension can be selected in a range of 1 x10 ² to 1 x10 ¹⁰ copies of RNA/ml, preferably 1 x10 ⁴ to 1 x10 ⁶ copies of RNA/ml, even more preferably 1 x10 ⁴ to 1 x10 ⁵ copies of RNA/ml.

According to one embodiment of the method of the invention, the volume of the virus suspension, for each package prepared, can be selected in a range of 2.0 ml to 200000.0 ml, preferably 2.0 ml to 200.0 ml. According to the present invention, the hydrostatic pressure can be increased at a speed selected in a range varying from 10 to 400 Mpa/minute, preferably from 50 to 300 Mpa/minute, even more preferably from 200 to 250 Mpa/minute,

In one embodiment of the method according to the present invention, the virus can be selected from coronaviruses such as SARS- CoV-2, MERS-CoV and SARS-CoV, preferably SARS-CoV-2, respiratory viruses such as Influenza A and B and Respiratory Syncytial Virus, viruses causing exanthematous diseases such as chicken pox, HHV6, HHV7, coxsackievirus A and B, German measles and measles, HIV, hepatitis virus or Ebola virus.

The present invention relates, moreover, to a method for preparing an antiviral vaccine based on an inactivated virus, said method comprising an inactivation step according to the method as defined above.

The subject matter of the present invention further relates to an inactivated virus obtainable according to the method as defined above. The inactivated virus according to the present invention preserves the initial protein structure of the active virus (i.e. not yet treated) responsible for the immune response in the host individual, and thus maintains the antigenic reactivity of the active virus. Preferably, the inactivated virus is a coronavirus, in particular SARS-CoV-2.

The present invention also relates to a pharmaceutical composition comprising the virus as defined in claim 9 together with one or more pharmaceutically acceptable excipients and/or adjuvants.

According to the present invention, the pharmaceutical composition can be a vaccine.

The present invention relates, moreover, to the use of an inactivated virus as defined above for the preparation of a vaccine.

The present invention will now be described, by way of non-limiting illustration, with particular reference to some illustrative examples and to the figures of the appended drawings, in which:

- figure 1 shows a schematic representation of the HPP technology applied for the step of inactivating the virus in the preparation of a vaccine. EXAMPLE 1 : Method for preparing a vaccine based on inactivated SARS-CoV-2 according to the present invention

Method for preparing the viral suspension

The viral suspension used in the inactivation tests (carried out in replicate with an identical portion of viral suspension pre-processed with identical methods) was prepared by collecting the supernatant from cell cultures (Vero E6 cells) infected with the SARS-CoV-2 lineage-81 , originally isolated from biological material left over at the end of a COVID- 19 diagnostic process and anonymised, prior to use in cell cultures, with a procedure that does not allow the source patient to be traced. The biological sample used in the study thus consists in a viral suspension that does not contain any genetic information of the individual from whom the virus was drawn. The Vero E6 cells were cultured in MEM medium supplemented with 5% foetal bovine serum, 1% l-glutamine and antibiotics. The initial growth density was no less than 1x10 ⁵ /cm ² and the minimum surface was 25 cm ² . The cell monolayer was incubated at 37 in the presence of 5% CO2 for 48 hours prior to infection. The infective viral load inoculated was no less than 1 x10 ⁴ copies of RNA/ml. The cell cultures were then incubated as described above for 72 hours. At the end of incubation, the viral load was quantified by RT PCR and the Ct data related to the N gene of SARS CoV-2 was converted into copies of RNA/ml, according to what is described in Correlating qRT-PCR, dPCR and Viral Titration for the Identification and Quantification of SARS-CoV-2: A New Approach for Infection Management. Brandolini M, Taddei F, Marino MM, Grumiro L, Scalcione A, Turba ME, Gentilini F, Fantini M, Zannoli S, Diani G, Sambri V. Viruses. 2021 May 28;13(6):1022. (doi: 10.3390/vl 3061022.)

The viral suspension collected as a supernatant from the infected cultures was briefly centrifuged at 1500 rpm in order to reduce the presence of cellular debris, re-evaluated from the viewpoint of the viral load present (see above), and then inserted into bags made of a sealable, flexible high-barrier multilayer plastic material suitable for undergoing an HPP treatment while minimising the risk of the pathogen leaking out into the outside environment. Each bag contained a volume of 2 ml of viral suspension.

High hydrostatic pressure (HHP) treatment

The treatments were carried out in a 350-litre high pressure system (Avure Technologies Inc., Erlanger, Kentucky, United States) at the “HPP Italia” plant in Traversetolo (Parma - Italy).

It should be specified that the process conditions depend to a very small degree on the HPP device used; in fact, HPP systems can generally differ in the number of intensifier pumps, which determines the speed at which the system reaches the setup pressure (that is, how steep the pressure increase curve will be as a function of time). However, this characteristic becomes secondary when the dwell time (i.e. the parameter that is always considered in identifying the terms of the treatment) starts from the moment at which the pressure setup value is reached; in other words, the speed at which the desired pressure value is reached is secondary for the purposes of the present invention (in the context of a process applied to products in the food industry, by contrast, speeding up the cycle time even by only a few tens of seconds has a direct impact on the costs of the service). For every sample produced, 2 ml of viral suspension of SARS-CoV-2 were introduced and sealed in a triple layer of pouches made of high barrier multilayer plastic material and transported in dry ice. The pouches subjected to different treatments were introduced into a pressure chamber whose free volume was filled with normal cold mains water (4 °C). Subsequently, through the action of special piston pumps, a high isostatic pressure was generated inside the pressure chamber at a speed of about 200 Mpa/minute; the increase in temperature due to the compression did not exceed 2-3°C/100 Mpa. This means that in the case of a treatment at 600 Mpa the temperature inside the pressure chamber was about 18-20 °C. For the purposes of the present invention, it was deemed advisable to keep the process temperature as low as possible in order to minimise the possibility of the surface protein structure of the virions undergoing major changes (it is in fact known that temperature is among the most effective physical parameters in the denaturation of proteins). The HPP treatments were carried out at 200, 300, 400, 500 and 600 Mpa, each for 5 minutes. This pressure dwell time, as already noted previously, is measured starting from when the setup pressure is reached.

The samples produced at least in quintuplicate (at least 5 pouches for every treatment condition) were the following:

Control (not treated by HHP; subjected to the same thermal conditions as the treated samples):

200 MPa (treated for 5 min with HHP at 200 MPa);

300 MPa (treated for 5 min with HHP at 300 MPa);

400 MPa (treated for 5 min with HHP at 400 MPa);

500 MPa (treated for 5 min with HHP at 500 MPa);

600 Mpa (treated for 5 min with HHP at 600 MPa).

The samples were subsequently kept in dry ice and transported to the microbiology laboratories, where specific tests were performed for the determination of:

1. viral inactivation;

2. protein characteristics of the suspensions treated with HPP;

3. antigenic characteristics of the suspensions treated with HPP.

Method of evaluating replication capacity

At the end of the treatments with HPP, the samples obtained were stored in dry ice and inoculated into Vero E6 cell cultures according to the standard protocol, as described above. The cultures were sampled (500 microlitres of supernatant) immediately after inoculation and every 24 hours up to 96 hours after inoculation: an RT PCR method (SeeGene AHPlex) was carried out on the samples in order to evaluate viral replication (according to the method described above). In addition, the cultures were observed every 24 hours for the presence of an evident cytopathic effect due to replication of the virus.

Results obtained

The results obtained demonstrated that the HPP treatment with values of 500 and 600 Mpa was able to completely inhibit the infective (growth) capacity of the SARS-CoV-2 virus for the Vero E6 cell system. On the other hand, the viral suspension not treated with HPP fully maintained the infective (growth) capacity for the Vero E6 cell system.

In detail, the viral suspensions not treated with HPP demonstrated a high viral replication capacity in the Vero E6 cell cultures as early as 24 hours after inoculation, as was observed, moreover, for the viral suspensions treated with HPP values of up to 400 Mpa. The suspensions treated with HPP at 500 and 600 Mpa, by contrast, demonstrated that following these treatments the virus lost its ability to replicate in the Vero E6 cell cultures, a clear and unambiguous demonstration of total loss of infective capacity.

Table 1 shows the numerical values of the viral load (copies/ml) of viral suspensions of SARS-CoV-2 subjected to different HPP treatments at different times after inoculation in Vero E6 cell cultures.

Table 1

In order to better investigate the morphological effects of the treatment on viral cells, the following characteristics were then evaluated:

- Protein characteristics of the suspensions treated with HPP: in order to evaluate the protein composition of the viral suspensions treated and not treated with HPP, the same were subjected to analysis by SDS PAGE (Maizel JV. SDS polyacrylamide gel electrophoresis. Trends Biochem Sci. 2000 Dec;25(12):590-2.) and subsequent image analyses of protein separations, which demonstrated that the protein pattern does not undergo any variation following treatment with HPP (unreported data);

Antigenic characteristics of the suspensions treated with HPP: the possible modifications of viral antigenicity induced by treatment with HPP were studied with the western blotting technique (Dowlatshahi S, Shabani E, Abdekhodaie MJ. Serological assays and host antibody detection in coronavirus-related disease diagnosis. Arch Virol. 2021 Mar;166(3):715- 731.). The reactivity of the individual antigenic components was evaluated vis-a-vis a pool of human sera (anonymised prior to use for the test) deriving from patients affected by a SARS-CoV-2 infection at various clinical stages (based on IgG response) left over at the end of normal diagnostic practice. In this case as well, no differences in antigenic reactivity were observed between preparations treated and not treated with HPP, further confirming the absence of modifications of the in vitro immune response between viral suspensions treated with HPP and infective ones (control).

In addition to the results regarding inactivation of the virus, in terms of loss of replication capacity, the first qualitative microstructural tests showed a high degree of maintenance of the virus morphology in terms of tertiary and quaternary protein structure. This preliminary result confirms the high potential of the method adopted and of the vaccine thus obtained.