Poxviruses are complex enveloped viruses having a diameter comprised between 200 and 300 nm that distinguish them principally by their unusual morphology, their large DNA genome and their cytoplasmic site of replication. The genome of several members of Orthopoxviruses, including two strains of vaccinia virus (VV): the Copenhagen Vaccinia Virus strain (GOEBEL et al., 1990, Virol. 179, 247-266 and 517-563; JOHNSON et al., 1993, Virol. 196, 381-401), the Wyeth strain (OSBORNE J D et al. Vaccine. 2007 Dec. 17; 25(52):8807-32) and the modified Vaccinia Virus Ankara (MVA) strain (ANTOINE et al., 1998, Virol. 244:365-396), have been mapped and sequenced. VV has a double-stranded DNA genome of about 192 kb coding for about 200 proteins of which approximately 100 are involved in virus assembly. MVA is a highly attenuated Vaccinia Virus strain generated by more than 500 serial passages of the Ankara strain of Vaccinia Virus on chicken embryo fibroblasts (MAYR et al., 1975, Infection 3:6-16). The MVA virus was deposited at Collection Nationale de Cultures de Microorganismes (CNCM) under depositary N° I-721. Determination of the complete sequence of the MVA genome and comparison with the Copenhagen VV genome allow the precise identification of the alterations which occurred in the viral genome and the definition of seven deletions (I to VII) and numerous mutations leading to fragmented ORFs (Open Reading Frame) (ANTOINE et al., 1998, Virology 244:365-396).
MVA is used as a prophylactic vaccine against smallpox and recombinant MVA is currently the subject of many preclinical and clinical studies for prophylactic and therapeutic vaccination against many types of targets, including cancer (melanoma, non-small cell lung carcinoma, renal cell carcinoma, prostate cancer, colorectal cancer, notably), viral (hepatitis B or C, HIV notably), bacterial (tuberculosis notably) and parasitic diseases (malaria notably) (see GOMEZ et al. Current Gene Therapy, 2008, 8:97-120).
Oncolytic vaccinia viruses are also under preclinical and clinical development (see KIRN et al. Nat Rev Cancer, 2009 January, 9(1):64-71).
In both cases, a live vaccinia virus is used.
A live vaccinia virus prophylactic or therapeutic vaccine is generally not administered to the patient just after production and purification, and thus needs to be stored for days, weeks or even months, without losing its potency.
Like all live viruses, live vaccinia viruses have natural instability, which is further increased by the fact that vaccinia virus is an enveloped virus (enveloped viruses are known to be less stable than non-enveloped viruses, see BURKE C J et al. Crit Rev Ther Drug Carrier Syst. 1999, 16(1):1-83; and REXROAD et al. Cell Preservation Technology. June 2002, 1(2):91-104), has a big size (brick shape of 200 to 300 nm), a large genome and is known to be particularly sensitive to UV damage, see LYTLE et al. J. Virol. 2005, 79(22):14244). Moreover, vaccinia virus envelop is even more complex than that of other enveloped viruses. Stabilizing vaccinia virus is thus particularly challenging. Attempts to stabilize vaccinia virus have been made. In most cases, a freeze-dried formulation has been proposed (BURKE C J et al. Crit Rev Ther Drug Carrier Syst. 1999, 16(1):1-83). Indeed, while the performance of freeze-drying may induce some loss of viral titer, once freeze-dried, low temperature and absence of movement and interaction between compounds in freeze-dried state make freeze-dried viruses generally more stable than viruses in liquid state. For instance, EP1418942 discloses vaccinia virus formulations for freeze-drying comprising a substantially pure vaccinia virus, a disaccharide, a pharmaceutically acceptable polymer and a buffer that is not a phosphate buffer. WO2014/053571 discloses other freeze-dried MVA formulations comprising polyvinylpyrrolidone (PVP) or derivatives thereof, at least one sugar (in particular sucrose), at least two different amino acids (in particular sodium glutamate and L-arginine), at least two pharmaceutical acceptable salts (in particular NaCl, Na2HPO4 and KH2PO4), wherein at least one of said salts is a phosphate salt and, optionally a pharmaceutical acceptable buffer (in particular Tris).
However, freeze-drying is expensive, needs specific equipment and freeze-dried formulations need to be reconstituted before administration. Moreover, freeze-drying involves a freezing step that may lead to some virus aggregation, in particular at high virus titers, which is not suitable for injectable administration. It would thus be very useful to have stable liquid vaccinia virus formulations available.
Previous attempts to stabilize vaccinia virus in the liquid state have not been very successful, since log loss superior to 1 log10 after less than 1 hour at 50° C. were observed in most cases (see BURKE C J et al. Crit Rev Ther Drug Carrier Syst. 1999, 16(1):1-83).
Evans et al disclosed stable liquid adenovirus (non-enveloped DNA virus) formulations buffered between pH 6 and pH 8, comprising a salt (generally NaCl), a sugar (sucrose in most cases), an inhibitor of free radical oxidation (notably EDTA, ethanol or an EDTA/ethanol combination), a non-ionic surfactant and divalent salts (see EVANS et al. J Pharm Sci. 2004 October, 93(10):2458-75; and U.S. Pat. No. 7,456,009). Preferred formulations generally also comprise histidine. Parameters identified as essentials for stability include the presence of an inhibitor of free radical oxidation (in particular EDTA, ethanol, an EDTA/ethanol combination, and/or histidine), and presence of a non-ionic surfactant. The presence of divalent salts is also identified as important for increasing adenovirus stability.
The usefulness of non-ionic surfactants for stabilization purpose has also been documented for papilloma virus (see SHI et al. J Pharm Sci. 2005 July, 94(7):1538-51). US2007/0161085 tested various liquid formulations for stabilization of influenza virus (enveloped RNA virus). Most stable formulations included arginine and gelatin. In this study, EDTA was shown to have no effect on influenza virus stability. A low amount of surfactant, in addition to arginine and gelatin, was found to be beneficial.
U.S. Pat. No. 7,914,979 relates to formulation for stabilization of enveloped Newcastle disease virus, comprising a non-reducing saccharide such as sucrose. Preferred compositions also contain an amino acid selected from lysine and arginine. In contrast, EDTA is indicated to have a negative effect on stability and is preferably absent from the formulation.
In WO2014/029702, various types of formulations have been tested for stabilization of four canine viruses: two small and medium non-enveloped viruses (canine parvovirus and canine adenovirus type 2) and two enveloped viruses of the paramyxoviruses family (canine distemper virus and canine parainfluenza virus). Results show that enveloped viruses are more difficult to stabilize than non-enveloped viruses, and that the optimal formulation significantly varies between viruses, even for two enveloped viruses of the same paramyxoviruses family (canine distemper virus and canine parainfluenza virus). In addition, it is indicated in Example 1 that while sucrose—in particular at a concentration of 17-25%—and amino acids (such as arginine and methionine), are efficient stabilizers, free radical scavengers (such as EDTA) do not significantly change the stability profile, although they might somewhat contribute to the stability.
The above description of prior art clearly illustrates that designing a stable liquid formulation for a particular virus is a difficult task, since many stabilizers candidates are known in the art and since their stabilizing effect greatly varies depending on the specific virus to be stabilized. In addition, as explained above, due to its enveloped nature, its large size and its DNA genome, vaccinia virus is particularly difficult to stabilize, notably in the liquid state.