CATIONIC NANOPARTICLES FOR ENHANCING INFECTIOUS CAPACITY OF LIVE VIRUSES

Abstract

A combination of cationic nanoparticles and viruses and uses thereof. The use of nanoparticles for enhancing the infectious capacity of a live virus, preferably a non-enveloped live virus.

Claims

1. Use of cationic nanoparticles for enhancing the infectious capacity of live virus.

2. The use according to claim 1, wherein said virus is selected from the group consisting of Adenoviridae, Caulimoviridae, Rudiviridae, Papillomarividae, Phycodnaviridae, Tectiviridae, Papovaviridae, Circoviridae, Parvoviridae, Birnaviridae, Reoviridae, Astroviridae, Caliciviridae, Picornaviridae, Potyviridae, Poliomarividae, Hepeviridae, Arteriviridae, Anelloviridia, Papillomarividae, Paramyxoviridae, Togaviridiae, Herpesviridae, Orthomyxoviridae, Flaviviridae, Hepadnaviridae, Rhabdoviridae, Poxviridae, Filoviridae, Retroviridae, Coronaviridae, Baculoviridae, Reoviridae and phages and bacteriophages and their combinations.

3. The use according to claim 1, wherein said virus is a recombinant or a defective or an attenuated virus.

4. The use according to claim 1, wherein said virus is a non-enveloped virus.

5. The use according to claim 4, wherein said non-enveloped virus is selected from the group consisting of Adenoviridae, Caulimoviridae, Myoviridae, Siphoviridae, Podoviridae, Rudiviridae, Papillomarividae, Phycodnaviridae, Tectiviridae, Papovaviridae, Circoviridae, Parvoviridae, Birnaviridae, Reoviridae, Astroviridae, Caliciviridae, Picornaviridae, Potyviridae, Poliomarividae, Hepeviridae, Arteriviridae, Anelloviridiae and their combinations.

6. The use according to claim 1, wherein the cationic nanoparticles cover the virus.

7. The use of cationic nanoparticles to improve the stability of the viral preparation, in a range of temperature from +1 C. to 45 C.

8. The use according to claim 1, wherein said nanoparticles are selected from cationic polysaccharide nanoparticles, from PLA or PGA or PLGA nanoparticles coated with cationic compounds, from chitosan and its derivatives or from cationic micelles or cationic liposomes.

9. The use according to claim 8, wherein said nanoparticles are selected from cationic polysaccharide nanoparticles, especially from cationic maltodextrin nanoparticles such as porous maltodextrin with or without a lipid core nanoparticle such as NP+ or DGNP, or from PLA or PGA or PLGA nanoparticles coated with cationic compounds, such as PEI, chitosan and its derivatives, such as trimethylamoniumchitosan.

10. A combination product consisting essentially of cationic nanoparticles and live virus.

11. The combination product according to claim 10, wherein said virus is selected from the group consisting of Adenoviridae, Caulimoviridae, Rudiviridae, Papillomarividae, Phycodnaviridae, Tectiviridae, Papovaviridae, Circoviridae, Parvoviridae, Birnaviridae, Reoviridae, Astroviridae, Caliciviridae, Picornaviridae, Potyviridae, Poliomarividae, Hepeviridae, Arteriviridae, Anelloviridia, Papillomarividae, Paramyxoviridae, Togaviridiae, Herpesviridae, Orthomyxoviridae, Flaviviridae, Hepadnaviridae, Rhabdoviridae, Poxviridae, Filoviridae, Retroviridae, Coronaviridae, Baculoviridae, Reoviridae and phages and bacteriophages and their combinations.

12. The combination product according to claim 11, wherein said nanoparticles are selected from cationic polysaccharide nanoparticles, from PLA or PGA or PLGA nanoparticles coated with cationic compounds, from chitosan and its derivatives or from cationic micelles or cationic liposomes.

13. The combination product according to claim 12, wherein said nanoparticles are selected from cationic polysaccharide nanoparticles, especially from cationic maltodextrin nanoparticles such as porous maltodextrin with or without a lipid core nanoparticle such as NP+ or DGNP, or from PLA or PGA or PLGA nanoparticles coated with cationic compounds, such as PEI, chitosan and its derivatives such as trimethyl-chitosan.

14. The use of a combination product as defined in claim 10 in a method of virus production.

15. A method for producing viruses comprising the steps of: a) incubating a host cell culture with a combination product as defined in claim 10; b) harvesting the viruses produced by the host cell.

16. The method of preparing the combination product as defined in claim 13, said method comprising a step of incubating live viruses with an excess of cationic nanoparticles, wherein said excess means that the quantity of cationic nanoparticles is preferably at least 10 times larger than the quantity of infectious virus particles (ratio weight/weight).

17. The combination product according to claim 10, for use in a method for treating cancer, cardiovascular diseases, neurodegenerative disorders and infectious diseases.

18. The combination product according to claim 10, for use as a vaccine.

19. The combination product according to claim 10, for use in gene therapy.

20. The use of the combination product according to claim 10, in a method of protein production.

21. A pharmaceutical composition comprising the combination product as defined in claim 10 and at least one pharmaceutically acceptable excipient.

Description

FIGURES

[0042] FIG. 1: Example of size (Z-Average), poly-dispersity index (PDI) and Zeta potential of cationic nanoparticles.

[0043] PLGA PEI: PLGA nanoparticles coated with PEI. PLGA Chitosan: PLGA nanoparticles coated with Chitosan. NP+: cationic maltodextrin nanoparticle. DG70: NP+ with lipid core. Liposome +: Cationic liposome. PLGA: Poly(Lactic-co-Glycolic Acid). PEI: Poly(Ethylenelmine).

[0044] FIG. 2: Example of size and Zeta potential of DG70 cationic nanoparticle, viruses and combination products at the mass ratio 1/3.

[0045] DG70: cationic maltodextrin nanoparticle with lipid core. GMB: Gumboro virus, NDV: Newcastle Disease Virus, Polio: Poliovirus-1, Reo: Reovirus, Rota: Rotavirus SA-11, BVDV: Bovine viral diarrhea virus, RSV: Respiratory syncytial virus, HSV: Herpes Simplex Virus 1.

[0046] FIG. 3: Example of size and Zeta potential of DG70 cationic nanoparticle, killed viruses and combination products at the ratio 1/10 (w/w). DG70: cationic maltodextrin nanoparticle with lipid core. GMB: Gumboro virus, Polio: Poliovirus-1, Reo: Reovirus, Rota: Rotavirus SA-11.

[0047] FIG. 4: Fold induction of UV-inactivated virus transfection alone or in combination product. PLGA PEI: PLGA nanoparticles coated with PEI. PLGA Chitosan: PLGA nanoparticles coated with Chitosan. NP+: cationic maltodextrin nanoparticle. DG70: NP+ with lipid core. Liposome +: Cationic liposome. PLGA: Poly(Lactic-co-Glycolic Acid). PEI: Poly(EthyleneImine). Polio: Poliovirus-1, HSV: Herpes Simplex Virus, BVDV: Bovine Viral Diarrhea Virus, RSV: Respiratory Syncitial Virus, Rota: Rotavirus, Reo: Reovirus, NDV: Newcastle Disease Virus, GMB: Gumboro virus.

[0048] FIG. 5: Study of chlorpromazine (CPZ) on gumboro associated or not with NP on killed virus endocytosis.

[0049] FIG. 6: CPE of DG70 cationic nanoparticles, poliovirus-1 and the related combination products. Hep-2 cells were infected with various dilutions of poliovirus-1, alone or in combination with nanoparticles, ranging from 10.sup.5 to 10.sup.4 TCID50/mL. The CPE was evaluated after 6 days. Data are from 2 independent experiments and are expressed as mean+SD. cells: untreated cells, nano: cationic maltodextrin nanoparticle with lipid core (DG70).

[0050] FIG. 7: Kinetics of the viral titer in cells infected with poliovirus-1 alone or in combination with DG70-nanoparticles at various virus TCID50/ml. Hep-2 cells were infected with various dilutions of poliovirus-1, alone or in combination product, ranging from 10.sup.1 to 10.sup.4 TCID50/mL. Supernatants were collected at different times post-inoculation. Poliovirus titer was determined by limiting dilution assay for 50% tissue culture infection doses in Hep2 cell cultures by the method of Reed-Muench.

[0051] FIG. 8: Fold increase of the infectious capacity of virus alone versus the combination products. Supernatant of infected cells with virus alone or combination products were used to re-infect cells. The CPE were calculated and the fold increase between virus and combination products were expressed as log 10. PLGA(): uncoated anionic PLGA nanoparticles. PLGA Chitosan: PLGA nanoparticles coated with Chitosan. PLGA PEI: PLGA nanoparticles coated with PEI. NP+: cationic maltodextrin nanoparticle. DG70: NP+ with lipid core. Liposome +: Cationic liposome. PLGA: Poly(Lactic-co-Glycolic Acid). PEI: Poly(Ethylenelmine). Polio: Poliovirus-1, CPV: Canine ParvoVirus. Rota: Rotavirus SA-11, HSV: Herpes Simplex Virus 1, RSV: Respiratory Syncitial Virus, BVDV: Bovine viral diarrhea Virus. Tox: Cell toxicity. Inhib: Inhibitory effect of the combination product compared to the virus alone.

[0052] FIG. 9: Percentage of VP-1 positive cells after infection for 6 or 18 hours with poliovirus-1 or DG70-poliovirus-1 combination products. Numbers express the total number of cells/the number of VP1+ cells (% of VP1+ cells). DG70: cationic maltodextrin nanoparticle with lipid core. Polio: Poliovirus-1. MOI: Multiplicity of Infection. Percentages refer to infected cells. Grey blocks refer to the presence of apparent lysis plaques.

[0053] FIG. 10: Representative microphotography of the detection of virus by VP1 immunofluorescence. Cells were infected at a multiplicity of infection of 0.6 for 18 h and anti-VP1 immunostaining (lower circles) was performed. Nucleus are stained by Hoescht (upper circles).

[0054] FIG. 11: Detection of viral RNA by Q-PCR. Cells were infected at a multiplicity of infection of 7.10.sup.2 and RNA were collected after 48 h. Numbers express the absolute value of Ct relative to the common non-coding region of enteroviruses. DG70: cationic maltodextrin nanoparticle with lipid core. Polio: Poliovirus-1.

[0055] FIGS. 12A, 12B: Study of the stabilization of virus against thermal denaturation. A: 2 h30 at 55 C.; B: 24 h at 45 C.

EXAMPLES

Material and Methods

[0056] 1/ Synthesis of Nanoparticles: [0057] Cationic maltodextrin nanoparticles: Maltodextrins are dissolved in 2 N sodium hydroxide with magnetic stirring at room temperature. Addition of epichlorhydrin and GTMA yields a cationic polysaccharide gel that is then neutralized with acetic acid and crushed using a high pressure homogenizer (Emulsiflex C3, France). The nanoparticles thus obtained are purified by tangential flow ultra-filtration (Centramate Minim II, PALL, France) using a 300 kDa membrane (PALL, France). [0058] DG70 nanoparticles: Porous maltodextrin-based with lipid core nanoparticles (DGNP) were prepared as described previously (Patent WO2014041427). The cationic maltodextrin nanoparticles obtained as described above are mixed with dipalmitoyl phosphatidyl glycerol (DPPG) above the gel-to-liquid phase transition temperature to produce DG70. [0059] Anionic PLGA nanoparticles: Negative PLGA nanoparticles (PLGA()) are produced by nanoprecipitation (Le Broc-Ryckewaert D et al., Int J Pharm., 2013). The PLGA copolymer is dissolved in acetone/ethanol (85:15) mixture composing the organic phase then injected in aqueous phase under stirring. Organic solvents are eliminated by vacuum evaporation. [0060] Cationic PLGA coated with PEI: PLGA nanoparticles are produced by nanoprecipitation. The PLGA copolymer is dissolved in acetone/ethanol (85:15) mixture composing the organic phase. These nanoparticles are cationised by injecting the dissolved PLGA copolymer in aqueous phase supplemented with 10% (w/w) Polyethylenimine (PEI) under stirring. Organic solvents are eliminated by vacuum evaporation. [0061] Cationic PLGA coated with Chitosan: PLGA nanoparticles are produced by nanoprecipitation. The PLGA copolymer is dissolved in acetone/ethanol (85:15) mixture composing the organic phase. These nanoparticles are cationised by injecting the dissolved PLGA copolymer in aqueous phase supplemented with 10% (w/w) Chitosan solution under stirring. Organic solvents are eliminated by vacuum evaporation. [0062] Cationic liposomes: DPPC/DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phosphocholine) liposomes are prepared by solubilizing DPPC and DPPE in ethanol, the solution is then injected with a syringe in water under stirring at 80 C. Liposomes are then purified by filtration, residual ethanol is eliminated under vacuum.

[0063] 2/ Cell Lines

[0064] Hep-2 cell line: Hep-2 cells were provided by BioWhittaker (Vervier, Belgium). The cell line, well adapted for enteroviruses culture, was grown in Eagle's minimum essential medium (MEM) supplemented with 10% inactivated fetal bovine serum (FBS), 1% L-glutamin and penicillin (100 U/ml)-streptomycin (100 mg/ml) and fungizone (0.25 mg/ml; Invitrogen, Saint Aubin, France) in an atmosphere of 5% CO2 and a humidified air at 37 C.

[0065] CMT-U27 cell line: Canine mammary tumor (CMT-U27) cell line was derived from a primary tumor (infiltrating ductal carcinoma). CMT-U27 cell line (a gift from Associated Professor Eva Hellmen) was obtained from the Uppsala University, Sweden, cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, penicillin-streptomycin (50 IU/mL) in an atmosphere of 5% CO2 and a humidified air at 37 C. These cells are used to produce the Canine Parvovirus.

[0066] CRFK cell line: Monolayers of Crandell Rees Feline Kidney (CRFK) cells (ATCC no. CCL-94) were grown in Dulbeco Minimum Essential Medium (MEM) supplemented with 10% Fetal Bovine Serum (FBS), 1% penicillin and streptomycin and non-essential amino acids at 37 C. and 5% CO.sub.2. This cells are used to product the Canine Parvovirus.

[0067] Vero cell line: These cells were provided by ATCC (ATCC CCL-81). The cell line, was grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% inactivated fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin Streptomycin in an atmosphere of 5% CO2 and a humidified air at 37 C.

[0068] MA 104 cell line: cells were provided by ATCC (ATCC CRL-2378.1). The cell line, was grown in Eagle Minimum Essential Medium (MEM) supplemented with 10% inactivated fetal bovine serum (FBS), 1% L-glutamine and 1% Penicillin-Streptomycin in an atmosphere of 5% CO2 and a humidified air at 37 C.

[0069] MDBK cell line: cells were provided by ATCC (ATCC CCL-22). The cell line, was grown in Eagle Minimum Essential Medium (MEM) supplemented with 10% horse serum (HS), 1% L-glutamine, 1% non-essential amino acids and 1% Penicillin-Streptomycin in an atmosphere of 5% CO2 and a humidified air at 37 C.

[0070] Raw 264.7 cell line: The Raw cells (ATCC TIB-71) are macrophage cells of Mus musculus. The cell line was grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% inactivated fetal bovine serum (FBS), 1% L-glutamine, 1% non-essential amino acids and 1% penicillin Streptomycin in an atmosphere of 5% CO2 and a humidified air at 37 C.

[0071] 3/ Live Viruses

[0072] Poliovirus-1: The monovalent oral poliovirus (Poliovirus 1) used in this example was provided by Eurovir Hygiene-Institut (Luckenwalde, Germany) with a virus titer at 10.sup.6 TCID50/mL and stored at 20 C.

[0073] Canine Parvovirus: Canine parvovirus (CPV), a single strand DNA virus and a significant worldwide canine pathogen belonging to the family Parvoviridae, is a highly contagious and a principal etiological agent of hemorrhagic enteritis in dogs. The strain used in this study was from ATCC (ATCC VR-2017) and was grown in CRFK cells and CMT-U27 cells.

[0074] Rotavirus: Simian rotavirus (ATCC VR-1565) strain SA-11, is a double stranded RNA virus of the Reoviridae family. Rotavirus causes diarrheal disease in children. The recommended hosts are MA-104 (ATCC CRL-2378-1).

[0075] Human herpesvirus type 1: Herpes simplex virus type 1 (HSV-1) is a member of the family Herpesviridae, that infects humans. This is an enveloped DNA virus. The strain used in this study was from ATCC (ATCC VR-733). The host cells are Vero cells (ATCC CCL-81)

[0076] Bovine viral diarrhea virus: BVDV (NBL2) causes one of the most significant infectious diseases in the livestock industry worldwide due to its high prevalence, persistence and clinical consequences. BVDV is single-stranded RNA enveloped viruses. The strain used in this study was from ATCC (ATCC VR-534). The host cells are MDBK cells (ATCC CCL-22)

[0077] Murine norovirus: Murine norovirus S99 Berlin (MNV) is a species of norovirus affecting mice. It is a non-enveloped virus with a linear positive-sense RNA genome. The host cells are Raw 264.7 cells (ATCC TIB-71).

[0078] Human respiratory syncytial virus: Respiratory syncytial virus (RSV) of the family Pneumoviridae causes respiratory tract infections during infancy and childhood. It is an enveloped virus, single-stranded RNA. The host cells are Hep-2 cells (BioWhittaker, Vervier, Belgium).

[0079] 4/ Killed Virus

[0080] Killed virus are virus that has been inactivated and did no longer show infectious capacity.

[0081] Viruses were produced in cell lines as described in section 3/ Live Virus and were UV-inactivated for 30 min under the Microbiological safety workbench (UV lamp). The size and the purity of the virus is determined with the Zetasizer Nano ZS. Gumboro, Newcastle Disease Virus (NDV) and Reovirus purified killed viruses were kindly provided by Intervet (MSD Sante Animale, France).

[0082] 5/ Labeling of Killed Virus

[0083] Killed virus are covalently labeled with fluorescein. Viral protein concentration is determined by the microBCA method. Briefly, 1 mg of FITC (Fluorescein IsoThioCyanate, dissolved in anhydrous DMSO) was added to 10 mg of viral proteins solubilized in 0.1M bicarbonate buffer (pH 9.5), and the solution was mixed for 6 h in the dark at room temperature. The preparation was purified by gel filtration on a PD-10 Sephadex desalting column (Sigma-Aldrich) and exclusion fractions were collected.

[0084] 6/ Combination Product

[0085] The combination of cationic nanoparticles and viruses is carried out by mixing both components in a relevant culture medium for the test on cell lines.

[0086] 7/ Size and Zeta Potential Analysis

[0087] The hydrodynamic diameter of cationic nanoparticles, viruses or the combination products was measured in 15 mM NaCl by dynamic light scattering using a Zetasizer Nano-ZS instrument (Malvern Instruments, Orsay, France). The zeta potentials of nanoparticle preparations were determined in water (ZetaSizer NanoZS analyzer, Malvern Instrument).

[0088] 8/ Cytometry Analysis on Killed Viruses

[0089] Cells were treated with the combination products (section 6 of the Material and Methods) comprising fluorescently labelled killed viruses. After 3 hours, cells were collected and cell fluorescence was analysed on an Accuri c6 flow cytometer (BD Biosciences, Erembodegem, Belgium).

[0090] 9/ CPE

[0091] Cytopathogenic effect (CPE) is a structural change in host cells that are caused by viral infection. The infecting virus causes lysis of the host cell through changes in cell morphology. Common examples of CPE include rounding of the infected cell, fusion with adjacent cells to form syncytia, and the appearance of nuclear or cytoplasmic inclusion bodies. CPE were determined using an inverted microscope.

[0092] 10/ Progeny and Virus Titration

[0093] Viruses were serially diluted in presence or not of nanoparticles in the relevant medium from 10.sup.1 to 10.sup.12 in eight replicates in 96-well plates. Then cells were incubated for 5 days in a 5% CO2 atmosphere at 37 C. Afterwards the plates were examined using an inverted microscope to evaluate the extent of the virus-induced cytopathic effect in the cell culture (CPE). Calculation of estimated virus concentration was carried out by the Spearman-Krber method and expressed as log.sub.10 TCID.sub.50. Supernatants from each well were collected and used to infect nave cells. The progeny test is performed to determine the MOI (Multiple of infection=number of infecting virus per one cell). The progeny is a virus titration. After several days of incubation, the virus have infected the cells and have produced a virus titer (TCID50/mL).

[0094] 11/ Determination of Viral RNA Content by Q-RT PCR

[0095] Poliovirus positive strand RNA was quantitated by QRT-PCR. Total RNA was extracted with Tri-Reagent (Sigma-Aldrich) following manufacturer's instructions. Total RNA was measured by a quantitative RT-QPCR for RNA with the Affinity script QPCR cDNA synthesis kit and the brilliant II QPCR kit (Agilent technology, France). Positive strand specific RT was carried out on extracted RNA by using the reverse primer at 42 C. for 15 min. PCR was performed with universal cycle conditions (10 min at 95 C., 40 cycles of 30 s at 60 C.) on a Mx3000p (Agilent technology, France). The following primers, used to detect Poliovirus RNA, were located within the enterovirus 5-nontranslated region, which is highly conserved among enterovirus serotypes: forward (5-CCC TGA ATG GGG CTA ATC), reverse (5-ATT GTC ACC ATA AGC AGC CA) and probe (5-VIC-AAC CGA CTA CTT TGG GTG TCC GTG TTT-TAMRA) (Applied Biosystems, ThermoFisher Scientific, France). Results were expressed as cycle threshold (Ct) which is inversely proportional to RNA level.

[0096] 12/ Determination of Viral Protein Content by Immunofluorescence

[0097] After washing with PBS, Hep-2 cells infected by Poliovirus were fixed with fresh 4% paraformaldehyde and permeabilized with chilled methanol/acetone. Nonspecific sites were blocked with rabbit serum/anti-Fc receptor solution (Miltenyibiotec). Cells were first labelled with primary antibodies, mouse anti-enterovirus VP1 anti-body (clone 5D8/1 Dako), then with rabbit anti-mouse alexa Fluor 488 (Molecular Probes). Nuclei were stained by Hoescht dye solution (Sigma, France). Slides were mounted and visualized by using a Zeiss LSM 710 confocal laser-scanning microscope equipped with argon and helium-neon lasers.

[0098] 13/ Evaluation of Clathrin Endocytosis of Gumboro Virus Associated or not with DG70

[0099] Vero cells were treated with the combination products (section 6 of the Material and Methods) comprising fluorescently labelled killed viruses. Cells were treated with or without 15 g/ml of chlorpromazine for 3 hrs. Cells were then collected and cell fluorescence was analysed on an Accuri c6 flow cytometer (BD Biosciences, Erembodegem, Belgium).

Results

[0100] 1/ Cationic Nanoparticles Synthesis and Characterisation

[0101] Different cationic nanoparticles were synthetized and analysed by dynamic light scattering. Among produced cationic nanoparticles: PLGA coated with PEI or Chitosan, cationic maltodextrin (NP+) and with lipid core (DG70), cationic liposome or chitosan. Results are depicted in FIG. 1.

[0102] 2/ Study on Killed Virus

[0103] a/ Combination product: Formulation of cationic nanoparticle and virus UV-inactivated virus (killed virus) were associated to cationic nanoparticles and the size and the zeta potential of the resulting combination products, so called formulations, were determined by dynamic light scattering.

[0104] Formulations with 3 times more nanoparticles (1/3 mass ratio, see FIG. 2) or with 10 times more nanoparticles (1/10 mass ratio, see FIG. 3) than viruses were analysed. The combinations have a greater size compared to cationic nanoparticles alone or virus alone which confirms the association. In addition, the zeta potentials of the viruses are negative while the combination products are positive, suggesting that cationic nanoparticles cover the virus.

[0105] In live virus experiments (section 3), the ratio virus/cationic nanoparticles is at least 1/1000 reinforcing the coating of viruses by cationic nanoparticles.

[0106] b/ Transfection of Killed Viruses

[0107] Combination products of cationic nanoparticle with UV-inactivated and fluorescently-labeled viruses were produced. The cationic nanoparticles used in this study were mainly DG70 nanoparticles. Killed virus (5 g) and cationic nanoparticles (15 g) were added to a medium with 10% serum before incubation with cells. The amount of viruses in the cells was analyzed by flow cytometry and representative data are summarized in FIG. 4.

[0108] Compared to UV-inactivated virus alone, the combination products, according to the invention, highly increase the virus entry into the cells. This could increase the infectious capacity of a live virus.

[0109] c/ Mechanisms of Endocytosis:

[0110] The endocytosis of gumboro virus was evaluated by FACS in presence of a clathrin inhibitor (chlorpromazine) after 3 h of incubation in Vero cells. The FIG. 5 is a representative study of virus endocytosis where we found that cationic nanoparticle mainly increases the virus endocytosis via the clathrin pathway, same results were obtained with all the virus tested.

[0111] 3/ Study on Live Viruses

[0112] a/ Effect on CytoPathogenic Effect

[0113] The cytopathogenic effect (CPE) is defined as the change in cell structure and viability due to a viral infection, typically a lysis plaque. The CPE of cationic nanoparticles, viruses or combination products are analysed with an inverted microscope. As described in FIG. 6, cationic nanoparticles increase the CPE of the virus by 4 log 10 TCID50/ml meaning a higher efficacy of at least 10000.

[0114] b/ Effect on Virus Production

[0115] i/ Effect on Progeny of Poliovirus

[0116] The viral shedding refers to the expulsion and release of virus progeny following successful reproduction during a host-cell infection. This allows to determine the amount of infectious viruses and their capacity of using the cellular machinery to reproduce themselves. A critical step is the entry into the cells.

[0117] To compare the infectious capacity of viruses versus the related combination products (nanoparticles combined with virus), the cells were infected with viruses alone or viruses combined with cationic nanoparticles (first round). Then the supernatants of the cells were collected and reused to infect non-infected cells (second round). The lysis of the later cells reveals the infectious capacity of the viruses or the combination products. As described in FIG. 7, the capacity of infectious of poliovirus is increased by several log 10 compared to the viruses alone. For example, the poliovirus-1 combined with DG70 cationic nanoparticles is 3 to 4 log 10 (=1000 to 10000) more infectious than the poliovirus-1 alone.

[0118] In FIG. 8 we summarize the increase of the infectious capacity observed by different combinations of cationic nanoparticles and non-enveloped virus. Poliovirus-1, Canine ParvoVirus and Rotavirus SA-11 combined with PLGA PEI cationic nanoparticles are 4 log 10 more infectious than viruses alone while the PLGA PEI cationic nanoparticles does not increase the infection capacity of enveloped virus. Then, an uncoated anionic PLGA nanoparticle was tested and no increase of the virus infectious capacity was observed.

[0119] The results confirm that the use of cationic nanoparticles increase the infectious capacity of non-enveloped live viruses.

[0120] iii/ Effect on Viral RNA and Protein Productions

[0121] In order to confirm the increase in the production of virus, measures of viral RNA and protein was performed by Q-PCR and immunofluorescence against VP1, one of the main protein of poliovirus-1.

[0122] Detection of VP1 protein compared to virus alone showed that, the DG70 combination product induced a higher and earlier viral expression (FIG. 9 and FIG. 10). At the nucleic acid level, the detection of the common non-coding region of enteroviruses RNA revealed a decrease of 5.46 cycle threshold by virus alone and the related DG70 cationic nanoparticle combination product. This corresponds to a 44-fold increase in the viral RNA production (FIG. 11) and means that cationic nanoparticles enhance the production of viruses at the protein and nucleic acid levels.

[0123] iv/ Study of Virus Protection Against Thermal Denaturation

[0124] The ability of cationic nanoparticles to protect viruses from thermal denaturation has been tested. Polioviruses were incubated either at 45 C. for 24 h or at 55 C. for 2 h30 in presence or not of DGNP. At 55 C. after 2 h30 incubation no protection was observed even in presence of DGNP (FIG. 12,A), while we observed a partial protection with NP at doses of virus corresponding at 1000 TCID50/ml when viruses were combined with nanoparticles at 45 C. for 24 h (FIG. 12,B). This result suggests that the combination provides protection of the virus against thermal denaturation.