Diluent for cell-associated alphaherpesvirus vaccine

Abstract

The present invention relates to the use of a diluent for the in-use stabilisation of cells infected with a cell-associated alphaherpesvirus. Contrary to the long-standing practice of incorporating a considerable amount of peptone into the diluent for such virus-infected cells, it was found that a reduction of the amount of protein in the diluent improved the in-use stability of alphaherpesvirus-infected cells. Whereby the best stability was even obtained using a protein-free diluent. This effect was especially pronounced for recombinant HVT viruses expressing a heterologous insert. Being protein-free is highly advantageous for the production of the diluent, in respect of costs, safety, and consistency of production.

Claims

1. A method for the in-use stabilization of cells infected with a cell-associated alphaherpesvirus, comprising admixing said infected cells with a protein-free diluent that comprises between 75 and 400 mM sugar and a phosphate buffer.

2. The method of claim 1, wherein the cell-associated alphaherpesvirus is of a genus selected from the group consisting of Mardivirus and Iltovirus.

3. The method of claim 1, wherein the cell-associated alphaherpesvirus is selected from the group consisting of Marek's disease virus and herpesvirus of turkeys.

4. The method of claim 1, wherein the cells are avian fibroblasts.

5. The method of claim 1, wherein the diluent has a pH in the range from about 7.0 to about 7.8.

6. The method of claim 1, wherein the sugar is sucrose.

7. The method for the in-use stabilization of cells infected with a cell-associated alphaherpesvirus of claim 1, wherein the protein-free diluent comprises between 100 and 300 mM sugar and a phosphate buffer.

8. A method for the in-use stabilization of cells infected with a cell-associated alphaherpesvirus, comprising admixing said infected cells with a protein free diluent that comprises a sugar and a phosphate buffer; wherein the phosphate buffer has a pH in the range of about 7.0 to 7.5, and comprises about 1 to about 4 mM KH.sub.2PO.sub.4, and about 5 to about 9 mM Na.sub.2HPO.sub.4; wherein the sugar is sucrose having a concentration of about 100 to 350 mM; wherein the diluent further comprises about 50 to 250 mM sodium and about 0.01-0.02 mg/ml phenolsulfonphtalein; and wherein the cell-associated alphaherpesvirus is selected from the group consisting of Marek's disease virus and herpesvirus of turkeys.

9. The method of claim 8, wherein the cells are avian fibroblasts.

10. A vaccine against cell-associated alphaherpesvirus; wherein the vaccine comprises cells infected with said alphaherpesvirus; wherein the cells are suspended in a protein-free diluent that comprises between 75 and 400 mM sugar and a phosphate buffer.

11. The vaccine of claim 10, wherein the vaccine is for avians; and wherein the cell-associated alphaherpesvirus is selected from the group consisting of an Infectious Laryngotracheitis virus, a Marek's disease virus, and a herpesvirus of turkeys.

12. The vaccine of claim 10, wherein the cells are avian fibroblasts.

13. A method of preparing the vaccine of claim 10, comprising admixing cells infected with a cell-associated alphaherpesvirus and the protein-free diluent.

14. The method of claim 13, wherein the cells are avian fibroblasts.

15. The vaccine of claim 10, wherein the cells are suspended in a protein-free diluent that comprises between 100 and 300 mM sugar and a phosphate buffer.

Description

EXAMPLES

Example 1: Outline of Stability Assays

(1) The base of the diluent for the use according to the invention is formed by phosphate buffer and sucrose.

(2) The phosphate buffer is used at 10 mM, and is set at a certain pH level by selecting the composition of the two phosphates. For example for a pH of 7.3 the diluent contained: 0.324 mg/ml of KH.sub.2PO.sub.4, and 1.356 mg/ml of Na.sub.2HPO.sub.4.2H.sub.2O.

(3) Sucrose was used at 50 mg/ml (i.e. 150 mM), some diluent samples tested had a higher sucrose content of 92.5 mg/ml (i.e. 270 mM).

(4) Phenolsulfonphtalein was used at 0.01 or 0.02 mg/ml.

(5) To some diluent samples tested magnesium was added at 1 mM of MgCl.sub.2.

(6) In some diluent samples tested CaCl.sub.2) was added to 0.133 mg/ml.

(7) In some diluent samples tested NaCitrate was added to 1 mM.

(8) In some diluent samples tested, peptone inclusion was used: NZ-amine AS, from Kerry Inc., pancreatic digest of casein, used at 1-14 mg/ml.

(9) Positive control for the stability studies was complete CEF culture medium comprising 1% v/v newborn calf serum and an antibiotics mixture.

(10) In-use stability incubations were performed by incubating samples of HVT-infected CEFs at room temperature (20-25° C.) for up to 4 hours. Next the samples were immediately titrated on overnight dishes with CEFs.

(11) The virus used for these stability assays was a recombinant HVT vector virus, construct HVP360, as described in WO 2016/102647. This comprises a double insert of heterologous genes: the NDV-F gene and the IBDV-VP2 gene. This recombinant virus had a reduced in-use stability as compared to non-recombinant HVT or MDV, in standard MDV diluent comprising 1.4% w/v peptone.

(12) Titration assays were standard: CEFs from 10 day old chicken embryos were prepared and seeded on 6 cm dishes. After attachment overnight (38° C., 5% CO.sub.2), next day, the dished were inoculated with dilutions of incubated samples from the stability assays. The dishes were incubated again until cpe became visible, usually after about 3 days. Titres were read by scoring cpe, preferably by using immuno-fluorescense by way of an antibody against HVT virus.

Example 2: Tests of Amounts of Peptone in the Diluent

(13) In an initial experiment, the effect of peptone amount was tested. Samples of recombinant HVP360-infected CEFs were incubated for 4 hours at room temperature in diluent compositions comprising different amounts of NZ-amine. The diluent used in these experiments had a pH of 7.4, and included magnesium- and calcium ions. One series of samples were incubated in diluent plus 1% v/v NCS, to mimic cell-culture medium.

(14) After incubations, samples were titrated on CEFs, in triplo, and the difference between titre before (t=0 hours) and after incubation (t=4 hours) was calculated as the ‘delta’. Standard deviation of titres was typically about 0.1. All titres are in 10 Log plaque forming units per ml.

(15) TABLE-US-00001 TABLE 1 Effect of different amounts of peptone on in-use stability of rec. HVT in CEF Titre in 10Log PFU/ml delta @ Diluent added added 2 t = 0 hr t = 4 hr PB, pH 7.4, Mg, Ca NZamine, 1 mg/ml 6.14 −0.18 sucrose NZamine, 7 mg/ml 6.17 −0.29 NZamine, 14 mg/ml 6.18 −0.56 1% NCS 6.18 −0.11 PB = phosphate buffer

(16) As was clear from these results, the presence of peptone could not mimic the effect of NCS, on the contrary: where NCS gave the smallest titre loss of all samples tested, the presence of peptone in the amount of the standard MDV diluent, 14 mg/ml (1.4% w/v), had the most negative effect: 7 mg/ml (0.7% w/v) was already better, and best in this experiment was the use of diluent with 1 mg/ml peptone (0.1% w/v). In conclusion: less peptone gave less titre loss.

Example 3: Tests of Low Amounts of Peptone

(17) A follow-up experiment was done to investigate the use of low amounts of peptone. This time the diluent did not contain magnesium or calcium, but different pH values were tested. Titrations were done in duplo.

(18) TABLE-US-00002 TABLE 2 Effect of low amounts of peptone on in-use stability of rec. HVT in CEF Titre in 10Log PFU/ml delta @ Diluent pH added t = 0 hr t = 4 hr PB, sucrose 7.2 — 5.6 −0.23 7.4 5.5 −0.14 7.2 NZamine, 1 mg/ml 5.5 −0.47 7.4 5.5 −0.34 7.2 NZamine, 3 mg/ml 5.5 −0.62 7.4 5.6 −0.57 Nobilis Diluent CA 5.4 −1.05 Culture medium + 5.6 −0.02 1% NCS

(19) Several conclusions could be drawn from these results: worst losses (highest delta after 4 hrs. at 25° C.) were found for this recombinant HVT construct when kept in a standard commercial MDV diluent (containing 14 mg/ml peptone) least losses were found for complete culture medium a basic diluent of phosphate buffer with sucrose was quite effective at reducing titre loss of recombinant HVT in CEF, for up to 4 hours at room temperature magnesium and calcium were not required diluent at pH 7.4 was more effective at reducing losses than diluent at pH 7.2 the lower the amount of peptone, the better, with the protein-free diluent as most effective.

Example 4: Further Variations of the Diluent

(20) In continuation of the experiment in Example 3 above, further variations of the diluent compositions were tested: addition of 0.1 mg/ml magnesium; correction to iso-osmotic value by adding salts: either 3.16 mg/ml NaCl; or the combination of 0.17 mg/ml KCl and 2.92 mg/ml NaCl. Alternatively iso-osmotic values were produced by increasing sucrose to 92.5 mg/ml, and no further salts.

(21) Also inclusion of 1 mM citrate in the diluent was tested.

(22) In-use stability results of these different compositions showed that: a low amount of magnesium can provide some further reduction of titre loss; a similar further reduction could be reached using increased sucrose concentration. the different ways to create iso-osmotic values had similar effects the inclusion of 1 mM citrate in the diluent, next to phosphate buffer and sucrose, enhanced the in-use stability of rec. HVT virus.

Example 5: Animal Trial

(23) To confirm that the cell-associated alphaherpesvirus was still effective as a vaccine after in-use stabilisation using the diluent according to the invention, animal trials are in preparation to test for protective effect against a challenge infection with MDV, or with NDV or IBDV.

(24) The basic set-up of the experiments will be a division over several test groups with appropriate controls.

(25) For a test of the protective effect against a challenge with MDV, it is common to apply the MDV challenge by way of shedder birds.

(26) Protection against NDV or IBDV, will be tested using appropriate virulent virus from those viral species.

(27) For a shedder trial, typically some 1140 fertilised chicken eggs will be used; about 240 of those will be used to generate birds that will serve as shedders. The rest will be divided over a number of test groups and two control groups: one receiving a control vaccination, and one receiving no vaccination.

(28) A brief outline of the planned experiments is as follows:

(29) General Protocol

(30) A 2-4 hr in-use stability incubation will be applied to CEFs infected with a rec. HVT, whereby the cells are taken up into a diluent for the invention. Vaccine viruses will be titrated prior to the start of the study to establish dilution for target doses. Chickens will be obtained as 200 embryonated eggs at day 14 of embryo development (ED), and 900 unset, embryonated eggs (ED 1). Eggs will be sorted and set according to: 240 (ED 14) for shedder treatment 150 (ED 1) for unvaccinated contacts 750 (ED 1) for vaccinate treatments; 125 to be in ovo vaccinated at ED 18 At ED 18, eggs for shedders will be candelled and placed in hatching trays. At hatch, shedder chickens will be eyedrop vaccinated against Newcastle disease and infectious bronchitis, neck-tagged and inoculated intra-abdominally with 200-400 PFU of virulent MDV serotype 1 and placed at 50 per pen. Birds will be provided food and water ad libitum and will be monitored and documented daily. On ED 18 for control and vaccinates, eggs will be divided over the treatment groups, vaccinated in ovo, and placed in hatching trays. All vaccines used will be back-titrated. At hatch, unvaccinated contacts and vaccinates will receive NDV/IBV eyedrop vaccine, will be neck-tagged and placed in contact with shedders. Birds will be provided food and water ad libitum and will be monitored and documented daily. At day 42 for the shedder birds, they will be euthanized and scored for MDV related lesions. At day 29 post-placement, all unvaccinated contacts and vaccinates will receive a booster vaccine for NDV and IBV, by spray. At day 49 post-placement, all remaining birds will be euthanized and scored for MDV related lesions.
Scoring of Chickens

(31) All mortality, clinical signs (paralysis, torticollis, red-leg), and lesions will be recorded.

(32) All lesions at termination (necropsy) will be recorded.

(33) Data will be reported as % affected by MDV, treatment survival curve, and protective index

(34) Protective Index

(35) Protective indices (PI) will be based on Witter method:
PI=(% MD in unvaccinated)−(% MD in vaccinated)/(% MD in unvaccinated)×100

(36) MD=signs of Marek's disease

(37) Housing:

(38) Birds will be housed in a single broiler house divided into 4 pens with automatic feeders and nipple waterers, with single radiant heating and air-handling systems.

(39) Monitoring

(40) Birds will be monitored daily for overall health, food, air circulation and water. Any culls or birds found dead, after two weeks post-placement, will be necropsied and lesions recorded. Birds succumbing during the first two weeks of life will be considered as “non-specific” mortality.

(41) Duration

(42) Experiment duration will be 63 days. Shedders will be placed 2 weeks prior to the vaccinates and will be co-housed for 28 days. Vaccinates/contacts will be kept for 7 weeks (49 days).

Example 6: Results of the Animal Experiment of Example 5

(43) The vaccination-challenge experiment described in Example 5 was performed in the middle of 2018. Unfortunately a technical failure in the climate-handling equipment of the animal facility caused a massive heat-stress event for the birds in the different treatment groups. This caused a large number of the birds to die, and the remaining ones to have severely disrupted immune responses, which did not allow a meaningfull analysis of the data. A re-run of the experiment is now being planned.

Example 7: In-Use Stability Testing of Further Cell-Associated Alphaherpesviruses

(44) Preferred Diluent

(45) The preferred composition of the diluent for use in the stabilisation according to the invention was used for determining the in-use stability of cells infected with further cell-associated alphaherpesviruses, both from commercial vaccines and from laboratory strains.

(46) The preferred version of the invention diluent is protein free, and has pH 7.1 after preparation, which shifts to 7.0 after heat sterilisation.

(47) TABLE-US-00003 TABLE 3 Composition of preferred invention diluent g/l mM phosphate buffer KH.sub.2PO.sub.4 0.451 3.3 Na.sub.2HPO.sub.4 1.19 6.7 sugar sucrose 50 146 sodium sodium-chloride 3.16 54 pH indicator phenolsulfonphtalein 0.01 0.03 water for injection — To 1 litre —
Vaccines Tested

(48) The various viruses tested are listed in Table 4. Virus was thawed from cold storage (−140° C.) and diluted in either the standard diluent Nobilis™ CA diluent, or in the preferred diluent for the invention. Dilutions for titrations were prepared as indicated in Table 4, and the t=0 samples for virus titration were titrated on plated CEF cells immediately. After 4 hours storage of the dilution at room temperature (about 21° C.), the dilutions were sampled again for titration on CEF to determine the end point of the in-use stability.

(49) TABLE-US-00004 TABLE 4 Overview of experimental conditions of in-use stability testing of MDV viruses Test sample type dilution 1:x no. replicates Nobilis ® Rismavac MDV1 80.000 2 Innovax ® ILT HVT + 2 inserts 100.000 2 Innovax ® ND HVT + 1 insert 500 4 Innovax ® ND-IBD HVT + 2 inserts 75.000 6 SB1 MDV2 1.000 4 FC126 HVT 40.000 4
Virus Titrations

(50) Petri dishes (diameter 6 cm) were seeded with CEFs at a density of 1.1×10{circumflex over ( )}5 cells/cm.sup.2 in 4 ml culture medium with antibiotics. These dishes were incubated overnight at 38° C. and 5% CO.sub.2. After about 24 hours the CEF cells had reached a confluence of approximately 80%. 100 μl of each of the samples taken in the stability test was added to the dishes; the number of replicates is indicated in Table 4. Dishes were incubated for a further 3-4 days until virus plaques were visible.

(51) IFT

(52) Plaques on the dishes were visualised using immuno-fluorescence assays. At the time plaques became clearly visible, the cells on the dishes were fixated using cold (−20° C.) 96% ethanol for 3-5 minutes. Subsequently, dishes were washed three times using standard wash buffer. 3 types of monoclonal antibodies were used as first antibody, a specific one for each of the virus types tested here: HVT, MDV1, or MDV2. The monoclonal antibodies were diluted to the appropriate strength in wash buffer, added to the plates, and incubated for one hour at 37° C. Thereafter, dishes were washed three times with wash buffer. A conjugate Goat-anti-Mouse-Alexa448 (1:1.000) as second antibody, and a counter stain with Evans Blue (1:1.500) were added to the dishes in wash buffer and incubated for another hour at 37° C. The dishes were then washed three times with wash buffer. Finally, 44% glycerol in PBS was added to the dishes. Virus plaques were counted using a fluorescence microscope, and virus titers of the test samples were calculated in pfu/ml.

(53) Results:

(54) The results of the in-use stability test of the different alphaherpesviruses are presented in Table 5, as the titres in pfu/ml at the start of the test, and after 4 hours at room temperature. The column with the delta indicates the difference in titre between the two time points. The two panels of Table 5 allow the comparison of the results of the standard diluent, to those of the preferred invention diluent.

(55) TABLE-US-00005 TABLE 5 Results of in-use stability testing of different alphaherpesviruses. Nobilis Diluent CA Invention diluent (10Log pfu/ml) (10Log pfu/ml) Test sample T = 0 T = 4 hr Δ T = 0 T = 4 hr Δ Nobilis ® Rismavac 6.0 5.8 0.2 6.0 5.8 0.2 Innovax ® ILT 6.9 6.5 0.4 6.8 6.4 0.4 Innovax ® ND 4.3 3.1 1.2 4.2 3.6 0.7 Innovax ® ND-IBD 7.1 6.7 0.4 7.0 6.7 0.3 SB1 5.4 4.8 0.6 5.4 5.1 0.3 FC126 6.8 6.2 0.6 6.7 6.4 0.4

(56) As can be clearly observed, the loss of titre in the invention diluent was either the same or less compared to the standard diluent. This effect occurred for all the three virus types tested: HVT, MDV1 and MDV2. Loss of titre was in most cases no more than 0.410 Log pfu/ml, and usually less.

(57) For one sample, Innovax ND, the loss in titre of 0.710 Log pfu/ml was quite large in the invention diluent, and exceeded the normal maximal loss of titre of 0.410 Log pfu/ml. However for this sample the loss in titre in the standard diluent was even much worse at 1.210 Log pfu/ml. As this was also the virus that needed the least dilution, this was probably not a good quality sample to begin with, and not representative of its kind, although it does show an impressive difference in stabilising capacity between the invention diluent and the standard diluent.