Patent Application
20110091500
This invention relates to a vaccine composition comprising one or more inactivated bluetongue virus (BTV) serotypes prepared from live attenuated bluetongue viruses with the addition of an adjuvant.
Bluetongue disease (BT) is an arthropod-borne viral disease of sheep and cattle, caused by one or many of the 24 known serotypes of the bluetongue virus (BTV). The virus has been recognized as an important a etiological agent of disease in sheep in South Africa, and until 1943 was believed to be restricted to Africa, south of the Sahara. The disease has since been identified in several countries outside Africa, such as Cyprus, Israel, the USA, Portugal, Pakistan, India, Italy, France, Spain, China, Malaysia, Bulgaria, Australia, Argentina and most recently Kazakhstan as well as North African countries including Morocco and Tunisia. In 2006, the disease was reported for the first time in some northern European countries (Germany, Belgium and The Netherlands) and has since spread to even more European countries. BTV commonly occurs between latitudes 35° S and 40° N, but the virus has also been detected further north at beyond 48° N in Xinjiang, China, western North America and in Kazakhstan (Dungu et al., 2004).
The factors contributing to the spread of BTV include animal migration and importation, extension in the distribution of its major vector, Culicoides imicola, involvement of novel Culicoides spp. vector(s), the ability of the virus to overwinter in the absence of adult vectors, and its persistence in healthy reservoir hosts such as cattle and some wild ruminants. The eradication of BTV from endemic regions of Africa is virtually impossible due to the role played by the widely distributed Culicoides spp. midge vectors, the multiplicity of serotypes that may circulate at any point in time, and the presence and ubiquitous distribution of reservoir species, both known and unknown. However, most indigenous breeds of sheep in sub-Saharan Africa are resistant to the disease.
Strategies for the control of BT depend on whether they are aimed at outbreaks of the disease in endemic areas or in areas where the disease is not usually present. In the latter case, the aim is usually eradication, whereas in endemic areas attempts can only be made to limit the occurrence of the disease and its economic impact through vaccination.
A BTV vaccine was initially developed in South Africa more than 50 years ago and has been improved to currently include 15 of the 24 serotypes known to occur in Southern Africa (Verwoerd & Erasmus, 2004). The current vaccine consists of live attenuated, cell-adapted, plaque-purified BTV serotypes in three pentavalent vaccines, which are administered separately at 3-week intervals.
Some concerns about the current vaccine have been raised in recent years. These include:
The need for safer BTV vaccines has been more critical during the recent European outbreaks due to the high susceptibility to BT of sheep and cattle in the affected countries, which may result in post-vaccination reactions when the currently available BTV vaccine is used.
There is therefore a need for an alternative vaccine which does not pose the same risks described above to sheep and cattle.
According to a first embodiment of the invention, there is provided a vaccine composition including one or more inactivated BTV serotypes from the same live attenuated serotype.
The composition may include Serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24, or a combination of any two or more of these serotypes.
The BTV may be chemically inactivated. Binary ethylenimine (BEA/BEI) and/or formaldehyde may be used to inactivate the BTV.
The composition may also include any adjuvant, such as Montanide ISA 70, ISA 206, IMS 2214 or aluminium hydroxide and saponin.
The BTV composition may be used for eliciting an immune response against the target BTV serotype(s) in a vaccinated animal. Typical animals include ruminants, such as sheep, cattle, goats and wild ungulates.
The composition may be administered to the animal as a single dose or as a first dose followed by one or more booster doses.
The composition may be formulated for intra-muscular or sub-cutaneous administration.
According to a second embodiment of the invention, there is provided a method of producing a composition substantially as described above, the method comprising the step of inactivating a live, attenuated BTV.
The method may also include an earlier step of attenuating the BTV.
According to a third embodiment of the invention, there is provided a method of preventing Bluetongue disease and/or eliciting an immune response to bluetongue virus in an animal, the method comprising administering to the animal a composition substantially as described above.
According to a fourth embodiment of the invention, there is provided a method of generating antibodies specific for a BTV antigen, the method comprising the step of administering a composition described above to an animal and recovering the antibodies.
The invention describes the preparation of an inactivated bluetongue virus (BTV) composition, and in particular a vaccine, using an attenuated live BTV vaccine strain as master seed.
The composition can be administered to an animal to elicit an immune response against the bluetongue virus serotype(s) included in the vaccine.
Non-replicating virus vaccines are considered to be safer than live attenuated vaccines, as they do not pose the same risks as described earlier in this specification. Inactivated vaccines are among the most suitable non-replicating vaccines. Up until now, it has been assumed that an inactivated vaccine has to be produced from a wild type strain in order to maintain its immunogenicity to elicit a strong immune response. However, the applicant has now found that a much better virus production yield is obtained when using an attenuated strain than when using a wild-type strain. Inactivated vaccines can be obtained from the inactivation of wild-type viruses, grown in cell culture, chemically inactivated and formulated with an appropriate adjuvant. The use of live attenuated viruses makes it easier and quicker to generate inactivated vaccines, given the fact that vaccine seed materials are readily available.
The safety and efficacy of inactivated vaccines depends on the purity, innocuity, residual inactivant and toxicity, antigen load, adjuvant selected, as well as handling and administration of the vaccine by the user. Incomplete inactivation may cause a problem with virulent wild-type viruses as they may cause disease in a vaccinated animal. Validated inactivation procedures must be followed, in particular with large production volumes to ensure complete inactivation. Inactivation kinetics must be determined for large-scale production processes.
The applicant currently manufactures a BTV vaccine comprising live attenuated viruses of 15 BTV serotypes, sold as Onderstepoort Bluetongue vaccine (Reg. No. G 0358 (Act 36/1947). The generation of each vaccine strain has been a complex process of virus passages in rodents, eggs, cell culture as well as plaque selection, and testing in sheep after each passage. Table 1 below summarizes the passage history of all BTV vaccine strains contained in the current vaccine.
After 2 to 3 annual immunizations, most sheep are immune to all serotypes in the vaccine.
Different inactivated BTV vaccines that were generated from corresponding live attenuated BTV vaccines are described in the examples below. These examples, however, are not to be construed as limiting in any way either the spirit or scope of the invention.
The titres obtained after harvest of the 3 different BT viruses evaluated, before and after PEG concentration, are shown in Table 2.
Table 3 provides an illustration of the sampling conducted during the inactivation process, with equivalent virus titre at each stage. The inactivation kinetics of the 3 viruses is summarized in
Inactivation with BEI results in first order kinetics inactivation, i.e. the inactivation process occurs linearly. This is in contrast with formalin inactivation that results in second order inactivation and with some viruses, incomplete inactivation. Thus, with BEI the end point can be determined. This is important for vaccines such as FMD (Foot and Mouth disease), for which there are maximum residual virus titres permissible in inactivated vaccines.
The aziridine compound binary ethylenimine (BEI) is produced by the cyclization of bromoethylamine (BEA) which occurs under alkaline conditions. The cyclization process causes a significant drop in pH from 13.5 to 8.5, which can be demonstrated visually by using the indicator B-napthol violet. This indicator was not used in this experiment. The pH values were taken manually throughout the cyclization process.
BEI is used at very low concentrations, but must nevertheless be handled with gloves as it is toxic. Residual BEI in inactivated vaccine was neutralized before use, by the addition of 0.2% of final volume of a 50% sodium thiosulphate solution.
The efficacy of different formulations of inactivated BTV vaccines (iBTV-4) was evaluated in guinea pigs. Two BEI inactivated BTV-4 vaccines containing different adjuvants (aluminium hydroxide gel and Montanide IMS® adjuvant) were evaluated for immunogenicity.
Serum samples of the 5 bled GP in each group were pooled as one sample for testing. Table 5 below summarizes results obtained during the experiment.
Only day 28 post vaccination (Day 7 post booster) samples were tested using a commercial AGID test from VMRD (Inc). All samples tested were positive, except the negative control and pre serums.
These are shown in
All serological results indicated that the inactivated BTV4 vaccine was immunogenic in guinea pigs, as was the case with the live attenuated BTV4 vaccine.
Neutralizing antibodies, which are an indication of protection to BTV infection, were triggered at a protective level with all the formulations used, though with varying titres. No neutralizing antibodies were detectable before the booster dose. Similar results were also observed with the live vaccine, used here as a standard.
Detectable neutralizing antibodies were recorded from 14 days post-booster, persisting at different titres depending on the formulation. The aluminium hydroxide formulation yielded titres similar to those observed with the live vaccine.
The aim of the present study was to assess the ability of the formulated inactivated BTV4 vaccine to trigger an immune response that could be correlated to protection. Since a single antigen payload was used (antigen in a dose equivalent to 107 pfu/dose), it is not possible to attribute the lower SNT titres observed with the IMS 2214 formulations to poorer protection ability. Furthermore, the ability of a specific adjuvant to trigger a stronger immune response may vary between animal species: a poorer response in guineas pig may be different in sheep or cattle.
The ELISA results showed that the guinea pigs vaccinated with all different formulations seroconverted and developed increasing antibody titres. Once again, animals vaccinated with the AL-BTV4 showed higher titres than those vaccinated with the different formulations of the inactivated vaccine.
AGID tests were done only on serum from day 14 post-booster and found to be positive.
It was concluded that the inactivated BTV4 vaccine, generated from the live attenuated BTV4 vaccine, was capable of generating an immune response and protective neutralizing antibodies comparable to those generated with the live attenuated vaccine. The results also indicated the need to evaluate the vaccine in target animals, i.e. sheep, using different formulations.
The efficacy and safety of different formulations of the inactivated BTV4 vaccine on sheep were evaluated. The following adjuvants were used to formulate 4 different types of the iBTV4 vaccine:
The iBTV4 antigen produced as described earlier and stored at −80° C. was used to generate the 4 different formulations under the following mixing conditions:
Each formulation was used to immunize 5 groups of 3 sheep, according to different schedules, as described in Table 6 below. A further 16 sheep were used as controls.
All sheep were pre-bled 3 days prior to commencement of the study. Rectal temperatures of the sheep were monitored and recorded twice daily from 3 days prior to the first vaccination and for 28 days post-vaccination, 21 days post booster (where applicable) and 21 days post-challenge. Sheep were bled for serum and whole blood (5 ml each) on days 0, 3, 7, 10, 14, 18, 21, 24 and 28 post-vaccination(s) and challenge day, to determine neutralising antibody responses and viremia levels. Eight sheep from each group (total of 64) were maintained for a 6 month period, in an insect-free facility. The temperatures of these sheep were also monitored, and they were bled on days −3, 0, 3, 7, 10, 14, 18, 21, 24 and 28 post-vaccination(s) for serum and whole blood; and weekly on days 35, 42, 49 and 56, and twice monthly thereafter (days 60, 74, 98, 112, 126, 140, 154, 168 and 182) for serum only.
Body temperatures and other clinical signs of the sheep were monitored daily for 21 days post-vaccination and post-challenge. Clinical reaction index (CRI) (Huismans et al., 1987; Lacetera & Ronchi, 2004) was used to quantify the observed clinical reactions. Briefly, the CRI was calculated as the sum of the following:
Serum samples were taken weekly after vaccination/challenge for evaluation of seroconversion.
Table 7 below summarizes serum neutralization results obtained on sheep in different groups, monitored up to 133 days post initial vaccination.
The CRI were calculated only for the 3 subgroups that were challenged, i.e. animals vaccinated twice and vaccinated 28 days post-booster, and animals vaccinated once and challenged 28 and 49 days post-vaccination, respectively.
The results obtained with the different formulations of the iBTV4 indicated that the adjuvant used played a role in the onset of immunity, as illustrated by SNT results and the level of protection afforded as indicated by the CRI results. Detectable SNT titres, up to 1:64, were recorded after a single vaccination in sheep immunized with the ISA 70 formulation, and to a lesser extent with the IMS 2214 and aluminium hydroxide/saponin already on day 21 post-vaccination, while no detectable titres were seen in the aluminium hydroxide group and very low titres were seen in the ISA 206 group. The booster vaccination improved the SNT response in all groups, except for the aluminium hydroxide group. The situation of sheep vaccinated with the ISA 206 formulation improved following the booster, but to a lesser extent than the other groups. Overall the ISA 70 formulation seemed to be better than all the others, following a single vaccination or two vaccinations.
Animals that were challenged after a booster dose in all of the 5 groups were protected, as illustrated by the CRI results: none of them had a CRI higher than one, while the unvaccinated positive controls had a CRI varying between 2.5 and 3.
Protection following a single vaccination was better in the ISA 70 group and to a lesser extent in the aluminum hydroxide/saponin group, thus correlating with the SNT results.
The generation of clinical signs following a virulent challenge varies enormously between different serotypes, and depends on factors such as sheep susceptibility. BTV4 generally does not result in severe clinical signs, even in more susceptible sheep breeds, as seen in the Spanish and Portuguese BTV4 outbreaks (OIE disease information, 2006). This could explain the lower CRI observed in the positive unvaccinated controls used in the present study. It was, however, still possible to see a difference between the clinical responses in vaccinated and unvaccinated animals.
The results of the present study clearly indicated that the iBTV4 vaccine was immunogenic and protective, with the adjuvant playing an important role in improving the protection afforded. The SNT results correlated positively with the CRI results, confirming the value of each of the two parameters.
The ability of an inactivated BTV8 vaccine to protect sheep against a virulent challenge of the European BTV8 wild-type virus was evaluated after single and booster vaccine doses, together with the safety of the vaccine. The inactivated BTV8 vaccine was based on the attenuated BT Serotype 8 vaccine virus.
Ten adult Merino ewes aged 1-2 years were used in this trial. Bluetongue (BT) susceptibility was confirmed by a competitive ELISA (OIE, 2004) in the BT reference laboratories at the Onderstepoort Veterinary Institute (OVI) of South Africa. Animals were stabled in an insect free stable for the duration of the experiment.
Inactivated monovalent BTV8 vaccine was produced as described in previous reports. Briefly, BTV8 attenuated seed stock antigen was used to infect 4 BHK roller flasks. Harvested antigen was precipitated using PEG as described earlier. Concentrated antigen was inactivated using BEI. The iBTV8 vaccine was formulated using ISA 206 as adjuvant.
Vaccinated sheep were monitored daily for 21 days post-vaccination and 21 days post-challenge for temperature and clinical signs. All sheep were exposed to environmental stresses daily for 3-4 hours post-challenge.
The Netherlands outbreak virus isolate BTV8 (G) 5/10A 2006/01KC 3BHK received from the OIE Bluetongue reference laboratory at OVI was used as challenge dose. A virus suspension of an additional passage in BHK cells with a dose of 107 pfu/ml was administered by the intravenous route.
Two groups of sheep were used in this trial (Table 8). One group was vaccinated once and challenged 28 days post-vaccination. The other group was boosted 21 days after the first vaccination, and then challenged 28 days post-vaccination.
Sheep were monitored daily for 21 days post-vaccination and challenge, for body temperature and other clinical signs. Clinical reaction index (CRI) (Huismans et al., 1987; Lacetera & Ronchi, 2004) was used to quantify the observed clinical reactions, as described earlier herein.
Vireamia in sheep was determined according to OIE guidelines (OIE, 2004). In brief, a 25 cm3 Falcon flask was inoculated with 1 ml of blood and incubated for +/−30 min at 37° C. before blood was rinsed from the flask and maintenance medium added. Negative samples were passaged a further two generations on tissue culture.
Serum samples were collected weekly after vaccination and after challenge for serological evaluation. Neutralising antibodies were determined by the microtitre serum neutralization test using serotype BTV8 neutralizing antigen (OIE, 2004) (Table 9).
Although all vaccinated animals showed temperature reactions to differing degrees post-challenge, the sheep that received a booster dose showed less temperature reaction, and subsequently had a lower CRI than those in Group B1 (
Neutralizing antibody response to a single vaccination with iBTV was low (Table 9), and the highest response 28 days post-vaccination was 32. A booster inoculation (Group B2) gave results of between 32 and 128. This level of antibody should be sufficient to protect animals against challenge. SNT of Group B2 post-challenge showed a significant rise from values post-booster. Antibody response after a single vaccination and challenge showed a similar drastic increase in the antibody levels.
The limited ability of the iBTV vaccine formulated with ISA 206 to trigger a full protective immunity after one vaccination was comparable to results described above with the iBTV4 vaccine. Even though sheep vaccinated with the iBTV8 vaccine were protected against a virulent challenge with the European BTV8, and on the basis of results described in example B above, the applicant is planning to conduct a comparative trial using an ISA 70 adjuvanted iBTV8 vaccine.
The vaccine formulation of the invention can be made by formulating clarified virus with ISA70. The suspension BHK-21 cells from ASG are scaled up from spinner flasks to 1000 L to maximum cell density. The cell culture is transferred to a 2000 L bioreactor and adjusted/diluted to a start cell density of 1-1.5×106 cells/ml. The cells are infected with virus and the virus culture harvested after 48 hours. The virus is clarified using a dead-end filter and titrated to determine a pre-inactivation titre. The antigen is inactivated with 10 mM BEI for 38 hours and tested to confirm inactivation and sterility. Bulk vaccine is formulated using ISA70 as an adjuvant in 30% antigen: 70% ISA70 ratio.
The applicant has thus shown that the different inactivated BTV vaccines that it generated were immunogenic in guinea pigs and sheep, and protected sheep against a virulent challenge. The adjuvant used played an important role in triggering an early and solid immunity, protecting vaccinated animals after either a single vaccination or two vaccinations.
Further studies using various inactivated BTV serotype combinations and different adjuvants are currently in progress.
While the invention has been described in detail with respect to specific embodiments thereof, it will be appreciated by those skilled in the art that various alterations, modifications and other changes may be made to the invention without departing from the spirit and scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008/03366 | Apr 2008 | ZA | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2009/051590 | 4/16/2009 | WO | 00 | 12/14/2010 |
