Patent Application
20130177582
The invention relates to a therapeutic agent capable of treating and/or preventing rabbit haemorrhagic disease virus infection in rabbits, namely a recombinant parapoxvirus, characterized in that the VP60 major capsid protein from the rabbit haemorrhagic disease virus (RHDV) is expressed from a foreign nucleic acid.
Rabbit haemorrhagic disease virus (RHDV) is a highly contagious disease in wild and domestic rabbits, such as of the species Oryctolagus cuniculus, which was first reported in the People's Republic of China in 1984. RHDV subsequently spread throughout Europe during the years 1987 to 1989. Infected rabbits-adults usually die within 48 to 72 hours after infection of necrotizing hepatitis and haemorrhagic syndrome. Morbidity and mortality rates in a population can be as high as 90-100%.
The disease is responsible for significant economic losses in commercial rabbit production, as well as for a high mortality rate in wild rabbits. Commercial rabbit production across the world is an important industry, particularly in Asia and central Europe where small scale rabbit husbandry is well established. The highly contagious and fatal nature of RHDV has had profound economic effects throughout many countries. Additional conservational aspects, such as rabbit population control, in countries such as New Zealand and Australia have led to a significant international effort to understand and control the disease.
Work in many laboratories has led to the characterization of the RHD virus as a calicivirus on the basis of capsid morphology, nucleic acid type and other physical characteristics. The virus particle consists of an un-enveloped icosahedral 35-40 nm diameter capsid, composed primarily of a major 60 kDa polypeptide species. The virus is encoded by a positive sense single strand RNA genome of approximately 7.4 kb and is organized as two open reading frames that code for the major 60 kDa (VP60) capsid protein, a putative protein of 12 kDa, and three non-structural proteins, including an RNA polymerase and a protease. The viral capsid results from the multimerization of approximately 180 copies of a single VP60 protein.
Various efforts have been made to produce vaccines for the treatment and prevention of RHDV infection in rabbit populations, most of which rely on administration of the VP60 capsid protein. VP60 capsid protein has been produced in various expression systems, such as yeast (Boga et al, Journal of General Virology (1997), 78, 2315-2318), Baculovirus/cell culture systems (Laurent et al, Journal of Virology, Oct. 1994, 6794-6798, Marin et al., Virus Research 39 (1995) 119-128) and Pichia pastoris (O. Farnós et al., Antiviral Research 81 (2009) 25-36). Recombinantly produced VP60 protein has been subsequently applied as an effective vaccine. However the production of recombinant protein is associated with various difficulties such as formation of structurally irrelevant protein aggregates, time consuming and complicated protein purification systems and difficulties producing sufficient quality and quantities for commercial production.
Recombinant VP60 has also been produced in potato plants and subsequently applied as a vaccine in leaf extracts (Castanón et al, Journal of Virology, May 1999, p. 4452-4455). Although effective in producing antibodies directed against VP60 in vaccinated rabbits, extract production from plants is comparatively slow compared to virus production in cell culture. Transgenic plants must be grown, harvested and processed, thus limiting output to a scale inappropriate for commercial application. Similarly pea-derived recombinant VP60 can lead to poor yield and thus limited commercial application (Mikschofsky H et al., Plant Biotechnol J. 2009 Aug;7(6):537-549). Similar complications are evident in related attempts to produce the VP60 antigen via expression in plants. Expression in carrots leads to insufficient expression levels for effective vaccination (Mikschofsky, H. et al, 2009, Journal of Agricultural Science 147, 43-49, Mikschofsky, H. et al, 2009, In Vitro Cell.Dev.Biol.Plant 45, 740-749). Expression tobacco plants using the cholera tocxin B as a functional adjuvant also revealed that expression levels of VP60 were sub-optimal, in addition to the further complications associated with production in a plant expression system (Mikschofsky, H. et al, 2009, Plant Sci. doi: 10.1016/j.plantsci.2009.03.010 (Cholera toxin B as a functional adjuvant in tobacco plants).
The use of viruses as RHDV vaccines offers a promising alternative to the administration of recombinant VP60 protein. Vaccinia based vaccines have been applied for RHDV (Bertagnoli et al., Vaccine, Vol. 14, No. 6, pp. 506-510, 1996), although long-term immunity induced by vaccinia virus can result in unsuccessful revaccination or reduced protection against vaccinia-encoded foreign antigens.
At the present time, due to the inability of the RHDV to be cultures in cell culture systems (therefore hindering viral-based vaccine production), present vaccines involve infection and sacrifice of animals in order to extract liver from which the RHDV can be subsequently isolated. This leads to inactivated or dead viral vaccines which are obtained via infection and sacrifice, a practice that has obvious disadvantages in terms of animal protection and financial limitations. RHDV won from infected animals (in the vaccines of the prior art) is subsequently inactivated before application in vaccination. This leads to a significant reduction in activity in comparison to the preferably living parapox-VP60 virus of the present invention. It also requires additional viral inactivation products during its manufacture and often additional adjuvants before application, in order to increase the immune response generated after vaccination.
The Parapoxvirus (PPV) presents a genus of the poxviridae, with the type-species PPV ovis virus (Orf virus, ORFV), which offers several potential advantages for use as a vaccine vector (Rziha et al., Journal of Biotechnology 73 (1999) 235-242). Arguments in favour of an ORFV vaccine vector include the restricted host range (sheep and goats), its tropism being restricted to the skin, the lack of systemic infection, a short-term vector-specific protective immunity and the exceptionally strong stimulation of fast innate cellular immune mechanisms at the site of infection (Büttner, M., and H. J. Rziha. 2002. J. Vet. Med. B Infect. Dis. Vet. Public Health 49:7-16.).
ORFV recombinants have been successfully administered as a vaccine against various virus infections, such as for Pseudorabies virus in pigs (Dory et al., Vaccine 24 (2006) 6256-6263, and van Rooij et al., Vaccine 28 (2010) 1808-1813) or in mice (Fischer et al., Journal of Virology, Sept. 2003, p. 9312-9323) as well as against Borna disease virus in rats (Henkel et al., J. of Virology 2005, p. 314-325).
The ORFV has however not been applied in rabbits or as a vaccine for RHDV. Despite recent advances in vaccines against RHDV, a strong demand remains for novel approaches towards controlling RHDV infection in both wild and commercial rabbit populations.
In light of the prior art the technical problem underlying the present invention is to provide an effective therapeutic agent capable of treating and/or preventing rabbit haemorrhagic disease virus infection in rabbits.
This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.
The invention therefore relates to a recombinant parapoxvirus comprising the VP60 major capsid protein from the rabbit haemorrhagic disease virus (RHDV). The Orf virus offers a restricted host range (sheep and goats) and tropism, and is thus a comparably safe viral vector.
In one embodiment the parapoxvirus of the present invention is characterized in that the VP60 major capsid protein from the rabbit haemorrhagic disease virus (RHDV) is expressed from a foreign nucleic acid sequence. The VP60 protein of the RHDV expressed from the recombinant Parapoxvirus acts to induce an antigen-specific immunity in treated animals which provides an effective protection from RHDV infection.
In one embodiment the parapoxvirus of the present invention is characterized in that the parapoxvirus is an Orf virus (ORFV), preferably the ORFV strain D1701, more preferably D1701-V-VP60n.
In one embodiment the parapoxvirus of the present invention is characterized in that the foreign nucleic acid sequence is a DNA sequence integrated into the parapoxvirus genome.
In one embodiment the parapoxvirus of the present invention is characterized in that the VP60 major capsid protein is encoded by a DNA sequence comprising the sequence according to SEQ ID No. 1 or SEQ ID No. 2.
In one embodiment the parapoxvirus of the present invention is characterized in that the VP60 major capsid protein comprises the amino acid sequence according to SEQ ID No. 3.
A further aspect of the present invention relates to the parapoxvirus as described herein for use as a medicament.
A further aspect of the present invention relates to the parapoxvirus as described herein for use as a vaccine in the therapeutic and/or prophylactic treatment of RHDV infection in a subject.
A further aspect of the present invention relates to a pharmaceutical composition comprising the parapoxvirus according to the present invention, a nucleic acid molecule encoding the VP60 protein according to SEQ ID No. 1 or SEQ ID No. 2 and/or the VP60 protein comprising the sequence according to SEQ ID No. 3 and a pharmaceutically acceptable carrier.
A further aspect of the present invention relates to use of the parapoxvirus as described herein as a vaccine in the therapeutic and/or prophylactic treatment of RHDV infection in a subject.
A further aspect of the present invention relates to a vaccine comprising the parapoxvirus as described herein, whereby the parapoxvirus is preferably alive. Living attenuated viruses are preferred. In one embodiment the vaccine as described herein can be characterised in that additional agents are present for enhanced vaccine function, such as adjuvants or immune stimulators. In a further embodiment the vaccine as described herein can be characterised in that the vaccine is a combination vaccine, comprising of vaccine-agents for additional diseases.
A further aspect of the present invention relates to a method of therapeutic and/or prophylactic treatment of RHDV infection in rabbits, characterised in that a therapeutically effective amount of the parapoxvirus according to the invention, the pharmaceutical composition of the invention or the vaccine as described herein is administered to a subject.
A further aspect of the present invention relates to a nucleic acid molecule encoding the rabbit haemorrhagic disease virus (RHDV) protein VP60 comprising the sequence according to SEQ ID No. 1 or SEQ ID No. 2. In one embodiment the nucleic acid molecule coding for VP60 is intended for use in the production of a recombinant parapoxvirus as described herein.
A further aspect of the present invention relates to a VP60 major capsid protein of the rabbit haemorrhagic disease virus (RHDV) comprising the sequence according to SEQ ID No. 3.
A further aspect of the present invention relates to a use of the nucleic acid molecule coding for VP60 and/or the VP60 major capsid protein in the manufacture of a medicament, preferably a vaccine, for the therapeutic and/or prophylactic treatment of RHDV infection in rabbits.
The subject of the present invention is intended to a rabbit. “Rabbit” is to be understood as mammals in the family Leporidae of the order Lagomorpha. Wild and domesticated rabbits are intended subjects of the present invention. There are eight different genera in the family classified as rabbits, including the European rabbit (Oryctolagus cuniculus) and cottontail rabbits (genus Sylvilagus), in addition to many other species of rabbit, included are also hares.
In one aspect, the present invention relates to a recombinant parapoxvirus, containing a
DNA sequence coding for the RHDV VP60 major capsid protein. According to the present invention, the recombinant parapoxvirus expresses gene products of the VP60 gene. Specific open reading frame(s) of VP60 are inserted into the parapoxvirus vector, and the resulting recombinant virus is used to immunize an animal. Expression of VP60 gene product results in an immune response in the treated animal targeted specifically against VP60. Thus, the recombinant virus of the present invention may be used in an immunological composition or vaccine to provide a means to induce an immune response which may, but need not be, protective. Said immune response may be therapeutic or prophylactic in its effect.
The combination of the VP60 with parapox virus enables VP60-containing virus to be produced in cell culture, something that was until now not successfully achieved. The present invention therefore enables production of VP60-vaccine without sacrifice of infected animals, as is currently carried out in the prior art. The parapoxvirus described herein is propagated effectively in Vero cells (Monkey kidney cell line).
The VP60 gene is expressed under control of an early parapoxvirus-promoter once integrated in the parapox genome. The VP60 gene product is therefore expressed after infection of the cells by the parapox virus, so that the VP60 gene is both transcribed and translated whilst in the host cells. However, this expression does not lead to the creation of infectious virus.
The active expression of recombinantly integrated gene products after infection represents a general principle of the poxviruses and is dependent on living vaccine. The recombinant virus of the present invention can however be applied as either living or dead virus, considering that the VP60 protein is expressed and leads to VP60 antigen production before application. Production of antibodies in host cells in response to the VP60 antigen occurs in response to application of either live or dead virus. In a preferred embodiment the recombinant virus of the present invention is applied to subjects as a living vaccine, as VP60 expression continues after infection.
A significant and unexpected advantage of the present invention is that subjects infected (treated or vaccinated) with the virus vaccine of the present invention do not produce virus particles, which cannot then be released from the treated subject. This represents a significant advantage for various reasons. Firstly, infection (vaccination) is limited to those subjects treated initially, without allowing uncontrolled propagation of the virus. The virus can therefore also not spread to other hosts, which is a potential concern despite the limited host range of the parapox virus. Secondly, because the virus is recombinant in nature (genetically modified) there is significant emphasis made by both the general public and by agricultural regulatory bodies that genetically modified products are not able to spread or be released into the environment. The fact that the recombinant viral vaccine of the present invention is not released from infected subjects represents therefore a surprising and advantageous aspect to the invention, which could not have been predicted in light of the prior art.
The present invention relates to all parapox viruses as vectors for the recombinant VP60 gene. In a preferred embodiment the parapoxvirus of the present invention is characterised in that the Parapoxvirus is an Orf virus (ORFV), preferably the ORFV-strain D1701, more preferably the strain D1701-V-VP60n. Derivatives of the ORFV-strain D1701 are intended to fall within the scope of the invention. The strain D1701 represents a highly attenuated virus originally isolated from Sheep. After serial cell culture passages the resulting avirulent D1701 strain was successfully used in the development of a live vaccine (Mayr, A. et al, 1981, Tbl. Vet. Med. B. 28, 535-552). The D1701 strain has been further characterised and examined for its properties as a vaccine vector (Cottone, R. et al, 1998, Virus Research 56, 53-67, Rziha, H.-J. et al, 2000, Journal of Biotechnology 83, 137-145, EP 0 886 679 B1). The strains described in Mayr et al (1981), Cottone et al (1998), Rziha et al (2000) and in EP 0 886 679 B1 are intended as preferred parapox vectors for the VP60 gene as described herein. These strains are highly attenuated, apathogenic and unable to reproduce in rabbits.
A further advantage of the recombinant parapoxvirus of the present invention is the immune stimulation effect that occurs during treatment of subjects. The parapoxvirus enhances the immune stimulatory effect, so that the virus vector itself acts a adjuvant for the VP-60 application. This unique and inventive combination of the parapox virus with the VP60 gene results in a synergistic effect, whereby the immune stimulatory properties of the parapoxvirus combine in a synergistic manner with VP60 antigen, which also induces an immune response (namely an antigen specific immune response), thereby providing a strong immune response towards the VP60 antigen. This combination of factors ultimately results in production of an effective vaccine. Until now, the approaches as described in the prior art that attempted to administer VP60 in various forms had never led to a sufficient immune response in order to provide effective protection against the virus. The unique combination of parapoxvirus and VP60 does however provide an effective solution to the long-felt need for a RHDV vaccine. As has been shown in the prior art, application of either parapoxvirus or VP60 antigen alone has no particular effect. The combination leads to surprising effectiveness, whereby each of the virus and VP60 produce an effect together that is greater than the sum of their parts, generating an immune response that provides lasting protection against the RHDV.
The administration procedure for recombinant virus of the present invention or expression product thereof, compositions of the invention such as immunological, antigenic or vaccine compositions or therapeutic compositions, can be administered via parenteral routes, such as intradermal, intramuscular or subcutaneous application methods. Such an administration enables a systemic immune response, or humoral or cell-mediated immune responses. The vaccine according to the present invention can also be incorporated in animal feed, enabling simple and effective immunisation of large populations.
The preferred methods of application relate to either sub-cutaneous application or intramuscular application, preferably by injection.
More generally, the inventive VP60 parapoxvirus recombinant, antigenic, immunological or vaccine parapoxvirus-VP60 (D1701-V-VP60n) compositions or therapeutic compositions can be prepared in accordance with standard techniques well known to those skilled in the pharmaceutical or veterinary art.
A therapeutically effective amount of such compositions can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts taking into consideration such factors as the age, sex, weight, species and condition of the particular subject, and the route of administration. The compositions can be administered alone, or can be co-administered or sequentially administered with compositions, e.g., with “other” immunological, antigenic or vaccine or therapeutic compositions thereby providing multivalent or “cocktail” or combination compositions of the invention and methods employing them. Again, the ingredients and manner (sequential or co-administration) of administration, as well as dosages can be determined taking into consideration such factors as the age, sex, weight, species and condition of the particular subject, and, the route of administration.
Examples of compositions of the invention include liquid preparations for orifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric, etc., administration such as suspensions, syrups or elixirs; and, preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration) such as sterile suspensions or emulsions.
In such compositions the recombinant parapoxvirus may be in admixture with a pharmaceutically acceptable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.
Short description of the figures:
In order to produce recombinant ORF viruses that express the VP60 protein of the RHDV (Rabbit Haemorrhagic Disease Virus), the entire VP60 gene (SEQ ID No. 1) was synthesised by the company “Mr. Gene” (Regensburg, Germany). In order to ensure correct expression of the recombinant VP60 the DNA sequence of the gene was optimised to remove early poxviral transcriptional stop motifs. The VP60 gene was provided in vector pMK-RQ, as shown in
Vero cells were infected with the beta-galactosidase (LacZ-gene positive) expressing ORFV strain D1701-VrV and subsequently transfected with the pdV-VP60n plasmid via nucleofection. After successful DNA recombination, the beta-galactosidase gene is exchanged by the VP60 gene of RHDV to generate white recombinant virus plaques that can be distinguished from the parental blue D1701-VrV strains.
VP60 gene positive plaques can therefore be identified using plaque-PCR after selection according to colour (
After propagation of the three plaque isolates, 3.3.4, 4.1.9 and 2.2.6, immunostaining of infected cells was carried out in order to test the expression of the VP60 protein. Either infected or uninfected cells were stained with a VP60-specific rabbit antiserum (
Correct insertion of the VP60 gene into the genome of the ORFV D1701-V was analysed by restriction enzyme digestion and Southern blot hybridisation, using all three recombinants. The results are shown in
The experimental results demonstrate correct insertion of the RHDV VP60 gene after exchange with the LacZ gene in all three plaque isolates of D1701-V-VP60. Further control hybridisations confirmed the correct gene insertion.
RNA was isolated at various time points after infection (2 to 32 hours). VP60 specific RNA was detected using Northern blot hybridisation and a VP60-specific probe (
Various specific antibodies were used to test protein expression of VP60 (60 kDa) by Western blot analysis (polyclonal rabbit anti-VP60 antiserum C1 from Prof. Dr. G.
Meyers, FLI Tübingen, polyclonal monospecific rabbit antiserum from Prof. B. Szewczyk, University of Gdansk, monoclonal antibody mAb1G8 from Dr. H. Schirrmeier, FLI Riems). Lysates were harvested at different time points (24 and 48 hours post infection). Lysates from uninfected cells were used as negative controls (ni) in addition to D1701-V infected cells. The cells were infected with an MOI of 1.0.
The results shown in
Immunofluorescence analysis using antibodies specific for the VP60 protein demonstrated globular-like VP60 expression in the cells infected with recombinant virus (
Immunization experiments were carried out with 16 rabbits (chinchilla bastard) four months old and were sourced from the animal facility of the Friedrich-Loeffler-Institut (FLI), Island of Riems. The experimental design consisted of using four groups of animals, each group containing four rabbits.
Group 1 was treated with a prime immunisation intramuscularly (IM), comprising administration of recombinant virus of 1×107 pfu/dosis (1 ml volume). Boosting treatments were subsequently carried out, the first boost occurring 28 days post vaccination, the second boost occurring 42 days post vaccination.
Group 2 was treated with a prime immunisation intramuscularly (IM), comprising administration of recombinant virus of 1×106 pfu/dosis (1 ml volume), although otherwise carried out identically to the treatment of group 1.
Group 3 was treated with a prime immunisation using the control substance 1 ID RicaVacc. No boosting treatments were applied.
Group 4 was the control group, no treatment was applied.
Serial serum samples were obtained before immunisation in addition to 7, 14, 21, 28, 35, 42, 49, and 57 days past vaccination.
Challenge infection with virulent RHDV was carried out to test the immunogenicity of the administered recombinant virus. One ml of virulent RHDV of the “Eisenhüttenstadt” strain (104.5 LD50/ml) was used to inoculate the rabbits intramuscularly (IM) at 57 days post vaccination.
Diagnostic investigation was carried out using the following measurements:
Serology Results: Serum was extracted at weekly intervals over 57 days post vaccination up until challenge. Levels of antibodies directed against the VP60 antigen were measured using an RHDV-antibody-ELISA test according to standard protocols (
Challenge results: Four animals of group 4 (controls) died within 48 hours after infection with virulent RHDV. All animals of groups 1, 2, and 3 survived the challenge infection. None of the immunised animals of groups 1, 2 or 3 demonstrated any clinical symptoms of RHDV infection. The pathomorphological analysis of the perished animals of group 4 revealed symptoms associated with infection with RHDV (haemorrhagic septicaemia and liver damage).
These results demonstrate that the recombinant virus of the present invention is capable of stimulating the production of anti-VP60 antibodies, and more importantly to mediate protection against lethal RHDV infection.
It was also demonstrated that the animals could be protected by vaccination with D1701-V-VP60n in the absence of detectable anti-VP60 serum antibodies.
The second set of animal experiments was designed to test the number of immunisations and the vaccination dose of D1701-V-vp60n, which is necessary to provide effective protection in rabbits against a challenge infection. Rabbits (n=4) were immunised once, twice or three times intramuscularly with 107, 106 or 105 PFU of the ORFV-recombinants before challenge infection with virulent RHDV (
The results demonstrate that all D1701-V-vp60n-immunised animals were completely protected against RHDV infection. It is also shown that in fact booster-immunisations with ORFV-recombinants are required for clear proof of specific antibodies in serum (
The animal experiments described herein provide evidence that a single intramuscular immunization with the lowest tested dose of ORFV-recombinants D1701-V-VP60n (105 PFU) provides effective protection against a challenge infection with virulent RHDV. The protective effect was comparable to an immunization with the commercially available RIKA-VACC® RHD, which is obtained from RHDV-infected liver material after inactivation. These examples therefore demonstrate the protective working of the recombinant VP60-expressing recombinant viruses as claimed.
In order to test the immune-modulating properties of virus preparations, 11-12 week old Balb/C mice were treated with 5×106 PFU per animal in an intraperitoneal immunisation. Animals used as negative controls were treated with PBS solution.
After 6, 12 or 24 hours blood from the treated animals was isolated, serum was extracted and the cytokine and chemokine expression was tested using ELISA kits. Furthermore, the spleens of each of the tested animals were isolated for testing the activation of NK (natural killer) cells in a lymphocyte-proliferation test.
34 cytokines and chemokines were measured using a multiplex ELISA system (BioRad, Germany). The results of this analysis are summarized in
The results demonstrated that 12 hours after immunisation the maximum expression of the measured cytokines appeared and decreased after 24 hours. Groups A, B and C represent different preparations of D1701 virus, whereby group D represents a vaccine from the prior art. Group C comprised a mixture of two different viral preparations.
As is shown in
The activity of NK-cells isolated from the spleen was also tested. The NK-cells were tested using both the chromium-release assay and by measuring cell proliferation of the splenocytes. The chromium-release assay demonstrated that 12 hours after application of the viral preparations a clear increase in the cytolytic capacity of the NK-cells of the groups C and D can be observed. 24 hours after immunisation the cytolitic activity was further increased.
In order to test splenocyte proliferation a cell proliferation test using a BrdU-ELISA test was applied. The splenocytes were cultured at 37° C. and 5% CO2 for three days before cell proliferation was stimulated. 6 hours after immunisation there was no significant increase in cell proliferation. However, after 12 hours the groups A and C demonstrated a clearly increased level of proliferation.
Together these immune stimulatory tests demonstrate that between 12 and 24 hours after immunisation activation of the innate immunity of the treated animals was observed. These results further demonstrate that the parapox viral vectors of the present invention provide an adjuvant effect when used as a vaccine.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10075174.2 | Apr 2010 | EP | regional |
| 10 2010 047 920.9 | Oct 2010 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2011/056840 | 4/29/2011 | WO | 00 | 3/21/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61333331 | May 2010 | US |
