WO1995027069A1

WO1995027069A1 - Alphavirus rna as carrier for vaccines

Info

Publication number: WO1995027069A1
Application number: PCT/EP1995/001080
Authority: WO
Inventors: Wilfried Dalemans; Claudine Bruck; Peter Torsten LILJESTRÖM
Original assignee: Smithkline Beecham Biologicals (S.A.); Bioption Ab
Priority date: 1994-03-31
Filing date: 1995-03-22
Publication date: 1995-10-12
Also published as: AU1951495A; GB9406498D0; ZA952556B

Abstract

Vaccine compositions are provided that employ alphavirus RNA molecules containing exogenous RNA sequences encoding an antigen for direct administration to a patient.

Description


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   ALPHAVIRUS RNA AS CARRIER FOR VACCINES. 



   The present invention is related to polynucleotides, in particular to recombinant polynucleotides which form part of a vector, especially an alphavirus vector. The invention also relates to the recombinant vectors and to pharmaceutical compositions comprising the vectors which are suitable for vaccine use. 



   WO 90/11092 ('Expression of exogenous polynucleotide sequences in a vertebrate') describes a pharmaceutical product comprising naked polynucleotide, operatively coding for a biologically active peptide in a suitable form for injection into a tissue to cause the cells ofthe tissue to express the said polypeptide. In particular it is claimed that the peptide may be immunogenic and the 'naked DNA' which codes for it may be used to vaccinate, for example, humans. 



   It has been reported that it is possible to immunise mice with 'naked DNA' to protect them from influenza virus (see Science, Volume   259, 19th   March 1993, page 1691). 



   In WO 92/10578 Garoff and Liljestrom described an expression system based on alphaviruses, in particular Semliki Forest Virus (SFV). SFV has a single stranded RNA genome ofpositive polarity and replication ofthis capped and polyadenylated RNA starts upon the initial translation ofthe 5' two thirds ofthe genomic RNA, producing a polyprotein which by autoproteolytic events is post-translationally cleaved into four non-structural proteins (nsPl - nsP4). These proteins are responsible for the replication ofthe plus strand genome into full length minus strands which later in infection are copied into new plus strand genomes. For further details, see also Biotechnology, Volume 9, December 1991, pages 1356 - 1361. 



   WO 92/10578 describes a DNA molecule encoding protein sequences being inserted into engineered variants ofthe cDNA of a positive stranded RNA virus genome from alphavirus which then, via RNA transcription and transfection into tissue culture cells, is used to produce recombinant virus particles for either immunisation or protein production. In relation to immunisation, the recombinant RNA genome (structural alphavirus protein genes replaced by heterologous gene) was cotransfected into target cells in vitro with another RNA directing expression of alphavirus structural proteins. This led to encapsidation ofthe recombinant RNA. 



  Resulting particles were used for vaccination. 



   The present approach, uses naked alphavirus RNA (with insertion of heterologous protein gene in place of structural alphavirus genes) for direct immunisation by administration to a mammal. This naked RNA may optionally admix with lipid for stabilisation purposes, but is not necessarily encapsulated by the lipid. Alternatively the RNA molecule may be delivered on an inert, in particular a 

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 gold, particle by means of a gene gun (see US Patent No. 5100792, 5036006 and
4945050). The inventors have demonstrated that it is possible to obtain good expression ofthe antigen in the muscle using RNA either stabilised by lipid, or without lipid being present.

   The term naked is used herein to distinguish the vaccine composition of the present invention f rom RNA molecules encapsidated by viral proteins, since in the present invention, RNA molecules are directly administered to the target tissue in vivo. 



   According to the present invention there is provided a vaccine composition comprising a naked RNA molecule derived from an alphavirus RNA genome and capable of efficient intracellular replication in an animal host cells, which RNA molecule comprises the complete alphavirus RNA genome regions, which are essential to replication ofthe said alpha virus RNA, and further comprises an exogenous RNA sequence capable of expressing its function in said host cell, said exogenous RNA sequence being inserted into the region ofthe RNA molecule which is non-essential to replication thereof, together with a suitable carrier, diluent or pharmaceutically acceptable excipient. 



   There is also provided a method ofpreventing or treating a viral infection in a mammal (especially a human) by administering an effective dose ofthe vaccine composition according to the invention. 



   There is further provided the use of a naked RNA molecule derived from an alphavirus RNA genome and capable of efficient intracellular replication in an animal host cells, which RNA molecule comprises the complete alphavirus RNA genome regions, which are essential to replication ofthe said alpha virus RNA, and further comprises an exogenous RNA sequence capable of expressing its function in said hos cell, said exogenous RNA sequence being inserted into the region ofthe RNA molecule which is non-essential to replication thereof, in therapy, more specifically for the preparation of vaccine composition for use in the preventment or treatment of. range ofinfections in a mammal. The vaccine composition according to the inventior may fmd application in anticancer vaccine therapy as well, wherein the exogenous sequence will encode a tumor antigen. 



   This is the first medical use of such naked RNA molecules and accordingly   th@   invention in one aspect provides an RNA molecule as herein described for use in medicine. 



   In a preferred embodiment the alphavirus is Semliki Forest Virus (SFV). 



   Preferably the naked RNA molecule which may be used in the invention has an exogenous RNA sequence which encodes a protein, a polypeptide or a peptide sequence defining an exogenous antigenic epitope or determinant. 

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   The naked RNA molecule which forms part ofthe composition according to the invention may be prepared according to the methods set forth in WO 92/10578. 



   In a particular embodiment the exogenous RNA sequence is derived from influenza haemagglutin in (HA) DNA, for example as illustrated in the Example below. In an alternative embodiment, the exogenous sequence is derived from a HSV DNA, in particular a sequence which codes for an HSVgD protein or derivative thereof. 



   To prepare such an embodiment there is required a vector (circular or linearised) comprising alphavirus DNA and an exogenous DNA fragment derived from the exogenous antigen of interest such as influenza HA or HSVgD. For example DNA corresponding to the coding sequence of influenza HA protein from strain A/PR/8/34 (Winter, G et al, 1981 Nature, 292,72-75) can be ligated to a linearised SFV plasmid such as pSFVl (Liljestrom, P and Garoff, H (1991), Biotechnology, 9, 1356-1361, prepared as described hereinbelow. Such vectors form a further aspect of the invention. 



   The compositions ofthe invention may be administered using the dosages and routes of administration described in WO 90/11092. It will be apparent however that the precise dosage will depend on factors such as the weight sex, mode of administration, general health ofthe patient and the condition to be treated. 



  Nonetheless for Intramuscular use the dosages to employed will typically be in the range of 0.05 g/kg to about 50 mg/kg, more typically from about 0.1to 10 mg/kg. 



  Subcutaneous, epidermal, intradermal or mucosal administration are also possible. 



   The following examples illustrate the invention. 



  EXAMPLE 1: GENERATION OF ANTI-INFLUENZA HA ANTIBODIES AND A PROTECTIVE RESPONSE UPON INTRAMUSCULAR INJECTION OF NAKED RECOMBINANT SFV-HA RNA IN MICE. 



  DNA construction. 



   Recombinant DNA technology was applied according to standard procedures. 



  Those are well-known by persons skilled-in-the art, and these general procedures are referred in Sambrook et al. (1). 



   The DNA corresponding to the coding sequence ofthe influenza haemagglutinin (HA) protein from strain A/PR/8/34 (2) was excised from pMS2 by restriction digest with HindIII, and protruding ends were blunt-ended by filling-in with Klenow polymerase. Remaining pUC plasmid DNA was further digested with PvuI, Ndel , and Bgll. These insert fragments were subsequently cloned into SmaI linearised   pSFVl    1 (3),   and transformed into E. coli XL1-Blue kompetent cells. 



  Recombinant colonies bearing the HA insert were detected by colony screening. First, 

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 colonies were hybridised to oligonucleotide WD19 (GGGGCAATCAGTTTCTGG) specific for the HA coding sequence. Subsequently, the colonies were re-hybridised to the oligonucleotide WD14 (GGCGGTCCTAGATTGGTG) specific for pSFV vector sequence. DNA of colonies hybridising to both oligonucleotides was prepared, and further analysed by   EcoRl   restriction digest. Clones with the HA insert in the right orientation were further analysed by restriction digests with   PvuII,   RsaI, BamHI+SphI, BglII+PstI. and BamHI+Eagl. A large-scale preparation of recombinant clone pSFV-HA14 was prepared and analysed by restriction digest with   EcoRl   and XhoI.

   The junctions created by ligation ofthe HA insert into pSFV 1 were analysed by DNA sequencing. Primers that were used were WD 14 (see above), WD18 (GCCTATACATATTGTGTC; corresponding to HA sequences), and WD19 (see above). The first 70 N-terminal amino acids ofthe HA protein were identical to the published sequence (2) except for a Cys to Ser mutation ofresidue 10 in the signal peptide sequence. Two additional, silent mutations were detected at the DNA level for residues   Val36   and Leu67. Sequencing ofthe 3' junction evidenced an additional CC dinucleotide preceeding the   HindIII-SmaI   junction, which is however outside the coding region. 



  RNA preparation. pSFV-HA14 DNA was linearised by restriction digest with SpeI, and purified. 



  Linear plasmid was incubated with SP6 RNA polymerase in the presence ofthe four ribonucleotides, Cap analogue, recombinant RNase-inhibitor, and the necessary buffer components according to (3). For in vivo application, the in vitro transcribed RNA was subsequently purified by Sephadex G50 chromatography, and concentration of the RNA was determined by UV absorbance. The integrity ofthe transcript was verified by agarose gel-electrophoresis. 



  In vitro expression of HA protein. 



   Four  g ofrecombinant pSFV-HA14 RNA was transfected into BHK cells (4.106 cells) by electroporation. Total cell lysate was harvested 16 hours after transfection, and proteins equivalent to 5.104 cells were separated by poly-acrylamide gel-electrophoresis. HA protein was evidenced by Western blot analysis with the monoclonal antibody H308 which is specific for the influenza HA protein. 



  Mouse injection with naked SFV-HA RNA. 



   30 g   of pSFV-HA14   RNA (per mice) was first incubated for 10 minutes at room temperature with 5 g of lipofectin reagent (GIBCO-BRL) and then adjusted to a final volume of 50 l ofphysiological saline solution (PBS). A sample ofthis 

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 preparation was analysed by agarose gel-electrophoresis, and the integrity ofthe RNA in the final preparation was confirmed. Twenty   CB6F1  mice (four groups of five mice) were injected into the quadriceps muscle, each with 50 l of RNA preparation (30 g RNA). 21 days post-injection, a first serum sample was taken (retroorbital; post-I) and mice were reinjected with 50 l RNA preparation now containing 22.9 g of RNA. 21 days after this booster injection, a second serum sample was taken (postII). 



  ELISA for anti-HA antibodies. 



   Serum samples from post-I and II bleedings were tested by an ELISA. 



  Microtiter plates were coated overnight at room temperature with a 1/8000 dilution (in PBS) of sucrose-purified Influenza virus A/PR/8 (lot 07/092/412). After rinsing four times with PBS   + 0.1 %   Tween20, plates were saturated with a solution of 1% gelatine in PBS (1 to 1.5 hour at 37 C). After rinsing (see above), the respective mouse serum samples were applied. These samples were diluted in PBS + 0.1% Tween20 + 0.1% gelatine (dilution starting at 1/25 or 1/100 for post-I and II samples, respectively, and serially diluting by a factor 3), and were incubated for 1.5 hours at 37 C. After four rinses, a 1/3000 dilution of a biotinylated anti-mouse antiserum (Amersham   RPN1001)   was applied and incubated for 1 hour at 37 C. After four rinses, plates were incubated with a 1/3500 dilution of Extravidine (Sigma E2886) (40 minutes, 37 C).

   After three rinses (see above), plates were once rinsed with water, and OPD substrate (Sigma 8787) was applied. Reaction was done at room temperature for 20 minutes (in the dark), and stopped by addition of 50% (vol) 2M H2S04. Read-out ofthe colour reaction was done at the wavelength of 492nm. Mouse S.31.3 had a titer of 74 EU/ml at post-I (figure 1), whereas the other 19 mice injected tested negative (figure 1 for mice S.31.1-2-4-5, other results not shown). The anti-HA ELISA titer of mouse S.31.3 increased to 176 EU/ml upon boosting (post-II sample; figure 2). 



  Positive control, influenza infected mice had an average titer of 7000-8000EU/ml in the same test (42 days post-infection), and naive mice tested negative (figure 3). 



  PROTECTION EXPERIMENTS Experiment 1
Three weeks after the second injection, the mice were challenged with   107.28   TCID50 ofthe homologous A/Pr/8 influenza strain given intranasally. Mice from both vaccination groups were sacrified at days 1,3,5 and 7 post challenge (5 mice per group and per day). Their lungs, trachea and turbinates were collected, triturated and stored at -80 C until assayed. Protection ofthe mice was assessed by quantifying the 

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 amount ofvirus present in the lungs at each time points mentioned above using a classical cell culture titration technique. 



   Results ofthe lung titration are given in the annexed table I   (log 10   TCID50/organ). They indicate that vaccination ofmice with RNA encoding HA is able to provide extensive protection against influenza replication in the lungs of vaccinated/challenged animals. 



   These results demonstrate for the first time that injection ofnaked recombinant RNA into animals can elicit protection against viral replication in an animal model. Since only one out ofthe twenty animals tested developed a detectable antibody response as monitored by ELISA, it seems that the observed protection is not mediated by pre-existing antibodies, but rather by priming ofthe immune system and possibly also by a cellular immune mechanism. 



  Experiment 2 Mice (Balb/c, 15 mice per group) were injected into the tibialis cranialis muscle with 10 g SFV RNA (control RNA prepared from   pSFVl  without insert) or 10 g SFVHA RNA (see above) contained in 50 l PBS. A booster injection was given three weeks later, and animals were challenged as described above. Non-immunised, challenged animals served as controls. ELISA analysis (see above) of serum samples taken 3 weeks after booster injection showed that all animals which received SFV-HA RNA had seroconverted. Virus titers in the lungs and trachea at days 3, 4, and 5 postchallenge (5 animals per point) were determined as described, and are represented in figure 4. These results show that a very consistent reduction in viral titer can be obtained upon immunisation with naked SFV-HA RNA. Protection against viral infection ofthe lungs was complete. 



  References. 



  1. Sambrook, J. et al.. (1989) "Molecular Cloning. A laboratory manual. Second edition." Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 



  2. Winter, G. et al..   (1981)   Nature, 292,72-75. 



  3.   Liljestrom,   P. and Garoff, H.. (1991) BioTechnology, 9, 1356-1361. 



  Example 2: Expression of beta-Galactosidase upon intramuscular injection of naked SFV3-lacZ RNA. 



  SFV3-lacZ RNA was prepared by in vitro transcription of SpeI linearised pSFV3-lacZ DNA (1). For intramuscular injection,   12g   ofthis RNA was resuspended in   lxPBS   buffer in a final volume   of 50 1   (per injection point). Two groups of each five mice 

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 were injected with 50 l RNA in the tibialis muscle and 50 l RNA in the quadriceps muscle ofthe opposite leg. Injection of this RNA did not lead to any visible adverse effect in the mice. In order to detect expression ofthe beta-Galactosidase protein, mice were sacrificed at day 1 and day 3 post-injection (5 mice per sampling) and the injected muscles were dissected. A lysate was prepared from both tibialis and quadriceps muscles, and beta-Galactosidase activity was assayed enzymatically (2). 



  Expression ofthe reporter gene was detected in all the injected tibialis muscles, both at day 1 and 3 post-injection. Mean enzymatic activity was 13.7 and 27.3U/mg protein, respectively. Expression in the quadriceps was detectable in 3 out of 5 mice at day 1 post-injection (mean activity 1.6U/mg protein), and in 1 of 5 mice at day 3. 



  References: 1.   Liljestrom   P and Garoff H (1991). Bio/Technology 9, 1356-1361. 



  2. MacGregor GR et al (1991). In: Gene transfer and expression protocols. Methods in molecular biology 7. Murray EJ (ed). Humana Press, Clifton, New Jersey. pp 217- 235. 



  Example 3 : Generation of antibodies against HSV2-gD by intramuscular injection of naked recombinant SFV-gD RNA. 



  Recombinant SFV-gD RNA was produced in a comparable way as described in example 1. Briefly, the fragment encoding the HSV2 gD protein (in a truncated form covering the extracellular part ofthe protein from amino acid 1 to 307) (1) was isolated from   pUC12-gDStop   clone 26 by EcoRI digestion. After filling-in the protruding ends, the fragment was ligated into SmaI digested   pSFV 1,   and recombinants were selected after transformation into E. coli. The recombinant plasmid, pSFV-gD, was linearised by partial digest with SpeI, and SFV-gD RNA was obtained after in vitro transcription. Biological activity ofthe RNA was confirmed by transfection into BHK21 cells revealing the expression of a truncated gD protein by Western blot. 



  Anaesthesised mice (Balb/c) were injected into the tibialis muscle with different amounts of SFV-gD RNA in PBS in a final volume of 50 l. Two booster injections were given after 1 and 5 months, respectively. Serum samples were taken at different time points after the RNA injections and antibodies directed against HSV-gD were quantified by a standard ELISA (see also example 1). Briefly, microtiter plates were coated with purified gD protein (1 g/ml), and subsequently saturated with PBS containing 4% newborn calf serum and 1% bovine serum albumin. Serum samples 

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 were applied at different dilutions, and thereafter incubated with a biotinylated antimouse IgG (Amersham RPN1177). Detection was performed by adding StreptavidinPOD (Amersham   RPN1051)   followed by OPDA (Sigma P8787).

   Specific anti-gD antibody titers were expressed as  g/ml total IgG by reference to a mouse total IgG standard curve. Figure 5 shows the results of SFV-gD naked RNA immunisation. All animals seroconverted, and a clear booster response can be observed. 



  Reference. 



  (1) McGeoch D.J. et al. (1987). J. Gen. Virol. 68, 19-38. 

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    TABLE 1 Protective effect of naked RNA vaccination against replication of influenza virus in the lungs of challenged mice   
 EMI9.1 
 
<tb> Day <SEP> 1 <SEP> Day <SEP> 3 <SEP> Day <SEP> 5 <SEP> Day <SEP> 7
<tb> p <SEP> SFV <SEP> IHA <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9
<tb> p <SEP> SFV <SEP> 1 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> 4.55 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> 4.05 <SEP> < 1.9 <SEP> < 1.9 <SEP> 2.3 <SEP> < 1.9 <SEP> 2.8 <SEP> 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9 <SEP> < 1.9
<tb> 
   (Units are log l 0 TCID50.

   Each data point represents an individual mouse. pSFVI : control mice injected with RNA without HA coding sequences.)

Claims

Claims 1. A vaccine composition comprising an alphavirus RNA molecule containing an exogenous RNA sequence encoding an antigen.

2. A vaccine composition as claimed in claim 1, wherein the RNA molecule is formulated with lipid.

3. A vaccine composition as claimed in claim 1 wherein the RNA molecule is absorbed on to an inert particle.

4. A vaccine composition as claimed in any of claims 1-3 wherein the exogenous RNA sequnce encodes an Herpes Simplex antigen.

5. A vaccine composition as claimed in any of claims 1-3 wherein the exogenous RNA sequnce encodes an Influenza antigen.

6. A vaccine composition as claimed in any of claims 1-3 wherein the exogenous RNA sequence encodes a tumor antigen.

7. An alphavirus RNA molecule comprising an exogenous RNA sequence encoding for an Herpes Simplex antigen or influenza antigen.

8. An alphavirus RNA molecule as claimed in claim 7 wherein the antigen is an HSVgD antigen or an influenza haemagglutinin antigen.

9. A DNA molecule corresponding to the RNA molecule of claim 7 or 8.