Title of Invention

VACCINE COMPOSITION COMPRISING MALARIA-DERIVED ANTIGENS AND AN ADJUVANT

Abstract A vaccine composition useful in the prevention of treatment of malaria comprises a plurality of malaria-derived antigens in combination with an adjuvant which is a preferential stimulator of TH1 cell response.
Full Text

Vaccioc Composition
The present invention relates to a novel vaccine cotnposhion and to its use \n medicine, parliculiuly in the prevention of malaria infections.
Malaria, is one of the world's major health problems with 2 to 4 million people dymp from tlie disease each year One of tlie most acnte fomiN of the disep.s-:: is jLiused h> the protozoan parasite. Plasmodium falciparum which »s responsible for most of the mortality attributable to Malana.
The Ufe cycle of’’. falciparum is complex, requiring two hosts, man and mosquito for completion. The infection of man is initiated by the inoculation of sporozoites in the saliva of an infected mosquito. The sporozoites migrate to the liver and there infect hepatocytes where they differentiate, via the exoerylhrocytic intracellular stage, into the merozoite stage which infects red blood cells (RBC) to initiate cyclical replication in the asexual blood stage. The cycle is completed by the differentiation of a number of mero7,oitcs in the RBC into '‘cw.al staye g;imctocytes which are ingested by the mosquito, where they develop tlirough a scries of stages in the midgut to produce sporo/oitcs which migrate to the salivary gland.
rhc sporozoite 5;inge ofF. faicipuTum has been identified as a potential target of u malaria vaccine. The major surface protein of the sporozoitc is known as circumsporozoite protein CCS Protein). This protein from suain 7G8 has been cloned. expressed and sequenced {Dame ‘ul Science 221 (1984) p593). The protein from strain 7G8 is characterised by having a central immunodominant repeat region comprising a temjpcptide Asn-Ala-Asn-Pro repeated 37 times but interspersed with four minor repeats Asn-Val-Asp-Pro In other strains the number of major and minor repeats vary as well as their relative position. This central portion is flanked by an N and C terminal portion composed of non-repetitive amino acid sequences designated as the repeatless portion of the CS protein.
It has been shown that irradiated spt)rozoites can provide significant protection against experimental human malana (Am. J. Trop. Med. Hyg. 24: 297-402. 1’)75). However. production difliculties makes the use of irradiated sporozoitc impractical frotn the point of view of prouucmg a vaccine.
Several gioups have proposed subiinii \accincs based on the circumsporo/oiie protein. Several of these vaccmes have undergone clinical testing, one is a synlhcuc

peptide, the other is a recombinant protein (Ballou sLal Lancet; i 1277 (1987) and Harrington eLalNature ‘2’:251 (mi)
These vaccines were successful in stimulating an onti-sporo’oite response Nonetheless, the magnitude of the response was disappointing, with some vaccinccs not making a response at all. Funhemiore. the absence of "boosting" of antibody levels on subsequent injections and results of in vitro lymphocyte proliferation assays suggested that l'-cc!is of most 'M' ihe’- volunteers did not recognise the immuno-dommant repeat Nonetheless, one vaccincc in each study did not deveh)p parasitemia.
International Patent Application No WO 93/10152 (Smithkline Beecham Biologicals) describes and claims a hybnd protein comprising substantially all the C-terminal portion of the CS protein, four or more tandem repeats of the immunodominant region, and the surface antigen from Hepatitis B virus (HBsAg). Preferably tlie hybrid protein comprises a sequence which contains at lea’t 160 amino acids vvHuch is substantially homologous to the C lerminal portion of the CS protein. The CS protein may be devoid of the last 12 amino-acids from the C tenmnai.
In particular there is described a protein which comprises a portion of the CS protein of P. Falciparum substantially as corresponding to amino acids 210-398 of P. falciparum 7G8 iused in frame via a linear imkcr to the N-tcmiinal of HBsAg. The linker may comprise a portion of prcS2 from HBsAg.
A particular embodiment described in WO 93/10152 is the hybrid protein designated RTS. This hybrid consists of:
° A methiomne-rcsidue, encoded by nucleotides 1059 to 1061, derived from the
Sacchromves ccrevisiae TnH3 gene sequence (nucleotides 1-1058 in ihis readmg frame make up the TDHJ promoter itselO (Musn A.M. etal ("iene 1981 l’ 1 r>-14";
Three amino acids. Met Ala Pu). derived from a nucleotide sequence i. U)62 to 1070) created by the cloning procedure used to construct the hybrid gene.
'‘ A btretch of 18’ amino acids, encoded by nucleotides 10’ 1 lo 16’’7
reprtisenting ammo acids 2\{) to >Q8 of the circumsporozoitc protein ((.SP) ‘‘f Plasmodium falciparum stram 7G8 (Dame iUil iupra)-

"‘ i\n amino acid (Axg) encoded by nucleotides 1638 to 1640. created by the
cloning procedure used to construct the hybrid gene.
‘ Four amino acids, Pro Val Tlir Asn, encoded by nucleotides 1641 lo 1652, and
representing the four carboxy icnninal rci’idues of the hepatitis D vinas (iid’v serotype) preS2 protein!*?).
'■ A siielch of 22o air:ino acids, encodea by nucleotides lo53 to 2’’ ‘M). and
specify'ing tlie S protein of hepatiti*; B virus (ad’ serotype).
WO 93/10152 further describes the expression of the hybrid protein in a recipient yeast strain which already carries in its genome several integrated copies of an hepatitis B S expression cassette. The resuUing strain synthcsises two polypeptides, S and RTS (or other hybrid protein of the invention), that spontai’eously co-assemble into mixed (for example RTS, S) lipoprotein particles. These particles, advantageously present tlae CSP sequences of the hybrid at their surface.
It IS an object of die present invention to confer mmiunity against P. falciparum and/or P. vivax infestations by immunizaiion with a composition comprising a plurality of antigens in combination with an adjuvant which is a preferential stimulator of THl cell *rsponsc.
Accordingly, the present invention provides a vaccine composition for use in the prevention or treatment of malaria, comprising a plurality of malana-denved antigens in combination with an adjuvant which is a preferential stimulator of THl cell response.
Preferably, at least one of the antigens is a hybrid protein as detlned above, such as RTS, more preferably in the fonn of mixed particles as defined above, such as RTS,S
A further aspect of the invention provides a vaccine composition for use in the prevention or treatment of malaria, comprising a plurality of malaria-derived antigens, characterised in that ai least one of the antigens is a hybrid protein as detlned above. such as RTS, more preferably in the form of mixed particles as defined above, such as RTS,S.
The amount of antigen present in each vaccine dose is selected as aii amount whieh induces an immunoprotectivc response without signitlcani, adverse side etfeels in

typical vaccines. Such amount will vary depending upon which specific iminunogens are employed. GeneralK, it is expected that each dose will compnse a total of 1-inOOfig of protein, prefembly 1-200 ‘g most preferably 10-lOOj.Lg. An optimal amount for a particular vaccine can be ascertained h\ stajidaid studies involving observation of immune rcsponse>i in subjects Following A fLirther aspect ol fhe invention lies in a method of treating a patient susceptible to Plasmodium infections by administering an effective amount of a vaccine as hereinbefore described.
Adjuvants which arc capable of preferential stmiulation of the Tfll cell response are described in International Patent Application Nos, WO 94/00153 and WO 95/17209.
A particular preferred adjuvant comprises QS2L an Hpic purified non-toxic fraction derived from the bark of Quillaja Saponaria Moiind, and 3 De-Oacylated monophosphoryl lipid A (3 0-MPL). optionally together with an oil in water emulsion.
3 De-0-acylated irionuphusphcryi upiu A is known trom GB 2220211 (Ribi). Chemically it is a mixture of 3 Dc-0-acylated monophosphoryl lipid A with 4, 5 or 6 acylatcd chains and is manufactured by Ribi Immiinochcm Montana. A preferred form of 3 De-0-acylated monophosphoryl lipid A is disclosed in International Patent Application No, 92/116556.
QS21 is a Hplc purified non toxic fraction of a saponin from the baik o( the South American tree QuilUja Saponaria Molma and its method of its production is disclosed (as QA21) m t;S patent No. 5.057,540.
A preferred oil-in-watcr emulsion comprises a meiabolisiblc oiL such as squalene, alpha tocopherol and twecn 80. AddiUonally the oil in water emulsion may contain span 85 and/or lecithin,
Hie ratio of QS21 3D-MPL will typically be in the order of 1 : 10 to 10. 1. preferably I : 5 to ‘ : 1 and often substantially II The preferred range foi optimal synergy i
preferably 10 \xg - 50 |.ig per dose. Typically the oil in water will comprise (Vuni 2 to 10% squalcne, from 2 to 10% alpha tocopherol and tfom 0.3 to 3% tueen 80. Preferably the ratio of sqaalene: alpha tocophen>l is equal or less than 1 as this provides a more stable emulsion. Span 85 may also be prescni at a level of I %. In some cases it may be advaniugeous thai the vaccmes of the present invennon will further conuun a stabiliser
Vaccine preparation is generally described m New Trends and Developmt’nts w. Vaccines, edited by Voller et al, Universuy Park Press, Baltimore, Maryland, I ‘ S. A. 1978. Encapsulation within liposonit;s is described, for example, by FuUcnon, U.S. Patent 4,235,877. Cot’jugation of protems to macromoleculcs is disclosed, for example, by Likliitc. U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757.
Mahiria-derived antigens useful in the present invention may be selected from the following;
1. A hybrid protein as defined above, sueh as RTS, more preferably in the form of mixed particles as defined above, such as RTS.S,
2. llic TRAP of a cloned isolate of-P. falciparum from Thailand kn.»wn as T/9/96. and proteins havii’g at least 30% homology thereto, and immunogenic derivatives including fragments thereof (described in International Patent Application Nos. WO »j/01496 and WO 91/11516 t3i Exploitation Limited), and WO 92/11868 (US Navy)).

3. ihe 16kD protein described m International Patent Application No, WO 91/18922. and proteins havmg at least 80% homology thereto, and immunogenic derivatives including fragments thereof
4. rhc apical membrane antigen-1 (AMA-1) of P. falciparum or P vivax, and proteins having at least 80% homology thereto, and immunogenic derivatives including fragments thereof
5. fhe eireumsporozoite protein (esp) of P falciparuin or P. vivax, and proteins having at least 80% homology thereto, and immunogenic derivatives mcluding fragments thereof

6, The MSP 1 of P. falciparum or P. vivax (US Patent No 4,837,016), and
proteins havmg at least S0% homology thereto, and immunogenic derivatives
includmg fragments thereof
7. Other cxocrN’throc}ac stage proiems and immunogenic derivatives including
fiagnients thereof.
8. Optionally, blood ‘lage proteins and immunogenic derivatives including
fiagmenls thereof
The temi "immunogenic derivative" encompasses any molecule such as a truncated or other derivative of the protein which retains the ability to induce an immune response to the protein following internal administration to a human* vSuch otlier derivatives can be prepared by the addition, deletion, substitution, or rearrangement otamino acids or by chemical modifications thereof
Immunogenic &agment*> of the protein, which may be useful in the preparation of subimit vaccines, may be prcpar%>d by expression of the appropriate gene tragmcnts or by peptide synthesis, for example using the Mcrrificld synthesis (The Peptides, Vol 2., Academic Press, NY, page 3).
The immunogenic derivative can be a hybrid, that is, a fusion polypeptide containing additional sequences which can carry oiu: or more epitopes for other Plasmodi’pi immunogens, or other non-piasmodium immimogens. Alternatively, the immunogenic derivative of the invention can be fused to a carrier polypeptide such Hepatitis B surface or core anlijjcn or to another currier which has immunosrimulating properties, as in the case of an adjuvant, or which otherwise enhances the immune response to the protein or derivative thereof, or which is useful in expressing, purifying or formulating the protein or derivative thereof
The proteins or immunogenic derivatives thereof which are useful m the invention may be chemically conjugated to a macromoleculc using a conventional linkint’ agciu such as glutiualdehydc (Gcerlings et af (1’88) J. Immunol. Methods, iUii. :i39 ?44)

The followmg Example illustraies the invention:
Example
Construction and expression of a recombinant TR.4P.
Thii; was prepared iis descMbed in W(J V()'01496.
Construction and expression of RTS,S.
This was prepared as described in WO 93/10152.
Adjuvantntion
Two adjuvant fonnulations were made each comprising the following oil in water emulsion component,
SB26: 5% squalcne 5% tocopherol 0 4% twccn 80; the particle size was 500 mn size SB62: 5% Squalene 5% tocopherol 2,0% twccn 80; the particle size vvas 180 mn
Preparation of Twcen 80 is dissolved in pho Preparation of emulsion SB26
This emulsion was prepared in an analogous manner uri)is)ng 0.4% lueen 80,
Other emulsions as dcnictcd m the Tahlo wore miide in an analogous manner.
To each 100m I of emulsion were added the two antigens (tOmg ot each antigen, equivalent to SO’'‘z per dosc) and mixed. This vvub combined with lOO’’/iT’l t>l 3D-

MPr. and lOOng/ml of QS21 to give the tmal fornmlaiion. Buffer was set accordiiig to salt content and pH.



Reference Example Vanous formulations of RTS,S
RTS.S is described m International patent application No, WO 9V101S2 and was formulated for vaccination of balb/c mice. Fi’'e animals were in each ‘ruup, 7 groups-of animals received tlie following fumiularions

Group 1 RTS..SSB62
Group 2 RTS,SQS21 3D’iVlPL
Group 3 RTS,S0S2l 3D-MPLSB62
Group 4 RTS,S3D-MPLAl(OH)3
Group 5 RTS.S AU0H)3
Group 6 Plain
Group 7 Negative control
(RTS,S - 5’g/dosc, 3 D-MPL S’g’dosc QS21 5ng/dose)

Enhanced lBG2a antibody response it mice is a measure of the ability of die adjuvant system to stimulate a THl type response.



WE CLAIM:
1. A vaccine composition for prevention or treatment of malaria, comprising a plurality of malaria-derived antigens selected from the group consisting of:
• a hybrid protein comprising substantially all the C-terminal portion of the CS protein, four or more tandem repeats of the immunodominant region, and the surface antigen from Hepatitis B virus (HBsAg);
• the TRAP of a cloned isolate of . falciparum from Thailand known as T/9/96, and proteins having at least 80% homology thereto, and immunogenic derivatives including fragments thereof;
• the 16kD protein described in International Patent Application No. WO 91/18922, and proteins having at least 80% homology thereto, and immunogenic derivatives including fragments thereof;
• the apical membrane antigen-1 (AMA-1) of P. falciparum or P, vivax, and proteins having at least 80% homology thereto, and immunogenic derivatives including fragments thereof;
• the circumsporozoite protein (csp) of P. falciparum or P. vivax, and proteins having at least 80% homology thereto, and immunogenic derivatives including fragments thereof;
• the MSP-1 of P. falciparum or P, vivax (US Patent No 4,837,016), and proteins having at least 80% homology thereto, and immunogenic derivatives including fragments thereof;

• other exoerythrocytic stage proteins and immunogenic derivatives including fragments thereof; and
• optionally, blood stage proteins and immunogenic derivatives including fragments thereof.
in combination with an adjuvant which is a preferential stimulator or THt cell response.
2. The vaccine composition according to claim 1, wherein the adjuvant comprises MPL.

3, The vaccine composition according to claim 1 or 3, wherein the adjuvant
comprises 0S21.
4. The vaccine composition according to any one of the preceding claims,
wherein the adjuvant comprises an oil-in-water emulsion.



Documents:

1724-mas-1997-abstract.pdf

1724-mas-1997-claims filed.pdf

1724-mas-1997-claims granted.pdf

1724-mas-1997-correspondence po.pdf

1724-mas-1997-correspondnece-others.pdf

1724-mas-1997-description(complete)filed.pdf

1724-mas-1997-description(complete)granted.pdf

1724-mas-1997-form 1.pdf

1724-mas-1997-form 26.pdf

1724-mas-1997-form 3.pdf

1724-mas-1997-form 5.pdf

1724-mas-1997-other documents.pdf

1724-mas-1997-pct.pdf


Patent Number 213122
Indian Patent Application Number 1724/MAS/1997
PG Journal Number 13/2008
Publication Date 28-Mar-2008
Grant Date 20-Dec-2007
Date of Filing 31-Jul-1997
Name of Patentee SMITHKLINE BEECHAM BIOLOGICALS S A
Applicant Address RUE DE L'INSTITUT 89, B-1330 RIXENSART,
Inventors:
# Inventor's Name Inventor's Address
1 COHEN JOSEPH SMITHKLINE BEECHAM BIOLOGICALS S A, RUE DE L INSTITUT 89,B-1330 RIXENSART,
PCT International Classification Number A61K 039/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 9616351.4 1996-08-02 U.K.