PLASMODIUM FALCIPARUM AND PLASMODIUM VIVAX VACCINE

Abstract

The present invention relates to a vaccine V comprising (A) at least one isolated polypeptide strand P comprising or consisting of at least nine consecutive amino acid moieties of the repetitive organellar protein, putative of Plasmodium falciparum or the hypothetical protein PVNG_04523 of Plasmodium vivax or a polynucleotide strand encoding for such polypeptide; and (B) at least one pharmaceutically acceptable carrier or excipient. Furthermore, the present invention refers to an antibody binding to the repetitive organellar protein,putative of Plasmodium falciparumor the hypothetical protein PVNG_04523 of Plasmodium vivax or a polynucleotide strand encoding therefor, to a method of generating such antibody and uses thereof.

Claims

1-23. (canceled)

24. A vaccine V comprising: (A) at least one isolated polypeptide strand P comprising or consisting of at least nine consecutive amino acid moieties of SEQ ID NO: 2, of SEQ ID NO: 3 or of SEQ ID NOs: 2 and 3, or a polynucleotide strand encoding for said polypeptide strand P; and (B) at least one pharmaceutically acceptable carrier or excipient.

25. The vaccine V of claim 24, wherein the isolated polypeptide strand P is obtained from heterologous expression.

26. The vaccine V of claim 24, wherein the isolated polypeptide strand P is obtained from heterologous expression in bacterial or eukaryotic cells.

27. The vaccine V of claim 24, wherein the at least one isolated polypeptide strand P comprises or consists of at least nine consecutive amino acid moieties of a sequence having at least 80% sequence homology to a peptide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

28. The vaccine V of claim 24, wherein said vaccine V comprises an adjuvant supporting immunologic stimulation.

29. The vaccine V of claim 24, wherein said vaccine V comprises at least one adjuvant supporting immunologic stimulation selected from the group consisting of alum and an immunostimulatory peptide.

30. The vaccine V of claim 24, wherein the at least one isolated polypeptide strand P consists of or comprises a peptide strand having at least 80% sequence homology to sequence SEQ ID NO: 2 or SEQ ID NO: 3 obtained from heterologous expression.

31. The vaccine V of claim 24, wherein the at least one isolated polypeptide strand P consists of or comprises a peptide strand having a sequence SEQ ID NO: 2 or SEQ ID NO: 3 obtained from heterologous expression.

32. The vaccine V of claim 24, wherein the at least one isolated polypeptide strand P is a truncated version of SEQ ID NO: 2 comprising or consisting of a fraction of SEQ ID NO: 2 truncated by at least 100 amino acid moieties in length and comprising at least two peptide sequences selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

33. The vaccine V of claim 24 wherein the at least one isolated polypeptide strand P is a truncated version of SEQ ID NO: 2 comprising or consisting of a fraction of SEQ ID NO: 2 truncated by at least 100 amino acid moieties in length and comprising all of the peptide sequences SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

34. The vaccine V of claim 24, wherein said vaccine V comprises or consists of: (A) at least one isolated polypeptide strand P comprising or consisting of a peptide strand having at least 80% sequence homology to a sequence SEQ ID NO: 2 or SEQ ID NO: 3 obtained from heterologous expression; and (B) at least one adjuvant supporting immunologic stimulation and optionally one or more further pharmaceutically acceptable carriers.

35. The vaccine V of claim 24, wherein said vaccine V comprises or consists of: (A) at least one isolated polypeptide strand P comprising or consisting of a peptide strand of sequence SEQ ID NO: 2 or SEQ ID NO: 3 obtained from heterologous expression; and (B) at least one adjuvant supporting immunologic stimulation selected from the group consisting of alum and an immunostimulatory peptide, and optionally one or more further pharmaceutically acceptable carriers.

36. The vaccine V of claim 24, wherein the polynucleotide strand encoding for said polypeptide strand P is double or single stranded DNA or double or single stranded RNA, or an analogue of double or single stranded DNA or double or single stranded RNA.

37. The vaccine V of claim 24, wherein the polynucleotide strand encoding for said polypeptide strand P is a plasmid.

38. A method for preventing malaria in a patient, wherein the patient is administered with a sufficient amount of a vaccine V of claim 24.

39. A method for preparing an antibody AB binding to Plasmodium falciparum or Plasmodium vivax comprising the following steps: (i) providing: (a) a vaccine V according to claim 24, and (b) an organism O suitible for generating antibodies; (ii) administering the organism O with the vaccine V; (iii) waiting until the subjected organism of step (ii) shows an immune response against the antigens of the vaccine V; (iv) obtaining antibody-generating cells C of the organism O of step (iii); (v) optionally hybridizing the antibody-generating cells of step (iv) with myeloma cells obtaining immortalized antibody-generating cells C1; (vi) optionally isolating the nucleotide encoding for the antibody AB of interest and transfer it to another antibody-generating cell type C2 suitible for expressing the antibody AB; (vii) cultivating the antibody-generating cells C, C1 or C2 of any of steps (iv) to (vi) under conditions enabling the production of the antibody AB; and (viii) isolating the antibody AB from step (vii).

40. An antibody or antibody fragment AB binding a polypeptide strand P SEQ ID NO: 2 or SEQ ID NO: 3 or a polynucleotide strand encoding for said polypeptide strand P with a dissociation constant of not more than 1000 nM.

41. An antibody or antibody fragment AB binding a polypeptide strand P SEQ ID NO: 2 or SEQ ID NO: 3 or a polynucleotide strand encoding for said polypeptide strand P with a dissociation constant of not more than 1000 nM, wherein said antibody or antibody fragment AB is obtained from a method of claim 39.

42. The antibody or antibody fragment AB of claim 40, wherein said antibody or antibody fragment AB is a monoclonal humanized antibody.

43. The antibody or antibody fragment AB of claim 40, wherein said antibody or antibody fragment AB bears a binding affinity to a polypeptide strand P of SEQ ID NO: 2 or SEQ ID NO: 3 of a dissociation constant of not more than 100 nM, not more than 50 nM, not more than 20 nM, not more than 10 nM, or not more than 5 nM.

44. A method for treating or preventing malaria in a patient, wherein the patient is administered with a sufficient amount of an antibody or antibody fragment AB of claim 40.

45. A method for diagnosing malaria in a patient, wherein the patient is administered with a sufficient amount of an antibody or antibody fragment AB of claim 40.

46. A method for staining Plasmodium falciparum or Plasmodium vivax in vitro, said method comprising the steps of: (i) providing (a) an optionally fixed sample S containing Plasmodium falciparum or Plasmodium vivax, and (b) an optionally stained antibody or antibody fragment AB according to claim 40; (ii) contacting the sample S with the optionally stained antibody or antibody fragment AB; (iii) optionally contacting the treated sample of step (ii) with a second antibody or antibody fragment AB2 which is stained and selectively binds to the antibody or antibody fragment AB.

Description

EXAMPLES

Example 1

Protective Effect of the Immunization with Pch ROPE

[0220] Immunization experiment with Pch ROPE in Balb/c mice.

[0221] A cDNA library of a Plasmodium chabaudi 96V was constructed in pBluescript SK (Werner et al. 1998). From the cDNA library the recombinant plasmid pBluescript SK (-) 70 was isolated by screening. It contains part of the Pch ROPE sequence coding for amino acids 1283-1516 (234 amino acids) as an insert.

[0222] The insert was sub cloned in the pGEX-1T vector, transformed in E. coli JM 109 bacteria and expressed as a glutathione-S-transferase fusion protein in E. coli JM 109. The fusion protein was isolated using glutathione-agarose beads, followed by thrombin cleavage to obtain the 234 amino acid Pch ROPE protein fragment in a pure form, called rec 700.

[0223] Mice were immunized at day 0 and day 21 with:

[0224] 7.5 g rec 700

[0225] 60 g Alum

[0226] in 200 l PBS pH 7.4

[0227] At day 35, mice were infected with 510.sup.6 Plasmodium chabaudi 96V parasitized erythrocytes. Balb/c mice were immunized only two times and without any supplementation of an adjuvant such as muramyl peptide (MDP).

[0228] Results

[0229] All of the non-immunized mice died. 50% of the immunized Balb/c survived and 50% died. The fact that a high number of Balb/c mice survived an otherwise deadly infection with this extremely virulent Plasmodium chabaudi 96V strain using a weak immunization protocol, was surprising.

[0230] It is noteworthy in this context to mention that in other experiments, not a single untreated mouse survived an infection with the Plasmodium chabaudi 96V strain over a period of about four years of conducting regular experiments with this strain (Watier et al., 1992).

[0231] Particularly surprising was the finding that a protective effect of the immunization could still be observed when adjuvants like muramyl peptide (MDP) were omitted. Such adjuvants are typically considered to be a relevant component to illicit a stronger immune response against an antigen used as a vaccine.

[0232] Furthermore it was recently shown that, in the case of malaria, alum (used in above immunization) is a comparably poor adjuvant for fighting diseases like malaria (Leslie, 2013). This shows that ROPE is a particularly effective immunogen. It is indicating a very high protection capacity of the ROPE protein when used as a malaria vaccine. Moreover, ROPE is very effective for generating specific antibodies.

[0233] It will be understood that an infection with Plasmodium chabaudi in rodents is a well-established model system for infections with Plasmodium falciparum or Plasmodium vivax in humans. For this reason, it is reasonable to assume that ROPE has a protective effect for mammals (including humans) immunized with Plasmodium falciparum or Plasmodium vivax recombinant ROPE against infection with these parasites. As shown immunization was highly efficient. Ideally a single immunization step using a strong immunization protocol would be sufficient to confer protection.

Example 2

Further Development of a Pf ROPE or Pv ROPE Malaria Vaccine

[0234] Steps towards the development of a Pf ROPE or Pv ROPE malaria vaccine include generation and testing of antibodies against ROPE fragments, testing of antibodies against ROPE peptide-microarrays covering the entire ROPE sequence, and preclinical and clinical trials.

[0235] a.) Generation and Testing of Antibodies Against ROPE Fragments

[0236] Synthesizing parts of Plasmodium falciparum (Pf) ROPE as peptides, raising soluble scFvs (single chain fragment variable) antibodies against these peptides and testing the capacity to block invasion of human red blood cells by Plasmodium falciparum in vitro in culture. Antibodies showing the strongest inhibition will be used to produce antibodies that can be used for passive immunization in humans, similar to an anti-malaria drug.

[0237] Several peptides are produced from the Pf ROPE amino acid sequence. To analyze the Pf B cell epitopes, the Pf ROPE sequence was analyzed using the PROTEAN subroutine in the DNASTAR package. This subroutine uses (Wang et al., 2016): [0238] Predicted alpha-regions (Gamier and Robson, 1989; Chou and Fasman, 1978) [0239] Hydrophilicity (Kyte and Doolittle, 1982) [0240] Flexibility (Karpus and Schulz, 1985) [0241] Surface probability (Emini et al., 1985) [0242] Antigenicity (Jameson and Wolf, 1988).

[0243] Based on this analysis the following peptides with good hydrophilicity, high accessibility, high flexibility, and strong antigenicity were selected as the antigen epitopes as shown in Table 1.

TABLE-US-00009 TABLE1 EpitopesderivedfromROPE Amino acid position Length in (amino Sequence ROPE Sequence acids) No. 331-356 KQEKEKEKEKEREKEKEREKEKEKEY 26 SEQIDNO:7 420-447 KNLKTELEKKEKELKDIENVSKEEINKL 28 SEQIDNO:8 520-560 SKKEKEYNQYKNTYIEEINNLNEKLEE 41 SEQIDNO:9 TNKEYTNLQNNYTN 812-840 KEEYEDKMNTLNEQNEDKMNSLKEEY 29 SEQIDNO:10 ENK 953-996 KGLKKEVEEKEHKRHSSFNILKSKEKFF 44 SEQIDNO:11 KNSIEDKSHELKKKHE 1050-1079 KDKSKEKIKDKENQINVEKNEEKDLKKK 30 SEQIDNO:12 DD 1277-1305 EDEKKRNLNEINNLKKKNEDMCIKYNEMN 29 SEQIDNO:13 1452-1479 KTNKENEEKIINLTSQYSDAYKKKSDES 28 SEQIDNO:14 1497-1521 SNNNIRTNEYKYEEMFDTNIEEKNG 25 SEQIDNO:15 1581-1605 GNISNKNENNNKKNNTCDGYDEKVT 25 SEQIDNO:16

[0244] It is reasonable that these peptides or fragments thereof are particularly suitable as a polypeptide strand P usable in a vaccine V.

[0245] Human antibodies are generated against above Pf ROPE peptides by phage display using human antibody gene libraries (Kugler et al. 2015).

[0246] In brief, biotinylated Pf ROPE peptides are immobilized on streptavidin coated microtiter plates. The libraries are incubated with the peptides, non-binding antibody phage particles removed by rigid washing steps. The bound antibody phages are eluted by trypsin and re-amplified using E. coli XL1-Blue and the M13-K07 helper phage. Subsequently, two further panning rounds are performed. Monoclonal antibodies are produced as soluble scFvs (single chain fragment variable) antibodies, using the phage display vector pHAL30 and identified by screening ELISA on the immobilized Pf ROPE peptides. This step is needed to discard non-specific binders of the corresponding antibody phage particles.

[0247] For further tests the monoclonal scFvssingle chain fragment variable-antibodies are re-cloned into the bivalent scFv-Fc format and produced in mammalian cells. The mammalian vector pCSE2.6-hIgG-Fc-XP and HEK293 6E cells are used. This is an IgG like bivalent molecule and also effector functions are established. (Jager et al., 2013). These IgG-like antibodies are used to test for their capacity to block invasion of human red blood cells by Plasmodium falciparum in vitro in culture. An in vitro growth inhibition activity assay (GIA) is used to measure the efficacy of the soluble scFvs antibodies directed against our peptides in blocking merozoite invasion (Kennedy et al., 2002).

[0248] Peptides identified as protective in the context of this in vitro inhibition can be used for the production of chemically synthesized vaccines, either as single peptides or as a fusion of several peptides or can be used as diagnostics.

[0249] b.) Testing of Antibodies Against ROPE Fragments in Microarrays

[0250] Peptide microarrays covering the entire Pf ROPE sequence are prepared. Sera from malaria patients are used to identify the immunodominant parts of the Pf ROPE protein during the course of an infection with Plasmodium falciparum.

[0251] The whole amino acid sequence of the target protein is retrieved from a public database and translated into 15-mer peptides with a peptide-peptide overlap of e.g. 12 amino acids. The peptide arrays with the corresponding peptides are produced by the company PEPperPRINT GmbH (Heidelberg, Germany) in a laser printing process on glass slides, coated with a PEGMA/PMMA graft copolymer, which are functionalized with a Ala-Ala-linker.

[0252] A layer of amino acid particles, containing Fmoc-amino acid pentafluorophenyl esters, is printed layer after layer onto the functionalized glass slides, with intermittent melting (i.e. coupling) steps at 90 C. and chemical washing and capping steps (Stadler et al., 2008), based on the same principle as Merrifield's solid-phase peptide synthesis. Peptides are generated in duplicates on the arrays, which are screened for IgG and IgM responses in human sera.

[0253] Therefore, peptide microarrays are placed in incubation trays (PEPperPRINT GmbH, Heidelberg, Germany) and blocked for 30 min at room temperature with western blot blocking buffer MB-070 (Rockland, USA). Then, sera are diluted 1:1000 in PBS buffer with 0.05% Tween 20 pH 7.4 (PBS-T) and 10% blocking buffer, incubating the sera for 16 h at 4 C. and 50 RPM orbital shaking. Peptide microarrays are washed three times shortly with PBS-T, followed by an incubation with a 1:2500 dilution of the secondary fluorescently labeled antibody, together with a control antibody for 30 min at room temperature. The peptide microarrays are washed with PBST and rinsed with deionized water. After drying in a stream of air, fluorescent images are acquired using an Odyssey Imaging System (LI-COR, USA) at 700 nm. Image analysis and quantification is performed with the PepSlide Analyzer software (Sicasys Software GmbH, Heidelberg, Germany).

[0254] c.) Further Preclinical and Clinical Trials with Recombinant Pf ROPE Protein

[0255] The result of this mapping will determine which part of the long Pf ROPE protein (1979 amino acids) will be expressed as a recombinant protein in E. coli and be used as an anti-malaria vaccine in Aotus monkeys and once proven to be efficient in human trials.

[0256] The DNA sequence coding for the entire Pf ROPE protein or parts of it is amplified by PCR of genomic Pf DNA and cloned into the pET-21a (+)-plasmid at the multiple cloning site (MCS). The MCS is under the control of a T7 promoter and flanked by a T7- and a HIS-tag. The recombinant pET-21a (+)-plasmid containing a sequence coding for Pf ROPE is transformed into E. coli BL21(DE3), a strain that allows high-efficiency protein expression of any gene that is under the control of a T7 promoter. The expressed recombinant Pf ROPE protein carries a histidine-tag at its C-terminus and is purified on a Nickel-column.

[0257] The recombinant Pf ROPE protein is mixed with a pharmaceutically acceptable carrier or excipient. A vaccine is obtained. This is applied to Aotus monkeys and once proven to be efficient in human trials.

REFERENCES

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