U.S. patent application number 12/647674 was filed with the patent office on 2010-11-18 for recombinant protein containing a c-terminal fragment of plasmodium msp-1.
This patent application is currently assigned to INSTITUT PASTEUR. Invention is credited to JOHN W. BARNWELL, SHIRLEY LONGACRE-ANDRE, KAMINI MENDIS, FARIDABANO NATO, CHARLES ROTH.
Application Number | 20100291133 12/647674 |
Document ID | / |
Family ID | 9489188 |
Filed Date | 2010-11-18 |
United States Patent
Application |
20100291133 |
Kind Code |
A1 |
LONGACRE-ANDRE; SHIRLEY ; et
al. |
November 18, 2010 |
RECOMBINANT PROTEIN CONTAINING A C-TERMINAL FRAGMENT OF PLASMODIUM
MSP-1
Abstract
The invention relates to a recombinant protein fabricated in a
baculovirus system, of which the essential constitutive polypeptide
sequence is that of a C-terminal fragment of 19 kilodalton (p19) of
the surface protein 1 (protein MSP-1) of the merozoite parasite of
the Plasmodium type, particularly Plasmodium falciparum, which is
infectious for humans, said C-terminal fragment remaining normally
anchored at the surface of the parasite at the end of its
penetration phase into human erythrocytes, in the occurrence of an
infectious cycle. Said recombinant protein is applicable to the
production of vaccines against malaria.
Inventors: |
LONGACRE-ANDRE; SHIRLEY;
(PARIS, FR) ; ROTH; CHARLES; (RUEIL-MALMAISON,
FR) ; NATO; FARIDABANO; (ANTONY, FR) ;
BARNWELL; JOHN W.; (STONE MOUNTAIN, GA) ; MENDIS;
KAMINI; (COLUMBO 8, LK) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, L.L.P.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
INSTITUT PASTEUR
PARIS
NY
NEW YORK UNIVERSITY
NEW YORK
|
Family ID: |
9489188 |
Appl. No.: |
12/647674 |
Filed: |
December 28, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11144833 |
Jun 6, 2005 |
7696308 |
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|
12647674 |
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|
09125031 |
Mar 10, 1999 |
6958235 |
|
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PCT/FR97/00290 |
Feb 14, 1997 |
|
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|
11144833 |
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Current U.S.
Class: |
424/191.1 ;
424/193.1; 424/272.1 |
Current CPC
Class: |
A61P 33/06 20180101;
C07K 16/205 20130101; Y02A 50/30 20180101; A61K 39/015 20130101;
C07K 14/445 20130101; Y02A 50/412 20180101; A61K 39/00 20130101;
C12N 2799/026 20130101 |
Class at
Publication: |
424/191.1 ;
424/272.1; 424/193.1 |
International
Class: |
A61K 39/015 20060101
A61K039/015; A61K 39/385 20060101 A61K039/385; A61P 33/06 20060101
A61P033/06 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 14, 1996 |
FR |
96/01822 |
Claims
1-45. (canceled)
46. A vaccine composition comprising: 1) a first recombinant
protein comprising: a) a 19 kilodalton (p19) C-terminal fragment of
a surface protein 1 of a merozoite form (MSP-1) protein of a
Plasmodium falciparum parasite, wherein said C-terminal fragment
remains anchored via a glycosylphosphatidylinositol group to the
surface of said Plasmodium falciparum parasite at an end of its
penetration phase into human erythrocytes during an infectious
cycle; or b) a portion of said 19 kilodalton (p19) C-terminal
fragment of a surface protein 1 of a merozoite form (MSP-1)
protein, which can induce an immune response and which can inhibit
parasitemia in vivo in a host infected with said Plasmodium
falciparum parasite and contains at least one of the two epidermal
growth factor (EGF) regions; and 2) a second recombinant p19
protein from a Plasmodium parasite that is homologous to Plasmodium
falciparum.
47. The vaccine composition according to claim 46, wherein said
first recombinant protein further comprises, upstream of said 19
kilodalton (p19) C-terminal fragment or fragment of said 19
kilodalton (p19) C-terminal fragment, a polypeptide containing less
than 50 amino acids of a C-terminal end of p33 of a MSP-1 protein
of a Plasmodium parasite.
48. The vaccine composition according to claim 46, wherein said p19
C-terminal fragment or said portion of said p19 C-terminal fragment
is deprived of an anchoring sequence of the native protein, which
is normally implicated in the induction of anchoring the native
protein to the cell membrane of a host in which it is
expressed.
49. The vaccine composition according to claim 47, wherein said
polypeptide contains less than 35 amino acids.
50. A vaccine composition according to claim 46, wherein the first
recombinant protein is produced from a transformed insect cell
deposited at the CNCM with registration number I-1661 or I-1662 on
Feb. 1, 1996.
51. The vaccinating composition according to claim 46, wherein said
recombinant protein is conjugated to a carrier molecule.
52. The vaccinating composition according to claim 46, further
comprising a signal peptide, which is from a surface protein 1 of a
merozoite form (MSP-1) of a Plasmodium parasite.
53. The vaccinating composition according to claim 52, wherein said
signal peptide is from Plasmodium vivax.
54. The vaccinating composition according to claim 52, wherein said
signal peptide is from Plasmodium falciparum.
55. The vaccinating composition according to claim 46, wherein said
second recombinant p19 protein from a Plasmodium parasite that is
homologous to Plasmodium falciparum is from Plasmodium vivax.
56. The vaccinating composition according to claim 46, wherein said
second recombinant p19 protein from a Plasmodium parasite that is
homologous to Plasmodium falciparum is from Plasmodium
cynomolgi.
57. The vaccinating composition according to claim 46, further
comprising alum.
58. The vaccinating composition according to claim 52, further
comprising alum.
59. A vaccinating composition which comprises: 1) a first
recombinant protein comprising: a) a sequence comprising thirty-two
amino acids of a surface protein 1 of a merozoite form (a MSP-1
protein) of a Plasmodium vivax from Met.sub.1 to Asp.sub.32; and b)
a 19 kilodalton C-terminal fragment of a surface protein 1 of a
merozoite form (MSP-1) of Plasmodium falciparum comprising an amino
acid sequence from Asn at amino acid position 3 to Ser at amino
acid position 95 of SEQ ID NO: 1 or a 19 kilodalton C-terminal
fragments of a surface protein 1 of a merozoite form (MSP-1) of
Plasmodium falciparum comprising an amino acid sequence from Asn at
amino acid position 3 to Ile at amino acid position 116 of SEQ ID
NO: 4; and 2) a second recombinant p19 protein from a Plasmodium
parasite that is homologous to Plasmodium falciparum.
60. The vaccine composition according to claim 59, wherein said
second recombinant p19 protein parasite that is homologous to
Plasmodium falciparum is from Plasmodium vivax.
61. A vaccinating composition which comprises: 1) a first
recombinant protein comprising: a) a sequence comprising thirty-two
amino acids of a surface protein 1 of a merozoite form (a MSP-1
protein) of a Plasmodium vivax from Met.sub.1 to Asp.sub.32; and b)
a 19 kilodalton C-terminal fragment of a surface protein 1 of a
merozoite form (MSP-1) of Plasmodium cynomolgi comprising an amino
acid sequence from Lys.sub.276 to Ser.sub.380 as shown in SEQ ID
NO: 11 or a fragment thereof which can induce an immune response
and which can inhibit parasitemia in vivo in a host infected with
said Plasmodium falciparum parasite; and 2) a second recombinant
p19 protein from a Plasmodium parasite that is homologous to
Plasmodium cynomolgi.
62. The vaccine composition according to claim 61, wherein said
second recombinant p19 protein parasite that is homologous to
Plasmodium cynomolgi is from Plasmodium vivax.
63. The vaccine according to claim 46, wherein said portion of said
p19 C-terminal fragment has a molecular eight of from 10 to 25
kDa.
64. The vaccine according to claim 46, wherein said portion of said
p19 C-terminal fragment has a molecular eight of from 10 to 15
kDa.
65. The vaccine composition according to claim 46, wherein said
second recombinant p19 protein is from Plasmodium vivax and wherein
said vaccine composition further comprises: a) a p42 fragment from
Plasmodium falciparum, wherein said from Plasmodium falciparum is
deprived of its most hypervariable regions, and b) a p42 fragment
from Plasmodium vivax, wherein said from Plasmodium vivax is
deprived of its most hypervariable regions.
66. The vaccine composition according to claim 65, further
comprising alum.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 11/144,833 filed Jun. 6, 2005, now U.S. Pat.
No. 7,696,308, which is a continuation of U.S. application Ser. No.
09/125,031 filed Mar. 10, 1999, now U.S. Pat. No. 6,958,235, which
is a 371 application of PCT/FR97/00290 filed Feb. 14, 1997
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to novel active principles for
vaccines derived from the major surface protein in merozoite forms
of a Plasmodium which is infectious for mammals, especially humans,
more generally termed MSP-1.
[0004] 2. Discussion of the Background
[0005] MSP-1 has already been the subject of a number of studies.
It is synthesised in the schizont stage of Plasmodium type
parasites, in particular Plasmodium falciparum, and is expressed in
the form of one of the major surface constituents of merozoites
both in the hepatic stage and in the erythrocytic stage of malaria
(1, 2, 3, 4). Because of the protein's predominant character and
conservation in all known Plasmodium species, it has been suggested
that it could be a candidate for constituting anti-malarial
vaccines (5, 6).
[0006] The same is true for fragments of that protein, particularly
the natural cleavage products which are observed to form, for
example during invasion by the parasite into erythrocytes of the
infected host. Among such cleavage products are the C-terminal
fragment with a molecular weight of 42 kDa (7, 8) which is itself
cleaved once more into an N-terminal fragment with a conventional
apparent molecular weight of 33 kDa and into a C-terminal fragment
with a conventional apparent molecular weight of 19 kDa (9) which
remains normally fixed to the parasite membrane after the
modifications carried out on it, via glycosylphosphatidylinositol
(GPI) groups (10, 11).
[0007] It is also found at the early ring stage of the
intraerythrocytic development cycle (15, 16), whereby the
observation was made that the 19 kDa fragment could play a role
which is not yet known, but which is doubtless essential in
re-invasive processes. This formed the basis for hypotheses formed
in the past that that protein could constitute a particularly
effective target for possible vaccines.
[0008] It should be understood that the references frequently made
below to the p42 and p19 proteins from a certain type of Plasmodium
are understood to refer to the corresponding C-terminal cleavage
products of the MSP-1 protein of that Plasmodium or, by extension,
to products containing substantially the same amino acid sequences,
obtained by genetic recombination or by chemical synthesis using
conventional techniques, for example using the "Applied System"
synthesiser, or by "Merrifield" type solid phase synthesis. For
convenience, references to "recombinant p42" and "recombinant p19"
refer to "p42" and "p19" obtained by techniques comprising at least
one genetic engineering step.
[0009] Faced with the difficulty of obtaining large quantities of
parasites for P. falciparum and the impossibility of cultivating P.
vivax in vitro, it has become clear that the only means of
producing an anti-malaria vaccine is to resort to techniques which
use recombinant proteins or peptides. However, MSP-1 is very
difficult to produce whole because of it large size of about 200
kDa, a fact which has led researchers to study the C-terminal
portion, the (still unknown) function of which is probably the more
important.
[0010] Recombinant proteins concerning the C-terminal portion of
the P. falciparum MSP-1 which have been produced and tested in the
monkey (12, 40, 41) are: [0011] a p19 fused with a
glutathione-S-transferase produced in E. coli (40); [0012] a p42
fused with a glutathione-S-transferase produced in E. coli (12);
[0013] a p19 fused with a polypeptide from a tetanic anatoxin and
carrying auxiliary T cell epitopes produced in S. cerevisiae (12);
[0014] a p42 produce in a baculovirus system (41).
[0015] A composition containing a p19 protein fused with a
glutathione-S-transferase produced in E. coli combined with alum or
liposomes did not exhibit a protective effect in any of six
vaccinated Aotus nancymai monkeys (40).
[0016] A composition containing a p42 protein fused with a
glutathione-S-transferase produced in E. coli combined with a
Freund complete adjuvant did not exhibit a protective effect in two
types of Aotus monkeys (A. nancymai and A. vociferans) when
administered to them. The p19 protein produced in S. cerevisiae
exhibited a protective effect in two A. nancymai type Aotus monkeys
(12). In contrast, there was no protective effect in two A.
vociferans type Aotus monkeys.
[0017] Some researchers (Chang et al.) have also reported
immunisation tests carried out in the rabbit using a recombinant
p42 protein produced in a baculovirus system and containing one
amino acid sequence in common with P. falciparum (18). Thus these
latter authors indicate that in the rabbit that recombinant p42
behaves substantially in the same way as the entire recombinant
MSP-1 protein (gp195). This p42 protein in combination with a
Freund complete adjuvant has been the subject matter of a
vaccination test in a non-human primate susceptible to infection by
P. falciparum, Aotus, lemurinus grisemembra (40). The results
showed that 2 of 3 animals were completely protected and the third,
while exhibiting a parasitemia which resembled that of the
controls, had a longer latent period. It is nevertheless risky to
conclude to a protective nature in man of the antibodies thus
induced against the parasites themselves. It should be remembered
that there are currently no very satisfactory experimental models
in the primate for P. vivax and P. falciparum. The Saimiri model,
developed for P. falciparum and P. vivax, and the Aotus model for
P. falciparum, are artificial systems requiring the parasite
strains to be adapted and often requiring splenectomy of the
animals to obtain significant parasitemia. As a result, the
vaccination results from such models can only have a limited
predictive value for man.
[0018] In any event, what the real vaccination rate would be which
could possibly be obtained with such recombinant proteins is also
questionable, bearing in mind the discovery--reported below--of the
presence in p42s from Plasmodiums of the same species, and more
particularly in the corresponding p33s, of hypervariable regions
which would in many cases render uncertain the immunoprotective
efficacy of antibodies induced in individuals vaccinated with a p42
from a Plasmodium strain against an infection by other strains of
the same species (13).
[0019] It can even be assumed that the high polymorphism of the
N-terminal portion of p42 plays a significant role in immune
escape, often observed for that type of parasite.
[0020] The aim of the present invention is to produce vaccinating
recombinant proteins which can escape these difficulties, the
protective effect of which is verifiable in genuinely significant
experimental models or even directly in man.
[0021] More particularly, the invention provides vaccinating
compositions against a Plasmodium type parasite which is infectious
for man, containing as an active principle a recombinant protein
which may or may not be glycosylated, whose essential constituent
polypeptide sequence is: [0022] either that of a 19 kilodalton
(p19) C-terminal fragment of the surface protein 1 of the merozoite
form (MSP-1 protein) of a Plasmodium type parasite which is
infectious for man, said C-terminal fragment remaining normally
anchored to the parasite surface at the end of its penetration
phase into human erythrocytes in the event of an infectious cycle;
[0023] or that of a portion of that fragment which is also capable
of inducing an immune response which can inhibit in vivo
parasitemia due to the corresponding parasite; [0024] or that of an
immunologically equivalent peptide of said p19 fragment or said
portion of that fragment; and said recombinant protein further
comprises conformational epitopes which are unstable in a reducing
medium and which preferably constitute the majority of the epitopes
recognised by human antiserums formed against the corresponding
Plasmodium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1A illustrates the nucleotide and amino acid sequences
of the synthetic gene (Bac 19) and the "native gene" (PF19) of P.
falciparum described by Chang et al;
[0026] FIG. 1B illustrates the nucleotide and amino acid sequences
of the synthetic gene (Bac 19) and the "native gene" (PF19) of the
Uganda Palo Alto isolate of P. falciparum;
[0027] FIG. 1C illustrates the PfMSP1.sub.P19A recombinant protein
sequence before cutting out the signal;
[0028] FIG. 1D illustrates the PfMSP1.sub.P19A recombinant protein
after cutting out the signal sequence;
[0029] FIG. 2A is an immunoblot using SDS-PAGE of the soluble
recombinant PfMSP1.sub.P19 antigen purified by immunoaffinity of
the presence (reduced) or absence (non-reduced) of
-mercaptoethanol;
[0030] FIG. 2B is an immunoblot with human antiserum of recombinant
purified MSP-1 P19 from P. vivax and P. cynomolgi under non-reduced
(NR), reduced only in the charging medium (R) and irreversibly
reduced (IR) conditions;
[0031] FIG. 3A is an immunoblot of the soluble PvMSP1.sub.P42
recombinant antigen in the presence of protein fractions derived
from merzoites of P. falciparum and separately isoelectric focusing
in the presence (reduced) or absence (nonreduced) of
-mercaptoethanol;
[0032] FIG. 3B is a graph illustrating the results of an ELISA
inhibition technique of P. vivax MSP-1 P42 and P19 antigens by the
antiserum of individuals with an acquired immunity to P. vivax;
[0033] FIG. 4 are nucleotide sequences. The underlines
oligonucleotides originate from P. vivax and are used as primers in
a PCR reaction. The lower portion of FIG. 4 illustrates the percent
identity between two isolates of P. vivax and P. cynomolgi;
[0034] FIG. 5 are curves illustrating the variation in the measured
parasitemia as the number of parasited red blood cells per
microliter of blood as the function of time passed after infection.
Curve A corresponds to the average values observed in three
vaccinated monkeys and curve B corresponds to the average values in
five controls;
[0035] FIG. 6A is a graph illustrating the parasitemia observed in
non-vaccinated control animals as a function of time after
injection;
[0036] FIG. 6B is a graph illustrating the parasitemia observed in
control animals which contained a saline solution also contain
Freunds adjuvant as a function of time after injection;
[0037] FIG. 6C is a superposition of FIGS. 6A and 6B;
[0038] FIG. 6D is a graph illustrating parasitemia at the end of
vaccination with p42 as a function of time;
[0039] FIG. 6E is a graph illustrating parasitemia in animals
vaccinated with p19 alone as a function of time;
[0040] FIG. 6F is a graph illustrating parasitemia in animals with
a mixture of P42 and P19 as a function of time;
[0041] FIG. 6G is the data obtained to produce the graphs in FIGS.
6A to 6F;
[0042] FIG. 7A is an immunoblot illustrating the in vivo response
of monkeys to injections of p19 with Freunds adjuvant (1), with
alum (2) and in the form of liposomes (3);
[0043] FIG. 7B is an immunoblot illustrating the in vivo responses
of a squirrel monkey after three injections with p19 with Freunds
adjuvant, with alum and in the form of liposomes;
[0044] FIG. 8A is a graph illustrating the percent parasitemia
versus days post infection of six monkeys which were immunized with
recombinant MSP-1(p19) six months earlier;
[0045] FIG. 8B is a graph illustrating the percent parasitemia
versus days post infection of six monkeys that were immunized with
normal saline and an adjuvant;
[0046] FIG. 8C is a graph illustrating the percent parasitemia
versus days post infection of monkeys that were used as
controls;
[0047] FIG. 8D is the data obtained to produce the graphs of FIGS.
8A to 8C;
[0048] FIG. 9A is a graph illustrating the percent parasitemia
versus days post infection of 2 macaques immunized with recombinant
p19 and alum;
[0049] FIG. 9B is a graph illustrating the percent parasitemia
versus days post infection of 2 macaques immunized with recombinant
p19 and alum;
[0050] FIG. 9C is a graph illustrating the percent parasitemia
versus days post infection of a macaque immunized with p19;
[0051] FIG. 9D is a graph illustrating the percent parasitemia
versus days post infection of 3 control macaques immunized with
physiological water and alum;
[0052] FIG. 9E is the data obtained to generate the graphs in FIGS.
9A to 9D;
[0053] FIG. 10A is a graph illustrating the percent parasitemia
versus days post infection in a squirrel monkey immunized with
MSP-1 p19 and alum;
[0054] FIG. 10B is a graph illustrating the percent parasitemia
versus days post infection in a squirrel monkey immunized with
MSP-1 p19 and Freunds;
[0055] FIG. 10C is a graph illustrating the percent parasitemia
versus days post infection in a squirrel monkey immunized with
MSP-1 p19 with liposomes;
[0056] FIG. 10D is a graph illustrating the percent parasitemia
versus days post infection in a squirrel monkey immunized with alum
as the control;
[0057] FIG. 10E is a graph illustrating the percent parasitemia
versus days post infection in a squirrel monkey immunized with
Freunds as the control;
[0058] FIG. 10F is a graph illustrating the percent parasitemia
versus days post infection in a squirrel monkey immunized with
liposomes as the control;
[0059] FIG. 10G is a graph illustrating the percent parasitemia
versus days post infection in a squirrel monkey immunized with
physiological water as the control.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0060] The presence of such conformational epitopes could play an
important role in the protective efficacy of the active principle
of the vaccines. They are particularly found in the active
principles which exhibit the other characteristics defined above,
when they are produced in a baculovirus system. If needs be, it is
mentioned below that the expression "baculovirus vector system"
means the ensemble constituted by the baculovirus type vector
itself and the cell lines, in particular cells of insects
transfectable by a baculovirus modified by a sequence to be
transferred to these cell lines resulting in expression of that
transferred sequence. Preferred examples of these two partners in
the baculovirus system have been described in the article by
Longacre et al. (19). The same system was used in the examples
below. It goes without saying, of course, that variations in the
baculovirus and in the cells which can be infected by the
baculovirus can be used in place of those selected.
[0061] In particular, the recombinant protein is recognised by
human antiserums formed against the corresponding Plasmodium or
against a homologous Plasmodium when it is in its non reduced state
or in a reduced non irreversible state, but is not recognised or is
only recognised to a slight extent by these same antiserums when it
is irreversibly reduced.
[0062] The unstable character of these conformational epitopes in a
reducing medium can be demonstrated by the test described below in
the examples, in particular in the presence of
.beta.-mercaptoethanol. Similarly, the examples below describe the
experimental conditions applicable to obtain irreversible reduction
of the proteins of the invention.
[0063] From this viewpoint, the recombinant protein produced by
Longacre et al. (14) can be used in such compositions. It should be
remembered that S. Longacre et al. succeeded in producing a
recombinant p19 from the MSP-1 of P. vivax in a baculovirus vector
system containing a nucleotide sequence coding for the p19 of
Plasmodium vivax, in particular by transfecting cultures of insect
cells [Spodoptera frugiperda (Sf9) line] with baculovirus vectors
containing, under the control of the polyhedrin promoter, a
sequence coding for the peptide sequences defined below, with the
sequences being placed in the following order in the baculovirus
vector used: [0064] a 35 base pair 5' terminal fragment of the
polyhedrin signal sequence, in which the methionine codon for
initiating expression of this protein had been mutated (to ATT);
[0065] a 5'-terminal nucleotide fragment coding for a 32 amino acid
peptide corresponding to the N-terminal portion of MSP-1, including
the MSP-1 signal peptide; [0066] either a nucleotide sequence
coding for p19, or a sequence coding for the p42 of the MSP-1
protein of Plasmodium vivax, depending on the case, these sequences
also being provided with ("anchored" forms) or deprived of (soluble
forms) 3' end regions of these nucleotide sequences, whose end
C-terminal expression products are reputed to play an essential
role in anchoring the final p19 protein to the parasite membrane;
[0067] 2 TAA stop codons.
[0068] For p42, the sequences derived from the C-terminal region of
MSP-1 extend consequently from amino acid Asp 1325 to amino acid
Leu 1726 (anchored form) or to amino acid Ser 1705 (soluble form)
and for p19, the sequences extend from amino acid Ile 1602 to amino
acid Leu 1726 (anchored form) or to amino acid Ser 1705 (soluble
form) it being understood that the complete amino acid sequences of
p42 and p19, whose initial and terminal amino acids have been
indicated above follow from the gene of the Belem isolate of P.
vivax which has been sequenced (20).
[0069] Similar results were obtained using, in the same vector
systems, nucleotide sequences coding for the p42 and p19 of
Plasmodium cynomolgi. The interest in P. cynomolgi is twofold: it
is a parasitic species very close to P. vivax which is infectious
for the macaque. It can also infect man. Further, access to the
natural hosts of P. cynomolgi, rhesus monkeys and toque macaques,
is also possible, to test the efficacy of the protection of MSP-1
from P. cynomolgi in natural systems. The rhesus monkey is
considered to be one of the most representative species for immune
reactions in man.
[0070] In particular, excellent results have been obtained in
vaccination tests carried out using the toque macaque with two
recombinant polypeptides: soluble p42 and, in particular, soluble
p19 derived from P. cynomolgi, respectively produced in a
baculovirus system and purified on an affinity column with
monoclonal antibodies recognising the corresponding regions of the
native MSP-1 protein. The following observations were made: six
monkeys immunised with only p19 (three monkeys) and the p19 and p42
together (three monkeys) all exhibited practically sterile immunity
after challenge infection. The results obtained in the three
monkeys immunised with p42 were less significant. Two of them were
as above, but since the third exhibited a lower parasitemia than
the controls immunised with a PBS buffer in the presence of Freund
adjuvant (3 monkeys) or not immunised (3 monkeys), it was less
clear.
[0071] A second challenge infection showed that the monkeys which
had received p19 alone were protected for at least six months. A
second vaccination test with p19 in combination with alum in this
system (toque macaque P. cynomolgi) exhibited significant
protection for 2 of the 3 monkeys. This is the first time that
MSP-1 or another recombinant antigen has demonstrated a protective
effect in the presence of alum (42).
[0072] The particularly effective test results carried out with the
macaque with recombinant polypeptides produced in a baculovirus
system using a recombinant p19 from P. cynomolgi showed that
recombinant polypeptides respectively containing recombinant p19s
from other Plasmodiums must behave in the same manner. They are
more meaningful for malaria in man than the results from tests
carried out with P. vivax or P. falciparum in their "artificial
hosts".
[0073] Baculovirus recombinant proteins derived from a C-terminal
MSP-1 portion (p19) have a very significant antimalarial protective
effect in a natural system, which constitutes the most
representative model for evaluating the protective effect of MSP-1
for man.
[0074] The protective effect obtained can be further improved if
the p19 form is deprived of the hypervariable region of the
N-terminal portion of p42, the effect of which can be deleterious
in natural situations in which the vaccinated subject is confronted
by a great deal of polymorphism. Further, p19 appears to possess
specific epitopes which are not present in p42.
[0075] The 19 kDa C-terminal fragment, the sequence of which is
present in the active principle of the vaccine, can be limited to
the sequence for the p19 itself, in the absence of any polypeptide
sequence normally upstream of the p19 sequence in the corresponding
MSP-1 protein. Clearly, though, the essential constituent
polypeptide sequence of the active principle can also comprise a
polypeptide sequence for the C-terminal side belonging to the 33
kDa (p33) N-terminal fragment still associated with the p19 in the
corresponding p42, before natural cleavage of the latter, if the
presence of this fragment does not modify the immunological
properties of the active principle of the vaccine. As will be seen
below, in particular in the description of the examples, the
C-terminal sequences of the p33 in various strains of the same
species of Plasmodium (see the C-terminal portion of the peptide
sequences of "region III" in FIG. 4 (SEQ ID NOS:11-14)) also have a
degree of homology or substantial conservation of the sequence, for
example of the order of at least 80%, in different varieties of
Plasmodiums which are infectious for man, such that they do not
fundamentally modify the vaccinating properties of the active
principle (the sequence ofo which corresponds to region IV in FIG.
4), in particular using the hypothesis which follows from this
figure; that the presumed cleavage site between the p19 and region
III of the p33 is located between the leucine and asparagine
residues in a particularly well conserved region (LNVQTQ) (SEQ ID
NO:15).
[0076] Normally the C-terminal polypeptide sequence of the p33,
when it is present, comprises less than 50 amino acid residues, or
even less than 35, preferably less than 10 amino acid residues.
[0077] In contrast, the essential constituent polypeptide sequence
of the active principle of the vaccine need not comprise all of the
sequence coding for p19, naturally providing that the latter
retains the ability to induce antibodies which protect against the
parasite. In particular, the molecular weight of the "fragment
portion" is 10 to 25 kDa, in particular 10 to 15 kDa. Preferably,
this polypeptide fragment portion contains at least one of the two
EGF (Epidermal Growth Factor) regions.
[0078] Clearly, the skilled person could distinguish between active
fragments and those which would no longer be so, in particular
experimentally by producing modified vectors containing inserts
with different lengths originating from the p19, respectively
isolated from the fragments obtained from the sequence coding for
p19, by reaction with appropriate restriction enzymes, or by
exonucleolytic enzymes which would be kept in contact with the
fragment coding for p19 for differing periods; the capacity of the
expression products from these inserts in the corresponding
eukaryotic cells, in particular in insect cells, transformed by the
corresponding modified vectors, to exert a protective effect can
then be tested, in particular under the experimental conditions
which are described below in the examples. In particular, the
expression products of these inserts must be able to inhibit a
parasitemia induced in vivo by the corresponding whole
parasite.
[0079] Thus, the invention includes all vaccinating compositions in
which the essential constituent polypeptide sequence of the active
principle is constituted by a peptide which can induce a cellular
and/or humoral type immunological response equivalent to that
produced by p19 or a fragment as defined above, provided that the
addition, deletion or substitution in the sequence of certain amino
acids by others would not cause a large modification of the
capacity of the modified peptide--hereinafter termed the
"immunologically equivalent peptide"--to inhibit said
parasitemia.
[0080] The p19 fragment can naturally also be associated at the
N-terminal side or the C-terminal side or via a peptide bond to a
further plasmoidal protein fragment having a vaccinating potential
(such as Duffy binding protein from P. vivax (29) or EBA-175 from
P. falciparum (30) and (31), one region of which is specifically
rich in cysteine), provided that its capacity to inhibit
parasitemia normally introduced in vivo by the corresponding
parasite is not altered but is amplified.
[0081] Upstream of the N-terminal end of p19, the fragment coding
for p19 or a portion thereof can also contain a peptide sequence
which is different again, for example a C-terminal fragment of the
signal peptide used, such as that for the MSP-1 protein. This
sequence preferably comprises less than 50 amino acids, for example
10 to 40 amino acids.
[0082] These observations pertain in similar fashion to the p19s
from other Plasmodium, in particular P. falciparum, the dominant
species of the parasites, responsible for one of the most serious
forms of malaria.
[0083] However, the techniques summarised above for producing a
recombinant p19 from P. vivax or P. cynomolgi in a baculovirus
system are difficult to transpose unchanged to producing a
recombinant p19 of P. falciparum in a satisfactory yield, if only
to obtain appreciable quantities which will allow immunoprotective
tests to be carried out.
[0084] The invention also provides a process which overcomes this
problem to a large extent. It also becomes possible to obtain much
higher yields of P. falciparum p19--and other Plasmodium where
similar difficulties are encountered--using a synthetic nucleotide
sequence substituting the natural nucleotide sequence coding for
the p19 of Plasmodium falciparum in an expression vector of a
baculovirus system, this synthetic nucleotide sequence coding for
the same p19, but being characterized by a higher proportion of G
and C nucleotides than in the natural nucleotide sequence.
[0085] In other words, the invention follows from the discovery
that expression of a nucleotide sequence coding for a p19 in a
baculovirus system is apparently linked to an improved
compatibility of successive codons in the nucleotide sequence to
express with the "cellular machinery" of the host cells
transformable by the baculovirus, in the manner of that observed
for the natural nucleotide sequences normally contained in these
baculovirus and expressed in the infected host cells; hence the
poor expression, or even total absence of expression of a native P.
falciparum nucleotide sequence; hence also a possible explanation
of the more effective expression observed by Longacre et al. (14)
for the p19 of P. vivax in a baculovirus system and, as the
inventors have also shown, of the P. cynomolgi sequence from
corresponding native p19 nucleotide sequences, because of their
relatively much higher amounts of G and C nucleotides than those of
the native nucleotide sequences coding for the p19 of P.
falciparum.
[0086] The invention thus more generally provides a recombinant
baculovirus type modified vector containing, under the control of a
promoter contained in said vector and able to be recognised by
cells transfectable by said vector, a first nucleotide sequence
coding for a signal peptide exploitable by a baculovirus system,
characterized by a second nucleotide sequence downstream of the
first, also under the control of the promoter and coding for the
peptide sequence: [0087] either of a 19 kilodalton (p19) C-terminal
fragment of the surface protein 1 of the merozoite form (MSP-1
protein) of a Plasmodium type parasite other than Plasmodium vivax
which is infectious for man, said C-terminal fragment remaining
normally anchored to the parasite surface at the end of its
penetration phase into human erythrocytes in the event of an
infectious cycle; [0088] or of a portion of that peptide fragment
provided that the expression product from the second sequence in a
baculovirus system is also capable of inducing an immune response
which can inhibit in vivo parasitemia due to the corresponding
parasite; [0089] or of an immunologically equivalent peptide of
said C-terminal peptide fragment (p19) or said peptide fragment
portion by addition, deletion or substitution of amino acids not
resulting in a large modification of the capacity of said
immunologically equivalent peptide to induce a cellular and/or
humoral type immunological response similar to that produced by
said p19 peptide fragment or said portion of said fragment; and
said nucleotide sequence having, if necessary, a G and C nucleotide
content in the range 40% to 60%, preferably at least 50%, of the
totality of the nucleotides from which it is constituted. This
sequence can be obtained by constructing a synthetic gene in which
the natural codons have been changed for codons which are rich in
G/C without modifying their translation (maintaining the peptide
sequence).
[0090] The nucleotide sequence, provided by a synthetic DNA, may
have at least 10% of modified codons with respect to the natural
gene sequence or cDNA while retaining the characteristics of the
natural translated sequence, i.e., maintaining the amino acid
sequence.
[0091] It is not excluded that this G and C nucleotide content
could be further increased provided that the modifications
resulting therefrom as to the amino acid sequence of the
recombinant peptide--or immunologically equivalent
peptide--produced do not result in a loss of immunological
properties, or protective properties, of the recombinant proteins
formed, in particular in the tests which will be described
below.
[0092] These observations naturally apply to other Plasmodium hich
are infectious for man, in particular those where the native
nucleotide sequences coding for corresponding p19s would have T and
A nucleotide contents which are poorly compatible with effective
expression in a baculovirus system.
[0093] The sequence coding for the signal used can be that normally
associated with the native sequence of the Plasmodium concerned.
But it can also originate from another Plasmodium, for example P.
vivax or P. cynomolgi or another organism if it can be recognised
as a signal in a baculovirus system.
[0094] The sequence coding for p19 or a fragment thereof in the
vector under consideration is, in one case, deprived of the
anchoring sequence of the native protein to the parasite from which
it originates, in which case the expressed protein is generally
excreted into the culture medium (soluble form). It is also
remarkable in this respect that under the conditions of the
invention, the soluble and anchored forms of the recombinant
proteins produced, in particular when they are from P. falciparum
or P. cynomolgi or P. vivax, tend to form oligomers, this property
possibly being at the origin of the increased immunogenicity of the
recombinant proteins formed.
[0095] The invention also concerns vectors in which the coding
sequence contains the terminal 3' end sequence coding for the
hydrophobic C'-terminal end sequence of the p19 which is normally
implicated in the induction of anchoring the native protein to the
cell membrane of the host in which it is expressed. This
3'-terminal end region can also be heterologous as regards the
sequence coding for the soluble p19 portion, for example
corresponding to the 3'-terminal sequence from P. vivax or from
another organism when it codes for a sequence which anchors the
whole of the recombinant protein produced to the cell membrane of
the host of the baculovirus system used. An example of such
anchoring sequences is the GPI of the CD59 antigen which can be
expressed in the cells of Spodoptera frugiperda (32) type insects
or the GPI of a CD14 human protein (33).
[0096] The invention also, naturally, concerns recombinant
proteins, these proteins comprising conformational epitopes
recognised by human serums formed against the corresponding
Plasmodium.
[0097] In general, the invention also concerns any recombinant
protein of the type indicated above, provided that it comprises
conformational epitopes such as those produced in the baculovirus
system, in particular those which are unstable in a reducing
medium.
[0098] The invention also, naturally, concerns said recombinant
proteins, whether they are in their soluble form or in the form
provided with an anchoring region, in particular to cellular hosts
used in the baculovirus system.
[0099] The invention also encompasses oligomers spontaneously
produced in the baculovirus systems used or produced a posteriori,
using conventional protein oligomerisation techniques. The most
commonly used technique involves glutaraldehyde. However, any
conventional system for bridging between the respective amine and
carboxyl functions in proteins can be used. As an example, any of
the techniques described in European patent application EP-A-0 602
079 can be used.
[0100] The term "oligomer" means a molecule containing 2 to 50
monomer units, each of the monomer units containing p19 or a
fragment thereof, as defined above, capable of forming an
aggregate. The invention also encompasses any conjugation product
between a p19 or a p19 fragment as defined above, and a carrier
molecule--for example a polylysine-alanine--for use in producing
vaccines, via bonds which are covalent or otherwise. The
vaccinating compositions using them also form part of the
invention.
[0101] The invention still further concerns vaccine compositions
using these oligomeric or conjugated recombinant proteins,
including proteins from Plasmodium vivax, these observations also
extending to oligomers of these recombinant proteins.
[0102] The invention also encompasses compositions in which the
recombinant proteins defined above are associated with an adjuvant,
for example an alum. Recombinant proteins containing the C-terminal
end region allowing them to anchor to the membrane of the cells in
which they are produced are advantageously used in combination with
lipids which can form liposomes appropriate to the production of
vaccines. Without being limiting, lipids described, for example, in
the publication entitled "Les liposomes aspects technologique,
biologique et pharmacologique" [Liposomes: technological,
biological and pharmacological aspects] by J. Delattre et al.,
INSERM, 1993, can be used.
[0103] The presence of the anchoring region in the recombinant
protein, whether it is a homologous or heterologous anchoring
region as regards the vaccinating portion proper, encourages the
production of cytophilic antibodies, in particular IgG.sub.2a and
IgG.sub.2b type in the mouse which could have a particularly high
protective activity, so that associating the active principles of
the vaccines so constituted with adjuvants other than the lipids
used to constitute the liposome forms could be dispensed with. This
amounts to a major advantage, since liposomes can be lyophilised
under conditions which enable them to be stored and transported,
without the need for chains of cold storage means.
[0104] Other characteristics of the invention will become clear
from the following description of examples of recombinant proteins
of the invention and the conditions under which they can be
produced. These examples are not intended to limit the scope of the
invention.
Description of the Construction of PfMSP1.sub.p19S (Soluble)
(Soluble p19 from P. Falciparum)
[0105] The recombinant construction PfMSP1.sub.p19S contains the
DNA corresponding to 8 base pairs of the leader sequence and the
first 32 amino acids of the MSP-1 of Plasmodium vivax from Met, to
Asp.sub.32 (Belem isolate; Del Portillo et al., 1991, P. N. A. S.,
88, 4030) followed by GluPhe due to the EcoR1 site connecting the
two fragments. This is followed by the synthetic gene described in
FIG. 1, coding the Plasmodium falciparum MSP1.sub.p19 from
Asn.sub.1613 to Ser.sub.1705 (Uganda-Palo Alto isolate; Chang et
al., 1988, Exp. Parasitol., 67, 1). The construction is terminated
by two TAA stop codons. This construction gave rise to a
recombinant protein which was secreted in the culture supernatant
from infected cells.
[0106] In the same manner and for comparison, a recombinant
construction was produced under conditions which were similar to
those used to produce the p19 above, but working with a coding
sequence consisting of a direct copy of the corresponding DNA of
the P. falciparum strain (FUP) described by Chang et al., Exp.
Parasit. 67, 1; 1989. The natural gene copy (from asparagine 1613
to serine 1705) was formed from the native gene by PCR.
[0107] FIG. 1A shows the sequences of both the synthetic gene
(Bac19) (SEQ ID NO:1) and the "native gene" (PF19) (SEQ ID
NO:3).
[0108] It can be seen that 57 codons of the 93 codons of the native
sequence coding for the p19 from P. falciparum were modified (the
third nucleotide in 55 of them and the first and third nucleotides
in the other 2 codons). New codons were added to the 5' end to
introduce the peptide signal under the conditions indicated above
and to introduce an EcoRI site for cloning, and similarly two stop
codons were added which were not present in the P. falciparum p19
to obtain expression termination signals. The individual letters
placed above successive codons correspond to the respective
successive amino acids. Asterisks (*) show the stop codons.
Vertical lines indicate the nucleotides which are the same in the
two sequences
Description of the PfMSP1.sub.p19A Construction (Anchored GPI)
(Anchored p19 of P. falciparum)
[0109] The PfMSP1.sub.p19A construction had the characteristics of
that above except that the synthetic sequence (FIG. 1B) (SEQ ID
NOS: 4 and 6) codes for the MSP1.sub.p19 of Plasmodium falciparum
(Uganda-Palo Alto isolate) from Asn.sub.1613 to Ile.sub.1726
followed by two TAA stop codons. This construction gave rise to a
recombinant protein which was anchored in the plasma membrane of
infected cells by a glycosyl phosphatidyl inositol (GPI) type
structure.
[0110] FIG. 1C represents the PfMSP1.sub.p19S recombinant protein
sequence before cutting out the signal sequence (SEQ ID NO:7).
[0111] FIG. 1D represents the PfMSP1.sub.p19S recombinant protein
sequence after cutting out the signal sequence.
[0112] The amino acids underlined in FIGS. 1C and 1D originate from
the EcoR1 site used to join the nucleotide sequences derived from
the N-terminal portion of the MSP-1 of P. vivax (with signal
sequence) (SEQ ID NO:9) and the MSP-1.sub.p19 of P. falciparum.
[0113] FIG. 2--The soluble recombinant PfMSP1.sub.p19 antigen
purified by immunoaffinity was analysed by immunoblot using
SDS-PAGE in the presence (reduced) or absence (non reduced) of
.beta.-mercaptoethanol. Samples were charged onto gel after heating
to 95.degree. C. in the presence of 2% SDS. Under these conditions
only covalent type bonds (disulphide bridges) can resist
disaggregation. The left hand blot was revealed with a monoclonal
antibody which reacted with a linear epitope of natural p19. The
right hand blot was revealed with a mixture of 13 human antisera
originating from subjects with acquired immunity to malaria due to
Plasmodium falciparum. These results show that the recombinant
baculovirus molecule can reproduce conformational epitopes in the
form of a polymer the majority of which are recognised by human
antiserum.
[0114] Immunoblot Analysis with Human Antiserum of Recombinant
Purified MSP-1 p19 from P. vivax and P. cynomolgi Under Non Reduced
(NR), Reduced Only in the Charging Medium (R) and Irreversibly
Reduced (IR) Conditions
[0115] This work was based on the idea that the baculovirus
expression system correctly reproduced the conformational epitopes
present in vivo on the C-terminal portion of MSP-1 in large
amounts. The best means of measuring this property (which may be
the only possible means in the absence of native purified proteins
corresponding to p19) was to study the reactivity of the
recombinant proteins with the antiserum of individuals exposed to
malaria, this reflecting the native proteins as "seen" by the human
immune system.
[0116] Thus soluble recombinant PvMSP-1 p19 and PcMSP-1 p19
antigens purified by immunoaffinity were analysed by immunoblot
using SDS-PAGE (15%) in the presence (reduced) or absence (non
reduced) of DTT. Samples were charged onto gel after heating to
95.degree. C. in the presence of 2% SDS. The irreversible reduction
was carried but as follows: the protein was resuspended in 0.2 M
Tris-HCl, pH 8.4, 100 mM DTT, 1.0% SDS and heated for 30 minutes at
70.degree. C. After diluting with water, acrylamide was added to a
final concentration of 2 M and the mixture was incubated under
nitrogen in the dark for 1 hour at 37.degree. C. The immunoblot was
revealed with a mixture of 25 human antisera originating from
subjects with an acquired immunity to malaria due to Plasmodium
vivax. V and C respectively designate proteins derived from the
MSP-1 of P. vivax and P. cynomolgi. It should be noted that
irreversibly reduced recombinant proteins exhibited no reactivity
with human antiserum while non irreversibly reduced proteins or non
reduced proteins exhibited good reactivity. (The non reduced Pv
MSP-1 p19 was a little weak since in its glycosylated state it does
not bind well to nitro-cellulose paper). These results show that
recognition of baculovirus MSP-1 p19 molecules by human antiserum
is largely if not completely dependent on conformational epitopes
sensitive to reduction which are reproduced in this system.
[0117] FIG. 3--The soluble PvMSP1.sub.p42 recombinant antigen
(Longacre et al., 1994, op. Cit.) was incubated for 5 hours at
37.degree. C. in the presence of protein fractions derived from
merozoites of P. falciparum and separated by isolectrofocussing.
The samples were then analysed by immunoblot in the presence
(reduced) or absence (non reduced) of .beta.-mercaptoethanol.
Isolectrofocussing fractions 5 to 12, and two total merozoite
extracts made in the presence (Tex) or absence (T) of detergent,
were analysed. The immunoblot was revealed with monoclonal
antibodies specific for MSP1.sub.P42 and .sub.p19 of P. vivax. The
results suggest that there is a proteolytic activity in the P.
falciparum merozoites which can be extracted with detergent.
Digestion of p42 in certain fractions appear to cause
polymerisation of the digestion products (p19); this polymerisation
is probably linked to the formation of disulphide bridges since in
the presence of .beta.-mercaptoethanol, the high molecular weight
forms disappear in favour of a molecule of about 19 kDa (Tex-R).
The p19 polymerisation observed in these experiments could thus be
an intrinsic property of this molecule in vivo.
[0118] FIG. 3B: The Differential Contribution of p42 and p19
Antigens to the P. vivax Anti-MSP-1 Human Response
[0119] Recognition of P. vivax MSP-1 p42 and p19 antigens by the
antiserum of individuals with an acquired immunity to P. vivax was
compared using the ELISA inhibition technique as follows: a mixture
of 25 human antisera originating from subjects with an acquired
immunity to malaria due to P. vivax was diluted to 1:5000 and
incubated for 4 hours at ambient temperature either alone, or in
the presence of a 1 mM purified P. vivax recombinant p42 or p19.
This mixture was transferred to a microliter well which had been
coated for 18 hours at 4.degree. C. with 500 ngml.sup.-1 of
purified absorbed recombinant p42 or p19, and incubated for 30
minutes at ambient temperature. After washing with PBS containing
0.1% of Tween 20, a goat anti-mouse IgG conjugated with peroxidase
was added and the mixture was incubated for 1 hour at 37.degree. C.
The enzymatic activity was revealed by reading the optical density
at 492 nm. The percentage inhibition was calculated based on values
of 100% of antiserum activity with the coated antiserum on the
microtitre plate in the absence of a competing antigen. Statistical
data were calculated using a Statview program. Each bar represents
the average percentage inhibition of a pair of competing/absorbed
antigens based on 4 to 12 determinations; the vertical lines
correspond to a 95% confidence interval. Asterisks (*) designate
the antigens produced in the presence of tunicamycin, thus with no
N-glycosylation. The important parameters of these measurements
were the dilution of the antiserum by 1:5000 which is in the region
which is sensitive to ELISA curves and the competing antigen
concentrations of 1 mM which includes competition by low affinity
epitopes. Thus these data reflect the maximum resemblance between
the two compared antigens. The results show that the majority, if
not all of the p42 epitopes recognised by the human antiserum are
present on the p19 since in the presence of the latter, the
reactivity of the human antiserum against p42 is inhibited as much
as by the p42 antigen itself. In contrast, however, about 20% of
the p19 epitopes recognised by human antiserum were not or were not
accessible on the p42, since the reactivity of the human antiserum
against the p19 was much less inhibited by p42 than by p19 itself.
Such specific epitopes of p19 can be constituted or revealed only
after cleaving the p42 into p19 and p33. These results were not
affected by glycosylation showing that the effect is really due to
a difference between the peptide components of p19 and p42 and not
to a difference in glycosylation. These results underline the fact
that p19 has a distinct immunological identity to p42.
Description of the PcMSP1.sub.p19S (Soluble) Construction (Soluble
p19 of P. cynomolgi)
[0120] The DNA used for the above construction was obtained from a
clone of the Plasmodium cynomolgi ceylonesis strain (22-23). This
strain had been maintained by successive passages through its
natural host (Macaca sinica) and cyclic transmissions via
mosquitoes (27).
[0121] Blood parasites in the mature schizont stage were obtained
from infected monkeys when the parasitemia had attained a level of
5%. They were then purified using the methods described in (25).
The DNA was then extracted as described in (26).
[0122] A 1200 base pair fragment was produced using a PCR reaction
using the oligonucleotides underlined in FIG. 4 originating from P.
vivax. The 5' oligonucleotide comprised an EcoRI restriction site
and the 3' oligonucleotide comprised two synthetic TAA stop codons
followed by a BglII restriction site. This fragment was introduced
by ligation and via these EcoRI and BglII sites into the
pVLSV.sub.200 plasmid already containing the signal sequence for
the MSP-1 protein of P. vivax (19). The new plasmid
(pVLSV.sub.200C42) was used to analyse the DNA sequences.
[0123] The P. cynomolgi and the corresponding P. vivax sequences
were aligned. The black arrows designate the presumed primary and
secondary cleavage sites. They were determined by analogy with
known sites in P. falciparum (27, 28). The vertical lines and
horizontal arrows localise the limits of the four regions which
were studied. Region 4 corresponded to the sequence coding for the
P. cynomolgi p19. Glycosylation sites are boxed and the preserved
cysteines are underlined. The lower portion of FIG. 4 shows the
percentage identity between the two isolates of P. vivax and P.
cynomolgi.
[0124] The recombinant construction PcMSP1.sub.p19S contains the
DNA corresponding to 8 base pairs of the leader sequence and the
first 32 amino acids of the MSP-1 of Plasmodium vivax from
Met.sub.1 to Asp.sub.32 (Belem isolate; Del Portillo et al., 1991,
P. N. A. S., 88, 4030) followed by GluPhe, due to the EcoR1 site,
connecting the two fragments. This is followed by the sequence
coding for the Plasmodium cynomolgi MSP1.sub.p19 from Lys.sub.276
to Ser.sub.380 (Ceylon strain). The construction was terminated by
two TAA stop codons. This construction gave rise to a recombinant
protein which was secreted in the culture supernatant of infected
cells.
Purification of Recombinant PfMSP1.sub.p19 Protein by
Immunoaffinity Chromatography with a Monoclonal Antibody
Specifically Recognising the p19 of Plasmodium falciparum
[0125] The chromatographic resin was prepared by binding 70 mg of a
monoclonal antibody (obtained from a G17.12 hybridoma deposited at
the CNCM [National Collection of Microorganism Cultures] (Paris,
France) on the 14 Feb. 1997, registration number 1-1846; this
G17.12 hybridoma was constructed from X63 Ag8 653 myeloma producing
IgG 2a/k recognising the P. falciparum p19) to 3 g of activated
CNBr-Sepharose 4B (Pharmacia) using standard methods detailed in
the procedure employed by Pharmacia. The culture supernatants
containing the soluble PfMSP1p19 were batch incubated with the
chromatographic resin for 16 hours at 4.degree. C. The column was
washed once with 20 volumes of 0.05% NP40, 0.5 M of NaCl, PBS; once
with 5 volumes of PBS and once with 2 volumes of 10 mM of sodium
phosphate, pH 6.8. Elution was carried out with 30 ml of 0.2 M
glycine, pH 2.2. The eluate was neutralised with 1 M sodium
phosphate, pH 7.7 then concentrated by ultrafiltration and dialysed
against PBS. To purify the anchored PfMSP1p19, all of the washing
and elution solutions contained a supplemental 0.1% of
3-(dimethyl-dodecylammonio)-propane sulphonate (Fluka).
Recombinant Plasmodium Vivax (p42 and p19) MSP1 Vaccination Test in
the Squirrel Monkey Saimiri Sciureus
[0126] This vaccination test was carried out on male non
splenectomised 2 to 3 year old Saimiri sciureus boliviensis
monkeys. Three monkeys were injected 3 times intramuscularly at 3
week intervals with a mixture of about 50 to 100 .mu.g each of
recombinant soluble PvMSP1.sub.p42 and .sub.p19 (19), purified by
immunoaffinity. Complete and incomplete Freund adjuvant was used as
follows: 1.sup.St injection: 1:1 FCA/FIA; 2.sup.nd injection: 1:4
FCA/FIA; 3.sup.rd injection: FIA. These adjuvant compositions were
then mixed 1:1 with the antigen in PBS. Five control monkeys
received the glutathione-S-transferase (GST) antigen produced in E.
coli using the same protocol. The challenge infection was carried
out by injecting 2.times.10.sup.6 red blood cells infected with an
adapted Plasmodium vivax strain (Belem) 2.5 weeks after the final
injection. The protection was evaluated by determining parasitemia
daily in all animals by examining smears stained with Giemsa.
[0127] The curves in FIG. 5 show the variation in the measured
parasitemia as the number of parasited red blood cells per
microlitre of blood (up the ordinate, logarithmic scale) as a
function of the time passed after infection (in days). Curve A
corresponds to the average values observed in the three vaccinated
monkeys; curve B corresponds to the average values in the five
control monkeys.
[0128] An examination of the Figure shows that the effect of the
vaccination was to greatly reduce the parasitmisa.
Recombinant Plasmodium Cynomolgi (p42 and p19) MSP1 Vaccination
Test in the Toque Macaque Macaca Sinica
[0129] Fifteen captured monkeys were used as follows: (1) 3 animals
injected with 100 .mu.g of soluble PcMSP1.sub.p42; (2) 3 animals
injected with 35 .mu.g (1.sup.st injection) or 50 .mu.g (2.sup.nd
and 3.sup.rd injections) of soluble PcMSP1.sub.p42; (3) 3 animals
injected with a mixture of PcMSP1.sub.p42 and p19; (4) 3 animals
injected with adjuvant plus PBS; (5) 3 animals not injected.
Complete and incomplete Freund adjuvant was used in the protocol
described above. Injections were intramuscular at 4 week intervals.
The challenge infection was made by injecting 2.times.10.sup.5 red
blood cells infected with Plasmodium cynomolgi 4 weeks after the
last injection. Protection was evaluated by determining parasitemia
daily in all animals by examining the parasitemia with Giemsa.
Parasitemia were classified as negative only after counting 400
smear fields. The parasitemia were expressed as a percentage of
parasitised red blood cells.
[0130] FIGS. 6A-6G show the results obtained. Each of them shows
parasitemia (expressed as the percentage of parasitised red blood
cells along the ordinate on a logarithmic scale) observed in the
challenge animals as a function of the time after infection (in
days along the abscissa).
[0131] The results relate to: [0132] in FIG. 6A; non vaccinated
control animals; [0133] FIG. 6B relates to animals which received a
saline solution also containing Freund adjuvant; [0134] FIG. 6C is
a superposition of FIGS. 6A and 6B, with the aim of highlighting
the relative results resulting from administration of Freund
adjuvant to the animals (the variations are clearly not
significant); [0135] FIG. 6D provides the results obtained at the
end of vaccination with p42; [0136] FIG. 6E concerns animals
vaccinated with p19 alone; [0137] finally, FIG. 6F concerns animals
vaccinated with a mixture of p19 and p42.
[0138] The p42 certainly induced a certain level of protection.
However, as shown in FIGS. 6E and 6F, the protection conferred by
the recombinant p19 of the invention was considerably better.
[0139] The hypothesis can be formulated that the improved
protection resulting from secondary cleavage of p42 which is
accompanied by revealing free cysteine which, as a result, forms
intermolecular bridges giving rise to p19 multimers which are
highly characteristic of this form in recombinant proteins of the
three species tested.
[0140] The numbers used to produce graphs (6A-6F) are given in FIG.
6G.
P. Cynomolgi Toque Macaque Vaccination Test; Second Challenge
Infection of Monkeys Vaccinated with p19 Alone and Controls (FIG.
8)
[0141] Six months later, with no other vaccination, the 3 macaques
which received the p19 MSP-1 alone with FCA/FIA (FIG. 6E) and the 3
macaques which received a saline solution containing Freund
adjuvant (FIG. 6B) and 2 new unaffected unvaccinated monkeys
underwent a new challenge infection by injecting 1.times.10.sup.6
red blood cells infected with Plasmodium cynomolgi. Protection was
evaluated by determining parasitemia daily in all animals by
examining Giemsa smears. The parasitemia were classified negative
only after counting 400 smear fields. The parasitemia were
expressed as the percentage of parasitised red blood cells (the
figures used to produce graphs 8A-C are given in FIG. 8D). The six
immunised animals which underwent challenge infection six months
earlier had no detectable parasitemia except for 1 animal in each
group which exhibited a parasitemia of 0.008% for 1 day (FIGS. 8A
and 8B). The two unaffected controls exhibited a conventional
parasitemia with a maximum of 0.8% and for 21 days (FIG. 8C). Thus
the 3 animals vaccinated with the MSP-1 p19 were also protected six
months later than the 3 controls which exhibited a complete
conventional infection after the first challenge infection, despite
the absence of or a very slight parasitemia after the first
challenge infection. These results suggest that the protection
period for p19 is at least six months.
Vaccination Test with p19 in Association with Alum in the P.
Cynomolgi Toque Macaque System (FIG. 9)
[0142] The previous positive protection results were obtained using
complete (FCA) or incomplete (FIA) Freund adjuvant. However, the
only adjuvant which is currently allowed in man is alum. For this
reason, we carried out a vaccination test with P. cynomolgi MSP-1
p19 in the toque macaque in the presence of alum as the adjuvant.
Six captured macaques were used as follows: (1) 3 animals injected
with 4 doses of 50 mg of recombinant P. cynomolgi MSP-1 p19 with 20
mg of alum; (2) 3 animals injected 4 times with physiological water
and 10 mg of alum. The injections were intramuscular at 4 week
intervals. The challenge infection was made by injecting
2.times.10.sup.5 red blood cells infected with P. cynomolgi 4 weeks
after the last injection. Protection was evaluated by daily
determination of parasitemia in all animals by examining Giemsa
smears. The parasitemia were classified negative only after
counting 400 smear fields. Parasitemia were expressed as the
percentage of parasitised red blood cells. The results of this
experiment were as follows. 2 of the 3 macaques immunised with
recombinant p19 with alum had about 30 times less total parasitemia
during the infection period (FIGS. 9A and 9B) than the 3 control
macaques immunised with physiological water and alum (FIG. 9D)
after the challenge infection. The third macaque immunised with p19
(FIG. 9C) was not very different from the controls. For the
vaccination test using Plasmodium cynomolgi p19 in the toque
macaque, macaca sinica, described in FIG. 9, the data used to
produce the graphs (9A-9D) are given in (FIG. 9E). While the
results are a little less spectacular than the preceding results
(FIGS. 6, 8), this is the first time that significant protection
has been observed for recombinant MSP-1 with alum.
[0143] FIG. 10: Vaccination Test with a Recombinant Plasmodium
falciparum p19 in the Squirrel Monkey
[0144] Twenty Saimiri sciureus guyanensis (squirrel monkeys) of
about 3 years old raised in captivity were used as follows: (1) 4
animals injected with 50 mg of soluble Pf MSP-1 p19 in the presence
of Freund adjuvant as follows: 1.sup.St injection: 1:1 FCA/FIA;
2.sup.nd injection: 1:4 FCA/FIA; 3.sup.rd injection: FIA. These
adjuvant compositions were then mixed with 1:1 antigen in PBS; (2)
2 control animals received Freund adjuvant as described for (1)
with only PBS; (3) 4 animals injected with 50 mg of soluble Pf
MSP-1 p19 in the presence of 10 mg of alum (Alu-Gel-S, Serva); (4)
2 control animals received 10 mg of alum with only PBS; (5) 4
animals injected with about 50-100 mg of GPI anchored Pf MSP-1 p19
reconstituted into liposomes as follows: 300 mmoles of cholesterol
and 300 mmoles of phosphatidyl choline were vacuum dried and
resuspended in 330 mM of N-octylglucoside in PBS with 1.4 mg of Pf
MSP-1 p19, GPI. This solution had been dialysed against PBS with
adsorbent Bio-Beads SM-2 (Bio-Rad) and the liposomes formed were
concentrated by centrifuging and resuspended in PBS The 1.sup.st
injection was made with fresh liposomes kept at 4.degree. C. and
the 2.sup.nd and 3.sup.rd injections were made with liposomes which
had been frozen for preservation; (6) 2 animals injected with
control liposomes made in the same way, in the absence of the p19,
GPI antigen as described for (5); (7) 2 animals injected with
physiological water. Three intramuscular injections were made at 4
week intervals. The challenge infection was made by injecting
1.times.10.sup.6 red blood cells infected with Plasmodium
falciparum. Protection was evaluated by determining parasitemia
daily in all animals by examining the Giemsa smears. Parasitemia
were expressed as the percentage of parasitised red blood cells.
The results of this vaccination test are shown in FIGS. 10,
A-G.
[0145] The groups immunised with p19 in Freund adjuvant or liposome
demonstrated similar parasitemia to the control groups after a
challenge infection (one animal (number 29) vaccinated with p19 in
Freund adjuvant died several days after challenge infection for
reasons independent of vaccination (cardiac arrest". Irregularities
in administration of the antigen in these 2 groups (poor Freund
emulsion, congealed liposomes) did not allow the significance of
these results to be completely evaluated. In the alum group, 2
animals showed total parasitemia for the duration of the infection
about 4 times less than the controls, 1 animal about 3 times less
and 1 animal was similar to the controls. This experiment was a
little difficult to interpret due to the variability in the
controls, probably due to the strain of parasite used for the
challenge infection which would not have been quite adapted to the
non splenectomised Saimiri model developed only recently in
Cayenne. However, the real effect with alum, although imperfect, is
encouraging in that our antigens seem to be the only recombinant P.
falciparum MSP-1 versions which currently have shown a certain
effectiveness in combination with alum.
Vaccination Test with a Recombinant Plasmodium falciparum p19 in
the Squirrel Monkey (Same Test as for FIG. 10)
[0146] Monkeys bred in captivity were injected intramuscularly with
1 ml of inoculum twice at 4 week intervals as follows: (1) 4
animals injected with 50 .mu.g of soluble PfMSP1p19 in the presence
of Freund adjuvant as follows: 1.sup.st injection: 1:1 FCA/FIA;
2.sup.nd injection: 1:4 FCA/FIA; and mixed then 1:1 with the
antigen in PBS; (2) 4 animals injected with 50 .mu.g of soluble
PfMSPp19 in the presence of 10 mg of alum; (3) 4 animals injected
with about 50 .mu.g of GPI anchored PfMSP1p19 reconstituted into
liposomes composed of 1:1 molar cholesterol and phosphatidyl
choline. The animals were bled 17 days after the second
injection.
[0147] Red cells from a squirrel monkey with 30% parasitemia due to
P. falciparum (with the mature forms in the majority) were washed
with PBS and the residue was diluted 8 times in the presence of 2%
SDS and 2% dithiothreitol and heated to 95.degree. before being
charged onto a polyacrylamide gel of 7.5% (separation gel) and 4%
(stacking gel). After transfer to nitrocellulose, immunoblot
analysis was carried out with antisera as follows: (1) pool of
antisera of 4 monkeys vaccinated with soluble PfMSP1p19 in Freund
adjuvant, twentieth dilution; (2) pool of antisera of 4 monkeys
vaccinated with soluble PfMSP1p19 in alum adjuvant, twentieth
dilution; (3) pool of antisera of 4 monkeys vaccinated with
anchored PfMSP1p19 in liposomes, twentieth dilution; (4) monoclonal
antibody, which reacts with a linear epitope of PfMSP1p19, 50
mg/ml; (5) SHI90 antisera pool originating from about twenty
monkeys repeatedly infected with P. falciparum and which had become
unaffectable by any subsequent infection with P. falciparum, five
hundredth dilution; (6) antiserum pool of unaffected monkeys (never
exposed to P. falciparum), twentieth dilution.
[0148] The results show that the 3 antiserum pools of monkeys
vaccinated with PfMSP1p19 reacted strongly and specifically with
very high molecular weight complexes (diffuse in the stacking gel)
and present in parasite extracts containing more mature forms.
These results support the hypothesis that a specific aggregate of
PfMSP1p19 is present in vivo comprising epitopes which are
reproduced in recombinant PfMSP1p19 molecules synthesised in the
baculovirus system, in particular oligomeric forms thereof.
[0149] FIG. 7 also illustrated these results. It shows immunoblots
produced on gel. The first three gel tracks illustrate the in vivo
response of monkeys to injections of p19 [(1) with Freund adjuvant,
(2) with alum, (3) in the form of a liposome] and in particular the
existence of high molecular weight complexes supporting the
hypothesis of in vivo aggregation of p19 in the form of an
oligomer, specific to the maturation stage (when p42 is cut into
p19 and p33).
[0150] This vaccination test also comprises a third injection
identical to the previous injections. The injection with Freund
adjuvant contained only FIA.
[0151] There were two animal controls for each group, namely: 2
control animals injected with PBS and Freund adjuvant; 2 control
animals injected with PBS and alum; 2 control animals injected with
liposomes without protein; and two control animals injected with
PBS without adjuvant. Protection was evaluated as described
above.
[0152] FIG. 7B: The data for this Figure were derived from the
squirrel monkey P. falciparum I vaccination test (FIG. 10 below).
The numbers correspond to the individual monkeys noted in FIG. 10.
The techniques and methods for this Figure were the same as for
FIG. 7 except that the individual antiserum for each monkey was
tested after three injections the day of the proof injection and
the SHI antiserum was diluted by 1:250. The results show that the
antiserum for 4 monkeys vaccinated with p19 and alum reacted
strongly and specifically with very high molecular weight complexes
while the monkeys of other groups vaccinated with p19 and Freund
adjuvant or liposomes showed only a little reactivity with these
complexes. Since the monkeys vaccinated with p19 and alum were also
the best protected, this reactivity with the high molecular weight
complexes appeared to indicate a protective effect, despite one
monkey in the group not being protected with respect to the
controls and that another was only partially protected.
[0153] The invention also, of course, concerns other applications,
for example those described below with respect to certain of the
examples, although these are not limiting in character.
[0154] Therapy
[0155] The recombinant molecule PfMSP1p19 can be used to produce
specific antibodies which can possibly be used by passive transfer
for therapeutics for severe malaria due to P. falciparum when there
is a risk of death.
[0156] Diagnostics
[0157] The recombinant molecules PvMSP1p42, PvMSP1p19 and PfMSPp19
derived from baculovirus can and have been used to produce specific
murine monoclonal antibodies. These antibodies, in combination with
polyclonal anti-MSP1p19 antisera originating from another species
such as the rabbit or goat can form the basis of a
semi-quantitative diagnostic test for malaria which can distinguish
between malaria due to P. falciparum, which can be fatal, and
malaria due to P. vivax, which is generally not fatal. The
principle of this test is to trap and quantify any MSP-1 molecule
containing the p19 portion in the blood.
[0158] In this context, the advantages of the MSP1p19 molecule are
as follows:
[0159] (i) it is both extremely well conserved in the same species
and sufficiently divergent between different species to enable
specific species reactants to be produced. No cross reaction has
been observed between antibodies derived from PfMSP1p19 and
PvMSP1p19;
[0160] (ii) the function of MSP1p19, while not known with
precision, seems to be sufficiently important that this molecule
does not vary significantly or is deleted without lethal effect for
the parasite;
[0161] (iii) it is a major antigen found in all merozoites and thus
it must in principle be detectable even at low parasitemia and
proportionally to the parasitemia;
[0162] (iv) since the recombinant MSP1p19 molecules derived from
baculovirus appear to reproduce more of the native structure of
MSP1P19, the antibodies produced against these proteins will be
well adapted to diagnostic use.
[0163] The microorganisms identified below have been deposited
under Rule 6.1 of the Treaty of Budapest of 1 Feb. 1996, in the
Collection Nationale de Cultures de Microorganisms (C.N.C.M.) of
Institut Pasteur at 28, rv du Dr. Roux 75724, Paris Cedex 15, under
the following registration numbers:
TABLE-US-00001 Identification Date of Registration reference
Deposit numbers PvMSP1p19A Feb. 1, 1996 I-1659 PvMSP1p19S Feb. 1,
1996 I-1660 PfMSP1p19A Feb. 1, 1996 I-1661 PfMSP1p19S Feb. 1, 1996
I-1662 PcMSP1p19S Feb. 1, 1996 I-1663 G17-12 Hybridoma Feb. 14,
1997 I-1846
[0164] The invention also concerns the use of these antibodies,
preferably fixed to a solid support (for example for affinity
chromatography) for the purification of type p19 peptides initially
contained in a mixture.
[0165] Purification means bringing this mixture into contact with
an antibody, dissociating the antigen-antibody complex and
recovering the purified p19 type peptide.
[0166] The invention also concerns vaccine compositions, also
comprising mixtures of proteins or fragment, in particular mixtures
of the type: [0167] P. falciparum p19 and P. vivax p19; [0168] P.
falciparum p19 and P. falciparum p42, the latter if necessary being
deprived of its most hypervariable regions; [0169] P. vivax p19 and
P. vivax p42, the latter if necessary being deprived of its most
hypervariable regions; [0170] P. falciparum p19 and P. falciparum
p42, the latter if necessary being deprived of its most
hypervariable regions, and P. vivax p19 and P. vivax p42, the
latter if necessary being deprived of its most hypervariable
regions.
[0171] In the present case, the most hypervariable regions are
defined as regions I or region II and all or part of region III,
the portion of region III which is preferably deleted being that
which is juxtaposed to region II (the conserved portion being
located to the side of the C-terminal of p33, close to the p19).
Regions II and III are illustrated in FIG. 4.
[0172] The invention is not limited to the production of human
vaccine. It is also applicable to the production of veterinary
vaccine compositions using the corresponding proteins or antigens
derived from parasites which are infectious for mammals and
products under the same conditions. It is known that infections of
the same type, babesiosis, also appear in cattle, dogs and horses.
One of the antigens of the Babesia species has a high
conformational homology (in particular in the two EFG-like and
cysteine-rich domains) and functional homology with a protein
portion of MSP-1 [(36), (37) and (38)].
[0173] Examples of veterinary vaccines using a soluble antigen
against such parasites have been described (39).
[0174] It goes without saying that the p19s used in these mixtures
can also be modified as described in the foregoing when considered
in isolation.
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[0216] The invention also concerns hybridomas secreting specific
antibodies selectively recognising the p19 of a MSP-1 protein in
the merozoite form of a Plasmodium type parasite which is
infectious for man other than Plasmodium vivax and which does not
recognise Plasmodium vivax.
[0217] In particular, these hybridomas secrete monoclonal
antibodies which do not recognise Plasmodium vivax and which
specifically recognise Plasmodium falciparum p19.
[0218] The invention also concerns a hybridoma characterized in
that it produces a specific antibody which specifically recognises
the p19 of P. vivax and the p19 of P. cynomolgi. A F10-3 hybridoma
has been constructed from the X63 Ag8 653 myeloma producing IgG
2b/k recognising the p42 glycoprotein of Plasmodium vivax.
Sequence CWU 1
1
151291DNAArtificial SequenceSYNTHETIC 1gaa ttc aac atc tcg cag cac
caa tgc gtg aaa aaa caa tgt ccc gag 48Glu Phe Asn Ile Ser Gln His
Gln Cys Val Lys Lys Gln Cys Pro Glu1 5 10 15aac tct ggc tgt ttc aga
cac ttg gac gag aga gag gag tgt aaa tgt 96Asn Ser Gly Cys Phe Arg
His Leu Asp Glu Arg Glu Glu Cys Lys Cys 20 25 30ctg ctg aac tac aaa
cag gag ggc gac aag tgc gtg gag aac ccc aac 144Leu Leu Asn Tyr Lys
Gln Glu Gly Asp Lys Cys Val Glu Asn Pro Asn 35 40 45ccg acc tgt aac
gag aac aac ggc ggc tgt gac gca gac gcc aaa tgc 192Pro Thr Cys Asn
Glu Asn Asn Gly Gly Cys Asp Ala Asp Ala Lys Cys 50 55 60acc gag gag
gac tcg ggc agc aac ggc aag aaa atc acg tgt gag tgt 240Thr Glu Glu
Asp Ser Gly Ser Asn Gly Lys Lys Ile Thr Cys Glu Cys65 70 75 80acc
aaa ccc gac tcg tac ccg ctg ttc gac ggc atc ttc tgc agc taa 288Thr
Lys Pro Asp Ser Tyr Pro Leu Phe Asp Gly Ile Phe Cys Ser 85 90 95taa
291295PRTArtificial SequenceSYNTHETIC 2Glu Phe Asn Ile Ser Gln His
Gln Cys Val Lys Lys Gln Cys Pro Glu1 5 10 15Asn Ser Gly Cys Phe Arg
His Leu Asp Glu Arg Glu Glu Cys Lys Cys 20 25 30Leu Leu Asn Tyr Lys
Gln Glu Gly Asp Lys Cys Val Glu Asn Pro Asn 35 40 45Pro Thr Cys Asn
Glu Asn Asn Gly Gly Cys Asp Ala Asp Ala Lys Cys 50 55 60Thr Glu Glu
Asp Ser Gly Ser Asn Gly Lys Lys Ile Thr Cys Glu Cys65 70 75 80Thr
Lys Pro Asp Ser Tyr Pro Leu Phe Asp Gly Ile Phe Cys Ser 85 90
953279DNAPlasmodium falciparum 3aacatttcac aacaccaatg cgtaaaaaaa
caatgtccag aaaattctgg atgtttcaga 60catttagatg aaagagaaga atgtaaatgt
ttattaaatt acaaacaaga aggtgataaa 120tgtgttgaaa atccaaatcc
tacttgtaac gaaaataatg gtggatgtga tgcagatgcc 180aaatgtaccg
aagaagattc aggtagcaac ggaaagaaaa tcacatgtga atgtactaaa
240cctgattctt atccactttt cgatggtatt ttctgcagt 2794354DNAArtificial
SequenceSYNTHETIC 4gaa ttc aac atc tcg cag cac caa tgc gtg aaa aaa
caa tgt ccc gag 48Glu Phe Asn Ile Ser Gln His Gln Cys Val Lys Lys
Gln Cys Pro Glu1 5 10 15aac tct ggc tgt ttc aga cac ttg gac gag aga
gag gag tgt aaa tgt 96Asn Ser Gly Cys Phe Arg His Leu Asp Glu Arg
Glu Glu Cys Lys Cys 20 25 30ctg ctg aac tac aaa cag gag ggc gac aag
tgc gtg gag aac ccc aac 144Leu Leu Asn Tyr Lys Gln Glu Gly Asp Lys
Cys Val Glu Asn Pro Asn 35 40 45ccg acc tgt aac gag aac aac ggc ggc
tgt gac gca gac gcc aaa tgc 192Pro Thr Cys Asn Glu Asn Asn Gly Gly
Cys Asp Ala Asp Ala Lys Cys 50 55 60acc gag gag gac tcg ggc agc aac
ggc aag aaa atc acg tgt gag tgt 240Thr Glu Glu Asp Ser Gly Ser Asn
Gly Lys Lys Ile Thr Cys Glu Cys65 70 75 80acc aaa ccc gac tcg tac
ccg ctg ttc gac ggc atc ttc tgc agc tcc 288Thr Lys Pro Asp Ser Tyr
Pro Leu Phe Asp Gly Ile Phe Cys Ser Ser 85 90 95tct aac ttc ttg ggc
atc tcg ttc ttg ttg atc ctc atg ttg atc ttg 336Ser Asn Phe Leu Gly
Ile Ser Phe Leu Leu Ile Leu Met Leu Ile Leu 100 105 110tac agc ttc
att taa taa 354Tyr Ser Phe Ile 1155116PRTArtificial
SequenceSYNTHETIC 5Glu Phe Asn Ile Ser Gln His Gln Cys Val Lys Lys
Gln Cys Pro Glu1 5 10 15Asn Ser Gly Cys Phe Arg His Leu Asp Glu Arg
Glu Glu Cys Lys Cys 20 25 30Leu Leu Asn Tyr Lys Gln Glu Gly Asp Lys
Cys Val Glu Asn Pro Asn 35 40 45Pro Thr Cys Asn Glu Asn Asn Gly Gly
Cys Asp Ala Asp Ala Lys Cys 50 55 60Thr Glu Glu Asp Ser Gly Ser Asn
Gly Lys Lys Ile Thr Cys Glu Cys65 70 75 80Thr Lys Pro Asp Ser Tyr
Pro Leu Phe Asp Gly Ile Phe Cys Ser Ser 85 90 95Ser Asn Phe Leu Gly
Ile Ser Phe Leu Leu Ile Leu Met Leu Ile Leu 100 105 110Tyr Ser Phe
Ile 1156342DNAPlasmodium falciparum 6aacatttcac aacaccaatg
cgtaaaaaaa caatgtccag aaaattctgg atgtttcaga 60catttagatg aaagagaaga
atgtaaatgt ttattaaatt acaaacaaga aggtgataaa 120tgtgttgaaa
atccaaatcc tacttgtaac gaaaataatg gtggatgtga tgcagatgcc
180aaatgtaccg aagaagattc aggtagcaac ggaaagaaaa tcacatgtga
atgtactaaa 240cctgattctt atccactttt cgatggtatt ttctgcagtt
cctctaactt cttaggaata 300tcattcttat taatactcat gttaatatta
tacagtttca tt 3427387DNAPlasmodium falciparumCDS(1)..(387) 7atg aag
gcg cta ctc ttt ttg ttc tct ttc att ttt ttc gtt acc aaa 48Met Lys
Ala Leu Leu Phe Leu Phe Ser Phe Ile Phe Phe Val Thr Lys1 5 10 15gaa
ttc aac atc tcg cag cac caa tgc gtg aaa aaa caa tgt ccc gag 96Glu
Phe Asn Ile Ser Gln His Gln Cys Val Lys Lys Gln Cys Pro Glu 20 25
30gaa ttc aac atc tcg cag cac caa tgc gtg aaa aaa caa tgt ccc gag
144Glu Phe Asn Ile Ser Gln His Gln Cys Val Lys Lys Gln Cys Pro Glu
35 40 45aac tct ggc tgt ttc aga cac ttg gac gag aga gag gag tgt aaa
tgt 192Asn Ser Gly Cys Phe Arg His Leu Asp Glu Arg Glu Glu Cys Lys
Cys 50 55 60ctg ctg aac tac aaa cag gag ggc gac aag tgc gtg gag aac
ccc aac 240Leu Leu Asn Tyr Lys Gln Glu Gly Asp Lys Cys Val Glu Asn
Pro Asn65 70 75 80ccg acc tgt aac gag aac aac ggc ggc tgt gac gca
gac gcc aaa tgc 288Pro Thr Cys Asn Glu Asn Asn Gly Gly Cys Asp Ala
Asp Ala Lys Cys 85 90 95acc gag gag gac tcg ggc agc aac ggc aag aaa
atc acg tgt gag tgt 336Thr Glu Glu Asp Ser Gly Ser Asn Gly Lys Lys
Ile Thr Cys Glu Cys 100 105 110acc aaa ccc gac tcg tac ccg ctg ttc
gac ggc atc ttc tgc agc taa 384Thr Lys Pro Asp Ser Tyr Pro Leu Phe
Asp Gly Ile Phe Cys Ser 115 120 125taa 3878127PRTPlasmodium
falciparum 8Met Lys Ala Leu Leu Phe Leu Phe Ser Phe Ile Phe Phe Val
Thr Lys1 5 10 15Glu Phe Asn Ile Ser Gln His Gln Cys Val Lys Lys Gln
Cys Pro Glu 20 25 30Glu Phe Asn Ile Ser Gln His Gln Cys Val Lys Lys
Gln Cys Pro Glu 35 40 45Asn Ser Gly Cys Phe Arg His Leu Asp Glu Arg
Glu Glu Cys Lys Cys 50 55 60Leu Leu Asn Tyr Lys Gln Glu Gly Asp Lys
Cys Val Glu Asn Pro Asn65 70 75 80Pro Thr Cys Asn Glu Asn Asn Gly
Gly Cys Asp Ala Asp Ala Lys Cys 85 90 95Thr Glu Glu Asp Ser Gly Ser
Asn Gly Lys Lys Ile Thr Cys Glu Cys 100 105 110Thr Lys Pro Asp Ser
Tyr Pro Leu Phe Asp Gly Ile Phe Cys Ser 115 120
1259330DNAPlasmodium falciparumCDS(1)..(330) 9gaa aca gaa agt tat
aag cag ctt gta gcc aac gtg gac gaa ttc aac 48Glu Thr Glu Ser Tyr
Lys Gln Leu Val Ala Asn Val Asp Glu Phe Asn1 5 10 15atc tcg cag cac
caa tgc gtg aaa aaa caa tgt ccc gag aac tct ggc 96Ile Ser Gln His
Gln Cys Val Lys Lys Gln Cys Pro Glu Asn Ser Gly 20 25 30tgt ttc aga
cac ttg gac gag aga gag gag tgt aaa tgt ctg ctg aac 144Cys Phe Arg
His Leu Asp Glu Arg Glu Glu Cys Lys Cys Leu Leu Asn 35 40 45tac aaa
cag gag ggc gac aag tgc gtg gag aac ccc aac ccg acc tgt 192Tyr Lys
Gln Glu Gly Asp Lys Cys Val Glu Asn Pro Asn Pro Thr Cys 50 55 60aac
gag aac aac ggc ggc tgt gac gca gac gcc aaa tgc acc gag gag 240Asn
Glu Asn Asn Gly Gly Cys Asp Ala Asp Ala Lys Cys Thr Glu Glu65 70 75
80gac tcg ggc agc aac ggc aag aaa atc acg tgt gag tgt acc aaa ccc
288Asp Ser Gly Ser Asn Gly Lys Lys Ile Thr Cys Glu Cys Thr Lys Pro
85 90 95gac tcg tac ccg ctg ttc gac ggc atc ttc tgc agc taa taa
330Asp Ser Tyr Pro Leu Phe Asp Gly Ile Phe Cys Ser 100
10510108PRTPlasmodium falciparum 10Glu Thr Glu Ser Tyr Lys Gln Leu
Val Ala Asn Val Asp Glu Phe Asn1 5 10 15Ile Ser Gln His Gln Cys Val
Lys Lys Gln Cys Pro Glu Asn Ser Gly 20 25 30Cys Phe Arg His Leu Asp
Glu Arg Glu Glu Cys Lys Cys Leu Leu Asn 35 40 45Tyr Lys Gln Glu Gly
Asp Lys Cys Val Glu Asn Pro Asn Pro Thr Cys 50 55 60Asn Glu Asn Asn
Gly Gly Cys Asp Ala Asp Ala Lys Cys Thr Glu Glu65 70 75 80Asp Ser
Gly Ser Asn Gly Lys Lys Ile Thr Cys Glu Cys Thr Lys Pro 85 90 95Asp
Ser Tyr Pro Leu Phe Asp Gly Ile Phe Cys Ser 100
10511379PRTPlasmodium cynomolgi 11Asp Gln Val Thr Thr Gly Glu Ala
Glu Ser Glu Ala Pro Glu Ile Ile1 5 10 15Val Pro Gln Gly Ile Asn Glu
Tyr Asp Val Val Tyr Ile Lys Pro Leu 20 25 30Ala Gly Met Tyr Lys Thr
Ile Lys Lys Pro Leu Glu Asn His Val Asn 35 40 45Ala Leu Asn Thr Asn
Ile Ile Asp Met Leu Asp Ser Arg Leu Lys Lys 50 55 60Arg Asn Tyr Phe
Leu Asp Val Leu Asn Ser Asp Leu Asn Pro Tyr Ser65 70 75 80Ile Pro
His Ser Gly Glu Tyr Ile Ile Lys Asp Pro Tyr Lys Leu Leu 85 90 95Asp
Leu Glu Lys Lys Lys Leu Leu Gly Ser Tyr Lys Tyr Ile Gly Ala 100 105
110Ser Val Asp Lys Asp Met Val Thr Ala Asn Asp Gly Leu Ala Tyr Tyr
115 120 125Gln Lys Met Gly Asp Leu Tyr Lys Lys His Leu Asp Glu Val
Asn Ala 130 135 140Cys Ile Lys Glu Val Glu Ala Asn Ile Asn Lys His
Asp Glu Glu Ile145 150 155 160Lys Lys Ile Gly Ser Glu Ala Ser Lys
Ala Asn Asp Lys Asn Gln Leu 165 170 175Asn Ala Lys Lys Glu Glu Leu
Gln Lys Tyr Leu Pro Phe Leu Ser Ser 180 185 190Ile Gln Lys Glu Tyr
Ser Thr Leu Val Asn Lys Val His Ser Tyr Thr 195 200 205Asp Thr Leu
Lys Lys Ile Ile Asn Asn Cys Gln Ile Glu Lys Lys Glu 210 215 220Thr
Glu Thr Ile Val Asn Lys Leu Glu Asp Tyr Ser Lys Met Asp Glu225 230
235 240Glu Leu Asp Val Tyr Lys Gln Ser Lys Lys Glu Asp Asp Val Lys
Ser 245 250 255Ser Gly Leu Leu Glu Lys Leu Met Asn Ser Lys Leu Ile
Asn Gln Glu 260 265 270Glu Ser Lys Lys Ala Leu Ser Glu Leu Leu Asn
Val Gln Thr Gln Met 275 280 285Leu Asn Met Ser Ser Glu His Arg Cys
Ile Asp Thr Asn Val Pro Glu 290 295 300Asn Ala Ala Cys Tyr Arg Tyr
Leu Asp Gly Thr Glu Glu Trp Arg Cys305 310 315 320Leu Leu Tyr Phe
Lys Glu Asp Ala Gly Lys Cys Val Pro Ala Pro Asn 325 330 335Met Thr
Cys Lys Asp Lys Asn Gly Gly Cys Ala Pro Glu Ala Glu Cys 340 345
350Lys Met Asn Asp Lys Asn Glu Ile Val Cys Lys Cys Thr Lys Glu Gly
355 360 365Ser Glu Pro Leu Phe Glu Gly Val Phe Cys Ser 370
37512380PRTPlasmodium vivax-like sp. 12Asp Gln Val Thr Thr Gly Glu
Ala Glu Ser Glu Ala Pro Glu Ile Leu1 5 10 15Val Pro Ala Gly Ile Ser
Asp Tyr Asp Val Val Tyr Leu Lys Pro Leu 20 25 30Ala Gly Met Tyr Lys
Thr Ile Lys Lys Gln Leu Glu Asn His Val Asn 35 40 45Ala Phe Asn Thr
Asn Ile Thr Asp Met Leu Asp Ser Arg Leu Lys Lys 50 55 60Arg Asn Tyr
Phe Leu Glu Val Leu Asn Ser Asp Leu Asn Pro Phe Lys65 70 75 80Tyr
Ser Pro Ser Gly Glu Tyr Ile Ile Lys Asp Pro Tyr Lys Leu Leu 85 90
95Asp Leu Glu Lys Lys Lys Lys Leu Leu Gly Ser Tyr Lys Tyr Ile Gly
100 105 110Ala Ser Ile Asp Lys Asp Leu Ala Thr Ala Asn Asp Gly Val
Thr Tyr 115 120 125Tyr Asn Lys Met Gly Glu Leu Tyr Lys Thr His Leu
Thr Ala Val Asn 130 135 140Glu Glu Val Lys Lys Val Glu Ala Asp Ile
Lys Ala Glu Asp Asp Lys145 150 155 160Ile Lys Lys Ile Gly Ser Asp
Ser Thr Lys Thr Thr Glu Lys Thr Gln 165 170 175Ser Met Ala Lys Lys
Ala Glu Leu Glu Lys Tyr Leu Pro Phe Leu Asn 180 185 190Ser Leu Gln
Lys Glu Tyr Glu Ser Leu Val Ser Lys Val Asn Thr Tyr 195 200 205Thr
Asp Asn Leu Lys Lys Val Ile Asn Asn Cys Gln Leu Glu Lys Lys 210 215
220Glu Ala Glu Ile Thr Val Lys Lys Leu Gln Asp Tyr Asn Lys Met
Asp225 230 235 240Glu Lys Leu Glu Glu Tyr Lys Lys Ser Glu Lys Lys
Asn Glu Val Lys 245 250 255Ser Ser Gly Leu Leu Glu Lys Leu Met Lys
Ser Lys Leu Ile Lys Glu 260 265 270Asn Glu Ser Lys Glu Ile Leu Ser
Gln Leu Leu Asn Val Gln Thr Gln 275 280 285Leu Leu Thr Met Ser Ser
Glu His Thr Cys Ile Asp Thr Asn Val Pro 290 295 300Asp Asn Ala Ala
Cys Tyr Arg Tyr Leu Asp Gly Thr Glu Glu Trp Arg305 310 315 320Cys
Leu Leu Thr Phe Lys Glu Glu Gly Gly Lys Cys Val Pro Ala Ser 325 330
335Asn Val Thr Cys Lys Asp Asn Asn Gly Gly Cys Ala Pro Glu Ala Glu
340 345 350Cys Lys Met Thr Asp Ser Asn Lys Ile Val Cys Lys Cys Thr
Lys Glu 355 360 365Gly Ser Glu Pro Leu Phe Glu Gly Val Phe Cys Ser
370 375 38013380PRTPlasmodium vivax-like sp. 13Asp Gln Val Thr Thr
Gly Glu Ala Glu Ser Glu Ala Pro Glu Ile Leu1 5 10 15Val Pro Ala Gly
Ile Ser Asp Tyr Asp Val Val Tyr Leu Lys Pro Leu 20 25 30Ala Gly Met
Tyr Lys Thr Ile Lys Lys Gln Leu Glu Asn His Val Asn 35 40 45Ala Phe
Asn Thr Asn Ile Thr Asp Met Leu Asp Ser Arg Leu Lys Lys 50 55 60Arg
Asn Tyr Phe Leu Glu Val Leu Asn Ser Asp Leu Asn Pro Phe Lys65 70 75
80Tyr Ser Ser Ser Gly Glu Tyr Ile Ile Lys Asp Pro Tyr Lys Leu Leu
85 90 95Asp Leu Glu Lys Lys Lys Lys Leu Ile Gly Ser Tyr Lys Tyr Ile
Gly 100 105 110Ala Ser Ile Asp Met Asp Leu Ala Thr Ala Asn Asp Gly
Val Thr Tyr 115 120 125Tyr Asn Lys Met Gly Glu Leu Tyr Lys Thr His
Leu Asp Gly Val Lys 130 135 140Thr Glu Ile Lys Lys Val Glu Asp Asp
Ile Lys Lys Gln Asp Glu Glu145 150 155 160Leu Lys Lys Leu Gly Asn
Val Asn Ser Gln Asp Ser Lys Lys Asn Glu 165 170 175Phe Ile Ala Lys
Lys Ala Glu Leu Glu Lys Tyr Leu Pro Phe Leu Asn 180 185 190Ser Leu
Gln Lys Glu Tyr Glu Ser Leu Val Ser Lys Val Asn Thr Tyr 195 200
205Thr Asp Asn Leu Lys Lys Val Ile Asn Asn Cys Gln Leu Glu Lys Lys
210 215 220Glu Ala Glu Ile Thr Val Lys Lys Leu Gln Asp Tyr Asn Lys
Met Asp225 230 235 240Glu Lys Leu Glu Glu Tyr Lys Lys Ser Glu Lys
Lys Asn Glu Val Lys 245 250 255Ser Ser Gly Leu Leu Glu Lys Leu Met
Lys Ser Lys Leu Ile Lys Glu 260 265 270Asn Glu Ser Lys Glu Ile Leu
Ser Gln Leu Leu Asn Val Gln Thr Gln 275 280 285Leu Leu Thr Met Ser
Ser Glu His Thr Cys Ile Asp Thr Asn Val Pro 290 295 300Asp Asn Ala
Ala Cys Tyr Arg Tyr Leu Asp Gly Thr Glu Glu Trp Arg305 310 315
320Cys Leu Leu Thr Phe Lys Glu Glu Gly Gly Lys Cys Val Pro Ala Ser
325 330 335Asn Val Thr Cys Lys Asp Asn Asn Gly Gly Cys Ala Pro Glu
Ala Glu 340 345
350Cys Lys Met Thr Asp Ser Asn Lys Ile Val Cys Lys Cys Thr Lys Glu
355 360 365Gly Ser Glu Pro Leu Phe Glu Gly Val Phe Cys Ser 370 375
38014281PRTArtificial SequenceSYNTHETIC 14Asp Gln Val Thr Thr Gly
Glu Ala Glu Ser Glu Ala Pro Glu Ile Val1 5 10 15Pro Gly Ile Tyr Asp
Val Val Tyr Lys Pro Leu Ala Gly Met Tyr Lys 20 25 30Thr Ile Lys Lys
Leu Glu Asn His Val Asn Ala Asn Thr Asn Ile Asp 35 40 45Met Leu Asp
Ser Ala Leu Lys Lys Ala Asn Tyr Phe Leu Val Leu Asn 50 55 60Ser Asp
Leu Asn Pro Ser Gly Glu Tyr Ile Ile Lys Asp Pro Tyr Lys65 70 75
80Leu Leu Asp Leu Glu Lys Lys Lys Leu Gly Ser Tyr Lys Tyr Ile Gly
85 90 95Ala Ser Asp Asp Thr Ala Asn Asp Gly Tyr Tyr Lys Met Gly Leu
Tyr 100 105 110Lys His Leu Val Lys Val Glu Ile Asp Lys Lys Gly Lys
Ala Lys Lys 115 120 125Glu Leu Lys Tyr Leu Pro Phe Leu Ser Gln Lys
Glu Tyr Leu Val Lys 130 135 140Val Tyr Thr Asp Leu Lys Lys Ile Asn
Asn Cys Gln Glu Lys Lys Glu145 150 155 160Glu Val Lys Leu Asp Tyr
Lys Met Asp Glu Leu Tyr Lys Ser Lys Val 165 170 175Lys Ser Ser Gly
Leu Leu Glu Lys Leu Met Ser Lys Leu Ile Glu Ser 180 185 190Lys Leu
Ser Leu Leu Asn Val Gln Thr Gln Leu Met Ser Ser Glu His 195 200
205Cys Ile Asp Thr Asn Val Pro Asn Ala Ala Cys Tyr Arg Tyr Leu Asp
210 215 220Gly Thr Glu Glu Trp Arg Cys Leu Leu Phe Lys Glu Gly Lys
Cys Val225 230 235 240Pro Ala Asn Thr Cys Lys Asp Asn Gly Gly Cys
Ala Pro Glu Ala Glu 245 250 255Cys Lys Met Asp Asn Ile Val Cys Lys
Cys Thr Lys Glu Gly Ser Glu 260 265 270Pro Leu Phe Glu Gly Val Phe
Cys Ser 275 280156PRTARTIFICIAL SEQUENCESYNTHETIC 15Leu Asn Val Gln
Thr Gln1 5
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