JPH0222300A - Immunogen conjugate, its production and generation of immune - Google Patents

Abstract

PURPOSE: To obtain a new titled conjugant being useful as a vaccine for malaria by covalent-bonding a carrier protein with a peptide forming an antigenic determinant of a circumsporosite protein through a spacer.

CONSTITUTION: An immunogenic conjugant is obtd. by covalent-bonding a carrier protein (pref. diphtheria toxin) with a peptide forming an antigenic determinant of a circumsporosoite protein through a spacer molecule (pref. adipic acid dihydrazide, etc.). In addition, as the above described peptide, there are a peptide with an amino acid arrangement of formula (wherein n is 1-50), etc., and they are obtd. ordinarily by synthesizing them in a solid phase resin system. in addition, for the above described covalent bonding, as a binding agent, 1- ethyl-3-(dimethylaminopropyl)carbodiimide is pref. used.

COPYRIGHT: (C)1990,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to the production of immunogenic conjugates and methods for their production. In particular, the invention relates to a conjugate vaccine characterized by a sporozoite surface coat protein linked via at least one spacer molecule to a carrier protein suitable for human vaccines.

BACKGROUND OF THE INVENTION Malaria, a debilitating and often fatal disease, is caused by infection with parasites of the Plamodium layer. Despite the strenuous efforts of national and international health organizations, malaria remains the number one cause of morbidity and mortality in tropical and subtropical regions of the world. In the 1982 table, 54 of the world's population
% of people live in areas where malaria is endemic and are therefore at risk of contracting the disease. It is estimated that between 2,500 and 30 million new cases of malaria are reported each year worldwide. In Africa alone, one million people die from malaria every year. These numbers have remained roughly constant over the past decade.

The etiological agent of human malaria is Plasmodium falciparum (P.
falcipanum, causing about 80 cases worldwide) and Plasmodium vivax (P, V
ivax). The developmental cycle of the malaria parasite is complex, undergoing several stages of differentiation in two hosts: mosquito and human.

The stage that infects humans is called sporozoite 9 and is transmitted to humans through mosquito bites. Sporozoites exist in the bloodstream for a short time before entering other cells. The first infection is thought to be a liver infection. After differentiation, this organ infects red blood cells, where it persists as an intracellular protozoan. When present in this form, the malaria parasite is isolated from the immune system and can mount only a weak, non-protective antibody response.

In principle, efforts aimed at eliminating malaria are directed at three targets: 1) eradication of the mosquito vector;
) treatment or control of infected persons; and 111) immunization of people at risk. Insecticides are the first promising option for control of the Anopheles mosquito vector.

However, the increased resistance to these 7rJ4 drugs, the harmful environmental impact, the cost of spraying, and the inaccessibility of thin areas, have significant limitations on the ability to treat malaria with insecticides.

Attempts to treat or control malaria using various antimalarial drugs have recently been hampered by the emergence of drug-resistant strains.

Resistance to chloroquine, the drug of choice, has spread widely, and a second drug using sulfonamide pyrimethamine has spread.
The same is true for the regimen. Recently, a new antimalarial drug, melfoquin, was introduced. There are also limitations to the use of melphoxy, as susceptibility to melfoxins is inconsistent among P. falciparum parasites in different geographic regions.

The first attempts to elicit an active immune response against malaria were made in the 1940s, when immunization with inactivated sporozoites from Plasmodium falciparum was shown to confer protection against avian malaria. Subsequent immunization of mice, monkeys, and humans with inactivated sporozoites was shown to confer protection against attack by the sporozoite stage of the parasite. The sporozoite immunogen used in the above study was mature sporozoites obtained from mosquito salivary glands. Although these studies have shown that anti-sporozoite antibodies are capable of conferring protection against malaria, the use of inactivated sporozoites as immunogens is impractical given the following: 1) Salivary gland 11) Sporozoites cannot be cultured in synthetic or semi-synthetic media;
and 111) High titers of anti-sporozoite antibodies can only be elicited by repeated intravenous immunizations or by the use of potent adipants, both methods being unsuitable as methods of human vaccine administration.

Currently, the expression of protective antigens by sporozoites is based on the sporozoite surface coat protein (csp), which is localized on the surface of the protozoan.
), J, F.

Young et al. Science, 228, 958 (1985). The gene containing the synthesis of C3P in Plasmodium falciparum has been cloned and sequenced. C8P is 41 TetraR
petido (composed of repeating units; 37 (ASN-
ALA-ASN-PRO) and four (ASN-PRO) flanked by two regions shared by P. falciparum and P.
VAL-ASP-PRO). Antibodies against the C3P(7)2 horn shared saleta region have been shown to have no protective effect. On the contrary, ASN-ALA-ASN-PR
Antibodies against the O region have been found to react with live sporozoites and protect them from entering liver cells, W. R. Barrow et al. Science 228, 996 (1).
985).

Based on these discoveries, malaria vaccine development efforts have focused around the production of antigens capable of eliciting antibody responses that recognize the ASN-ALA-ASN-PRO region of C3P. The gene segment encoding (ASN-VAL-ASP-PRO) in addition to 15 (ASN-ALA-ASN-PRO) has been synthesized and
The two copies are spliced together. This sequence is designated R32. R32 was spliced into plasmid 9, which encodes tetracycline resistance (T, TR). T in addition to R32. The fusion product encoding the 32 amino acids of the TR gene product is R32-TeT.
It is called 32. When used to immunize mice, R3
2-TeT32 recognizes C8P in live sporozoites,
generated an antibody response that protected sporozoite invasion into liver cells. The presence of the TeT32 gene product was required for the induction of an immune response. If the TeT32 region was removed, a strong oil-based adjuvant was required to induce an immune response. Subsequently, R32-TeTaz was mixed with Al (
OH) 3 (to act as an adipant) and administered intramuscularly to human volunteers in various amounts (10 μy to 1,000 μg). R32-TθT32 elicited only a weak anti-sporozoite immune response even at the highest dose tested. This weak immune response, combined with the relatively large amount of antigen required to generate it, makes it unsuitable as a human vaccine for public health use.

A different attempt to generate an anti-sporozoite immune response was
SN-ALA-ASN-PRO is the use of an artificially synthesized low molecular weight peptide 9 consisting of repeating units. Due to their low molecular weight, these petites themselves cannot act as immunogens. Veptibi is therefore covalently linked to a carrier protein to create a conjugate vaccine.

In one research example, “k petit” (ASN-ALA-A
consisting of 2 to 4 repeating units of SN-PRO) succinimidyl 4 (N-maleimido 9 methyl) cyclohexane-1-carboxylade (SMCC) and m-
Maleimidobenzoyl N-hibiloxysuccinimide ester (MBS) is used as a binding agent to bind to bovine serum albumin or thyroglobulin. W,R
, Barrow et al. 1985 (above sentence r: me). These conjugates were able to elicit an anti-sporozoite antibody response in mice when administered with a strong adipant. Again, these complexes are not suitable for human use for the following reasons; 1) antibodies raised against the carrier protein are likely to contain human proteins that share common determinants; ξ Reacts with protein or, in the case of bovine serum albumin, with ingested food. The two binders used (SMGC and MBS) have highly reactive groups, which would be expected to react with host tissues (these are likely to be incorporated into the zygote). ), and 1
11) The immunogenicity of these conjugates in the absence of adjuvant 9 or in the presence of adjuvant 9 suitable for human use has not been demonstrated.

European Patent Application No. 166.410 and US PC
T Application No. 84100144, U.S. POT Application No. 8510
No. 1733 and US PCT Application No. 8,610,627 describe general methods for attaching peptides to carrier proteins. Neisseria meningitidis bound to polysaccharides
A slide conjugate vaccine has also been made consisting of a protein (Seria meningitidis), Einhorn et al. Lancet, August 9, 1986.7)299.However, this type of protein is also not conjugated to a peptide. These and other documents referenced herein are incorporated herein by reference.

Genes involved in the production of P. vivax sporozoite surface coat proteins have also been identified. The repeating unit of the array is GLY:ASP:ARG:ALA
:ASP : GLY : (:, LU : PRO
: It is ALA.

SUMMARY OF THE INVENTION The present invention relates to immunogen preparations and vaccines made therefrom. A peptide derived from a P. falciparum sporozoite surface coat protein and/or a P. vivax sporozoite surface coat protein is covalently linked to a carrier protein through a spacer molecule. P. falciparum and P. vivax vaccines and P. falciparum - P. vivax bivalent vaccines are described.

In one embodiment, the invention provides (ASN: AL
A :ASN:PRO)n (in the formula, n=t-5o) is an amino acid having the composition of - using a binding agent such as dimethylaminopropylcarbodiimide hydrochloride (hereinafter abbreviated as EDEC) to bind CRM 1 g y , diphtheria toxin, toxin A1 choleragnoide, and a suitable carrier protein such as Neisseria meningitidis group B envelope protein. Binds to proteins.

In another embodiment, the present invention provides an amino acid having the following composition:
iwo: H-ASN:PRO:ASN:ALA:ASN
:PRO:ASN:ALA:ASN:PRO:ASN:
ALA-OH (hereinafter referred to as Mpep) was reacted with ammonium chloride at pH 4-6, and the product was hereinafter referred to as Mpep (
Abbreviated as N). Mpep or Mpep(N) uses a spacer molecule and a binding agent to bind CRM1g7, diphtheria toxin, toxin A1 choleragenoid and Neisseria meningitidis B.
It is bound to a carrier such as an extracellular membrane protein.

In yet other embodiments, the invention provides the following amino acid composition MET:ASP:PRO((ASN:
ALA: ASN: PRO) 15 (ASN: VA
L:ASP:PRO))2LEU:ARG (hereinafter R3
2-LeuARG) is attached to a suitable carrier protein using a spacer molecule and a binding agent.

In yet another embodiment of the invention, the peptide" (
GLY:ASP:ARG:ALA:ASP:GLY:G
LU:PRO:ALA)" (hereinafter abbreviated as "pep", where n = 1-50) is attached to a suitable carrier protein using a spacer molecule and a binding agent.

In yet another embodiment of the invention, a method is described for the anhydrous solubilization and detoxification of Gram-negative bacterial envelope proteins, thereby allowing the treated protein to be used as a carrier protein.

SUMMARY OF THE INVENTION The present invention is directed to immunization preparations comprising peptides that share as antigenic determinants the sporozoite surface membrane (asp) of Plasmodium falciparum or Plasmodium vivax. These novel immunizing agents are induced by a vaccine synthesized from a known amino acid sequence homologous to C8P of Plasmodium falciparum or Plasmodium vivax. These non-immunogenic properties render PETIT-9 conjugated to a carrier tannin using a spacer molecule and a binding agent, rendering it immunogenic and suitable as a vaccine for human use.

These vaccines, when administered parenterally, orally or via the nasopharyngeal route, are useful as active immunization against malaria caused by Plasmodium falciparum or Plasmodium vivax.

The composition of the conjugate vaccine is prepared as follows: The carrier protein is conveniently non-toxic, non-pyrogenic, insoluble, pharmaceutically suitable, and large enough to elicit a human immune response. Regardless of the antibody against the second infection, one should choose one that is stable at pH 2-12, hypoallergenic, and has a relatively large number of reactive groups for binding. Diphtheria toxin, CRM197
Carrier proteins such as gram-negative bacterial coat proteins, such as toxin A1 choleragnoids, and meningococcal group 8 coat proteins, can be used in the present invention.

Diphtheria toxin is caused by Corynebacterium diphtheriae.
The culture supernatant of PW 3 can be purified by ammonium sulfate precipitation, ion exchange chromatography and gel filtration to R; Holmes Infection Ambiimmunity 12:1
392 (1975).

Detoxification can be achieved by adding formalin and incubating at 35°C for one month to dissolve the toxin in the toxin solution. satisfy the needs determined by

CRMl157 (a non-toxic protein that immunologically cross-reacts with diphtheria toxin) was obtained from the culture supernatant of C. diphtheriae C7 (βtox-197) as described by Holmes (cited above, 1975) in R. refine.

Toxin A is Pseudomonas aeruginosa.
From the culture supernatant of S. uginosa) PA103, S.
As described by J. Krinno et al. [Infection and Immunity 39: 1072]
(1983)), filtration of ammonium sulfate precipitation and ion exchange chromatography. It is also used for other immunologically relevant cross-reactive substances.

Choleragenoid is produced by processing purified cholera toxin under acidic conditions.
CK, H., Wong et al., Biological Standard 95: 197.
(1977)). Escherichia c.
oli) B subunit thermolabile Other choleragenoid analogues such as blue-violet can also be used.

Neisseria meningitidis (Neis)
It can be purified from Seria menin 1tidis) as follows. The killed bacteria are homogenized in Tris-EDTA buffer. Centrifuge the supernatant at 100,000XF for 2 hours and collect the klets. The shell-containing pellet was suspended in 1-thiempigen BB at pH 8 and heated at 4°C.
Incubate for 16 hours. Sonicate the solution. Add ammonium sulfate to 5005'/L. The precipitate is dissolved in Tris-NaClEDTA, empigen buffer and sonicated. The sonicated solution is dialyzed against the above buffer.

Filter the solution through a 0.22 μM membrane filter. Mix with 3 volumes of cold ethanol and collect the precipitate.

Klet is replaced with sodium phosphate, NaC7! , Zwittergent buffer (7)F
(8). Other solubilized Gram-negative bacterial coat proteins can also be used. gonorrhea (N, gonorrhea)
aθ) or other meningococci (N, meningitiai)
s) such as Neisseria, fi (Neisseria)
It can also be used for convenient tan and ξri textures.

Deep peptide H-ASN with the following amino acid composition: PRO: ASN: ALA:
ASN: PRO: ASN: ALA: AS
N :PRO:ASN:ALA-OH (i.e. Mpe p
)l'! , which can be synthesized using a solid-phase resin system, Bachem F
Available from einchemikalen AG, Basel, Switzerland.

The binder used is conveniently an insoluble carbodiimide 9. An example of a convenient binder is 1-ethyl-3-(:)methylaminopropyl)carbodiimide" (EDEC), which has the structure:

At an advantageous rate, the binding agent generates a reactive intermediate of the carrier protein that enables reaction between the carrier protein and the surfactant molecules.

The sweeter molecules used are conveniently non-toxic, non-pyrogenic (7)H2-12) and have one or more reactive groups.

The sulfur molecule used is conveniently given the formula; NH2-N
It is a dicarboxylic acid dihuman 9radito 9 having the structure: HC:O-(OH2)n-Co-NH-NH2 (in the formula, n=x-zO).

Examples of convenient spacers are adipic acid dihibiradibi (AD) I), succinic anhydride, oxalic anhydride, maleic anhydride and phthalic anhydride, other suitable anhydrides or mono- or Jihibira:) Hi. The use of these spacers allows a high proportion of peptides to bind to the carrier protein.

In the case of meningococcal coat protein, succinic anhydride or other suitable anhydride treatment renders the protein water soluble. Note that this spacer/solubilizer treatment can be used to render Gram-negative bacterial envelope proteins, including proteins from all Neisseria species such as Neisseria meningitidis and Neisseria gonorrhoeae, into soluble, non-toxic carriers. Neisseria meningitidis protein or other Gram-negative outer membrane protein (OMP)
) are insoluble in the absence of surfactants or other solubilizing agents with polysaccharides or other hydrophilic regions.

OMP treated with anhydrous phosphoric acid is soluble and the complex remains soluble. The complexes showed lower pyrogenicity in rabbits compared to meningococcal proteins before treatment with phosphoric acid anhydride, indicating that these proteins were anagastric as well as solubilized and could be used as carriers. It allows the use of

Before coupling the peptide to a carrier, the peptide must be pretreated with a compound such as ammonium chloride to protect the carboxy groups. Additionally, the peptide must be treated with a compound such as succinic anhydride to protect the amino groups of the peptide. The use of compounds such as succinic anhydride in this method also contributes to the spacer function.

The malaria conjugate vaccine described below uses a 5-anhydride compound that immunologically crosses C8P of P. falciparum or P. vivax to facilitate the covalent attachment of the peptides to various carrier proteins. It is synthesized using acid and/or adipic acid dihydradiene (ADH) as a spacer molecule.

The conjugation conditions used as part of the present invention are effective for covalently linking malaria spot or R32Lθu ARG to a variety of carrier proteins.

The molar ratio of R peptide to carrier protein is 5:
1 to 18:1, while the ratio of R32-Le self to toxin A is 13:1.

As seen in the Western plots in Figures 1-4, conjugate binding occurs in a complex manner at various molecular weights.

The vaccine of the invention can be mixed with a suitable A:)S band such as aluminum hydroxide or aluminum phosphate.

The invention will be described in more detail below, without being restricted to the illustrated embodiments.

CRJA 1 g 7 is 0. I M NaPO,s,
Dialyze for a long time at pH 6.5.

Transfer the material to a 125 fflJ Erlenmeyer flask and raise the pH to 8.0.

Succinic anhydride is recrystallized by adding 70ml of boiling acetic acid to 10.9ml of succinic anhydride. When the solution is left to cool to 4°C, crystals form. After removing excess liquid and washing the crystals three times with anhydrous ether, the crystals are ground in a mortar and pestle and stored at -20°C under a nitrogen atmosphere.

450 η of recrystallized succinic anhydride is added to 30 η CRM197 in 30+ nl water, while stirring continuously. The pH is maintained at 8.2 by addition of NaOH.

After 2 hours, add another 450 μ of succinic anhydride to adjust the pH.
maintains 8.2 for 2 hours. These reactions were carried out at 25° C. and the product is hereinafter referred to as CRM197(S).

Transfer the reaction mixture to a dialysis tube and add 2 L of 061M N
Dialyze against aPO4 pH 6.5 buffer solution. Change the fluid 4 times. Removed from dialysis tube, 0.45μ
Sterilize through M filter and store at 4°C. The size is approximately 60rnl.

Dissolve 60 μ of Mpop in 12 ml of 2M ammonium chloride solution. In this process, the carboxy terminus of the peptide is protected, ie, Mpsp(N) is formed.

Therefore, in the subsequent steps described below, the reaction proceeds through the reactive groups of Ptibi 9 other than the carboxy terminus, thereby strongly limiting antigenic changes and self-polymerization of Ptibi.

250 to 1-ethyl-3-(:,)methylaminopropyl)carbodiimibihydrochloride (EDEC: ) is added to the 2Ptibi solution. pH was adjusted to 4.0 with 0.2 M HCl.
Lower it to 7. Monitor pH and keep it at 4.7. 30 minutes later,
A further 250 μ of EDEC is added to maintain the pH at 4.7. After another 30 minutes, add another 250 mg of EDEC to maintain the pH at 4.7. After another 3 hours, N.
The aOHT pH is increased to 6.9 and the mixture is stored at 4°C. Store frozen (-70°C) and lyophilize.

Ammonium chloride treated iptide (Mpep(N))
Dissolve in 12 ml of sterile water and purify twice with a Sephadex GIO column. This removes unreacted reagents. The fraction containing 4 peptides has UV absorption (OD22
0), collected and lyophilized.

CR1vf1g7(3) is covalently bound to Mpep(N) by the following method. 30rrMi succinic anhydride treated fjJ CRM197 (CRMx 97 (S
60 rnl of an aqueous solution in which )) is mixed with an IQ ml aqueous solution in which 30 ml of ammonium chloride-treated R petit vi is dissolved. 7) Reduce H to 5.5 with 2N HCl. Add 5007 ng of EDEC and slowly stir the mixture
Stir for an hour. Next, add soom9 EDEC and add 2
Stir for 0 minutes. Add 500 μ of EDEC again and add 20
Stir for a minute. Further, 500 μ of EDEC was added and stirred for 1 hour. At this point the pH is 5.4. The reaction mixture was transferred to a dialysis tube and (13)MNaPO4,7)H
Dialyze against 7.05. The solution is removed from the dialysis bag under aseptic conditions and force filtered through a 0.8 μM P,E filter, followed by a 0.45 μM filter. Sterile autoclave Amicon concentrator and YM 3
Concentrate the solution to 25 ml using a Q membrane. 0.22μ
Pass through M filter and store at 4°C.

choleragenoid” at 4°C (13) MNaPO4,
Dialyze for a long time against pH 6.5. 45 μm of dialyzed choleragenoid was placed in a 50 at Erle/Mayer flask and the pH was raised to 8.0. Add 200 mg of succinic anhydride and maintain pH at 8.2 with IN NaOH. 3
After 0 minutes, 200μ of succinic anhydride was added and lNNa
Return the pH to 8.2 with OH. After 1 hour, 200 μ of succinic anhydride is added and the pH is maintained at 8.1 for the next 1 hour. Transfer the solution to a dialysis bag, Q, l M NaP
Dialyze against O4, pH 6.5.

Mpep(N) was synthesized in Example 1. choleragenoi)-'' (S) from 45 to 250 mg is placed in a 250 mg Erlenmeyer flask along with desalted Mpep (N).

2 NHCe K Te PH 5.4 K lower, 50 on
Add yznoEDEC. After stirring gently for 30 minutes, an additional 500 μ of EDEC is added. Add 500 μ of EDEC twice more at 30 minute intervals and stir gently for 5 hours at 4°C. The reaction mixture was transferred to a dialysis tube (13
) Dialyze against MNaPO4, pH 7.05. The material is removed from the dialysis bag and stored at 4°C after passing through a 0.22 μM filter.

Place 900 N. meningitidis 2B protein in 37 ml of water into a 125 ml/Erlenmeyer flask.

Add 450 μ of succinic anhydride and bring the pH back to 9.2 with 1N NaOH. Neisseria meningitidis group B outer coat protein becomes a water-soluble protein by treatment with succinic anhydride. At this point the protein solution becomes very clear. 1 hour pH
Maintain 8.1. Then 450 m9 of succinic anhydride are added and kept at t) H8,O for 2 hours. The reaction mixture is gently stirred for an additional 3 hours, during which time the pH drops to 7.0. The mixture is dialyzed against Q, 1M NaPO4, pH 7.05 buffer. Even with detergent immobility, the protein remains soluble.

10m1 (7) 7X plus 30m1 Mpep (N)
is added to 40 μl of succinic anhydride-treated meningococcal 2B protein in an Ersinmeyer flask. Lower the pH to 5.5 with 2N HCl, add 500μ of EDEC,
Stir gently for 5 hours at 5°C. 3 more at 20 minute intervals
Add 500 times of EDEC and then stir for 1 hour. Transfer the reaction mixture to a dialysis tube, o, IM NaP
Dialyze against O4, pH 7.05. The material is removed from the dialysis bag, passed through a 0.22 μM filter, and stored at 4°C.

Example 4 Choleragenoid (ADH)-Mpep(S) (using adipic acid dihibirazide and succinic anhydride as S-R-Ser molecules) 13 μ of choleragenoid added to 5.4 ml of Q, 1 M NaPO4, pH 7.0 into a 25ml flask.

Add 300 µm of adipic acid dihuman 9-radito (ADH) to lower the pH to 5.2. Add 120 µm of EDEC and keep the pH at 5.5. After 30 minutes, Add EDEC (3) and adjust the pH to 5.
Keep it between 5-5.8.

After 1 hour, 120 μm of EDEC was added, stirred for 1 hour, and then transferred to a dialysis bag. The reaction mixture was (13) M NaP
O4.

Dialyze against pH 6.5. The resulting product is called choleragenoid (ADH).

20 ■ Mpep Q, l M NaP○45rnl
and raise the 7)H to 8.2 with IN NaOH. Add 150 μl of succinic anhydride and maintain pH at 8.0 with 1N NaOH. After 90 minutes, 150 μ of succinic anhydride is added and the pH is maintained at 8.0 for the next 60 minutes. 2.
After stirring gently for 5 hours, the pH becomes 7.0. The solution is purified on a Sephadex GIO column and the appropriate fractions are lyophilized. The resulting product is designated Mpep(S).

25m#) 0. t MNaPO4, pH 6.5 solution to 13~20 m9
of Mpep(S) is added. Reduce pH to 4.5.

Add 500 μm of EDEC and lower the pH to 4.5.

Add 500 μ of EDEC three times at 20 minute intervals, followed by further stirring for 20 minutes. Adjust the pH to 6.0 and
Stir at ℃ for 5 hours. Transfer the reaction mixture to a dialysis bag (
13) Dialyze against M NaPO4, pH 7.05. The contents are concentrated to 20 ml with a PMIQ Amicon filter and then filtered with a 022 μM filter.

soT! 50ml of R32-L eu ARCr
into a flask and adjust the pH to 8.0. 225■
of succinic anhydride is added and the pH is maintained at 8.0. 1
After an hour, 180 μ of conozic anhydride is added to adjust the pH, followed by a fresh 180 μ of conozic anhydride after 1 hour. The reaction solution is gently stirred for 5 hours.

Dialyze against 0.1 M NaPO4, pH 6.5. Remove from dialysis bag and filter through a 0.45 μM filter. This is called R32-Leu ARG (S).

R32-LeuARG (S) was placed in a 50 ml flask, the pH was lowered to 4.4, and 300 m EDEC
Add.

32 ml of ADH-binding toxin A (toxin A (ADH)) was gradually added over 30 minutes while stirring gently.

Once toxin A (ADH) has been added, the solution becomes cloudy. Add 300 μm of EDEC, followed by two more 300 μl of EDEC at 1 hour intervals.
Thirty minutes after adding the EC, the H was raised to 7.0, the mixture was transferred to a dialysis bag, and the H was raised to 7.0. I M NaPOH buffer p
Dialyze against H7,01.

put it on. Add 23 ml of 2M ammonium chloride solution. The pH of the solution is lowered to 4.5 with HCI. 500■
of EDEC and adjust 71)H to 4.5. 9
After 0 minutes, add 500 μ of EDEC and reduce 7) H to 4.5
lower to

After 60 minutes, 00 m9 of EDEC was added and the pH was 4.6.
maintain. One hour after the last EDF,C addition, the contents were transferred to a dialysis bag and treated with (13)M NaPO4, pH 6.
, 5.

30 rnl of R32-Leu ARC(N) are mixed with 31 ml of R32-Leu ARC(N).

Lower the pH to 5.45 with 0.5 ml of 2N HCI. Add 500 μ of EDEC every 30 minutes over 90 minutes. One hour after the last addition of EDEC, the contents are dialyzed against (1, IMNaP○4, pH 7,05 using a dialysis tube with a cant-off molecular weight of 50,000. Pass through a 0.2271M filter.

Colegenoids are combined with succinic anhydride as described above (Example 4). 90 μ of R32- in 23 ml of water
In a 125 ml sterile flask containing Le u ARG, meningococcal envelope protein (○MP) is treated with succinic anhydride as described in Example 3. This live release is called Neisseria meningitidis 2B(S).

27m7 containing 50■ tan/ξri quality! 25 ml of meningococcal 2B(S) solution previously dissolved in 3 ml tr) R20! Mix with pep. Lower the pH of the mixture. Add 100 μ of EDEC and stir for 4 hours. Next, 200q of EDEc is added. After 30 minutes, add 200 mg of EDEC and 25 to 25 Mpep. After 30 minutes, 200 μ of EDEC: is added. After 3 hours 200 μ of EDEC is added. The mixture is stirred for 3 hours and then dialyzed against 3 changes of 5 liters of PBS.

45m7 meningococcal OMP was prepared using the method described in Example 3 (
7) Perform succinic anhydride treatment by carefully lowering H to <6.10. Add 200 μ of EDEC:.

Add 50 μ of Vpep dissolved in water dropwise over 15 minutes. Lower pH to 5.5. 1 hour after 100■E
Add DEC. The solution becomes somewhat opalescent.

Three hours after the reaction has stopped, the solution is dialyzed against 5 liters of PBS.

Example 9 Toxin A (ADH) of lO prepared by the method described in Example 5
) of 2.7 ml ii and 50 ~ R32L eu
Mix ARG. Lower the pH to 4.80 with 1 MH (J) and add 100 mg of EDEC. After stirring at a constant speed for 20 minutes, add ~100 EDEC,
．． Lower it to 8. Reaction initiation 6(13)00 and 1404
After 0 minutes, add 100 μDEC. Constant stirring was maintained at 25° C., and 7 hours after the start of the reaction, dialysis was performed by exchanging 5 liters of PBS three times. The solution was ultrafiltered with an Amicon PM3Q membrane to a volume of 30 ml and the non-covalently bound R
Remove 32LθUARG and dialyze again against PBS. The final vaccine is filtered through a 0.22μ filter.

50 μ of toxin A (
ADH) solution to lower the pH of 138 ml to 4.830.

Dissolve 50 μMpep in 3 ml R20. Add 500 μl of the injection solution dropwise over 10 minutes. pH to 4
．． After lowering the temperature to 700, add 100 m9 of EDEC. Add 1.5 mJ of petit vi over 20 minutes.

Add 100m7 of EDEC followed by remaining 500μ
Add putide to g. After a total reaction time of 5 hours, the solution is dialyzed with 4 changes of 5 liters of PBS.

Prepare a 13.8 ml solution of 50 η of toxy7A (ADH) and lower the pH to 4.8 as described above. Add 200mg (7) EDEC. 50 μ of Vpep (n=2) are dissolved in 3 ml of R20.

Add 2 ml of peptide 9 solution dropwise over 20 minutes.

Add 100ml of EDF, C, and add the remaining ml dropwise over 10 minutes. 40 and 60 minutes after the start of the reaction, 10
Add 0 mg of EDEC.

3 hours after the start of the reaction, add 5 liters of PBS (changed 4 times)
Perform dialysis against.

The features of the invention will become fully clear from the following detailed description of the preferred embodiments of the invention, read in conjunction with the attached tables: Table A shows a list of some of the vaccines prepared. The composition is shown.

Table A Conjugate vaccine a) R32LeuARG (Sl Toxi7A (ADH
) b) R32LeuARG(N) is
l Cutin Composition Iptibi R32Le uARG Succinic Anhydride Carrier Toxin A Adipic Acid Dihibira:) Vid) Mpep about Neisseria meningitidis e) Mpep(N)CRM 197(S)f) Mp
ep(N) choleragenoid'(S)g) ”pop(S
choleragenoid” (ADH)h) R32LeuAR
G) Xin A (ADH) i) Mpep pulp 1 Zhuo Yan 2B
(S) j) Mpe pCRM + 97 (Slk) Mp
epDT (ADH) 1) Mpep tetanus (ADH) m) Mpep tetanus (ADH) n)
Vpsp Myelitis meningitidis 2B (S+ pep Mpep Mpep Mpep R32LeuARG Mpep Mpep Mpep Mpep Mpep Ammonium chloride Ammonium chloride Succinic anhydride ViVav (n=2 〕h2ba 1 product line) RM197 Cholera genoid 9 Cholera Noidtoxin A Meninikotsukuru OMP CRM 1 g 7 diphtheria toxoid tetanus toxoid 9 choleragenoid 9 meningococcal 2B t nobi-: ishi Dihibiradibi Succinic Anhydride Toxin A Choleragenoid Succinic Anhydride Choleragenoid CRM 197 Adipic Acid Dihydrito 9 Adipic Acid Dihibiradibi Dipic Acid Dihibiradibi Succinic Anhydride (n = 2) The chemical composition of the conjugate vaccine is shown as a molar ratio of .

Table 1 Composition of selected conjugate malaria vaccines Conjugate
, Ya ratio 1 (Ikupetibi/carrier protein) Ki'/7A(ADH)-R32-LeuARG(S
) 13:1 cov lagenoid is) - Mpep (
N) 12.5: 1 choleragenoid” (ADH)-Mpep(S) 5
: ICRMi97(S)-Mpep(N)
Table 2 shows the biological properties of the conjugate vaccine regarding its stability when used as a human vaccine as a second embodiment of the present invention. .

Table 2 Biological properties of petit viconjugate vaccine for malaria R32
-LeuARG (Polymerization) Neisseria meningitidis 2B (S) - Mpep (N) CRM1g7 (S) - Mpep (N) choleragenoid (S) - Mpe p (N) Non-toxic Sterile 0.3 Non-toxic Sterile 0.3 Non-Toxic Sterile 0 Non-Toxic Sterile 1 Conjugate (pre-absorbed with Al(OH)3) was dissolved in saline to a final concentration of 10 μji/ml, except for R32
-Leu ARG-choleragenoid conjugate is 2 μg/m
I made it to l. Each rabbit was administered 1 ml of the sample per kg of body weight intravenously.

Maximum increase in body temperature recorded 4 hours after administration of 210μ//kg (body weight). choleragenoid' (ADH) -M
pep(S) did not increase body temperature at 2 μA9 (body weight).

Conjugates 1)-p) and e) of Table A were all suitable (ie <0.2) at 50μ7kg.

3 mice (18-20y) and guinea pigs (250-3
00g) and 100 μs of each sample was intraperitoneally administered. There were no deaths and all animals gained weight. European Pharmacopoeia
Macopoeia) V, 2.1.5.

4 European Pharmacopoeia V, 2.1.1.

Table 3 shows the strength of the immune response in rabbits to the conjugate, vaccine malaria iptide portion.

Table 3 R32-L in rabbits after immunization with conjugate vaccine
euASG (polymerized) 48 tox 7A (AD
H) R32-LeuARG(S) 8107 choleragenoih”(s)-R32-LeuARG(N) 5
29 diphtheria toxoid” (ADH)-Mpep 3
126 Neisseria meningitidis B(S)-Mpep(N)
30050RM, 97(S)-Mpep(N)
1674 choleragenoi K(S)-Mpep(
N) 988 choleragenoi)-”(ADH
)-Mpep(S) 1452 (5712-15,
017) (53,4-851) 1. Rabbits (3 animals per group) at 4% (wt/υO) kl
(OH) 100 μl of vaccine in 3 days 0 and 28
The mice were immunized on the 42nd day, and serum was collected on the 42nd day. The coupling agent used was EDEC in all cases.

2. ELISA antibody titer is expressed in units (optical density x serum dilution/
ml). The antibody response cross-reacts with sporozoite surface coat protein (C3P) (detected using peptide antigen).

Table 4 shows the strength of the immune response in rabbits to the carrier protein portion of the conjugate vaccine.

Table 4. Immunoglobulin G antibody response to carrier protein in rabbits after vaccination with petivconjugate for malaria Normal rabbit serum Toxin A (ADH)-R32-LeuAGR(S) Choleragenide (S)-R32-LeuAGR(N ) diphtheria toxoid (ADH)-Mpep Neisseria meningitidis B (S
)-Mpep (N) CRMl g 7 (S) -Mpep (N)
Choleragenide (S) - Mpep (Company) 8.3 (1,2
-44) 126.6 (79-208) 10 (2,5-24,5) 157.3 (45-322) 205.3 (97-340) 25.6 (2,5-30) 1. Rabbit (3 animals per group 4% (wt/vol) A/
N○H) Vaccine 100 μy″’co day and 28 in 3
I was immunized on the day.

Serum was collected on day 42. The coupling agent used was EDEC in all cases.

2. ELISA antibody titer is in units (optical density x serum dilution/
ml). Antibody reactions are shown for the corresponding protein carrier.

Table 5 is a second list of vaccines and methods in Tables 3 and 4.
Table 5 is the second set of data.
(ADH)-R32LE (s) Cholera)
'(S)-R32LeuARG(N)DT(ADH)-
Mpep Neisseria meningitidis 2B (S)-Mpep (N) CRMt 97 (S)-Mpep (N) Choleragenoitope Sνpep (N) 80 1.25 0 6819 13.7 4.00 674 10.5 400 3019 14.8 400 2950 36.9 400 2000 44.6 100 1192 11.9 100 5.6 41.4 21.3 91.5 The titer shows the geometric mean.

The Kimoto hold rise represents the geometric mean bold rise.

***Fluorescence assay: Falciparum
m) Immobilize sporozoites from malaria on slide 9. Coincubate dilutions of rabbit antibodies with sporozoites. The slides are washed and a fluorescent goat anti-rabbit antibody is incubated with the sporozoites. After thorough washing, the slides are read with a fluorescence microscope bell for the maximum dilution of serum that gives a fluorescence reaction.

Table 6 shows the simultaneous administration of Mpe p conjugated to two different carrier proteins. Show the results.

Table 6 Anti-Mpep ELISA titer after immunization of rabbits with bivalent vaccine Mpep-ADH-CHOL 11066l
1066 (6300-19000) Large DH-tetanus toxoid 1, rabbits (3 animals) 100 on days 0 and 14
/μ9 vaccine by intramuscular injection. 28 serum
Samples were taken on day one. For the combined vaccine group, 50 μy of vaccine from each component was administered at two different sites.

Table 7 shows antibody responses in humans to synthetic peptides conjugated to carrier proteins.

Table 7 Antibody responses in humans after vaccination with Petito 9 against malaria conjugated with adjuvant Al(OH)3 suitable for use in humans Vaccine Sweat Ia Optical Density (ELISA) response Bora/Tia Number 2.5 3 25 6.5 7 6.5 8 8.5 8 Vaccine 1: Using meningococcal outer coat protein as a carrier (Mpep-MENING-B) Vaccine 2:
Choleragenoid was used as a carrier coupled to NANP via ADH. (Mpep-ADH-C
HOL) Vaccine 3: CRM197 (genetically induced diphtheria toxoid'') was used as carrier. (Mpep-CRM197) Vaccine 4:
Choleragenoid 9 was used as a carrier protein and coupled to Mpep by using both ADH and succinic anhydride.

(Mpep-ADH-3uc-CHOL) The conjugate described above was made suitable for human use. Therefore, the malaria antigen, carrier protein, spacer molecule and reaction conditions were combined to produce an acceptable final product. However, although the reagents themselves are neither toxic nor pyrogenic, - once covalently bound, one or more of the combinations may induce increased molecular weight or new antigenic determinants or reactions. Therefore, each conjugate vaccine was tested for toxicity and pyrogenicity. All conjugates were non-pyrogenic and pyrogenic. A dose of >2 μg/kg raises body temperature by less than 0.05°C. This dose level is similar to that for other vaccines for human use such as Meningococcal A and C. (0
05μIAy). The conjugate vaccine was nontoxic when administered at 100 μg intraperitoneally to either mice or guinea pigs. These studies were conducted according to international harmonized guidelines (European Pharmacopoeia). The findings here suggest that these conjugate vaccines are well tolerated in dogs when administered parenterally to humans.

The immune response induced in rabbits after immunization with the above malaria conjugate vaccine was then evaluated. First, they studied antibody responses to the malaria antigen component of the vaccine. The original (ie non-conjugated) R32-LeuARG was found to be non-immunogenic (48 mean ELISA units/me (serum)). However, R32-Leu ARG
and conjugates consisting of toxin A or collagenoubi are highly immunogenic (8107 and 3126 mean EL, respectively).
SA: s-=7t/ml (serum)). Similarly, the unconjugated form is putodo, i.e. ASN: PRO: ASN: ALA: AS
N: PRO: ASN: ALA: ASN
:PRO: ASN: Immunizing rabbits with ALA does not cause an antibody reaction.

Above, putito 9 is added to diphtheria toxoid 9, toxy/A
Conjugates prepared covalently to either N. meningitidis group B coat membrane protein, CRM t 97, or choleragenoid 9 can now raise antibodies against the carrier protein. However, when choleragenoid 9 was coupled to R32-LeuARG or the above Veptibi via succinic anhydride, a very weak anti-carrier response occurred.

This indicates that the method used to form these two conjugates abolished most of the immunodominant antigenic determinants expressed by choleragenoids.

Despite this, these two conjugates were able to elicit a good antimalarial peptide 9 response.

In order to demonstrate that the above conjugates are effective for use as active vaccines against malaria, an exhaustive attempt was made to evaluate the safety and immunogenicity of several conjugates in human volunteers. . Volunteers were immunized with the following conjugates: CRM1g7-Mpe p choleragenoid-Mpθp choleragenoid-Mpep (ADH) meningococcal B - Mpep These vaccines were immunized with Al(
○H) 3 and 100 μg of carrier protein was intramuscularly administered in 0.5 ml to each volunteer.

Reactions after vaccination are mild, temporary, and primarily localized pain. To demonstrate the absence of systemic toxicity,
The following analyzes were performed at the time of immunization and one week later: hemoglobin, hematocrit, red blood cell count, white blood cell count, blood cell count, serum glutamate-oxalacetate-transaminase, alkaline phosphatase, creatinine, and bilirubin. Vaccination did not change any of these parameters, indicating no toxicity.

A preferred embodiment of the invention has been described herein in which the epitopes forming the antigenic determinants of the sporozoite surface coat protein are replaced by a) N. gonorrhoeae or P. aeruginosa Bacterial hairs (uttachment or organelles) from microorganisms
ga-nelles), b) hepatitis B virus, C) H
TL■■/shiW virus (for example, gp 120),
d) internal image anti-idiotype antibodies;
It will be clear to those skilled in the art that conjugates can be formed using peptides that form antigenic determinants of. In particular, changes and modifications in the spacer molecules used, the number of amino acid repeat units in the 2-peptide 9 moiety, and modifications of the peptide to block reactive groups prior to covalent attachment to the carrier protein are of interest in the present invention. It is possible without departing from the spirit.

[Brief explanation of the drawing]

Figure 1 is a Western plot of malaria conjugate vaccine and carrier protein developed with monoclonal antibodies against malaria peptides. For each gel (the explanation is as follows = 1) R
32LeuARC 2) R32LeuARG(N) 3) R32LeuARG(S) 4) R32LeuARG(S)-Toxy A(ADH)
5) R32LeuARG (N) -Choleragenoid 9 (S) 6) Mpep (N) -Choleragenoid (S)
7) Mpep(S) --IL/Lagenoi)-"(
ADH) 8) Mpep(N)-Ntll Streptococcus B(S)
g) Mpep (N)-CRM197 (S) 10) Molecular standards 11) Toxin A (ADH) 12) Choleragenoids (S) 13) Choleragenoids true ADH) 14) Neisseria meningitidis B (S) 15) CRM197 (S) ) Figure 2 is a Western plot of a mixture of monoclonal antibodies against the carrier protein versus the vaccine. Each gel is described as follows: 1) R32LeuARG 2) R32LeuARG(N) 3) R32LeuARG(S) 4) R32LeuARG(S)-)Xi7A(AD
H) 5) R32LeuARG(N) -: I v lagenoid (S) 6) Mpep (N) - choleragenoid)' (S17) Mpep (S) - choleragenoid (
ADH) 8) Mpep (N) - Neisseria meningitidis B (S)
9) Mpep(N)-GRMt9t(S)10
) Molecular standards 11) Toxin A (ADH) 12) Choleragenoit 9 (S) 13) Choleragenoitha ADH) 14) Neisseria meningitidis B (S) 15) CRM197 (S) Figure 3 shows Vivax and Falciparum (Falciparum) <Western plot of Falciparum and Falciparum conjugate vaccines developed with monoclonal antibodies against Petibi. A description of each gel is as follows. 1) Neisseria meningitidis 2B (S) 2) cRM197 (S) 3) Choleragenoids (ADH) 4) Vpep (V=21 Neisseria meningitidis 2B (S) 5)
Vpep (V=2) CRM197(S)6)Vpe
p (V = 2): 'L'lagenoid' (ADH) 7) Vp
ep (V = 2) Covragenoid (ADH) 8) M
pep choleragenoid (S) 9) Mpep meningococcus 2
B(S) 10) Mpep CRM1g7 (S) FIG.
Vivax) and Falciparum (Falcipar)
um) Western plot of conjugate vaccine. Each gel is described as follows: 1) Neisseria meningitidis 2B (S) 2) CRM197 (S) Choleragenoids (ADH) Vpep (V=2) Neisseria meningitidis 28 (S) Vpep (V
= 2) CRM197 (S)Vpep (V=2
) Cholera 'f/i)' (ADH) Vpep (V=2
): yv lagenoid)” (ADH) Mpep choleragenoid 9 (S) Mpep meningococcus 2B (S) Mpep CRM 1 g 7 (S) Afu 3 Figure 4

Claims (36)

[Claims] (1) A method for producing an immunogenic conjugate, comprising: (i)
A method comprising covalently linking a carrier protein to (ii) a peptide forming an antigenic determinant of a circumsporozoid protein, via (iii) at least one spacer molecule. (2) said binding step covalently binds said at least one spacer molecule to said carrier protein and one of said peptide to form a complex A; 2. The method of claim 1, comprising covalently bonding the other of the two. (3) the binding step includes covalently binding a first spacer molecule to the carrier protein to form complex A, covalently binding a second spacer molecule to the peptide to form complex B; Claim 1 comprising covalently bonding A to complex B to form said conjugate.
Section method. (4) The method according to claim 1, wherein the peptide has the following amino acid sequence: (ASN:ALA:ASN:PBO)^n (where n is 1 to 50). (5) The peptide has the following amino acid sequence: (GLY:ASP:ARG:ALA:ASP:GLY:
GLU:PRO:ALA)^n, where n is from 1 to 50. (6) The peptide has the following amino acid sequence: MET:ASP:PBO [(ASN:ALA:ASN:
PBO)_1_5ASN:VAL:ASP:PRO)_
2. The method of claim 1 comprising: (7) The peptide has the following amino acid sequence: MET:ASP:PRO [(ASN:ALA:ASN:
PRO)_1_5ASN:VAL:ASP:PRO〕_
2LEU:ARG. (8) The method according to claim 1, wherein in the binding step, another protein forming the antigenic determinant of the sporozoite surface coat protein is directly bound to the carrier protein. (9) The method of claim 1, wherein the peptide is pretreated with an agent that selectively blocks amino or carboxyl groups before the binding step. (10) The method according to claim 9, wherein the peptide is pretreated with ammonium chloride. (11) The method according to claim 9, wherein the peptide is pretreated with succinic anhydride. (12) The carrier protein is diphtheria toxoid,
CRM197, toxin A, choleragenoid, Escherichia coli (
2. The method of claim 1, wherein the method is selected from the group consisting of B subunit of Escherichia coli heat-labile toxin and N. meningitidis group B coat membrane protein. (13) The method according to claim 1, wherein the carrier protein is a Gram-negative outer coat protein. (14) The carrier protein is Neisser
iameningitidis) coat membrane protein. (15) The at least one spacer molecule has the following composition: NH_2-NH-CO-(CH_2)_n-CO-NH
Claim 1, which is a dicarboxylic acid dihydrazide of -NH_2 (in the formula, n is 1 to 20)
Section method. 16. The method of claim 1, wherein the at least one spacer molecule is succinic anhydride, adipic dihydrazide, oxalic anhydride, phthalic anhydride, maleic anhydride, or a mixture thereof. (17) The method of claim 1, wherein the bonding step is carried out using a water-soluble carbodiimide as the binder. (18) The method according to claim 17, wherein the water-soluble carbodiimide is 1-ethyl-3-(dimethylaminopropyl)carbodiimide. (19) An immunogenic conjugate capable of inducing a protective response against malaria infection in mammals, the conjugate comprising (i
A conjugate consisting of (i) a carrier protein covalently bonded via (iii) at least one spacer molecule to (i) a peptide consisting of at least one antigenic determinant of a sporozoite surface coat protein. (20) The conjugate according to claim 19, in which the peptide has the following amino acid sequence: (ASN:ALA:ASN:PRO)^n (where n is 1 to 50). (21) The peptide has the following amino acid sequence: (GLY:ASP:ARG:ALA:ASP:GLY:
GLU:PRO:ALA)^n (where n is 1 to 50). (22) The peptide has the following amino acid sequence: MET:ASP:ALA [(ASN:ALA:ASN:
PRO)_1_5ASN:VAL:ASP:PRO〕_
2. The conjugate according to claim 19, consisting of: (23) The peptide has the following amino acid sequence: MET:ASP:PRO [(ASN:ALA:ASN:
PRO)_1_5ASN:VAL:ASP:PRO〕_
The conjugate of claim 19 consisting of 2LEU:ARG. (24) The carrier protein is diphtheria toxoid,
20. The conjugate of claim 19, which is selected from the group consisting of CRM197, toxin A, choleragenoid, B subunit of E. coli heat-labile toxin, and Neisseria meningitidis group B coat membrane protein. (25) The conjugate according to claim 19, wherein the carrier protein is a Gram-negative coat protein. (26) The conjugate according to claim 19, wherein the carrier protein is meningococcal outer coat protein. (27) A dicarboxylic acid dihydrazide in which at least one spacer molecule has the following composition: NH_2-NH-CO-(CH_2)_n-CO-NH
-NH_2 (where n is 1 to 20). (28) The conjugate of claim 19, wherein the at least one spacer molecule is succinic anhydride, oxalic anhydride, phthalic anhydride, maleic anhydride, adipic dihydraside, or a mixture thereof. (29) The conjugate according to claim 19, wherein another peptide forming an antigenic determinant of the sporozoite surface coat protein is directly bound to the carrier protein. (30) Claim 1 further including an adjuvant
9 term zygote. (31) The conjugate according to claim 30, wherein the adjuvant is selected from the group consisting of aluminum hydroxide and aluminum phosphate. (32) A method for producing immunity against malaria in a warm-blooded animal, which comprises administering the conjugate of claim 19 to the warm-blooded animal. (33) A method of forming a water-soluble non-toxic carrier protein for use in a conjugate vaccine, comprising treating the outer coat protein of a Gram-negative bacterium with an anhydride. (34) The method of claim 33, wherein the anhydride is selected from the group consisting of succinic anhydride, maleic anhydride, oxalic anhydride, and phthalic anhydride. (35) Gram-negative bacterium outer coat protein is Neisseria (Neisseria)
34. The method of claim 33, wherein the protein is from the genus Isseria. (36) At least two conjugates according to claim 19 are administered to a warm-blooded animal, each conjugate having a different carrier protein, producing immunity against malaria in the warm-blooded animal. Method.