JPS5810524A - Protective provocative proteinaceous antigen - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to antigenic substances, vaccines containing them, and their use for conferring immunity against malaria.

The malaria parasite is Plaamodium.
m) is a protozoan of the genus. Part of its complex life cycle is asexual replication within the host's red blood cells, which causes the disease symptoms known as malaria.

The vertebrate host, on the other hand, generates an effective inflammatory immune response against blood-borne parasites. Therefore, it is thought that the blood form is an antigen, which induces the production of specific and inflammatory antibodies in the infected or immunized host.During the asexual erythrocytic stage of replication, free parasites (merozoites) It recognizes the surface of red blood cells, attaches specifically to it, and damages the red blood cells by invasion of the capsule (repeatedly, forming schizonts. Following nuclear division, division of the cytoplasm occurs, A large number of intraerythrocytic merozoites are formed.

As the host's red blood cell membrane breaks down, merozoites are released into the plasma, where they attach to the host's red blood cells and become invasive.This is the only form in which free merozoites come into direct contact with antibody-containing blood cells. Therefore, it has been thought until now that antibodies against Plasmodium novoleai are present on the surface of free merozoites. C'ohen, Butcher and Orandall (nature, 196
9, 223, 368-671). These investigators reasoned that the effect of antibodies in this system was to block specific antigens on the merozoite surface that were involved in attachment to host red blood cells. Next, Mit
hell.

Butcher and Cohen (Immunolo
gy, 1975 e29.397-407) using pure merozoites emulsified in Efreund's complete 7-diupand.

There is no evidence that protection against malaria parasites is any different from primates against malaria parasites. In addition to findings in primate parasites, protective antibodies against malaria parasites have also been found. For example, antibodies against P. yoelii
and/or act on merozoites (Fre
eman and Parish: Rxperimen
tal parasit+o1ogy e 52:1
8-24, 1981)! ri211 Tsume I:lzo()
% heterologous monoclonal antibody vp, 'yo・lii has a protective effect when injected into stained mice (yraeman
.

'rrejaosisicz and Cross: N
ature e 198 L284: 566-36
8; Freeman, Holder.

TretdjdOailsWiCl and croaa
: in @The) Ioat-Invader I
interplay” (HoVan den Boa
ache-).

R15evisr /1iorth Ho1land
, 1980 e pp 121-124).

There have been many attempts to determine the diversity of protein antigens possessed by merozoites (for example, %I), eana et al.
arisitology, 1978e 77:3
3!1-644”.

Miller et al.; J@IEXp6 Mad, 1
980 e 151 near 50-798 and K11a
jian: Proc, Nat.

Acad, 8ci, 1980 s77:3695-3
699). However, antigens that can induce a host response or specific antigens that are specifically recognized by such a host response have never been identified or characterized.

The present invention was completed by isolating a series of antigens associated with the melodic form 1aK of these Delasmodium parasites and elucidating their properties.

That is, the present invention provides, as its first aspect, 11) molecules fi2. OX108~2.6X10'', (il) associated with a localized area of the erythrocyte merozoite morphology of the Zorazmodium parasite.Provides an inducible, linogenic antigen of Norasmodium genus production and its functional derivatives.

Molecular weight refers to the apparent molecular weight measured using polyacrylamide gel electrophoresis and standard molecular weight 41I fibers. The molecular weight of the antigen protein of the present invention is υ, K,
Laemmli (Nature, 1970 *
227:680-685). As commonly used standard molecular weight fibers (such as spectrin heterodimer (molecular weight 2.2 x 10' and molecular weight 2.4 x
10”).

The antigenic protein of the present invention has a molecular weight of approximately 2.35 x 10
It is also clear that the number 6 is particularly advantageous.

The antigenic protein of the present invention can be used for many Plasmodium spp.
For example, Plasmodium yoelii, P
, berghei.

It is known to occur in malaria parasites, ΩS, and P. chabaudi, and has also been found in primate malaria parasites, especially the human parasite, P. falci arum. In the "FJIa-related" boars used in this specification, the protein antigenic substances are not only derived from the merozoite form of Mala 1) parasitism (but also from the same or similar ainoacids derived from other sources). It is also meant to refer to the antigenically equivalent substance having the sequence 'J[.

The antigens of the invention are discovered late in the developmental cycle of the malaria parasite. That is, it is noticeable at the time when individual merozoites are formed during the process of red blood circle development. The antigen of the invention is not found in the ring and trophozoite stages. That is, P,f with a leopard 48 hour developmental cycle
In the case of M. alciparum1, the antigen is not degraded during the first 30 hours of development, and peak synthesis occurs during a period of about 36 to 45 hours, which is the key point for total protein synthesis.
It is slower than 56 hours.

As will be clear to those skilled in the art, all substances having the antigenicity of the proteinaceous substances defined above and fragments thereof, as well as antigens or fragments thereof) introduced into the substance, are identical to the maternal antigen. It has an advantageous immune system. Such substances are referred to herein as "mechanical derivatives."

The antigenic protein of the present invention can be produced by any one of the classification methods for producing this type of antigen. This woodcutter's method has %
Isolation of antigenic proteins from parasites, or anti! Chemical or biological production of proteins.

Included, for example, is expression from a host that has been suitably altered necrotically by complete or partial chemical or genetic manipulation.

The antigenic protein of the present invention is isolated from the merozoites of the parasite by means of a monoclonal antibody specific for the antigen of the present invention. Antigen separation methods using monoclonal antibodies have never been applied to the purification of malaria antigens.

That is, the present invention provides a second aspect of the present invention, in which (1) the solubilized red blood cells containing the merozoite form of Zolazmodium parasites are solubilized, and (1) the solubilized substance is contacted with a monoclonal antibody specific for a desired antigen. The present invention provides a method for producing an antigenic protein as defined above, comprising the steps of: 011) recovering the antigen from the antibody-antigen complex.

The present invention also provides the above-defined antigenic protein produced by the Jokiman process. Solubilization of merozoites can be performed by any method known in the art for this type of solubilization. Appropriate conditions can be used to solubilize the parasitic material without causing autolysis or degeneration. In particular, the bud-forming I11 substance cannot be solubilized by contacting it with a surfactant. As a surfactant, ionic,
Although nonionic surfactants can be used, nonionic surfactants are preferred. Examples of surfactants with this virtue include:
Non1det P 40, Triton x −
100*Br1j 99 (8M11, R respectively
ohm andHaas and ICIII (registered trademark), and Renew 30 (Honeywell
Examples include nopolyetherene tridecyl ether such as At1aa manufactured by Limlted, registered #B). The surfactant has a final #11 degree of 0.01 to 596
It is added in a v/v ratio.

The monoclonal antibody used in the method of the present invention can be produced by any method known in the art (for example, Milgtein:8cient1
fic American, 1980 m 243
(4): 56-64). This method involves treating mice against parasites, in this case malaria parasites.

For example, there is a method of immunization by infecting a person with Class 1 malaria parasite. Lymphocytes with an independent and strict ability to produce antibodies that are recognized to be heterologous antigenic determinants are separated and fused with mouse myeloid cells. It has antibody expression ability and clonogenicity of 1 fu. Screening for each hybridoma.

One or more cell lines expressing a monoclonal antibody specific for the antigenic determinant of interest are obtained and this cell line is used for large scale production of monoclonal antibodies.

This antibody is then used for the isolation and purification of the antigenic determinant of interest. Identification of antibodies that are % different from the antigens of the invention can be readily accomplished by simple tests known in the art. cell lines, monoclonal antibodies derived therefrom,
P, yoeli recognized by $5 and its antibodies
There are reports on the identification test λ of antigens from i (
Freeman et al.: rh.

Ho5t Invad@r Interplay, H,
Van aen Boschs I! .

gtsevtsr / North HOllan (l
Bioch@m1cal Preaa.

1980, pp 121-124), but I! f# A different antigen has not been isolated, and a given antigen has been isolated.
The monoclonal antibodies and cell lines used to test the monoclonal antibodies have only been partially characterized.
roa, and the aged cells are passed through the antibody-containing support. This is conveniently done using columns.

Antigenic proteins are recovered from antibody-antigen complexes by decomposing them. Conditions for carrying out this step are well known in the art.

Suitable conditions for maintaining protein immunity include thiocyanate ions such as potassium thiocyanate (K).
washing in the presence of a suitable surfactant.

The antigens described above can be used in vaccines to induce immunity against malaria in vertebrate hosts at risk of infection with Zolazmodium parasites. In this case, the antigenic protein can be mixed with a pharmaceutically acceptable carrier.

As an aspect of the Is3, the present invention provides a vaccine comprising the above-mentioned antigen and a pharmaceutically acceptable carrier.

Used as a pharmaceutically acceptable carrier, e.g.

Liquid media suitable for use as a vehicle for introducing the antigen tJ1 into a patient can be mentioned. The antigen protein may be contained in a carrier as a liquid solution.

It may also be suspended in solid form. It can also be solubilized by adding a pharmaceutically acceptable surfactant.

Adjupan-P used in vaccines to stimulate immune responses
can also be added to the vaccine, thereby increasing the effectiveness of the vaccine. Advantageous adjuvants which can be used in the present invention include Freund's complete 7 diepants, especially saponins, coryne-bacterium p.
Mention may be made of arvum (coparvax) and aluminum hydroxide, rum or mixtures of these or other known adjuvants.

The final concentration of antigen protein in the vaccine ranges from 0.2 to 5 Da/V, preferably from 0.5 to 215 Da/V! /L1. It is advantageous to manufacture m such that the range of T<K is preferably 1 breath/V. After 11 breasts, the vaccine can be filled into sterile containers and then sealed and stored at a low temperature, for example, 4° C., or it can be freeze-dried.

In order to induce immunity against malaria in a vertebrate host, an appropriately formulated vaccine Y is administered once or more than once. Single dose is 0.1 to 2, preferably 0
．． Particularly preferred is a vaccine with a weight of 0.2 to 11. A fourth aspect of the invention &X comprises administering to a host an effective amount of the above-mentioned vaccine. The vaccines of the present invention may be administered by any conventional method of vaccine administration, including oral and parenteral (including subcutaneous or intramuscular administration). It can be administered by injection.

Treatment is carried out by a single dose or multiple doses over a period of time of the vaccine.

The present invention will now be explained in more detail with reference to the following examples, which are not intended to limit the invention in any way.

Example 1 Induction of the hybridoma line P, yoelii immunoimmunized tri BALB/O mouse pancreatic cells, in the presence of polyethylene glycol,
PB-n81/1-Ag4-1 was fused with bone marrow noodle cells (Galfra et al.: Nature, 1977
#266:550-552), this cell Y.

RPMX medium (Litt) supplemented with 10% fetal bovine serum was used as a base.
lefield:8science, 1964, 1
45:709-710) and placed in 144 tissue culture wells of 2iU volume each. 10 days later,
The culture supernatant was tested for P. yoelii-specific antibodies by indirect immunofluorescence (ZIP). 143
Tests were conducted on each piece.

Thirty-eight cases showed a positive reaction. Antibodies specific to merozoite-related antigens were observed in culture number 77, but not to ring bodies or trophozoites. However, antibody binding was not uniformly observed.

localized to a region on or within each merozoite;
It was suggested that its existence was within the rose formation. Colonies derived from single cells were grown in semi-solid agar covered with a medium, and from this culture a clonal hybridoma cell line ay was produced. Continue, wzc 25,7
It was named 7. Cell line WIC! 25.77 is 19
July 16, 1981! It was entrusted to the Pasteur Institute as No. 161.

This hybridoma line was further grown as ascites in BALB/C mice, and fJ10 cape/V monospecific antibodies were found in the ascites and serum of these mice. Two other hybridoma lines, WIC2,5,37 and WIC25,86, also secrete antibodies specific for the same antigen when challenged by antibodies secreted by cell line WIC25,77.

Example 2 Preparation of P. yoelii-related antigens was grown to produce 80 to 90 antibodies, and red blood cells were collected and solubilized. 1 to inhibit proteolytic activity of this cell.
mM phenylmethylsulfonyl fluoride (PM8F
), 0.1 mM) silyl L-lysine chloromethyl ketone (TLCIC), 5 mM ethylene glycol-bis-(inoethyl ether)-N,N-tetraacetic acid al (]!G'l"A): M and Contains 5% iodoacetic acid. 50mM Tris, 5mM x 5-lendia (ytetraacetic acid CEDTA) = 1 qlr No.
n1dstP40 (FJ(8,0)) was degraded and solubilized at 4°C in buffer. The degraded cells were separated at 100,000 g, and about 70 parasite proteins (this fraction (Calculated from the ratio of 35B-methionine after in vitro introduction)
The supernatant containing .

Protein A-'8sparo was obtained from the ascites of a mouse containing the hybridoma line WIC25,7Z.
The immunoglobulins were purified by binding to se and performing a single step elution at pH 3.0. The immunoglobulin fraction was converted to cyanogen-promide-activated Bepharoa.
To.

Coupling was carried out using immunoglobulin 10 da/l swollen del. The immunoadsorbent was thoroughly washed and loaded onto a column, containing 10 mM 'I'ria, 1 mM EDTA.
, 1 RGTA, 1 iP40 (pH 8,0
). ``Supernatant liquid obtained as described above''
t-110mMTris*1mMBDTA,
1mMI! GTA, 1% NP 40 (pH 8
,0) and washed with this buffer. Non-specific binding was removed by washing with 5 column volumes of this buffer to which 5 M NaCjg was added. next,
Column'& 10mM 'I'riB, 1mM
KDTA.

ImM BGT human, 0.011iP40 (pH 8,0)
Washed with containing buffer. Specific elution was performed with the same buffer containing 2M potassium thiocyanate.

The bath material was again subjected to immunoadsorption clotgelography. Concentrate the specific eluate to 10 - of the original volume and
mM'['ria, 1 mM m1M'person, 1
mMBGTA, 0.1% HP 40-containing buffer (p
Dialyzed against H8,0'). This sample was applied to a column re-equilibrated with this buffer and 1jl of residual material (
1, Q mM Tri8゜1 mM IIDTA,
1mM IG'l'A, Q, ()j% NP
4 Q'j+5 and 2 M KBCM-containing buffer (P
) I・8.0).

Eluate Y: evaporated and dialyzed, antigen V%.

Example 5 Biochemical properties of p. yoelii related antigen Example 2
The antigenic substance obtained in 8D8 was subjected to lumi electrophoresis, and
．． Molecular weight labeled spectrin heterodimer (molecular weight 2.2 and 105
and 2.4 x 105)
5X10"t' was obtained.

Cells containing t were incubated in a medium containing highly active 558-methionine to introduce 3-methionine 7Y into proteins. 8 taphyloco with monoclonal antibody 25.77 and Protein A'It
Ill fiber antigen protein was obtained by precipitating the immune complex by immunoprecipitation with ccuaurel Ii cells.

When this material was subjected to YBDfl del electrophoresis, a single band with an apparent molecular weight of 2.35 x 105 was detected by autoradiography. r slices containing antigenic proteins in the presence of chymotrigisin. upon incubation, the peptide digestion products were released. These peptides Vp) (4,4) are developed by thin-layer electrophoresis and then chromatographed at right angles using butanol-acetic acid-water-pyridine. Figure 1 shows standard tests including peracid-staining and 1181-iodinated concanavalin A on proteins in acrylamide gels.
Lectin binding results showed that this protein was not glycosylated.
oae or wheat bran agglutinin - aepharoae
It was also confirmed that the size of the protein was not affected even in the presence of tunicamycin, a synthetic inhibitor of N-glycomolization of Y-proteins produced by the protein synthesis. O'Farrall (J, Biol.

Chem, 1975 * 250: 4007-
4021), the isoelectric point of the protein was measured using a two-dimensional r-luminescence electrophoresis system. When amphorin was used and one gradient Y pH was set to 3 to 10, the isoelectric point was within the range of pH 6±1.

Groups of 5% BALB/C mice were immunized with the antigenic protein obtained in Example 2 or the control preparesin using the following Skageer.

Group 1: 20 μg of antigen was emulsified in 25.77w70 India Complete Adjuvant (YC) and injected intraperitoneally (ip) at 0.21 Ij volume on day 0.

20 μg of antigen 25.77 was suspended in normal mouse serum 0.21 and injected intravenously (iv) on the 18th day.

20 μl of antigen suspended in saline 25.77 V No. 42
I had an IF injection on the same day.

Group 2: FCA was administered ip on day 0, k Y tv of 1M8 was administered on day 18, saline alone was administered yiv on day 42, and serum samples were taken on day 49.
F) was titrated and tested. The results are shown below.

Group Mouse IIF titer Antiserum 1
1) 1:1280 merozoite 2
>1:1280 #5 )1:1280 1 4 >1:1280 #5 >1:1280 #2 1 <1:40 -2
<1:40 - 5 <1:40 - 4 <1:40 - 5 <1:40 - The pattern of fluorescence staining using serum from mice immunized with antigen 25.77 was similar to that of monoclonal antibody 25.77. could not be distinguished from the pattern obtained using

Staining was restricted to merozoites from P, y□·111.

These results demonstrate the purity and immunological properties of the antigen 25.77 produced by the ipH2 method.Treatment: Protection against infection The antigen protein obtained in Example 2 or the control preparation N, of 5 BALB/O mice. Groups were immunized according to the following schedule.

Group 1: 12 μg of antigen 25.77 was emulsified in ff0A and injected ip at 0.2-vol on day 0.

#124 antigen 25.77 in 10A was injected ip on day #135.

Day 50 Antigen at 20 pH in 0.1 wLl saline 25.
I had one injection of 77.

Details 2: 2 μg of antigen 25.77 was taken in yOm and injected ip on day 0.

On day 35, 24 antigens 25.77 in 1 PaA were injected IP.

Antigen 2 of 2011/I in 0.1 Wll saline on day 50
5.77 was injected for 1 month.

Group 6: 10 ml saline was injected ip on day 0.

On the 35th day, 1 p of saline was injected in 10 people. On the 50th day, 1 p of saline was injected. On the 60th day, a serum sample was taken, and antibody titration and immunoprecipitation were performed. Analysis was carried out. On day 61, mice were given P, yoelii YM
104 '44 live red blood cells were injected YIV. The results of the immune response of each group to the immunized mice are shown in the first group. At the conditional doses employed, immunization with 12 ml of antigen 25.77 elicited a stronger antibody response than with 24 ml of antigen 25.77.

1 >1:10.240 merozoite 2
>1:640 #5 (
1:40 Non-specific Figure 2 shows the average parasitemia after administration of P. yoelii in each group of 5 immunized and control mice.

P, yoelii challenge results for each group Y: Figure 2 shows. The group showing a high antibody titer also had a large preservation effect against challenge.

All control mice died within 8 days after infection. Antigen specificity of serum from immunized mice VfP! The antibody response was determined by molecular weight 2 ml used for immunization, as examined by immunoprecipitation.
35,000 merozoite-specific antigens.

Red blood cells were administered intraperitoneally 2 to 4 times for immunization, followed by the same procedure.
A booster was administered to the q*v venous circle, and 6 days later, the pancreatic cells described in Ga 1 and P 5- Ha 1 / 1- Ag4
-1 Perform fusion of bone marrow noodle cells. P on IXP exam.

Antibodies that react with falciparum merozoites are 71
-, and from this culture, 11 hybridoma lines were cloned and grown as ascites in BALB/C mice. All of the obtained I. hybridoma lines secreted antibodies, which were expressed by P, yoel
ii A pattern similar to the IIF pattern exhibited by the antibody of Example 1 against merozoites YP, falciparum
% BALB/Crusy, P, falci shown for merozoites
Human immune serum 13t was obtained from the lysate of p5Lrum-infected cells.
Immunization was performed by intraperitoneally administering 1004 g of purified protein remaining on an affinity column containing immunoglobulins collected from A. Three weeks later, a booster of the same substance was administered intravenously, and 3
P3-NB1/I Ag in pancreatic cells after 1 day
Fusion of 4-1 cells was performed.

The schizont-enriched fraction of parasite-infected erythrocytes was prepared by centrifugation through a Petcal 1 layer and labeled with 368-methionine for 2 hours. Collect these cells,
It was decomposed and solubilized as described in Example 2 at 4°C. After centrifugation, supernatant solution V5 Tipolyacrylica 2 dollar 8D8
analyzed by r-lumi pneumophoresis.

Along with many small peptides, this quality has an apparent molecular weight of 2.05, 2.18, 2.25, 2.55, 2
．． 50 and 2.58 x 10 labeled polypeptides were the main components.

Serum % from 14 Cambian donors: in v
itr.

and tested by the P.ra1ciparum invasion inhibition test.During one eel invasion cycle, these sera had 16
It showed an effect of inhibiting arrogant talk for ~90m. Then these serum'
The k3IS8-methionine labeled polypeptide was subjected to an immunoprecipitation test. Immune serum precipitates at least 40 polypeptides, 10 of which have molecular weights of 2. OX
It was more than '10'. Especially 1 molecule 112.35
The X 10' polypeptide caused a strong precipitation in the case of a serum with a strong inhibitory effect, and only slightly precipitated in a serum with a low effect in the invasion inhibition test.

In vitro development of intrabulbar parasites yit56
There is no time [Treatment with sorbitol twice and synchronization is also 48
Temporal relationship of polypeptide synthesis during time cycles)!
/@I was able to defeat it. Schizont is the second sorbitol treatment 2
It was first noticed after 7 hours, and the first re-invasion occurred after 39 hours of eye opening. Polypeptide platform 1V for 30 seconds: 588-methionine tone was used every 6 hours. A peptide with a molecular weight of 2.35 x 10' that is recognized by the human immune system is synchronized with v! 6, 12.

It was not synthesized at 18-M and 24 hours (ring and trophozoite stages)'. Synthesis of this polypeptide in the receptacle was observed most frequently at 30 hours, but most synthesis was seen at 36, 42 and 45 hours. Since the peak of total protein synthesis during mitotic reproduction occurs after 36 hours of eye opening, this protein is synthesized in the late stage of merozoite formation, during the development of the merozoites.

Example 8 Vaccine The vaccine used for immunization has the following composition and can be produced by a conventional method.

Prescriber Antigen 2W9 Physiological Saline Total Volume 11Ij Prescription B Antigen 1Target o iy's, I Lml!
Physiological saline, total volume 1v

[Brief explanation of drawings]

Figure 1 shows P-7oelii-related antigens include 388-methy, t
Figure 2 shows the average parasitemia of 5 mice in each group immunized with antigen 25.77 or control (Group 1 -). 124 treatment, mu: 2nd group - 2 treatment %O: 3rd group 1 control). Agent Asamura Kōgai 4 Meikyo Weikyogi Soma Proceedings Amendment (Method) % Formula % Representation of the incident 1989 Patent Application No. b22”70 No. 2, Title of Invention Yonghyukkei, Ia Kenya 3. Person making the amendment □ Relationship to the case Patent applicant 4, Agent 5, Date of amendment order: 27/1988 8, Contents of amendment as shown in the attached sheet

Claims (1)

[Claims] (13(1) Apparent molecular weight 2.0 to 2.6 x 10
'The genus Derasmodium produces red blood cell merozoite forms associated with localized areas. Protection of Delasmodium parasites! ! In-derived protein antigen and its 1m dry derivative (23 apparent molecular weight f12.35 x 10' 64117) Antigen 133 IF-producing human derasmodium parasites according to claim 1 Antigen according to any one of paragraphs 1 or 2 (4) Parasites - Plasmodium falciparum (Plaamidae)
(1) solubilizing red blood cells containing the merozoite form of Delasmodium parasites, and (I) converting the solubilized substance into a final antigen; contacting with a specific monoclonal antibody to form an antibody-antigen complex; and (iii) recovering the antigen from the antibody-antigen complex. Antigen (6) according to any one of claims 1 to 4, which is prepared by chemical or biological regeneration. Antigen according to any one of paragraphs to paragraphs 4 to 4+73 by a method consisting of (a) isolation of the antigen from the red blood cell merozoite form of the parasite, 8 and (b) chemical or biological regeneration of the antigen. 1% liver Method for producing an antigen according to any one of claims 1 to 4 (8) Antigen according to any one of claims 1 to @6 , and a pharmaceutically acceptable carrier (9)
mfalclparum) Claims slFF1iil Vaccine according to Clause sI8+10 Claims @Claims 1 to Sg6 for use in inducing complete Q in a vertebrate host with a potentially infectious agent The antigen described in any one of Items 1 to 6 is administered to a host. Immunity against malaria in a potentially infected vertebrate host '4I: Method of inducing malaria The antigen described in claim 10 in the form of a vaccine The antigen is in the form of a vaccine in claim 1 1
Method described in Section 1