JP2001046061A - Weakened dengue virus and dengue fever vaccine using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a weakened dengue virus having genetically extreme stability without reversion to high virulent, useful for producing a vaccine, etc. by subjecting a dengue virus separated from a dengue fever patient of slight disease to passage to the sensitive cell of the dengue virus. SOLUTION: A dengue virus separated from a dengue fever patient of slight disease is subjected to passage to the sensitive cell of the dengue virus to give a weakened dengue virus such as ThNHp 11/93 strain, etc. A monofunctional or polyfunctional dengue fever vaccine is prepared by making the vaccine include the weakened dengue virus as an active ingredient in an amount to perform immunity. A mixed vaccine is preferably prepared by making the vaccine include the weakened dengue virus as one of plural active ingredients in an amount to preform immunity. The dose of the vaccine is 0.2-1.0 ml per adult.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an attenuated dengue virus, a method for preparing the same, and a dengue vaccine containing such an attenuated virus as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Dengue fever is an acute infectious disease caused by dengue virus infection. This virus is serologically classified into four types, type 1 to type 4, and is transmitted in the cycle between humans and mosquitoes, mainly in children, and its symptoms are classified into the following three types: fever and chills・ Mild dengue fever (D
F: Dengue Fever), after fever, bleeding tendency,
Dengue hemorrhagic fever (DH with plasma leakage and thrombocytopenia)
F: Dengue Hemorrhgic Feve
r) Furthermore, after DHF, extremely severe dengue shock syndrome (DSS: Dengu), which may lead to a loss of consciousness from a shock state associated with a rapid drop in blood pressure and death.
e Shock Syndrome). Regarding dengue vaccine, a total of 14 kinds of vaccines using attenuated strains of dengue virus, recombinant vaccinia virus, subunits produced by baculovirus or yeast, synthetic peptides, recombinant DNA, etc. as active ingredients are known. I have. However, none of these vaccines has yet reached practical application (The Jordan Re.
port 1998, page 96, National I
nstate of Health, USA).
In addition, among the above, what is known to be under clinical test is the type 2 dengue virus attenuated strain 16681-PDK5
3 monovalent live vaccine (Vacc
ine, 14, 329-336, 1996), and 1 to 1
Live tetravalent vaccine using each attenuated strain of each type 4 dengue virus (Vaccine, 17, 597-601, 199)
9).

[0003]

All of the attenuated strains of the live vaccines described above have been attenuated by passage of a virulent virus isolated from a DHF patient in cell culture, and have been attenuated from attenuated to virulent. May be reverted to
Live vaccines using such attenuated strains are always dangerous and their safety cannot be guaranteed. In addition, other vaccines than the live vaccine have not yet been confirmed for their safety and efficacy, and are still in the stage of preclinical testing.

[0004]

SUMMARY OF THE INVENTION The present invention provides a genetically extremely stable attenuated dengue virus which does not revert to virulent and a method for producing the same. Furthermore, the present invention provides a monovalent or multivalent live and inactivated dengue vaccine, and a mixed vaccine with other heterologous antigens, using such an attenuated virus as an active ingredient.

[0005]

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the following surprising findings by the inventor: (1) Mild dengue (D
F) The so-called DF-derived dengue virus isolated from patients is already an attenuated virus itself in the wild; (2) DF-derived dengue virus produces DF in both primary and re-infection, resulting in DHF and DSS No (D
DF in the first infection with HF-derived virus, and DH in the re-infection
F, DSS is caused by re-infection of DSS-derived virus); and (3) DF-derived dengue virus is more infectious and cytokine-inducing for human peripheral blood leukocytes (PBL) than both DHF-derived and DSS-derived viruses. Is significantly or significantly lower (described later in Examples).

Based on the above findings, the most significant features of the present invention reside in the dengue virus isolated from DF patients and its industrial use. According to the present invention, the following embodiments (1) to (5) are provided, respectively. (1) a method for preparing an attenuated dengue virus, wherein a dengue virus isolated from a mild dengue (DF) patient is passaged to its susceptible cells; (2) a dengue virus isolated from a mild dengue patient. An attenuated dengue virus prepared by subculturing into susceptible cells; (3) an attenuated dengue virus according to claim 2, wherein the prepared attenuated virus is the ThNHp11 / 93 strain; and (4) a dengue virus isolated from a mild dengue patient. Immunizes with an attenuated dengue virus prepared as an active ingredient by subculturing into susceptible cells A monovalent or multivalent dengue vaccine characterized in that it contains an amount of a monovalent or multivalent dengue virus; and (5) an attenuated dengue virus prepared by passing a dengue virus isolated from a mild dengue patient to its susceptible cells. Amount of immunity as one of the effects,
A combination vaccine characterized by containing.

Isolation and passage of dengue virus: Viruses can be isolated from body fluids, organ tissues, etc. of dengue patients using dengue virus-sensitive cells. A remarkable point of the present invention is to isolate dengue virus from DF patients. As a virus isolation material, a material derived from DF patients, particularly serum of DF patients is desirable. Examples of the sensitive cells include human-derived cells such as BSC-1 and HL-CZ, monkey-derived cells such as LLC-MK2 and Vero, primary monkey kidney cells, and BHK-2 derived from hamsters.
1. C6 / 36 derived from mosquitoes, brain cells of mice and hamsters, embryonated chicken eggs and the like can be used. Passage of the freshly isolated virus in dengue virus-sensitive cells is performed for the purpose of mass production of the virus particles. The cells used for the passage are not limited to one type, and two or more types of various cells can be used in combination. For example, C6
The virus passaged into / 36 cells can be passaged into Vero cells. Such a virus passage is carried out at a culture temperature of about 25-37.
At C, the virus production gradually increases with the virus acclimation to the cells by the passage, and the passage number is usually about 1 to 50.
The virus acclimation, that is, the virus production is maximized within the range of about 1 to 30 generations, preferably about 3 to 15 generations, and the mass production of the desired virus particles becomes possible. Such adapted viruses are eligible as seed viruses for vaccine production, and conversely, vaccines produced with this seed are excellent, safe and effective.

Measurement of virus infectivity: The virus antigen-bearing cell population in the susceptible cells can be measured as an index. For example, the number of foci formed in BHK-21 inoculated with a serially diluted sample virus solution, that is, the number of focus formation units (FFU) is counted, and
Per measured value, expressed in FFU / ml.

Induction of cytokines in human PBL by viral infection: healthy human peripheral blood lymphocytes (PBL)
After inoculation with the dengue virus, the culture is cultured, and the culture solution is collected over time to obtain a specimen. Using the supernatant of the low-speed centrifugation of each sample, the change in the amount of cytokine production in the BPL culture solution induced by virus infection is quantified. Human PBL is separated and procured from human venous blood. The cytokines to be quantified include B cell stimulating factor interleukin-6.
(IL-6), interleukin-1β (IL-1β) involved in the execution of cell death and the inflammatory response, tumor necrosis factor-α (TNF-α) that causes hemorrhagic necrosis, and the like are produced. The amount can be determined using a commercially available ELISA kit. In addition, the virus infectivity of the low-speed centrifugation supernatant, that is, the amount of virus produced in the infected PBL,
Measure FFU / ml.

[0010] Determination of the virus antigen positive rate of infected PBL:
It is measured by an indirect fluorescent antibody method using a low-speed centrifugal pellet of the infected PBL culture solution. For example, an anti-dengue virus highly immunized mouse antibody is used as a primary antibody, and a FITC (fluorescein isothioc) is used as a secondary antibody.
Yanate) -labeled anti-mouse IgG sheep antibody. Positive rate P% is calculated by counting the number n of cells positive for dengue virus antigen with respect to the total number N of cells, and P = 1
It is calculated from 00 × n / N.

[0011] Significant difference test: Significant difference can be estimated by the Student's t test for the above-mentioned cytokine production amount and chi-square test for the antigen positive rate.

Characteristics of the attenuated dengue virus derived from DF: This virus was isolated from human PB at a concentration of 1 × 10 ⁶ cells / ml.
After infecting L at a multiplicity of infection of 10 (FFU / cell),
The cultivation at 37 ° C. was continued for 4 days, during which time the virus antigen positive rate (infectivity to PBL) and the amount of Sato quine production measured about 4 times in total about every 24 hours from the infection were as follows:
Always within the following ranges: the antigen positive rate is about 40% or less,
IL-6 is less than about 2,200 pg / ml, and TNF-
α is about 1,000 pg / ml or less; or a significant difference in that these measurements are significantly lower than the DHF- and DSS-derived virus-infected PBLs tested simultaneously. Certainly, this is to be estimated stochastically at a risk rate of 5% or less by a significant difference test using chi square for antigen positive rate and Student t for cytokine production.

Preparation of vaccine: attenuated dengue virus strain,
For example, ThNH-p11 / 93 obtained in Example 1 described later.
By passing the strain to Vero cells, a seed virus for vaccine production is obtained. Ver.
After culturing in step (o), the culture solution is collected to prepare a virus suspension, and then the cells and the components in the suspension are removed by filtration, centrifugation, etc., to obtain a vaccine stock solution. From such a stock solution,
A live attenuated dengue vaccine or an inactivated dengue vaccine can be prepared. In addition, virus inactivation
For example, an inactivating agent such as formalin, β-propiolactone, glutardialdehyde, or the like is added to a virus suspension, mixed and reacted with a virus. When, for example, formalin is used as the inactivating agent, its addition amount is about 0.00
5-0.1% (V / V), inactivation temperature about 4-38 ° C,
The inactivation time is about 5-180 days. The obtained vaccine is provided in liquid or dry form in a sealed vial or sealed ampule. Subcutaneously inoculate 0.2 to 1.0 ml per person. In addition, this vaccine contains monovalent to type-4 dengue virus antigens as an active ingredient.
It is provided in the form of a monovalent dengue vaccine. Further, this vaccine is mixed with vaccines of other species, for example, vaccines of Japanese encephalitis, yellow fever, hepatitis B, influenza, rotavirus, poliovirus, etc., and tetanus toxoid, etc. Can also be provided. In addition, the above vaccine is regulated by the Pharmaceutical Affairs Law (the 1st Act of 1960).
No. 45) Notification No. 2 of the Ministry of Health and Welfare based on the provisions of No. 42, Paragraph 1
Manufactured in accordance with the rules set forth in No. 17, "Biological Product Standards", process control and quality control. For example, for dengue vaccine, criteria such as "Japanese encephalitis vaccine" and "Dried Japanese encephalitis vaccine" are applied, and after passing various tests related to safety, efficacy, etc., and after eligibility as a vaccine is confirmed Available on the market.

Hereinafter, embodiments, configurations and effects of the present invention will be specifically described with reference to examples and reference examples. However, the present invention is not limited to these.

[0015]

EXAMPLES Example 1 Virus isolation: Using a mosquito-derived cell line, C6 / 36, three strains of dengue type 2 virus were isolated from sera of dengue patients in the northeast of Thailand in 1993, respectively. That is, T
The hNH-p11 / 93 strain was obtained from Th
The -28/93 strain was from a DHF male patient confirmed by hospitalization, and the ThNH-7 / 93 strain was from a hospitalized DS.
Each was separated from S patients.

Example 2 Measurement of virus infectivity: The infectivity of the three strains obtained in Example 1 was measured by FFU using BHK-21 cells (Bi).
ken Journal, 21, 137-147, 19
78). For cell culture, flat bottom 96 wells (0.1 ml
/ Well) A microplate [manufactured by Nunc (Denmark)] was used. That is, the seed cell concentration was 1 × 10
⁴ / ml, and cultured at 37 ° C. for 2 days in a 5% (V / V) CO ₂ incubator, followed by a final concentration of 5% (V / V).
Two wells were used for each virus solution obtained by serially diluting fetal bovine serum 10-fold with the mixed Eagle's medium.
Each well was inoculated. After shaking the plate every 30 minutes for 2 hours at 37 ° C. to adsorb the virus to the cells, a final concentration of 0.5% (W / W) methylcellulose 4000 is added, and a mixed maintenance medium (0.1 ml / well) is added. And layer 3
The cells were cultured at 7 ° C for 3 days. The FFU count after the end of the culture is
It was performed by immunoperoxidase staining. First, the above overlay medium was removed, and the plate was placed in phosphate buffered saline (PB).
After washing with S), the cells were fixed with PBS containing 5% (V / V) formalin at a final concentration of 20 minutes, and then washed with PBS.
Next, a final concentration of 1% (W / W) Non at room temperature for 20 minutes.
The cells were contacted with PBS supplemented with idetP-40 to increase the permeability of the cells, blocked by Block Ace (Snow Brand Milk Products, Japan), and washed with PBS. This was reacted with a 500-fold diluted DHF patient serum for 60 minutes, and then with a 500-fold diluted HRPO-labeled anti-human IgG antibody [Cappel (USA)] for 60 minutes. The HRPO reaction was performed at a final concentration of 0.5 mg / ml in 3,3'-diam.
inobenzidine 4 hydrochloric acid [Sigma Che
medical (USA)] and 0.02% (V / V) H ₂
The O ₂ using a substrate containing 5 minutes to perform. Next, after washing the plate with tap water, brown foci were counted by microscopy to calculate the infectious titer FFU / ml. The virus infectivity titers (× 10 ⁶ FFU / ml) used in the following examples were 1.5 to 8.8 for the ThNH-p11 / 93 strain, respectively.
5, ThNH-28 / 93 strains are 1.8 to 18, and Th
The strain of NH-28 / 93 was 1.36 to 39.

Example 3 (1) Preparation of PBL: PBL material was collected from three healthy subjects. However, donor 1 has a history of dengue type 1 fever, and donor 2 has a history of dengue type 3 fever.
Had no known history of infection. PBL is Lymphopre
Separation was performed by density gradient centrifugation using p [Nycomed Pharma AS (Norway)]. That is, 10 ml of heparin-added venous blood was collected from each of the above donors, and an equal volume of RPMI 1640 medium [Gibco
BRL (USA)], and then mixed with 15 ml of L
ymphoprep, 1,500 rpm at room temperature
and centrifuged for 30 minutes. The PBL fraction was collected and L
In order to remove ymphoprep, Hanks solution [B
io Whitaker (USA)], the pellet was collected, and the cells were washed. After counting the concentration of the collected cells, the cells were divided into four equal parts, and each equal part was washed again with Hanks' solution to obtain PBL.

(2) Virus Infection to PBL: Example 1
And the anti-dengue virus serum (10,000-fold dilution) collected from each of the above PBL donors
And reacted at 37 ° C. for 1 hour. After the reaction, each virus was infected to each of the four divided PBLs at a multiplicity of infection of 10%.
(FFU / cell) and cultured at 37 ° C. for 2 hours in a CO ₂ incubator. Next, the infected cells were
Wash twice with ks solution and bring the cell concentration to 1 × 10 ⁶ cells / ml
After adjusting with a medium, the culture was continued at 37 ° C.
The medium was a 6-volume AIM-V medium [Gibco B
RL Life-Technology (USA)],
3 volumes of Iscove's modified Dulb
ecco's medium [Gibco BRL Life-T
technology (USA)] and one volume of heat-inactivated AB serum [Sigma Chemical Company (USA)]
Manufactured]. As a control, the supernatant of uninfected C6 / 36 cell culture was used instead of virus. During post-infection culture, 0.2 ml of the PBL culture was sampled over time and centrifuged at 4 ° C. at 1,500 rpm for 5 minutes, and the supernatant contained cytokines, ie, IL-6, IL-1β,
And TNF-α, and the cells in the pellet fraction were used for dengue virus antigen detection. Cytokine quantification was performed using an ELISA kit [Edogen (USA)] according to the manual. In addition, the detection of dengue virus antigen was performed by using anti-dengue type 2 virus / mouse hyperimmune ascites as the primary antibody and FI as the secondary antibody.
TC-labeled anti-mouse IgG hidge antibody [Cappel L
laboratory (USA)].

(3) Results: Positive rate of dengue type 2 antigen in infected PBL: Positive rate (%) fluctuated with time due to differences depending on the PBL donor. However, that of the DF-derived virus ThNH-p / 93,
DHF in the range of 7.4 to 35.0% (≦ 40%)
It was estimated that there was a significant difference at a risk rate of 5% or less, which was always the lowest and significantly lower than those of both virus of origin and DSS. Table 1 shows the results.

[0020]

[Table 1]

Production of IL-6 in infected PBL: I
L-6 production (pg / ml) was 12
It was almost plateau for ~ 210 hours. The production amount of the DF-derived virus ThNH-p / 93 ranges from 1,767 to
It is in the range of 1,977 pg / ml (≦ 2,200 pg / ml) and is always the lowest and significantly lower than those of both DHF-derived and DSS-derived viruses, with a significant difference at a risk rate of 5% or less. Was estimated. Table 2 shows the results.
Shown in

[0022]

[Table 2]

Production of IL-1β in infected PBL:
The amount of IL-1β produced (pg / ml) was 1
It reached a maximum at 2 hours, reached 350 to 700 pg / ml (180 pg / ml for the control), disappeared and became undetectable at 72 to 96 hours. During this time, the virus-infected PBL always showed a higher value than the control. But,
Regarding the amount of IL-1β production, no correlation or significant difference was observed between dengue fever symptoms, that is, DF, DHF and DSS.

Production of TNF-α in infected PBL:
The amount of TNF-α production (pg / ml) was 1
It reached a maximum in 2 hours, disappeared in 48 to 72 hours, and became undetectable. The production amount of the DF-derived virus ThNH-p / 93 was 345 to 672 pg / ml (≦ 1,000 p
g / ml), which is always the lowest and much lower than those of both DHF-derived and DSS-derived viruses, and it is estimated that there is a significant difference at a risk rate of 5% or less. Table 3 shows the results.

[0025]

[Table 3]

Example 4 Difference in Gene Level between Virulent and Attenuated: Reference Example 1 described below shows the difference in amino acids encoded by each gene of the three highly virulent to attenuated dengue virus strains obtained in Example 1. ing. Of these amino acids, which amino acid substitution caused the difference between virulent and attenuated was analyzed by site-directed mutagenesis. These include cDNA clones of full-length genomic RNA of type 2 dengue virus producing infectious dengue virus particles (Virology, 230-30).
8, 1997), and “TaKaRa LA PCR
In vitro Mutagenesis Kit "
(Manufactured by Takara Shuzo) was used. First, a virus (R) in which the 16th amino acid of PrM was arginine was obtained from infectious RNA obtained by transcription of the clone. Next, after preparing a virus (R → I) in which the arginine was mutated to isoleucine, the virus (R → I) was further backmutated to obtain a virus (I → R). The infectivity to PBL of these three viruses was measured in the same manner as described in Example 3 by indirect immunofluorescence on cells three days after infection. However, the infectivity was determined by counting the number of dengue virus antigen-positive cells in a total of 20 visual fields, and expressing the average value per visual field, the number of positive cells / field. The significant difference was based on the Student's t test. Virus (R) and virus (I →
Both infectivity of R) was significantly lower than that of the virus (R → I), and it was confirmed that there was a significant difference at a risk rate of 5% or less. In addition, there was no significant difference between the virus (R) and the virus (I → R). Table 4 shows the results.
This means that the R → I or I → R mutation of the 16th amino acid of PrM is a very important marker between both attenuated and potent.

[0027]

[Table 4]

[0026]

[Reference Example] Reference Example 1 Difference in amino acid sequence encoded by dengue virus genome: Reported by the present inventors (Virology, 244,
458-466, 1998). Table 5 shows the main differences in the amino acid sequences encoded by the genomes of the three virus strains derived from DF, DHF and DSS, respectively, for a total of 11 amino acids.

It should be noted that in Table 5, the D
F is ThNH-p11 / 33 strain, DHF is ThNH-2
The 8/93 strain and DSS mean each virus of the ThNH-7 / 93 strain. PrM related to “genomic region”
Indicates a pre-M protein (M is a matrix), and NS indicates a region on the genome occupied by a gene encoding each of the nonstructural proteins. The number related to the “amino acid number” is an amino acid number counted from the N-terminal to the C-terminal in the protein encoded by each gene, with methionine at the start codon as the first. ThN from DF
Such a difference in amino acid sequence between the H-p11 / 33 strain and the other two strains causes a difference in attenuated to virulent pathogenicity, and the action / function shown in Example 3 above, ie, ,
It is understood that differences are manifested as differences in sensitivity to human PBL (antigen positive rate) and cytokine inducibility (production amount). In particular, from the results described in Example 4, the difference in the 16th amino acid (arginine or isoleucine) of PrM of dengue virus is extremely important for distinguishing between attenuated and virulent,
In addition, this can reduce dengue virus from virulent to attenuated,
Alternatively, a technique is provided that can optionally convert attenuated to highly poisoned. Therefore, the significance of Table 5 can be used for genetic diagnosis for predicting or determining the degree of dengue fever or attenuated or highly toxic, for example, diagnosis using RT-PCR, and further described in Example 4. In addition, the present invention provides a method for interchangeability between attenuated and virulent viruses by genetic engineering and an attenuated or virulent virus prepared by the method.

[0028]

[Table 5]

[0029]

Industrial Applicability The present invention provides an attenuated dengue virus strain which is superior and safer and is genetically stable as compared with conventional dengue vaccine strains, a method for preparing the same, and an extremely safe attenuated strain containing such attenuated strain as an active ingredient. It provides an effective and effective dengue vaccine and ensures the prevention and strong development of dengue prevention worldwide. Although the dengue endemic area is limited to tropical to subtropical,
In a world where international exchange is thriving, it will contribute to the health of all humankind, not just those living in endemic areas, and will also bring the gospel to the global epidemic control system and international activities.

Claims (5)

[Claims] 1. A method for preparing an attenuated dengue virus, wherein a dengue virus isolated from a mild dengue patient is passaged to its susceptible cells. 2. An attenuated dengue virus prepared by passing a dengue virus isolated from a mild dengue patient to a susceptible cell. 3. The prepared attenuated virus is ThNHp11.
The attenuated dengue virus according to claim 2, wherein the strain is / 93 strain. 4. A monovalent or polyvalent dengue vaccine containing an attenuated dengue virus prepared as an active ingredient in an amount sufficient to produce immunity by subculturing a dengue virus isolated from a mild dengue patient into a susceptible cell. 5. A mixed vaccine containing an attenuated dengue virus prepared by subculturing a dengue virus isolated from a mild dengue patient to a susceptible cell in such an amount as to provide immunity as one of a plurality of active ingredients. .