Patent Application
20250076299
The present invention relates to a method for functional evaluation of antibody, which uses pseudovirus.
Dengue virus, which belongs to the genus Flavivirus of the Flaviviridae family, is a +strand RNA virus, and its genome consists of three structural protein regions (C, prM, E) and seven non-structural protein regions (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). Dengue virus includes four types of viruses (DENV-1 to DENV-4) and they are biologically classified as serotypes. Dengue virus is transmitted by mosquitoes. When a dengue virus-carrying mosquito bites a human, the injected virus first infects dendritic cells, monocytes, and lymphocytes in the skin and undergoes primary proliferation. The virus then travels through the bloodstream to various organs in the body and undergoes secondary proliferation. The condition in which the proliferated virus is present in the blood is called viremia, and when a mosquito sucks such blood, it becomes a new virus-carrying mosquito and can transmit the virus to other humans.
Dengue virus becomes epidemic mainly in tropical/subtropical regions where the main mosquito vector, Aedes aegypti, lives. In recent years, it is known that the Aedes albopictus, which lives in temperate regions including Japan, also transmits dengue virus. When a domestic dengue virus epidemic occurred mainly in the Kanto region in 2014, the Aedes albopictus was the vector animal. The population at risk of being infected with dengue virus is estimated to be 3.9 billion people, and 390 million people, which is one-tenth the number, are infected with some kind of dengue virus every year. Even when humans are infected with the dengue virus, many of the infections are inapparent infections, and only about 100 million of them actually develop symptoms.
As described above, even though many infected people show inapparent infection, some of the infected people may develop dengue fever, a transient febrile disease. The main symptoms of dengue fever are relatively mild, including fever, headache, muscular pain, and joint pain. Most cases of dengue fever are mild, but a part of patients with dengue fever develop a severe form, dengue hemorrhagic fever. The symptoms of dengue hemorrhagic fever (plasma leakage, bleeding tendency) are severe and can lead to death if proper treatment is not provided. It has been epidemiologically proven that first infection often causes dengue fever and the second infection with a dengue virus of a serotype different from the first infection increases the risk of developing dengue hemorrhagic fever. It is said that a third or subsequent infection does not occur.
Although the mechanism of severe progression in dengue viral infection has not been completely elucidated, it is known that higher levels of viremia tend to result in severe progression. Antibody-dependent enhancement (ADE) is one of the leading theories for the onset mechanism of severe progression, and induces hyperviremia. ADE is a phenomenon in which antibodies, which are supposed to neutralize viruses, actually worsen the viral infection. Antibodies that have bound to virus promote viral invasion into cells via Fcγ receptor on the cell surface and increase infection.
It has been reported that the number of severe cases increased in the vaccinated group because antibodies induced by inoculation with dengue vaccine (Dengvaxia) showed ADE. In particular, when this vaccine was inoculated to people who were negative for dengue antibodies, they experienced a pseudo-initial infection and the risk of severe progression at next infection increased. For this reason, only those who are positive for dengue antibodies can be vaccinated with Dengvaxia at present. Previous studies from overseas have shown epidemiological reports stating that ADE confirmed in vitro is correlated with severe progression in vivo.
Simple rapid kits (immunochromatography, ELISA method) are commercially available for antibody test in infectious diseases. They are all qualitative and quantitative chemical test methods and do not reveal biological functional activities of antibody. The outline of the ADE test is to examine how much infection of cells can be increased by adding antibodies in the presence of the minimum necessary amount of virus. A method for evaluating ADE using fixed cells has been reported (Non Patent Literature 1). Existing ADE tests can examine biological functions of antibodies, but require live dengue virus as the assay antigen. Dengue virus is classified as a Category IV infectious disease under the Infectious Disease Control Act. Therefore, biosafety level 2 (BSL2) equipment is required when handling Dengue virus. Furthermore, since this method requires at least three days to obtain results, it is realistically difficult to obtain results before patients hospitalized with dengue fever become seriously ill.
Single Round Infectious Particles (SRIPs) are one time infectious particles. SRIPs that retain the outer shell of a specific virus can be produced by using the outer shell gene of the virus when producing SRIPs (Non Patent Literatures 1 to 4). SRIP contains in its inside a self-replicating gene called replicon. Although replicon genes are replicated within SRIPs-infected cells, they cannot be amplified as particles (viruses) and can be safely handled because they do not cause cell destruction or reinfection. By encoding nanoluciferase as a reporter gene, infected cells can be measured based on luciferase activity. Since SRIPs bind to antibodies as pseudoviruses, they can be used as antigens for tests of antibody functions such as antibody neutralization activity and ADE activity. ADE activity measurement using SRIPs and the luciferase activity produced thereby has also been reported (Non Patent Literature 4), but this method also required three days for measurement.
Other functional antibody tests include neutralization tests. In the neutralization test, an antibody is added to a site where a large amount of antigen (virus) is present and how much virus can be reduced is determined. Neutralization tests also previously required a live virus as the assay antigen. A neutralization test using SRIPs as substitute antigens and detecting the luciferase activity produced by SRIPs that infected cells has been reported (Non Patent Literatures 5 to 6). However, it requires 3 days of culture since it takes time for the replicon to increase within the cells after the reaction with the neutralizing antibody.
Therefore, in order to generalize neutralization tests and ADE tests, there was a demand for a method in which safe antigens can be used and by which the results can be obtained more quickly.
The present inventors have conducted intensive studies of Single Round Infectious Particles (SRIPs) suitable as assay antigens for neutralization tests and ADE tests. As a result, they have found that the use of yellow fever virus (YFV) gene as the backbone of the replicon plasmid results in rapid uptake of SRIPs into cells and accelerated replication thereof, thereby shortening the time up to the detection, and successfully developed a rapid diagnostic method for antibody function.
That is, the present invention relates to the following.
Using the SRIPs having YFV replicon of the present invention, antibody function can be determined in a shorter period of time (1 to 2 days) than conventional (3 days), whereby rapid antibody tests can be performed. This allows for rapid determination of the possibility that test subjects develop ADE due to reinfection, as well as its use for property evaluation of antibodies (neutralization activity evaluation) in the development of pharmaceutical products and evaluation of induction potency of ADE active antibodies of vaccine.
The present invention relates to single round infectious virus particles (SRIPs) containing a gene having a region encoding a labeled protein and a region encoding non-structural (NS) proteins 1 to 5 of the yellow fever virus genome, a capsid protein of a virus, and an outer shell protein (Envelope) of an evaluation target virus.
SRIPs in the present specification have a region encoding a labeled protein and a region encoding non-structural (NS) proteins 1 to 5 of the yellow fever virus genome in the gene thereof. The “labeled protein” is not particularly limited as long as it is a protein capable of labeling cells infected with the aforementioned SRIPs. Preferably, it can be expressed only in cells infected with the aforementioned SRIPs. The labeled protein is preferably a fluorescent protein or a luminescent protein. Specifically, green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), fluorescent proteins improved from these fluorescent proteins (see https://www.fpbase.org/), luciferase, and nanoluciferase can be mentioned.
Yellow fever virus is a virus that belongs to the genus Flavivirus of the Flaviviridae family, and its genome is a single-stranded plus RNA. In the present specification, therefore, the “gene” may be DNA or RNA. In the virus particles of the genus Flavivirus, a nucleocapsid in which a capsid protein (also called core protein) is bound to a single-stranded RNA is surrounded by membrane protein and envelope proteins. One reading frame is present on single-stranded RNA, and three types of structural proteins (C, prM, E) and seven types of non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) are encoded. The “non-structural (NS) proteins 1 to 5 of the yellow fever virus genome” means these seven types of non-structural proteins. Proteins with mutations in a part (e.g., 1 to 5 amino acids) of these proteins are also included in the non-structural (NS) proteins 1 to 5 of the yellow fever virus genome as long as they are considered to be substantially the same as these proteins and the effects of the present invention are not affected.
The gene of SRIPs in the present specification optionally has, where necessary, a region other than a region encoding the aforementioned labeled protein and a region encoding non-structural (NS) proteins 1 to 5 of the yellow fever virus genome. However, in order to acquire the function of one time infectivity, it does not contain gene regions encoding at least (i) a capsid protein, and (ii) one or both of a membrane protein and an envelope protein. That is, since the virus particles in the present specification have the Envelope of the evaluation target virus, they can infect cells that are infected by the evaluation target virus, whereas they cannot proliferate within the infected cells because the gene thereof does not encode capsid protein, and/or Envelope and membrane protein precursor.
For example, a region encoding a labeled protein and a region encoding non-structural (NS) proteins 1 to 5 of the yellow fever virus genome may be linked to transcriptional regulatory regions that can be expressed in cells used in infection evaluation tests. Such transcription regulatory region optionally contains a promoter. The promoter is not particularly limited as long as it has activity in the aforementioned cells. For example, it may be a constitutive promoter, and specifically, cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, CAG promoter (promoter having cytomegalovirus enhancer, chicken β-actin promoter, and rabbit β-globin gene splicing acceptor), ubiquitin promoter, Simian virus 40 (SV40) promoter, and elongation factor 1α (EF-1α) promoter can be mentioned. A promoter is linked upstream of the aforementioned protein-encoding region so as to directly control the expression of the region.
The “capsid protein of a virus” that the SRIPs in the present specification has is a protein that binds to the aforementioned RNA gene to form a nucleocapsid. The virus from which the capsid protein is derived is not particularly limited as long as it belongs to the Flaviviridae family. Examples thereof include yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV), and YFV and DENV are preferred.
The outer shell protein (Envelope) is a protein that constitutes the outer shell of virus, and has the functions of binding to receptors and membrane fusion. The evaluation target virus from which the Envelope is derived is not particularly limited as long as it belongs to the aforementioned Flavivirus genus, and may be, for example, DENV. DENV includes four kinds of type 1 to type 4, which are called DENV-1, DENV-2, DENV-3, and DENV-4. Throughout the present specification, DENV may be any one of DENV-1 to DENV-4, a plurality of them, or all of them.
SRIPs in the present specification may further contain a membrane protein in addition to those mentioned above.
SRIPs in the present specification can be obtained by introducing a replicon vector containing a gene having a region encoding a labeled protein and a region encoding non-structural (NS) proteins 1 to 5 of the yellow fever virus genome, a vector containing a gene encoding a capsid protein, and a vector containing a gene encoding Envelope and a gene encoding a membrane protein precursor (prM) into the same cell (
The neutralization activity of antibody can be measured in a short time by infecting Fcγ receptor-non-expressing cells with the aforementioned SRIPs in the presence of a test antibody. Therefore, the present invention provides a method for determining neutralization activity of an antibody, including contacting Fcγ receptor-non-expressing cells with the aforementioned SRIPs in the presence of a test antibody, culturing the aforementioned cells for 24 to 72 hr, and measuring a label in a culture medium resulting from the aforementioned culture, wherein when the measured label is less than the label measured in negative control cells similarly contacted with SRIPs in the absence of the antibody or in the presence of a negative control antibody, the test antibody is determined to have neutralization activity on the aforementioned evaluation target virus.
The Fcγ receptor-non-expressing cell may be any cell that does not express Fcγ receptors, and is preferably an epithelial cell, such as human or primate (monkey, chimpanzee, etc.) cell. A specific example of the Fcγ receptor-non-expressing cell is Vero cell.
The aforementioned SRIPs may be brought into contact with Fcγ receptor-non-expressing cells in the presence of a test antibody by adding the aforementioned SRIPs to the culture medium of the Fcγ receptor-non-expressing cells after or simultaneously with the addition of the test antibody to the culture medium, or by adding the test antibody and the aforementioned SRIPs mixed in advance to the culture medium of the Fcγ receptor-non-expressing cells. For example, a test antibody and SRIPs are mixed in advance, and the mixture is allowed to stand at about 37° C. for 1 to 6 hr (preferably, 2 hr) and then contacted with Fcγ receptor-non-expressing cells. As the test antibody, an antibody composition as well as a purified antibody can be used. For example, serum derived from a test subject can be used as is or used after dilution. For dilution, it can be diluted 5120-fold or less, 640-fold or less, 320-fold or less, 160-fold or less, 80-fold or less, 40-fold or less, or 10-fold or less.
The time of contact of SRIPs in the presence of a test antibody is not less than 24 hr and less than 72 hr, preferably 24 to 48 hr.
The label can be measured as appropriate according to the kind of the selected label. For example, luminescence can be measured using a luminometer.
Neutralization activity is determined by comparing the measured level of label with the level of label in the negative control. A negative control is an Fcγ receptor-non-expressing cell infected with SRIPs under the same conditions as the test for the test antibody except that neutralization activity is not present. Specifically, the negative control may be an Fcγ receptor-non-expressing cell infected with SRIPs in the absence of a test antibody, or an Fcγ receptor-non-expressing cell infected with SRIPs in the presence of an antibody that evidently does not have neutralization activity (e.g., antibodies that do not bind to the evaluation target virus). The level of the label of the negative control can also be obtained by performing the above-mentioned neutralization activity evaluation method simultaneously with the test antibody, or may be obtained by performing the above-mentioned neutralization activity evaluation method separately from the test antibody. When the level of the label is low as compared with the negative control, the test antibody can be determined to have the neutralization activity. For example, when patient serum is used as the test antibody and the level of the label in the presence of the serum is lower than that of a negative control, the patient can be determined to have an antibody with neutralization activity.
Throughout the present specification, the “level” means an indicator of quantified abundance and includes, for example, concentration, amount, or an indicator that can be used in place of these. Therefore, the level may be a measured value itself such as fluorescence intensity, or may be a value converted to concentration. In addition, the level may be an absolute numerical value (abundance, abundance per unit area, etc.), or may be a relative numerical value compared to a comparison control set as necessary.
The antibody-dependent enhancement ability (ADE) of antibody can be measured in a short time by infecting Fcγ receptor-expressing cells with the aforementioned SRIPs in the presence of a test antibody. Therefore, the present invention provides a method for determining antibody-dependent enhancement ability of an antibody, including contacting Fcγ receptor-expressing cells with the aforementioned SRIPs in the presence of a test antibody, culturing the aforementioned cells for 30 min to 24 hr, and measuring a label in the aforementioned culture medium, wherein when the level of the aforementioned measured label is higher than that measured in a negative control cell, the test antibody is determined to have antibody-dependent enhancement ability for the aforementioned evaluation target virus.
The Fcγ receptor-expressing cell may be any cell that expresses Fcγ receptors, and is preferably a cell of the immune system, such as human or primate (monkey, chimpanzee, etc.) cell. A specific example of the Fcγ receptor-expressing cell includes Mylc cell (MiCAN Technologies, Inc., Japan) and K562 cell.
The aforementioned SRIPs may be brought into contact with Fcγ receptor-expressing cells in the presence of a test antibody by adding the aforementioned SRIPs to the culture medium of the Fcγ receptor-expressing cells after or simultaneously with the addition of the test antibody to the culture medium, or by adding the test antibody and the aforementioned SRIPs mixed in advance to the culture medium of the Fcγ receptor-expressing cells. As the test antibody, an antibody composition as well as a purified antibody can be used. As the antibody composition, for example, serum derived from a test subject can be used as is or used after dilution. For dilution, it can be diluted 10-fold or more, 40-fold or more, 160-fold or more and/or 10240-fold or less, 2560-fold or less, 640-fold or less, or may be any combination of these upper limit values and lower limit values, for example, 10-fold or more and 10240-fold or less, or 40-fold or more and 2560-fold or less. SRIPs obtained by the above-mentioned method can be diluted as appropriate and used in this method. In this case, the dilution rate can be 2-fold or more, and may be, for example, 10-fold or more, 100-fold or more, or 1000-fold or more.
Cell culture can be performed, for example, on a plate capable of cultivating immune cells, such as a 96-well plate. The number of cells to be cultured when using a 96-well plate can be set to not less than 6.9×10 cells/well, not less than 2.1×102 cells/well, not less than 6.2×102 cells/well, not less than 1.9×103 cells/well, not less than 5.6×103 cells/well, or not less than 1.7×104 cells/well. The number of cells to be cultured can be appropriately determined according to the dilution rate of SRIPs and the cells to be used. For example, when Mylc cells are used, the number of cells on a 96-well plate is preferably set to not less than 1.9×103 cells/well.
The time for contacting SRIPs with cells in the presence of a test antibody (cell culture time in the presence of the test antibody and SRIPs) is 30 min to 24 hr, or may also be, for example, 2 to 24 hr, 5 to 24 hr, 30 min to 16 hr, 2 to 16 hr, or 5 to 16 hr.
Label can be measured as appropriate according to the kind of the label selected. For example, luminescence can be measured using a luminometer.
ADE ability is determined by comparing the measured level of label with the level of label in the negative control. A negative control is an Fcγ receptor-expressing cell infected with SRIPs under the same conditions as the test for the test antibody except that neutralization activity is not present. Specifically, the negative control may be an Fcγ receptor-expressing cell infected with SRIPs in the absence of a test antibody, or an Fcγ receptor-expressing cell infected with SRIPs in the presence of an antibody that evidently does not have ADE ability (e.g., antibodies that do not bind to the evaluation target virus). The level of the label of the negative control can also be obtained by performing the above-mentioned ADE ability evaluation method simultaneously with the test antibody, or may be obtained by performing the above-mentioned ADE ability evaluation method separately from the test antibody. When the level of the label is high as compared with the negative control, the test antibody can be determined to have the ADE ability. For example, when patient serum is used as the test antibody and the level of the label in the presence of the serum is higher than that of a negative control, the patient can be determined to have an antibody with ADE ability.
As described above, the SRIPs of the present invention can be used for the measurement of the functions (neutralization activity, ADE ability) of an antibody. Therefore, the present invention relates to a composition for determining antibody function containing the aforementioned SRIPs as an active ingredient. The antibody function here is a function related to viral infection and is typically neutralization activity or antibody-dependent enhancement ability. The composition of the present invention can be a composition for use in the aforementioned neutralization activity evaluation method or ADE ability evaluation method. The composition can contain components such as stabilizer, buffer, and the like in addition to the aforementioned SRIPs.
Also, the present invention relates to a kit for determining antibody function, containing the aforementioned SRIPs. The antibody function here is a function related to viral infection and is typically antibody-dependent enhancement ability or neutralization activity. The kit may further contain a negative control antibody or a standard antibody, and/or an Fcγ receptor-non-expressing cell (when the antibody function is neutralization activity) or an Fcγ receptor-expressing cell (when the antibody function is ADE). The kit of the present invention may further contain an outer box, container, diluent, culture medium, and/or instructions. In the kit for determining antibody function of the present invention, different components may be packaged in separate containers and included in one kit, or only some components (including at least the aforementioned SRIPs) may be included in the kit, and other components may be provided separately from the kit.
The present invention is explained in more detail in the following using Examples, but they are not intended to limit the scope of the present invention. The documents cited throughout the present specification are incorporated in their entirety in the present specification by reference.
According to the procedures of a previous report (Matsuda, M. et al., Sci Rep; 8:16624(2018)), luciferase-expressing SRIPs were produced based on the prM/E gene information of dengue type 1 virus, dengue type 2 virus (JX042506), dengue type 3 virus (EU081222), and dengue type 4 virus (AY618988). Schematic diagram of SRIPs production process is shown in
Specifically, a YFV subgenomic replicon plasmid was constructed using the 17D-204 strain of yellow fever virus (YFV) (
RNA was extracted from Vero cells infected with YFV and reverse transcribed into cDNA. A cDNA encoding 101aa mature capsid (C) was amplified from the obtained cDNA. The obtained fragment was cloned into pCAGGS to construct a plasmid pCAG-YF-C expressing the YFV capsid (
(3) Construction of prME-Expression Plasmid
Plasmid pcD1MEYG1 encoding pre-membrane/membrane (prM/M) of dengue type 1 virus (DENV-1) (D1/Hu/Saitama/NIID100/2014) and envelope (E) protein was prepared by a previously reported method (Konishi et al., Vaccine; 24(12): pp. 2200-2207 (2006)). Plasmids pcD2ME, pcD3MESG, pcD4METh encoding pre-membrane/membrane (prM/M) of each of dengue type 2 virus (DENV-2) (D2/New Guinea C/1944), dengue type 3 virus (DENV-3) (D3/SG/05K4454DK1/2005), and dengue type 4 virus (DENV-4) (D4/Thailand/0476/1997), and envelope (E) proteins were prepared by a previously reported method (Yamanaka, A. et al., (2014), Matsuda, M. et al., (2018) supra) (
293T cells were proliferated in a 10 cm dish and cotransfected with three kinds of plasmids using polyethyleneamine (
K562 cells which are human leukemia cells, and Mylc cells which are immortalized myeloid cells (MiCAN Technologies, Inc.) were used as Fcγ receptor-expressing cells. In addition, Vero cells (Fcγ receptor-non-expressing), which are commonly used in dengue virus infection experiments, were used as Comparative Example. As a culture medium, RPMI-1640 was used for K562 cells, α-modified Eagle Minimum Essential Medium (αMEM) was used for Mylc cells, and Dulbecco's modified Eagle medium (DMEM) was used for Vero cells, and 10% fetal bovine serum (FBS) was added to each and used. 40 μL of the step-diluted D1-SRIPs were inoculated into each cell (0.25 to 1.0×105 cells), allowed to adsorb for 2 hr at 37° C., and then 100 μL of warmed culture medium was dispensed into each well and further cultured at 37° C. After 24 to 48 hr, the cells were washed once with 300 μL of phosphate-buffered saline (PBS) per well and lysed with 30 μL of Passive Lysis 5× Buffer (E1941) of Promega KK. After thorough stirring, the cell lysate (10 μL) and Promega KK's luciferase substrate, Nano-Glo (registered trademark) Luciferase Assay System (N1110) (10 μL) were mixed in a white microplate, and luciferase activity was measured with a 96-well plate compatible luminometer GloMax (registered trademark) Navigator System of Promega K. K. Similarly, K562 cells and Mylc cells were infected with D2-SRIPs, D3-SRIPs, and D4-SRIPs and the luciferase activity thereof was measured.
The results of D1-SRIPs are shown in
Similarly, the results of D2-SRIPs, D3-SRIPs, and D4-SRIPs are shown in
Vero cells were passaged in a 96-well microplate (0.25×105 cells per well). The next day, 75 μL of 10 to 10240-fold step-diluted dengue antibody-positive serum (2 samples: #1 and #2) was mixed with 75 μL of an equal volume of 100-fold diluted D1-SRIPs, reacted at 37° C. for 2 hr, and 40 μL of the serum-SRIP mixture was inoculated into Vero cells (Fcγ receptor-non-expressing cells) that were passaged the previous day and allowed to adsorb for additional 2 hr at 37° C. Thereafter, 100 μL of warmed 10% FBS-added DMEM was dispensed into each well, and intracellular luciferase activity was measured after 48 hr of culture at 37° C.
The results are shown in
As K562 cells, only quasi-adherent cells that adhered to the bottom of the culture flasks were selectively passaged and used in this test. First, 36 μL of diluted (1:500) dengue antibody-positive serum (#1) and 36 μL of an equal volume of D1-SRIP (1:10 to 1:1000 dilution) were mixed in a 96-well poly-L lysine-coated microplate and reacted for 2 hr at 37° C., and then mixed with 1×104 cells, 5×104 cells, or 1×105 cells of K562 cells (Fcγ receptor-expressing cells) (50 μL). After 24 hr of culture at 37° C., intracellular luciferase activity was measured. The same experiment was performed under serum-free conditions and used as Control.
The results are shown in
In the same manner as above, 36 μL of diluted (1:500) dengue antibody-positive serum (#1) and 36 μL of an equal volume of dengue type 1-SRIP (1:10 to 1:1000 dilution) were mixed in a 96-well poly-L lysine-coated microplate and reacted for 2 hr at 37° C., and then mixed with 1×104 cells, 5×104 cells, or 1×105 cells of Mylc cells (Fcγ receptor-expressing cells) (50 μL). After 24 hr of culture at 37° C., intracellular luciferase activity was measured. The same experiment was performed under serum-free conditions and used as Control.
The results are shown in
The above results indicate that the combination of Fcγ receptor-expressing cells (K562 and Mylc cells) and SRIPs can be used to measure ADE activity of dengue antibody in the serum. From the data of Fold enhancement (
36 μL of diluted (1:100) dengue antibody-positive serum (#1) and 36 μL of an equal volume of D2-SRIPs, D3-SRIPs, or D4-SRIPs (each 1:100 dilution) were mixed in a 96-well poly-L lysine-coated microplate and reacted for 2 hr at 37° C., and then mixed with 1.5×103 to 4.8×104 K562 or Mylc cells (Fcγ receptor-expressing cells) (50 μL). After 24 hr of culture at 37° C., intracellular luciferase activity was measured.
ADE activity of dengue antibody-positive serum (#1) against SRIPs of each serotype was shown by Fold enhancement (
Dengue antibody-positive serum (#1) was fixed to a 1:500 antibody dilution rate, D1-SRIPs antigen dilution rate was set to 1:100 or 1:1000, and ADE test using K562 or Mylc cells was performed with shorter incubation times (30 min to 16 h). Specifically, 36 μL of diluted (1:500) dengue antibody-positive serum (#1) and 36 μL of each equal volume of D1-SRIPs (1:10 or 1:1000 dilution) were mixed in a 96-well poly-L lysine-coated microplate and reacted for 2 hr at 37° C., and then mixed with 1.3×104 cells or 4.0×104 cells of K562 or Mylc cells (Fcγ receptor-expressing cells) (50 L). After 30 min, 1 hr, 2 hr, 5 hr, 7 hr, or 16 hr of culture at 37° C., intracellular luciferase activity was measured.
Dengue antibody-positive serum (#1) was fixed to a 1:500 antibody dilution rate, D1-SRIPs antigen dilution rate was set to 1:2 and 1:10, and ADE test was performed using Mylc and K562 cells by 5 hr culture. Specifically, 36 μL of diluted (1:500) dengue antibody-positive serum (#1) and 36 μL of equal volume of D1-SRIPs (1:2 or 1:10 dilution) were mixed in a 96-well poly-L lysine-coated microplate and reacted for 2 hr at 37° C., and then mixed with 6.9×101 to 1.5×105 Mylc cells or K562 (Fcγ receptor-expressing cells) (50 L). After 5 hr of culture at 37° C., intracellular luciferase activity was measured.
Dengue antibody-positive serum (#1) was fixed to a 1:500 antibody dilution rate, D1-SRIPs antigen dilution rate was set to 1:100 and 1:1000, and ADE test was performed using Mylc and K562 cells by 48 hr culture. Specifically, 36 μL of diluted (1:500) dengue antibody-positive serum (#1) and 36 μL of equal volume of D1-SRIPs (1:10 or 1:1000 dilution) were mixed in a 96-well poly-L lysine-coated microplate and reacted for 2 hr at 37° C., and then mixed with 6.9×101 to 1.5×105 Mylc cells or K562 (Fcγ receptor-expressing cells) (50 μL). After 48 hr of culture at 37° C., intracellular luciferase activity was measured.
Dengue antibody positive serum was step-diluted, reacted with each of D1-SRIPs to D4-SRIPs antigens, infected with Mylc and K562 cells, and ADE test was performed. The assay was performed under two different conditions below.
Using the SRIPs having YFV replicon of the present invention, antibody function can be determined in a shorter period of time than conventional, whereby rapid antibody tests can be performed. This application is based on a patent application No. 2021-089309 filed in Japan (filing date: May 27, 2021), the contents of which are incorporated in full herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-089309 | May 2021 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/016731 | 3/31/2022 | WO |
