US 2026/0167703 A1 · A METHOD FOR DETERMINING THE INFECTIVITY OF A VIRUS

Abstract

The present invention relates to a method for determining the infectivity of a virus, in particular a flavivirus. Further, a method for determining the titer of a virus-specific antibody in a sample is provided. In addition, the use of the method in the quality control of vaccines and in the diagnosis of an infection are provided as well. The present invention also provides a method for determining the proportion of infectious virus particles in a sample comprising infectious and non-infectious virus particles. The present invention further relates to a maturation assay for determining the average maturation degree. The present invention also relates to novel anti-dengue virus serotype 2 (DENV2) and anti-dengue virus serotype 4 (DENV4) antibodies and antigen binding fragments thereof being useful in the method of the invention. Further, nucleic acids encoding them and host cells comprising them are provided. In addition, the use of the antibodies in the prevention or treatment of dengue disease is provided. Also, diagnostic methods using them and kits comprising them are provided.

Claims38

Dependent claims

A method for determining the infectivity of a plaque-forming virus in a sample comprising the steps of:

(a) seeding cells from a virus-susceptible cell line in an assay plate and culturing the cells for a culture period;
(b) preparing serial dilutions of the virus-containing sample;
(c) adding the serially diluted samples to the cells seeded and cultured in step (a) and incubating the cells over a first incubation period;
(d) culturing the cells of step (c) over a second incubation period;
(e) lysing the cells of step (d);
and
(f) determining the presence and/or amount of a virus antigen of the virus in the lysate of step (e) in an immunoassay method, wherein the amount of the virus antigen is indicative for the infectivity of the virus in the sample.

4 more elements

A method for determining the average maturation degree of a population of flaviviruses in a flavivirus-containing sample, wherein the method comprises:

(a) coating at least one well of an assay plate with at least one primary antibody specific for a flavivirus envelope protein (E protein) antigen;
(b) contacting the at least one primary antibody coated on the at least one well of the assay plate of step (a) with the flavivirus-containing sample to allow binding of the virus antigen to the primary antibody;
(c1) contacting the virus antigen bound to the primary antibody with (i) a secondary antibody specific for the E protein antigen, wherein the at least one secondary antibody is conjugated to a detectable label;
or
(c2) contacting the virus antigen bound to the primary antibody with the (i) secondary antibody specific for the E protein antigen and further contacting the at least one secondary antibody with a tertiary antibody being specific for the constant region of the at least one secondary antibody and conjugated to a detectable label;
(d) determining the amount of the E protein antigen in the sample;
(e) coating at least one well of an assay plate with at least one primary antibody specific for a flavivirus envelope protein (E protein) antigen;
(f) contacting the at least one primary antibody coated on the at least one well of the assay plate of step (e) with the flavivirus-containing sample to allow binding of the virus antigen to the primary antibody;
(g1) contacting the virus antigen bound to the primary antibody with (ii) a secondary antibody specific for a portion of the pr domain of the flavivirus prM protein, wherein the at least one secondary antibody is conjugated to a detectable label;
or
(g2) contacting the virus antigen bound to the primary antibody with (ii) a secondary antibody specific for a portion of the pr domain of the flavivirus prM protein and further contacting the at least one secondary antibody with a tertiary antibody being specific for the constant region of the at least one secondary antibody and conjugated to a detectable label;
(h) determining the amount of the prM protein in the sample;
and
(i) calculating the ratio of the amount of prM protein to the amount of the E protein which is indicative for the average maturation degree of the flaviviruses in the sample.

11 more elements

An antibody specific for Dengue virus serotype 2 envelope protein (DENV2 E protein) or an antigen binding fragment thereof, wherein

(i) the VH CDR1 region of the antibody or antigen binding fragment thereof is selected from the group consisting of SEQ ID NO:
106, SEQ ID NO:
109, and SEQ ID NO:
112, or a variant thereof having at least 85% identity;
(ii) the VH CDR2 region of the antibody or binding fragment thereof is selected from the group consisting of SEQ ID NO:
107, SEQ ID NO:
110, and SEQ ID NO:
113, or a variant thereof having at least 85% identity;
(iii) the VH CDR3 region of the antibody or antigen binding fragment thereof is selected from the group consisting of SEQ ID NO:
108, SEQ ID NO:
111, and SEQ ID NO 114, or a variant thereof having at least 85% identity;
(iv) the VL CDR1 region of the antibody or antigen binding fragment thereof is selected from the group consisting of SEQ ID NO:
127, SEQ ID NO:
129, and SEQ ID NO:
131, or a variant thereof having at least 82% identity;
(v) the VL CDR2 region of the antibody or antigen binding fragment thereof is selected from the group of an amino acid sequence consisting of RAS, QAS and LAS, or a variant thereof having at least 65% identity;
and
(vi) the VL CDR3 region of the antibody or antigen binding fragment thereof is selected from the group consisting of SEQ ID NO:
128, SEQ ID NO:
130, and SEQ ID NO:
132, or a variant thereof having at least 85% identity,
wherein the antibody or antigen binding fragment thereon does not cross-react with other dengue serotypes other than DENV2 and has one or more of the following properties:
(1) a binding activity for DENV2-VLP calculated as EC50 value of 38 ng/ml or less;
and/or
(2) a koff value of 1×10−4 sec−1 or less.

22 more elements

An antibody specific for Dengue virus serotype 4 envelope protein (DENV4 E protein) or an antigen binding fragment thereof, wherein

(i) the VH CDR1 region of the antibody or antigen binding fragment thereof is selected from the group consisting of SEQ ID NO:
1, SEQ ID NO:
4, SEQ ID NO:
7, SEQ ID NO:
10, SEQ ID NO:
13, SEQ ID NO:
16 and SEQ ID NO:
19, or a variant thereof having at least 85% identity;
(ii) the VH CDR2 region of the antibody or binding fragment thereof is selected from the group consisting of SEQ ID NO:
2, SEQ ID NO:
5, SEQ ID NO:
8, SEQ ID NO:
11, SEQ ID NO:
14, SEQ ID NO:
17 and SEQ ID NO:
20, or a variant thereof having at least 85% identity;
(iii) the VH CDR3 region of the antibody or antigen binding fragment thereof is selected from the group consisting of SEQ ID NO:
3, SEQ ID NO:
6, SEQ ID NO:
9, SEQ ID NO:
12, SEQ ID NO:
15, SEQ ID NO:
18 and SEQ ID NO:
21, or a variant thereof having at least 85% identity;
(iv) the VL CDR1 region of the antibody or antigen binding fragment thereof is selected from the group consisting of SEQ ID NO:
50, SEQ ID NO:
52, SEQ ID NO:
54 and SEQ ID NO:
56, SEQ ID NO:
58, SEQ ID NO:
60 and SEQ ID NO:
62 or a variant thereof having at least 82% identity;
(v) the VL CDR2 region of the antibody or antigen binding fragment thereof is selected from the group of an amino acid sequence consisting of RAS, EAS and RAF, or a variant thereof having at least 65% identity;
and
(vi) the VL CDR3 region of the antibody or antigen binding fragment thereof is selected from the group consisting of SEQ ID NO:
51, SEQ ID NO:
53, SEQ ID NO:
55 and SEQ ID NO:
57, SEQ ID NO:
59, SEQ ID NO:
61 and SEQ ID NO:
63, or a variant thereof having at least 85% identity,
wherein the antibody or antigen binding fragment thereof does not cross-react with dengue serotypes other than DENV4 and has one or more of the following properties:
(1) a binding activity for DENV4-VLP calculated as EC50 value of 80 ng/ml or less;
and/or
(2) a koff value of 1×10−4 sec−1 or less.

43 more elements

Dependencies are inferred from the claim text.

A method for determining the infectivity of a virus

Life of this patent

A method for determining the infectivity of a virus

Life of this patent

Abstract

Claims38