WO2020051328A1 - Assay for determining antibody response to dengue virus - Google Patents

Abstract

The present invention relates to an assay for determining the titer of neutralizing antibodies against each of dengue serotypes 1, 2, 3 and 4 in a subject.

Description

ASSAY FOR DETERMINING ANTIBODY RESPONSE TO DENGUE VIRUS

FIELD OF THE INVENTION

[0001] The present invention relates to an assay for determining the titer of neutralizing antibodies against each of dengue serotypes 1, 2, 3 and 4 in a subject.

BACKGROUND OF THE INVENTION

[0002] Vaccines for protection against viral infections have been effectively used to reduce the incidence of human disease. One of the most successful technologies for viral vaccines is to immunize animals or humans with a weakened or attenuated virus strain (a "live attenuated virus"). Due to limited replication after immunization, the attenuated virus strain does not cause disease. However, the limited viral replication is sufficient to express the full repertoire of viral antigens and can generate potent and long-lasting immune responses to the virus. Thus, upon subsequent exposure to a pathogenic virus strain, the immunized individual is protected from the disease. These live attenuated viral vaccines are among the most successful vaccines used in public health.

[0003] Dengue disease is a mosquito-borne disease caused by infection with a dengue virus. Dengue virus infections can lead to debilitating and painful symptoms, including a sudden high fever, headaches, joint and muscle pain, nausea, vomiting and skin rashes. To date, four serotypes of dengue virus have been identified : dengue-1 (DENV-1), dengue-2 (DENV-2), dengue-3 (DENV-3) and dengue-4 (DENV-4). Dengue virus serotypes 1-4 can also cause dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In the most severe cases, DHF and DSS can be life threatening. Dengue viruses cause 50-100 million cases of debilitating dengue fever, 500,000 cases of DHF/DSS, and more than 20,000 deaths each year, a large portion of which are children. All four dengue virus serotypes are endemic throughout the tropical regions of the world and constitute the most significant mosquito- borne viral threat to humans there. Dengue viruses are transmitted to humans primarily by Aedes aegypti mosquitoes, but also by Aedes albopictus mosquitoes. Infection with one dengue virus serotype results in life-long protection from re-infection by that serotype, but does not prevent secondary infection by one of the other three dengue virus serotypes. In fact, previous infection with one dengue virus serotype may lead to an increased risk of severe disease (DHF/DSS) upon secondary infection with a different serotype.

[0004] To date, only one vaccine, Dengvaxia®, has been licensed for use in protecting against dengue disease. However, clinical trials have shown that Dengvaxia® can enhance, rather than reduce, the risk of severe disease due to dengue infection in individuals who had not been previously infected by a dengue virus (seronegative populations). Therefore, Dengvaxia® is only recommended for use in individuals who had been previously infected with at least one dengue virus serotype (seropositive populations).

[0005] There is a need for an assay to quantify the level of neutralizing antibodies in a subject before and/or after vaccination with a dengue vaccine. This assay should also be able to differentiate between seropositive and seronegative subjects.

[0006] Brewoo et al. (2012) Vaccine 30: 1513-1520 describe a neutralization assay to determine neutralizing antibody titers. Roehring et al. (2008) Viral Immunology 21(2): 123-132 and the WHO "Guidelines for plaque reduction neutralization testing of human antibodies to dengue viruses", Immunization, Vaccines and Biologicals (2007) provide guidelines for a plaque-reduction neutralization test of human antibodies to dengue viruses. Putnak et al. (2008) Am. 1 Trap. Med. Hyg. 79(1): 115-122 compare three assays for measuring dengue virus neutralizing antibodies. Timiryasova et al. (2013) Am. 1 Trap. Med. Hyg. 88(5): 962-970 disclose the optimization and validation of a plaque reduction neutralization test for all four serotypes of dengue virus.

OBJECTS AND SUMMARY

[0007] It is an object of the present invention to provide an assay to quantify the level of neutralizing antibodies in a subject before and/or after vaccination with a dengue vaccine.

[0008] The present invention is therefore directed to a method for determining the titer of neutralizing antibodies against each of dengue serotypes 1, 2, 3 and 4 in a blood serum sample, the method comprising the steps of:

(a) seeding cells from a dengue-susceptible cell line on 96-well assay plates and culturing the cells for a culture period;

(b) preparing serial dilutions of the blood serum sample;

(c) separately mixing the serially diluted blood serum samples prepared in step (b) with dengue serotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4 to obtain separate mixtures for each dengue serotype and incubating the separate mixtures;

(d) adding the separate mixtures prepared in step (c) to the cells seeded and cultured in step (a) and incubating the cells with the separate mixtures;

(e) providing an overlay for the cells incubated in step (d) and incubating the cells for an incubation period of 40 to 75 hours;

(f) determining the number of plaques in each well and comparing the number of plaques in each well to an unneutralized control to determine the level of neutralizing antibodies against each of dengue serotypes 1, 2, 3 and 4.

[0009] In one embodiment different incubation periods are used in step (e) for the mixtures of different dengue serotypes.

[0010] In one embodiment the incubation period in step (e) for mixtures of dengue serotype 4 is shorter than the incubation period for mixtures of dengue serotypes 1, 2 and 3, preferably it is 46±2 hours.

[0011] In one embodiment the incubation period in step (e) for mixtures of dengue serotype 2 is longer than the incubation period for mixtures of dengue serotypes 1, 3 and 4, preferably it is 70±2 hours.

[0012] In one embodiment the dengue-susceptible cell line is selected from Vera cells, LLC-MK2 cells and BHK-21 cells.

[0013] In one embodiment the culture period in step (a) is 12 to 36 hours.

[0014] In one embodiment in step (c) the dengue serotype 1 is DENV-1 strain 16007, dengue serotype 2 is DENV-2 strain 16681, dengue serotype 3 is DENV-3 strain 16562 and dengue serotype 4 is DENV-4 strain 1036.

[0015] In one embodiment the separate mixtures in step (c) are incubated overnight at a temperature of 2°C to 8°C.

[0016] In one embodiment the overlay in step (e) is selected from the group consisting of methylcellulose, carboxymethylcellulose and agarose.

[0017] In one embodiment the cells are incubated at a temperature of 33°C to 35°C in step (e). [0018] In one embodiment the number of plaques in each well is determined using serotype-specific anti-dengue monoclonal antibodies.

[0019] In one embodiment said blood serum sample is obtained from a subject which has not been vaccinated with a dengue virus vaccine or is obtained from a subject which has been vaccinated with a dengue virus vaccine.

[0020] In one embodiment the dengue virus vaccine is a tetravalent dengue virus composition.

[0021] In one embodiment the dengue virus vaccine comprises a chimeric dengue serotype 2/1 strain, a dengue serotype 2 strain, a chimeric dengue serotype 2/3 strain, and a chimeric dengue serotype 2/4 strain.

[0022] In one embodiment each one of the four live attenuated dengue virus strains has attenuating mutations in the 5'-noncoding region (NCR) at nucleotide 57 from cytosine to thymine, in the NS1 gene at nucleotide 2579 from guanine to adenine resulting in an amino acid change at position 828 from glycine to asparagine, and in the NS3 gene at nucleotide 5270 from adenine to thymine resulting in an amino acid change at position 1725 from glutamine to valine.

[0023] In one embodiment the dengue virus vaccine comprises a live attenuated chimeric dengue serotype 1 virus, a live attenuated chimeric dengue serotype 2 virus, a live attenuated chimeric dengue serotype 3 virus and a live attenuated chimeric dengue serotype 4 virus.

[0024] In one embodiment the live attenuated chimeric dengue serotype 1 virus comprises a genome of an attenuated yellow fever virus whose prM-E sequence has been substituted with the prM-E sequence of dengue serotype 1, the live attenuated chimeric dengue serotype 2 virus comprises a genome of an attenuated yellow fever virus whose prM-E sequence has been substituted with the prM-E sequence of dengue serotype 2, the live attenuated chimeric dengue serotype 3 virus comprises a genome of an attenuated yellow fever virus whose prM-E sequence has been substituted with the prM-E sequence of dengue serotype 3 and the live attenuated chimeric dengue serotype 4 virus comprises a genome of an attenuated yellow fever virus whose prM-E sequence has been substituted with the prM-E sequence of dengue serotype 4.

[0025] The present invention further relates to the use of the method described herein for determining the dengue serostatus of a subject before vaccination with a dengue virus vaccine.

[0026] The present invention further relates to the use of the method described herein for analyzing a subject's antibody response after vaccination with a dengue virus vaccine.

[0027] The present invention further relates to a method for determining whether a subject is dengue- seronegative or dengue-seropositive, comprising the steps of:

(a) performing the method for determining the titer of neutralizing antibodies against each of dengue serotypes 1, 2, 3 and 4 as disclosed herein with a blood sample from said subject;

(b) calculating the MNT50 value based on the level of neutralizing antibodies; and

(c) determining that the subject is dengue-seronegative if the MNT50 value is below the lower limit of detection or determining that the subject is dengue-seropositive if the MNT50 value is above the lower limit of detection of the assay. DEFINITIONS

[0028] In describing the present invention, the following terms are to be used as indicated below. As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0029] As used herein, the term "dengue serotype" refers to a species of dengue virus which is defined by its cell surface antigens and therefore can be distinguished by serological methods known in the art. At present, four serotypes of dengue virus are known, i.e. dengue serotype 1 (DENV-1), dengue serotype 2 (DENV-2), dengue serotype 3 (DENV-3) and dengue serotype 4 (DENV-4).

[0030] As used herein, the term "tetravalent dengue virus composition" refers to a dengue virus composition comprising four different immunogenic components from the four different dengue serotypes DENV-1, DENV-2, DENV-3 and DENV-4, preferably comprising four different live, attenuated dengue viruses, each representing one dengue serotype, and which aims to stimulate immune responses to all four dengue serotypes.

[0031] As used herein, the term "live attenuated dengue virus" refers to a viable dengue virus which is mutated to provide reduced virulence. The live attenuated dengue virus can be a dengue virus in which all components are derived from the same dengue serotype or it can be a chimeric dengue virus having parts from two or more dengue serotypes.

[0032] A "virus strain" and in particular a "dengue virus strain" is a genetic subtype of a virus, in particular of a dengue virus, which is characterized by a specific nucleic acid sequence. A dengue serotype may comprise different strains with different nucleic acid sequences which have the same cell surface antigens. A dengue virus strain can be a dengue virus in which all components are derived from the same dengue serotype or it can be a chimeric dengue virus having parts from two or more dengue serotypes.

[0033] As used herein, "TDV-2" refers to a molecularly characterized and cloned dengue serotype 2 strain derived from the live attenuated DEN-2 PDK-53 virus strain. The PDK-53 strain is described for example in Bhamarapravati et al. (1987) Bulletin of the World Health Organization 65(2): 189-195. In one embodiment, the TDV-2 strain served as a backbone for the chimeric TDV-1, TDV-3 and TDV-4 strains, into which parts from the wildtype DEN-1, DEN-3 and DEN-4 strains were introduced.

[0034] A "chimeric dengue virus" or "chimeric dengue serotype strain" or "chimeric dengue strain" comprises parts from at least two different dengue serotypes. As used herein, the chimeric dengue virus does not include parts from a different flavivirus such as yellow fever virus, Zika virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, tick-borne encephalitis virus. In particular, the chimeric dengue virus described herein does not include parts from the yellow fever virus. As used herein, a "chimeric dengue serotype 2/1 strain" or "DENV-2/1 chimera" or "TDV-1" refers to a dengue virus chimeric construct which comprises parts from both DENV-2 and DENV- 1. In particular, in the chimeric dengue serotype 2/1 strain the prM and E proteins from DENV-1 replace the prM and E proteins from DENV-2 as detailed below. As used herein, a "chimeric dengue serotype 2/3 strain" or "DENV-2/3 chimera" or "TDV-3" refers to a dengue virus chimeric construct which comprises parts from both DENV-2 and DENV-

3. In particular, in the chimeric dengue serotype 2/3 strain the prM and E proteins from DENV-3 replace the prM and E proteins from DENV-2 as detailed below. As used herein, a "chimeric dengue serotype 2/4 strain" or "DENV-2/4 chimera" or "TDV-4" refers to a dengue virus chimeric construct which comprises parts from both DENV-2 and DENV-

4. In particular, in the chimeric dengue serotype 2/4 strain the prM and E proteins from DENV-4 replace the prM and E proteins from DENV-2 as detailed below. [0035] As used herein, "TDV" refers to a tetravalent live attenuated dengue vaccine that comprises a mixture of the four live attenuated dengue virus strains TDV-1, TDV-2, TDV-3 and TDV-4 expressing surface antigens from the four dengue serotypes DENV-1, DENV-2, DENV-3 and DENV-4, respectively. In one embodiment, TDV-1 has the nucleotide sequence according to SEQ ID No. 1 and/or the amino acid sequence according to SEQ ID No. 2. In one embodiment, TDV-2 has the nucleotide sequence according to SEQ ID No. 3 and/or the amino acid sequence according to SEQ ID No. 4. In one embodiment, TDV-3 has the nucleotide sequence according to SEQ ID No. 5 and/or the amino acid sequence according to SEQ ID No. 6. In one embodiment, TDV-4 has the nucleotide sequence according to SEQ ID No. 7 and/or the amino acid sequence according to SEQ ID No. 8.

[0036] As used herein, the term "dengue disease" refers to the disease which is caused by infection with dengue virus. Symptoms of dengue disease include sudden high fever, headaches, joint and muscle pain, nausea, vomiting and skin rashes. The term dengue disease also includes the more severe forms of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Symptoms of DHF include increased vascular permeability, hypovolemia and abnormal blood clotting mechanisms. Subjects with DHF may present with severe manifestations of plasma leakage and hemorrhage. When a subject with DHF experiences shock he or she will be categorized as having DSS. Symptoms of DSS include bleeding that may appear as tiny spots of blood on the skin and larger patches of blood under the skin. Prolonged shock is the main factor associated with complications including massive gastrointestinal hemorrhage that can lead to death.

[0037] As used herein, "preventing dengue disease" refers to preventing a subject from developing one or more symptoms of dengue disease because of an infection with a dengue virus. As used herein, the term "prophylactically treating dengue disease" is equivalent to "preventing dengue disease". In a particular embodiment, preventing dengue disease includes preventing DHS and/or DSS.

[0038] As used herein, "vaccinating" refers to the administration of a vaccine to a subject with the aim to prevent the subject from developing one or more symptoms of a disease. As used herein, "vaccinating against dengue disease" refers to the administration of a dengue vaccine composition to a subject with the aim to prevent the subject from developing one or more symptoms of dengue disease.

[0039] As used herein, "serostatus" refers to the amount of antibodies a subject has with respect to a certain infectious agent, in particular dengue virus. As used herein, "seronegative" or "serona^'ive" means that the subject does not have neutralizing antibodies against any one of dengue serotypes DENV-1, DENV-2, DENV-3 and DENV-4 in the serum. A seronegative or serona^'ive subject or subject population is defined by a neutralizing antibody titer of less than 10 for each one of the four dengue serotypes. A subject or subject population having a neutralizing antibody titer of equal to or more than 10 for at least one dengue serotype is defined as being "seropositive" with respect to said dengue serotype.

[0040] As used herein, a "neutralizing antibody titer" or "titer of neutralizing antibodies" refers to the amount of antibodies in the serum of a subject that neutralize the respective dengue serotype. The neutralizing antibody titer against DENV-1, DENV-2, DENV-3 and DENV-4 is determined in a serum sample of the subject using the method of the present invention. As used herein, the terms "geometric mean neutralizing antibody titer" and "GMT" refer to the geometric mean value of the titer of neutralizing antibodies against the corresponding dengue serotype in the serum of subjects in a subject population. The geometric mean value is calculated by a well-known formula.

[0041] As used herein, a "dengue-susceptible cell line" is a cell line which can be kept in culture in vitro and which can be infected with dengue virus. In the context of the present invention the dengue-susceptible cell line is capable of being lysed upon replication of the virus in the cells so that plaques are formed. Suitable dengue- susceptible cell lines include, but are not limited to, Vero cells, LLC-MK2 cells and BHK-21 cells. Preferably, the dengue-susceptible cell line is a Vero cell line.

[0042] As used herein, "culturing" means maintaining cells under conditions selected such that the cells remain viable and able to divide. Such conditions include temperature, pFH and culture medium. Typically, mammalian cells are cultured at a temperature of about 37°C.

[0043] As used herein, the "MNT₅o value" is the neutralizing titer at which a plaque reduction of 50% compared to a control not incubated with a blood serum sample is obtained. The MNT₅₀ value is calculated through linear interpolation using the equation MNT₅₀ = 10^A[(50-c)/m] where c = y intercept of regression line and m = slope of regression line.

[0044] As used herein, "lower level of detection" or "LLOD" means the lowest antibody titer that could be determined to be statistically different from a negative control.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 : Genetic structure of the four dengue strains contained in TDV. The solid red triangles indicate the three attenuating mutations present in the 5'NCR, NS1 and NS3 proteins. The TDV-1, TDV-3 and TDV-4 strains are chimeric viruses where the prM and E genes from dengue serotype 1, 3 and 4, respectively, are inserted into the TDV-2 backbone.

[0046] Figure 2: Schematic drawing illustrating the microneutralization test (MNT) used to determine the titer of neutralizing antibodies.

DETAILED DESCRIPTION

Denaue virus strains

[0047] The dengue virus is a single stranded, positive sense RNA virus of the family flaviviridae. The taxonomy is outlined in Table 1. The family flaviviridae includes three genera, flavivirus, hepacivirus and pestivirus. The genus flavivirus contains highly pathogenic and potentially hemorrhagic fever viruses, such as yellow fever virus and dengue virus, encephalitic viruses, such as Japanese encephalitis virus, Murray Valley encephalitis virus and West Nile virus, and a number of less pathogenic viruses.

Table 1. Dengue Virus Taxonomy of the GMO Parental Strain

[0048] The flavivirus genome comprises in 5¹ to 3¹ direction (see Figure 1):

a 5'-noncoding region (5'-NCR), a capsid protein (C) encoding region,

a pre-membrane protein (prM) encoding region,

an envelope protein (E) encoding region,

a region encoding nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) and

a 3¹ noncoding region (3'-NCR).

[0049] The viral structural proteins are C, prM and E, and the nonstructural proteins are NS1 to NS5. The structural and nonstructural proteins are translated as a single polyprotein and processed by cellular and viral proteases.

[0050] A dengue virus vaccine may comprise a dengue virus composition that comprises four live attenuated dengue virus strains: a molecularly characterized and cloned dengue serotype 2 strain derived from the live attenuated DEN-2 PDK-53 virus strain (TDV-2), and three chimeric dengue strains derived from the TDV-2 strain by replacing the structural proteins prM and E from TDV-2 with the corresponding structural proteins from the other dengue serotypes, resulting in the following chimeric dengue strains:

- a DENV-2/1 chimera (TDV-1),

a DENV-2/3 chimera (TDV-3) and

- a DENV-2/4 chimera (TDV-4).

[0051] The genetically modified tetravalent dengue vaccine TDV is based on a molecularly characterized and cloned dengue-2 virus strain (TDV-2). This attenuated TDV-2 strain was generated by cDNA cloning of the attenuated laboratory-derived DEN-2 PDK-53 virus strain that was originally isolated at Mahidol University, Bangkok, Thailand (Kinney et al. (1997) Virology 230(2): 300-308). DEN-2 PDK-53 was generated by 53 serial passages in primary dog kidney (PDK) cells at 32°C (Bhamarapravati et al. (1987) Bull. World Health Organ. 65(2): 189-195).

[0052] The attenuated DEN-2 PDK-53 strain (the precursor of TDV-2) was derived from the wild type virus strain DEN-2 16681 and differs in nine nucleotides from the wild type as follows (Kinney et al. (1997) Virology 230(2): 300- 308): a. 5'-noncoding region (NCR)-57 (nt-57 C-to-T): major attenuation locus

b. prM-29 Asp-to-Val (nt-524 A-to-T)

c. nt-2055 C-to-T (E gene) silent mutation

d. NS1-53 Gly-to-Asp (nt-2579 G-to-A): major attenuation locus

e. NS2A-181 Leu-to-Phe (nt-4018 C-to-T)

f. NS3-250 Glu-to-Val (nt-5270 A-to-T): major attenuation locus

g. nt-5547 (NS3 gene) T-to-C silent mutation

h. NS4A-75 Gly-to-Ala (nt-6599 G-to-C)

* nt-8571 C-to-T (NS5 gene) silent mutation

[0053] The three nucleotide changes located in the 5' noncoding region (NCR) (nucleotide 57) (mutation (i)), the NS-1 (amino acid 828 of SEQ ID NO. 4) (mutation (iv)) and NS-3 genes (amino acid 1725 of SEQ ID NO. 4) (mutation (vi)) form the basis for the attenuation phenotype of the DEN-2 PDK-53 strain (Butrapet et al. (2000) 1 Virol. 74(7): 3111-3119) (Table 2). These three mutations are referred to herein as the "attenuating mutations" and are comprised in TDV-1, TDV-2, TDV-3 and TDV-4. Table 2. Attenuating mutations in the common genetic backbone of all TDV strains

[0054] In one embodiment, TDV-2 comprises in addition to the three attenuating mutations one or more mutations selected from:

a) a mutation in the prM gene at nucleotide 524 from adenine to thymine resulting in an amino acid change at position 143 from asparagine to valine, and/or

b) a silent mutation in the E gene at nucleotide 2055 from cytosine to thymine, and/or

c) a mutation in the NS2A gene at nucleotide 4018 from cytosine to thymine resulting in an amino acid change at position 1308 from leucine to phenylalanine, and/or

d) a silent mutation in the NS3 gene at nucleotide 5547 from thymine to cytosine, and/or

e) a mutation in the NS4A gene at nucleotide 6599 from guanine to cytosine resulting in an amino acid change at position 2168 from glycine to alanine, and/or

f) a silent mutation in the prM gene at nucleotide 900 from thymine to cytosine.

[0055] The silent mutation in the NS5 gene at nucleotide 8571 from cytosine to thymine of DEN-2 PDK-53 is not present in the TDV-2 strain.

[0056] In another embodiment, TDV-2 comprises in addition to the three attenuating mutations one or more mutations selected from:

g) a mutation in the prM gene at nucleotide 592 from adenine to guanine resulting in an amino acid change at position 166 from lysine to glutamine, and/or

h) a mutation in the NS5 gene at nucleotide 8803 from adenine to guanine resulting in an amino acid change at position 2903 from isoleucine to valine.

[0057] In another embodiment, TDV-2 comprises in addition to the three attenuating mutations the mutations a) and g), preferably the mutations a), g), c), e) and h), more preferably the mutations a), g), c), e), h) and b), even more preferably the mutations a), g), c), e), h), b) and d), and most preferably the mutations a) to h). The nucleotide positions and amino acids positions of TDV-2 refer to the nucleotide sequence as shown in SEQ ID NO. 3 and amino acid sequence as shown in SEQ ID NO. 4.

[0058] The dengue virus structural envelope (E) protein and pre-membrane (prM) protein have been identified as the primary antigens that elicit a neutralizing protective antibody response (Plotkin 2001). For creation of the tetravalent dengue vaccine (TDV), TDV-2 was modified by replacing the nucleic acid sequence encoding the DENV-2 prM and E glycoproteins with the nucleic acid sequence encoding the corresponding wild type prM and E glycoproteins from the DENV-1, DENV-3, and DENV-4 wild type strains DENV-1 16007, DENV-3 16562 or DENV-4 1036 virus, respectively, (see Table 3) using standard molecular genetic engineering methods (Huang et al. (2003) 1 Virol. 77(21): 11436-11447). Table 3.Viral origin of prM/E gene regions of the TDV virus strains

[0059] A diagram of the of the four TDV strains comprised in the dengue vaccine composition is shown in Figure 1

[0060] The chimeric dengue strains TDV-1, TDV-3 and TDV-4 express the surface antigens prM and E of the DENV-1, DENV-3 or DENV-4 viruses, as depicted in Table 3 respectively, and retain the genetic alterations responsible for the attenuation of TDV-2. Thus, each of the TDV-1, TDV-3 and TDV-4 strains comprises the attenuating mutations described in Table 2.

[0061] In one embodiment, TDV-1 comprises in addition to the three attenuating mutations one or more mutations selected from:

c) a mutation in the NS2A gene at nucleotide 4018 from cytosine to thymine resulting in an amino acid change at position 1308 from leucine to phenylalanine, and/or

d) a silent mutation in the NS3 gene at nucleotide 5547 from thymine to cytosine, and/or

e) a mutation in the NS4A gene at nucleotide 6599 from guanine to cytosine resulting in an amino acid change at position 2168 from glycine to alanine, and/or

i) a silent mutation in the E gene at nucleotide 1575 from thymine to cytosine, and/or

j) a silent mutation in the junction site between the prM-E gene and the DEN-2 PDK-53 backbone at nucleotide 453 from adenine to guanine, and/or

k) a mutation in the junction site between the prM-E gene and the DEN-2 PDK-53 backbone at nucleotides 2381/2382 from thymine-guanine to cytosine-cytosine resulting in an amino acid change at position 762 from valine to alanine.

[0062] In another embodiment, TDV-1 comprises in addition to the three attenuating mutations one or more mutations selected from:

L) a mutation in the NS2A gene at nucleotide 3823 from adenine to cytosine resulting in an amino acid change at position 1243 from isoleucine to leucine, and/or

m) a mutation in the NS2B gene at nucleotide 4407 from adenine to thymine resulting in an amino acid change at position 1437 from glutamine to asparagine, and/or

n) a silent mutation in the NS4B gene at nucleotide 7311 from adenine to guanine.

[0063] In another embodiment, the TDV-1 strain comprises in addition to the three attenuating mutations the mutations I) and m), preferably the mutations I), m), c) and e), even more preferably the mutations I), m), c), e), d) and n), and most preferably the mutations I), m), c), e), d), n), i), j) and k). The nucleotide positions and amino acids positions of TDV-1 refer to the nucleotide sequence as shown in SEQ ID NO. 1 and amino acid sequence as shown in SEQ ID NO. 2. [0064] In one embodiment, TDV-3 comprises in addition to the three attenuating mutations one or more mutations selected from:

c) a mutation in the NS2A gene at nucleotide 4012 from cytosine to thymine resulting in an amino acid change at position 1306 from leucine to phenylalanine, and/or

d) a silent mutation in the NS3 gene at nucleotide 5541 from thymine to cytosine, and/or

e) a mutation in the NS4A gene at nucleotide 6593 from guanine to cytosine resulting in an amino acid change at position 2166 from glycine to alanine, and/or

j) a silent mutation in the junction site between the prM-E gene and the DEN-2 PDK-53 backbone at nucleotide 453 from adenine to guanine, and/or

k) a mutation in the junction site between the prM-E gene and the DEN-2 PDK-53 backbone at nucleotides 2375/2376 from thymine-guanine to cytosine-cytosine resulting in an amino acid change at position 760 from valine to alanine, and/or

o) a silent mutation in the prM gene at nucleotide 552 from cytosine to thymine, and/or

p) a mutation in the E gene at nucleotide 1970 from adenine to thymine resulting in an amino acid change at position 625 from histidine to leucine.

[0065] In another embodiment, TDV-3 comprises in addition to the three attenuating mutations one or more mutations selected from:

q) a mutation in the E gene at nucleotide 1603 from adenine to thymine resulting in an amino acid change at position 503 from threonine to serine, and/or

r) a silent mutation in the NS5 gene at nucleotide 7620 from adenine to guanine.

[0066] In another embodiments, TDV-3 comprises in addition to the three attenuating mutations the mutations p) and q), preferably the mutations p), q), c) and e), even more preferably the mutations p), q), c), e), d) and r), and most preferably the mutations p), q), c), e), d), r), j), k) and o). The nucleotide positions and amino acids positions of TDV-3 refer to the nucleotide sequence as shown in SEQ ID NO. 5 and amino acid sequence as shown in SEQ ID NO. 6.

[0067] In one embodiment, TDV-4 comprises in addition to the three attenuating mutations one or more mutations selected from:

c) a mutation in the NS2A gene at nucleotide 4018 from cytosine to thymine resulting in an amino acid change at position 1308 from leucine to phenylalanine, and/or

d) a silent mutation in the NS3 gene at nucleotide 5547 from thymine to cytosine, and/or

e) a mutation in the NS4A gene at nucleotide 6599 from guanine to cytosine resulting in an amino acid change at position 2168 from glycine to alanine, and/or

j) a silent mutation in the junction site between the prM-E gene and the DEN-2 PDK-53 backbone at nucleotide 453 from adenine to guanine, and/or

k) a mutation in the junction site between the prM-E gene and the DEN-2 PDK-53 backbone at nucleotides 2381/2382 from thymine-guanine to cytosine-cytosine resulting in an amino acid change at position 762 from valine to alanine, and/or

s) a mutation in the C gene at nucleotide 396 from adenine to cytosine resulting in an amino acid change at position 100 from arginine to serine, and/or

t) a silent mutation in the E gene at nucleotide 1401 from adenine to guanine, and/or

u) a mutation in the E gene at nucleotide 2027 from cytosine to thymine resulting in an amino acid change at position 644 from alanine to valine, and/or v) a mutation in the E gene at nucleotide 2275 from adenine to cytosine resulting in an amino acid change at position 727 from methionine to leucine.

[0068] In another embodiment, TDV-4 comprises in addition to the three attenuating mutations one or more mutations selected from:

w) a silent mutation in the C gene at nucleotide 225 from adenine to thymine, and/or

x) a mutation in the NS2A gene at nucleotide 3674 from adenine to guanine resulting in an amino acid change at position 1193 from asparagine to glycine, and/or

y) a mutation in the NS2A gene at nucleotide 3773 from adenine to an adenine/guanine mix resulting in an amino acid change at position 1226 from lysine to a lysine/asparagine mix, and/or

z) a silent mutation in the NS3 gene at nucleotide 5391 from cytosine to thymine, and/or

aa) a mutation in the NS4A gene at nucleotide 6437 from cytosine to thymine resulting in an amino acid change at position 2114 from alanine to valine, and/or

bb) a silent mutation in the NS4B gene at nucleotide 7026 from thymine to a thymine/cytosine mix, and/or cc) a silent mutation in the NS5 gene at nucleotide 9750 from adenine to cytosine.

[0069] In another embodiments, TDV-4 comprises in addition to the three attenuating mutations the mutation s), u) and v), preferably the mutations s), u), v), c), e), x), y) and aa), even more preferably the mutations s), u), v), c), e), x), y), aa) and w), even more preferably the mutations s), u), v), c), e), x), y), aa), w), d), z), bb) and cc), and most preferably the mutations s), u), v), c), e), x), y), aa), w), d), z), bb), cc), j), k) and t). The nucleotide positions and amino acids positions of TDV-4 refer to the nucleotide sequence as shown in SEQ ID NO. 7 and amino acid sequence as shown in SEQ ID NO. 8.

[0070] In a preferred embodiment, TDV-1 has the nucleotide sequence of SEQ ID NO. 1, TDV-2 has the nucleotide sequence of SEQ ID NO. 3, TDV-3 has the nucleotide sequence of SEQ ID NO. 5, and/or TDV-4 has the nucleotide sequence of SEQ ID NO. 7. In a further preferred embodiment, TDV-1 has the amino acid sequence of SEQ ID NO. 2, TDV-2 has the amino acid sequence of SEQ ID NO. 4, TDV-3 has the amino acid sequence of SEQ ID NO. 6, and TDV-4 has the amino acid sequence of SEQ ID NO. 8. In a further preferred embodiment, TDV-1 has a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 2, TDV-2 has a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 4, TDV-3 has a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 6, and TDV-4 has a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 8.

Table 4.Sequences of the TDV virus strains

[0071] Thus, in a particularly preferred embodiment, the dengue virus vaccine comprises the live attenuated dengue virus strains TDV-1, TDV-2, TDV-3 and TDV-4, wherein TDV-1, TDV-3 and TDV-4 are based on TDV-2 and comprise the prM and E regions of DENV-1, -3 and -4, respectively. In another particularly preferred embodiment, TDV-1 is characterized by the nucleotide sequence according to SEQ ID No. 1 and the amino acid sequence according to SEQ ID No. 2, TDV-2 is characterized by the nucleotide sequence according to SEQ ID No. 3 and the amino acid sequence according to SEQ ID No. 4, TDV-3 is characterized by the nucleotide sequence according to SEQ ID No. 5 and the amino acid sequence according to SEQ ID No. 6 and TDV-4 is characterized by the nucleotide sequence according to SEQ ID No. 7 and the amino acid sequence according to SEQ ID No. 8.

[0072] The E protein of DENV-3 has two fewer amino acids than the E protein of DENV-2. Therefore, the nucleotides and encoded amino acid backbone of TDV-2 starting after the E region of DENV-3 at nucleotide 2374 of SEQ ID NO. 5 and amino acid 760 of SEQ ID NO. 6 are 6 nucleotides less and 2 amino acids less than the original TDV-2 nucleotide and amino acid positions, respectively.

[0073] Preferably, in said embodiments the chimeric dengue serotype 2/1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2/3 strain is TDV-3 and the chimeric dengue serotype 2/4 strain is TDV-4. More preferably, TDV-1 is characterized by the nucleotide sequence according to SEQ ID No. 1 and the amino acid sequence according to SEQ ID No. 2, TDV-2 is characterized by the nucleotide sequence according to SEQ ID No. 3 and the amino acid sequence according to SEQ ID No. 4, TDV-3 is characterized by the nucleotide sequence according to SEQ ID No. 5 and the amino acid sequence according to SEQ ID No. 6 and TDV-4 is characterized by the nucleotide sequence according to SEQ ID No. 7 and the amino acid sequence according to SEQ ID No. 8.

[0074] Preferably, the chimeric dengue serotype 2/4 strain, preferably TDV-4, has the highest concentration in the dengue vaccine composition, followed by the chimeric dengue serotype 2/3 strain, preferably TDV-3, followed by the chimeric dengue serotype 2/1 strain, preferably TDV-1, followed by the dengue serotype 2 strain, preferably TDV-2. It is particularly preferred that the dengue serotype 2 strain has the lowest concentration of the four strains present in the dengue vaccine composition.

[0075] In another embodiment, the dengue vaccine comprises live attenuated chimeric dengue virus, wherein the virus backbone is derived from a yellow fever (YF) virus, in which case, the chimera is referred to herein as a "chimeric YF/dengue virus". Typically, in the chimeric YF/dengue virus the prM and E sequences of the attenuated yellow fever backbone are replaced with the prM and E sequences of a dengue serotype. Examples of dengue/YF chimeric viruses are described in patent application WO 98/37911. A tetravalent dengue virus vaccine comprising dengue/YF chimeric viruses wherein the prM and E sequences of the attenuated yellow fever backbone are replaced with the prM and E sequences of a dengue serotype is marketed under the name Dengvaxia®.

[0076] The chimeric YF/dengue virus may comprise the genomic backbone of the attenuated yellow fever virus strain YF17D (Theiler M . and Smith H.H. (1937) 1 Exp. Med., 65, p. 767-786) (viruses YF17D/DEN-1, YF17D/DEN-2, YF17D/DEN-3, YF 1 7D/DEN-4). Examples of YF17D strains which may be used as a genomic backbone include, but are not limited to, YF17D204 (YF-VAX®, Sanofi-Pasteur, Swiftwater, PA, USA; Stamaril®, Sanofi-Pasteur, Marcy I'Etoile, France; ARILVAX™, Chiron, Speke, Liverpool, UK; FLAVIMUN®, Berna Biotech, Bern, Switzerland; YF17D- 204 France (X15067, X15062); YF17D- 204,234 US (Rice et al., 1985, Science, 229: 726-733), or the related strains YF17DD (Genbank access number U17066), YF17D-213 (Genbank access number U17067) and the strains described by Galler et al. (1998, Vaccines, 16(9/10): 1024-1028). Any other attenuated yellow fever virus strain which may be used in humans may be used to construct chimeric YF/dengue viruses.

[0077] One example of a chimeric YF/dengue virus is the "Chimerivax™ dengue" or "CYD", a chimeric yellow fever (YF) virus which comprises the genomic backbone of an attenuated YF virus in which the sequences coding for the pre-membrane (prM) and envelope (E) proteins have been replaced by nucleic acid sequences encoding the corresponding structural proteins of a dengue virus. Construction of chimeric Chimerivax virus may be achieved in substantial accordance with the teaching of Chambers et al. (1999) J Virology 73(4):3095-3101. A chimeric dengue virus containing the prM and E sequences of a serotype 1 dengue fever strain (DEN-1) is referred to as "CYD-l or CYD DENI". A chimeric YF containing the prM and E sequences of a DEN-2 strain is referred as "CYD-2 or CYD DEN2". A chimeric YF virus containing the prM and E sequences of a DEN-3 strain is referred to as "CYD-3 or CYD

DEN3". A chimeric YF virus containing the prM and E sequences of a DEN-4 strain is referred to as "CYD-4 or CYD

DEN4". The preparation of these dengue Chimerivax™ viruses have been described in detail in international patent applications WO 98/37911 and WO 03/101397, to which reference may be made for a precise description of the processes for their preparation. The chimeras may be generated by using prM and E sequences from strains DEN 1

PU0359 (TYP1140), DEN2 PU0218, DEN3 PaH881/88 and DEN 4 1228 (TVP 980). Alternatively, other dengue fever virus strains may be used as a source of nucleic acids to facilitate construction of chimeric YF/dengue viruses. Alternatively, other dengue fever virus strains may be used as a source of nucleic acids to facilitate construction of chimeric viruses useful in the practice of the present invention.

Method for determining the titer of neutralizing antibodies

[0078] The present invention is directed to a method for determining the titer of neutralizing antibodies against each of dengue serotypes 1, 2, 3 and 4 in a blood serum sample, the method comprising the steps of:

(a) seeding cells from a dengue-susceptible cell line on 96-well assay plates and culturing the cells for a culture period;

(b) preparing serial dilutions of the blood serum sample;

(c) separately mixing the serially diluted blood serum samples prepared in step (b) with dengue serotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4 to obtain separate mixtures for each dengue serotype and incubating the separate mixtures;

(d) adding the separate mixtures prepared in (c) to the cells seeded and cultured in step (a) and incubating the cells with the separate mixtures;

(e) providing an overlay for the inoculated cells and incubating the cells for an incubation period of 40 to 75 hours;

(f) determining the number of plaques in each well and comparing the number of plaques in each well to an unneutralized control to determine the level of neutralizing antibodies against each of dengue serotypes 1, 2, 3 and 4.

[0079] The blood serum samples are obtained by collecting blood from a human subject and separating the serum from the other components of the blood. The blood serum sample may be obtained from a human subject which has not been vaccinated with a dengue virus vaccine, e.g. to determine whether the subject is seronegative or seropositive before vaccination. Alternatively, the blood serum sample may be obtained from a human subject which has been vaccinated with a dengue virus vaccine, e.g. to determine whether the subject has developed a neutralizing antibody response against the dengue virus vaccine. The dengue virus vaccine with which the subject has been vaccinated may be a tetravalent dengue virus composition as described above. In one embodiment, the blood serum sample is heat inactivated before use. In one embodiment, the blood serum sample is stored at a temperature of less than or equal to -60°C. In the method of the present invention serial dilutions of the blood serum samples are prepared. The serial dilution of the blood serum samples is the stepwise dilution of the blood serum samples according to a given dilution factor. In one embodiment, the blood serum samples are stepwise diluted two-fold from an initial 1 : 10 dilution. [0080] In one embodiment, the dengue-susceptible cell line used in step (a) is selected from Vero cells, LLC-MK2 cells and BHK-21 cells. Preferably, the dengue-susceptible cell line used in step (a) is a Vero cell line. The dengue- susceptible cell line is seeded on 96-well plates, i.e. a defined amount of the dengue-susceptible cell line is introduced into a well of a 96-well plate which contains a suitable growth medium for the dengue-susceptible cell line. Suitable growth media for dengue-susceptible cell lines are known to the skilled person and include DMEM with 10% fetal bovine serum. The dengue-susceptible cell line is seeded with a density of 1 to 4 x 10⁵ cells per ml, preferably of 1.5 to 3.5 x 10⁵ cells per ml, more preferably of 2 to 3 x 10⁵ cells per ml and most preferably of 2.5 x 10⁵ cells per ml. In some embodiments, the dengue-susceptible cell line is cultured for a culture period of 12 to 36 hours, preferably of 18 to 30 hours and most preferably of 20 to 24 hours. The culture period is calculated from the time the cells are seeded until the time the separate mixtures of the serially diluted blood serum samples with dengue serotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4 are added to the cells.

[0081] If the subject from which the blood serum sample has been obtained has been vaccinated, the dengue serotype strains with which the serially diluted blood serum samples are separately mixed are those strains from which the immunogenic components with which the subject has been vaccinated are derived. In one embodiment, the dengue serotype strains comprise one or more of the following : DENV-1 strain 16007, DENV-2 strain 16681, DENV-3 strain 16562 and DENV-4 strain 1036. In one embodiment, the subject has been vaccinated with a tetravalent dengue virus composition comprising a chimeric dengue serotype 2/1 strain comprising the prM and E genes of DENV-1 strain 16007, a dengue serotype 2 strain comprising the prM and E genes of DENV-2 strain 16681, a chimeric dengue serotype 2/3 strain comprising the prM and E genes of DENV-3 strain 16562, and a chimeric dengue serotype 2/4 strain comprising the prM and E genes of DENV-4 strain 1036.

[0082] The separate mixtures of the serially diluted blood serum samples with dengue serotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4 prepared in step (c) may be incubated overnight at a temperature of 2 to 8°C. Alternatively, The separate mixtures of the serially diluted blood serum samples with dengue serotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4 prepared in step (c) may be incubated for 1 to 2 hours at a temperature of 37°C.

[0083] In one embodiment, in step (c) the dengue serotype 1 is DENV-1 strain 16007, dengue serotype 2 is DENV-2 strain 16681, dengue serotype 3 is DENV-3 strain 16562 and dengue serotype 4 is DENV-4 strain 1036.

[0084] The separate mixtures of the serially diluted blood serum samples with dengue serotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4 prepared in step (c) are added to the dengue-susceptible cell line to allow for virus absorption. The cells are incubated with the separate mixtures of the serially diluted blood serum samples with dengue serotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4 for a period of 60 to 180 minutes, preferably for a period of 90 to 120 minutes. The cells are incubated with the separate mixtures of the serially diluted blood serum samples with dengue serotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4 for a period of 60 to 180 minutes, preferably for a period of 90 to 120 minutes at a temperature of 37°C.

[0085] The overlay provided in step (e) to the incubated cells serves to limit the virus diffusion within the plate which permits plaque formation. The overlay can be added to the cells either after aspiration of the separate mixtures of serially diluted blood serum samples with dengue serotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4 or without aspiration of these mixtures. Preferably, the overlay is added to the cells without aspiration of the separate mixtures of serially diluted blood serum samples with dengue serotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4. In one embodiment, the overlay in step (e) is selected from the group consisting of methylcellulose, carboxymethylcellulose and agarose. Preferably, the overlay is methylcellulose. More preferably, the overlay is 1% methylcellulose in DMEM. In some embodiments, the cells with the overlay are incubated at a temperature of 33°C to 35°C, preferably at a temperature of 34°C.

[0086] In one embodiment, different incubation periods are used in step (e) for the mixtures of different dengue serotypes. In some embodiments, the incubation period for mixtures of dengue serotype 4 is shorter than the incubation period for mixtures of dengue serotypes 1, 2 and 3, for example the incubation period for mixtures of dengue serotype 4 is less than 50 hours, preferably 46±2 hours. In some embodiments, the incubation period for mixtures of dengue serotype 2 is longer than the incubation period for mixtures of dengue serotypes 1, 3 and 4, for example the incubation period for mixtures of dengue serotype 2 is between 65 and 75 hours, preferably 70±2 hours. In some embodiments, the incubation period for mixtures of dengue serotypes 1 or 3 is between 60 and 70 hours, preferably 66±2 hours. In some embodiments, the incubation period for mixtures of dengue serotype is between 60 and 70 hours, the incubation period for mixtures of dengue serotype 2 is between 65 and 75 hours, the incubation period for mixtures of dengue serotype 3 is between 60 and 70 hours and the incubation period for mixtures of dengue serotype 4 is less than 50 hours. In some embodiments, the incubation period for mixtures of dengue serotype 1 is 66±2 hours, the incubation period for mixtures of dengue serotype 2 is 70±2 hours, the incubation period for mixtures of dengue serotype 3 is 66±2 hours and the incubation period for mixtures of dengue serotype 4 is 46±2 hours.

[0087] In one embodiment, the number of plaques in each well is determined using serotype-specific anti-dengue monoclonal antibodies. The skilled person knows how to prepare serotype-specific antibodies. Suitable approaches are described for example in Gentry et al. (1982) Am. 1 Trap. Med. Hyg. 31, 548-555; Henchal et al. (1985) Am. 1 Trap. Med. Hyg. 34, 162-169; and Henchal et al. (1982) Am. J. Trap. Med. Hyg. 31(4):830-6). For example, mice can be immunized with a specific dengue serotype and the B cells isolated from these mice can be fused with a fusion partner to prepare a hybridoma. Suitable serotype-specific antibodies are selected based on the binding of the antibodies to the serotype with which the mice were immunized and lack of binding to those serotypes with which the mice were not immunized. In one embodiment, the mice were immunized with a serotype selected from dengue 1 strain Hawaii, Envelope, dengue 2 strain New Guinea C, Envelope, isotype 1, dengue 3 strain H87, Envelope, isotype 2A, and dengue 4 strain H241, Envelope, isotype 1.

[0088] To determine the number of plaques, the overlay is removed from the cells and the cells are washed, e.g. with phosphate-buffered saline. After washing, the cells are fixed with methanol or acetone for 60 minutes at a temperature of less than or equal to -20°C. After washing the cells, the serotype specific anti-dengue monoclonal antibodies are added to the corresponding wells and incubated for 18±4 hours at 2-8°C, before the cells are washed and incubated with a labelled secondary antibody binding to the serotype specific anti-dengue monoclonal antibodies for 9o to 120 minutes at 37°C. After washing, the substrate for the enzyme attached to the labelled secondary antibody is added and incubated for an appropriate period. If the secondary antibody is labelled with peroxidase, the substrate may be 2-amino-9-ethyl carbazole (AEC) in DMSO and the incubation period is 20 minutes at room temperature.

[0089] The number of plaques may be determined visually or using a plaque counter such as the ViruSpot Plaque counter. The percentage neutralization reduction may be determined compared to the virus control and the MNT50 value may be calculated.

[0090] In one embodiment, the invention is directed to a method for determining the titer of neutralizing antibodies against each of dengue serotypes 1, 2, 3 and 4 in a blood serum sample, the method comprising the steps of:

(a) seeding Vero cells on 96-well assay plates and culturing the Vera cells for a period of 20 to 30 hours;

(b) preparing serial dilutions of the serum sample; (c) separately mixing the serially diluted serum samples with dengue serotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4 to prepare separate mixtures and incubating the separate mixtures overnight at a temperature of 2 to 8°C;

(d) incubating the cells seeded and cultured in step (a) with the separate mixtures prepared in step (c) in separate wells for 90 to 120 minutes;

(e) providing a methylcellulose overlay for the inoculated cells and incubating the cells for an incubation period of 40 to 75 hours at 34°C;

(f) determining the number of plaques in each well using serotype-specific anti-dengue monoclonal antibodies and comparing the number of plaques in each well to an unneutralized control to determine the level of neutralizing antibodies against each of dengue serotypes 1, 2, 3 and 4.

[0091] In one embodiment, the invention is directed to a method for determining the titer of neutralizing antibodies against each of dengue serotypes 1, 2, 3 and 4 in a blood serum sample, the method comprising the steps of:

(a) seeding Vero cells on 96-well assay plates and culturing the Vero cells for a period of less than 24 hours;

(b) preparing serial dilutions of the serum sample;

(c) separately mixing the serially diluted serum samples with dengue serotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4 to prepare separate mixtures and incubating the separate mixtures overnight at a temperature of 2 to 8°C;

(d) incubating the cells seeded and cultured in step (a) with the separate mixtures prepared in step (c) in separate wells for 90 to 120 minutes;

(e) providing a methylcellulose overlay for the inoculated cells and incubating the cells for the following incubation periods at 34°C:

- dengue serotype 1 : 66±2 hours,

- dengue serotype 2: 70±2 hours,

- dengue serotype 3: 66±2 hours,

- dengue serotype 4: 46±2 hours;

(f) determining the number of plaques in each well using serotype-specific anti-dengue monoclonal antibodies and comparing the number of plaques in each well to an unneutralized control to determine the level of neutralizing antibodies against each of dengue serotypes 1, 2, 3 and 4.

[0092] In one embodiment, the invention is directed to the use of said method for determining the dengue serostatus of a subject before vaccination with a dengue virus vaccine or for analyzing a subject's antibody response after vaccination with a dengue virus vaccine.

EXAMPLES

[0093] The following Examples are included to demonstrate certain aspects and embodiments of the invention as described in the claims. It should be appreciated by those of skill in the art, however, that the following description is illustrative only and should not be taken in any way as a restriction of the invention.

Example 1 : Preparation of the dengue virus strains.

[0094] The methods used to generate the chimeric dengue strains TDV-1, -3 and -4 were standard molecular cloning and DNA engineering methods and are described in Huang et al. (2003) 1 Virology 77(21): 11436-11447. The following well-known methods were used to construct and introduce the prM-E genes of dengue serotypes 1, 3 and 4 into the TDV-2 backbone: Reverse-transcriptase PCR (RT-PCR), PCR, restriction enzyme digestion, DNA fragment ligation, bacterial transformations by electroporation, plasmid DNA preparations, in vitro transcription by T7 RNA polymerase, and transfection of Vero cells by electroporation.

[0095] After growing and purifying the different dengue serotypes separately as described in Huang et al. (2013) PLOS Neglected Dis, 7(5):e2243, they are mixed in specified concentrations. The mixture of dengue serotypes is present in a dengue vaccine composition and combined with a composition of pharmaceutically acceptable excipients resulting in a dengue vaccine composition comprising 15% w/v a, a trehalose dihydrate, 1% w/v poloxamer 407, 0.1% w/v human serum albumin and 100 mM sodium chloride. The dengue vaccine composition is lyophilized and represents a lyophilized unit dose of TDV. The lyophilized unit dose is reconstituted with 37 mM aqueous sodium chloride solution and the reconstituted unit dose comprises 15% w/v a, a trehalose dihydrate, 1% w/v poloxamer 407, 0.1% w/v human serum albumin and 137 mM sodium chloride.

Example 2: Microneutralization test

[0096] Immunogenicity was measured by a microneutralization assay to each one of the four dengue serotypes with titers defined as the dilution resulting in a 50% reduction in plaque values (MNT50). Briefly, on day 1 Vero cells were seeded on 96-well assay plates in DMEM and 10% FBS at a density of 2.5 x 10⁵ cells/ml and incubated at 37°C for 24 hours. Human serum samples were heat inactivated and stored at < -60°C until use in the assay. The test sera were obtained from subjects immunized with the chimeric dengue strains TDV-1, -3 and -4 as described in Example 1 as well as with the TDV-2 strain described herein. Positive controls for the four dengue serotypes comprised human serum derived from patients who had natural dengue virus infection diluted into negative human serum to generate controls with high, medium and low titers of neutralizing antibodies to each dengue serotype. On day 2 serial dilutions of the heat-inactivated antibody-containing test and control sera samples (dilutions range 1 : 10 to 1 :20480) were prepared and mixed with a constant concentration of dengue viruses, in particular DENV-1 strain 16007, DENV-2 strain 16681, DENV-3 strain 16562 and DENV-4 strain 1036, (target 60-80 pfu/well) in 10% DMEM in a 96 well microtiter plate and incubated overnight at 2-8°C to enable the neutralization of the virus by the antibodies present in the sera. After the incubation the mixture of virus and antibodies was transferred onto the 96 well plates with Vero cells and the plates were incubated at 37°C for 90-120 minutes to infect the Vero cells. A 1% methylcellulose overlay in DMEM was applied to the plate to restrict spread of progeny virus and the plate was incubated for 46-70 hours at 34°C depending on the Dengue serotype:

DENV1 - 66±2 hours

DENV2 - 70±2 hours

DENV3 - 66±2 hours

DENV4 - 46±2 hours

[0097] After the incubation the cells were washed twice with PBS and fixed by adding cold methanol and incubating for 60 minutes at a temperature of < -20°C. After fixing the plates were dried and washed three times with washing buffer (lx PBS, pH 7.4 with 0.5% Tween), before 50 pi of serotype-specific anti-dengue mouse monoclonal antibodies in blocking solution (2.5% nonfat dry milk in PBST) per well were added and incubated with the cells for 18±4 hours at 2-8°C.

[0098] The monoclonal antibodies were made as described in Gentry et al. (1982) Am. 1 Trap. Med. Hyg. 31, 548-555; Henchal et al. (1985) Am. J. Trap. Med. Hyg. 34, 162-169; and Henchal et al. (1982) Am. J. Trap. Med. Hyg. 31(4):830-6). Briefly, the anti-DENV-1 HBD was made against dengue 1 strain Hawaii, Envelope, the anti-DENV- 2 was made against dengue 2 strain New Guinea C, Envelope, isotype 1, the anti-DENV-3 HBD was made against dengue 3 strain H87, Envelope, isotype 2A, and the anti-DENV-4 HBD was made against dengue 4 strain H241, Envelope, isotype 1.

[0099] After incubation, the plates were washed three times with washing buffer and 50 pi of a secondary peroxidase labelled goat anti-mouse IgG (H + L) (KPL Cat#074-1806) in blocking solution was added and incubated for 90 to 120 minutes at 37°C. Then the plates were washed three times with washing buffer and 50 mI of precipitant substrate (2-amino-9-ethyl carbazole (AEC) tablet in 2.5 ml DMSO, 47.5 ml 50mM acetate buffer and 250 mI hydrogen peroxide) were added and the mixture was incubated for 20 minutes at room temperature. Finally, the substrate was removed, the plates were rinsed with dH₂0 and dried.

[00100] Sample titers were calculated using the linear regression method and reported as MNT50 titers for each sample. Clinical data were reported as a geometric mean titer for all the individual MNT50 titers in each treatment group. Briefly, the number of infectious foci in each well was counted and the titer of neutralizing antibodies was determined by comparing the percent reduction of infectious foci centers in wells containing antibody (test samples) in comparison to wells containing virus alone. The MNT50 was calculated using the following linear regression equation :

MNT50 = 10^A[(50-c)/m]) where c = y intercept of regression line and m = slope of regression line

[00101] Each test sample was tested in triplicates and the titer was calculated from the average of the triplicates. A schematic drawing of the steps performed in this test is provided in Figure 2.

Example 3: Validation of the microneutralization test

[00102] The microneutralization test was validated by assessing the following parameters: interassay precision, limits of quantitation, lower limit of detection and linearity.

a) Interassay precision

[00103] Assay precision is an estimate of the variation due to random error as well as due to analysis on different days by different operators. To measure intermediate precision, eight dilutions of six high-titer anti-DENV serum samples from participants in clinical studies DEN-106 (NCT02193087) and DEN-204 (NCT02302066) were tested in singleton by three technicians on four separate days, for a total of 12 replicates each. The assays over the four days also included plates containing different Vero cell passages. Each high-titer sample was diluted 2- or 4-fold in negative human serum to obtain samples with titers across the range of the assay. The seventh dilution targeted the lower limit of quantitation (LLOQ) and the eighth dilution targeted below the LLOQ. The overall %GCV (percent geometric coefficient of variation) for intermediate precision of the diluted samples had to be < 60.

[00104] To evaluate intermediate precision further using incurred samples, a panel of 20 additional samples from vaccinated participants in the DEN-106 and DEN-204 clinical trials that had anti-dengue antibody titers across the range of the assay (<10 to 20480) were tested a minimum of 12 times. Three technicians tested the sample panel in a single batch on 4 separate days.

[00105] The overall %GCV and contribution of variance was calculated for the 12 replicates of each of the 20 samples for each Dengue serotype using variance component analysis using residual maximum likelihood (REML). Raw data were used to calculate the contribution of variance and overall intermediate precision (%GCV).

[00106] Variability estimates were obtained on the natural log (In)-transformed titers using the MIXED procedure in SAS. When estimating variability by sample for each serotype, a MIXED model containing random effects for analyst ' ' ’and run within analyst ¹ * ·^' · · ^¾was used. Variability was measured across the samples tested within the 12 precision runs and within the 12 accuracy runs. Variability estimates (reported as %GCV) were calculated as

[00107] To determine overall variability, all samples with geometric median titers (GMedT) within the quantifiable range for the assay were combined; those with a GMedT across the 12 assay runs that was outside the quantifiable range of the assay were excluded from the analysis. All remaining titers that were either < LLOQ or > ULOQ were used in the analysis. A MIXED model containing fixed effects for sample (and dilution where appropriate) and random

( CT ) {

^L

effects for analyst ' ^{A J}), day ^D , analyst by day ', and all other interaction

was used. The overall variability estimates (measured in %GCV) were calculated as

[00108] The results are shown in Table 5 below.

[00109] Table 5: Overall sample precision estimates for high-titer (intermediate precision) and range- of-titers (incurred) samples for all samples tested and for samples with GMedT within the assay LOQ

[00110] The overall %GCV ranged from 26.0 to 36.5 for the high-titer dilution samples, and from 30.6 to 77.1 for the incurred samples. After samples with GMedT outside the quantifiable range were excluded, the combined overall %GCV across both the dilution and incurred samples ranged from 28.9 to 48.1 across the serotypes (Table 1).

[00111] The overall %GCV met the acceptance criteria (<60%) for the high-titer dilution samples for all four serotypes and for DENV-2, -3 and -4 for the incurred samples. Overall %GCV for the DENV-1 incurred samples was > 60% (77.1%) which was most likely due to discrepant MNT50 values observed for two incurred samples on one of the test days as the result of a possible sample mix-up by one operator. The MNT50 values for these two samples in this run were < 10, while the other 11 replicates for these samples resulted in MNT50 values > 100. The titer assignment for the negative results (< 10) for these samples resulted in very high per sample %GCV and skewed the overall %GCV for the DENV-1 incurred sample analysis.

b) Limits of quantitation

[00112] The lower and upper limits of quantitation (LLOQ and ULOQ) defined the concentration range over which the assay was acceptably accurate and was able to precisely quantitate samples. The limits of quantitation (LOQ) were set based on acceptable performance of the assay by evaluating the precision profile of the test samples and the accuracy of the assay.

[00113] The final limits of quantitation were determined by using the following criteria :

i. The LLOQ had to be > 10.

ii. The ULOQ could not be > 10240 because 10240 was the second-to-last in the dilution series in which each sample was assayed by this method.

iii. The accuracy fold-bias predicted estimates had to be between 0.62-fold and 1.88-fold throughout the quantifiable range; and 80% of the samples with GMedT within the lower and upper range determined by locally weighted regression (LOESS) smoothing of the precision estimates had to have variability estimates <60% GCV.

iv. The ULOQ could not be higher than the geometric median of the sample with the highest titer used in the precision analysis or than the expected titer of the sample with the highest titer used in the accuracy analysis.

[00114] The limits of quantitation of this assay are summarized in Table 6.

[00115] Table 6: Limits of quantitation of the microneutralization assay

[00116] When using samples within the range of titers identified in Table 6, the assay met the acceptance criteria that the predicted bias between the GMT and the expected titers be between 0.62-fold and 1.88-fold. After applying the final limits of quantitation in Table 6, the proportion of samples with expected titers within the LOQs with bias results that were between 0.62 and 1.88-fold were > 80% for all serotypes, i.e. 92.3% for DENV-1, 87.0% for DENV- 2, 80.0% for DENV-3; and 88.6% for DENV-4.

c) Lower limit of detection (LLOD)

[00117] The LLOD was defined as the lowest antibody titer that could be determined to be statistically different from a blank at a stated confidence level. In other words, samples that did not contain neutralizing antibody could be distinguished from those that did contain neutralizing antibody at dilutions as low as 1 : 10. Briefly, 20 samples for each serotype (12 samples with titers > 10 and 8 samples with titers < 10) were tested in six independent runs by a minimum of two technicians on three different days. To evaluate the LLOD of the assay, the percentage of results falling above and below a titer of 10 was determined for each sample. For each sample, the following were determined : (i) GMedT; (ii) the percentage of individual results within 2- and 3-fold of the GMedT; (iii) variability in terms of GCV. GCV was also assessed across all samples. To meet the precision criteria, > 80% of the samples had to have > 80% of the replicate results within ±2-fold of the GMedT.

[00118] A lower limit of detection of 10 was confirmed to be acceptable for each dengue serotype and met the precision criteria : > 80% of the samples had > 80% of their replicates within ±2-fold and ±3-fold of the GMedT (see Table 7 below). [00119] Table 7: Percent of samples that met the serostatus and precision LLOD criteria

d) Linearity

[00120] Assay linearity was determined by testing eight dilutions covering the range of the assay of six high-titer positive anti-DEN human serum samples in singleton in a minimum of 12 independent assays. Samples were tested by three independent technicians on four separate days.

According to the protocol for inter-assay precision, the overall first order regression had to result in R2 > 0.95 and a slope in the range of -1 ± 0.2. The lack-of-fit/pure error analysis required a p-value >0.05 for the quadratic term. If the p-value of the quadratic term was <0.05 then the percentage difference between the 1st and 2nd order regression models for the predicted loglO GMT for each sample in the linear range had to be <5%, with the exception of samples with titers at or near, but not less than, the LLOD, for which p had to be <10%.

Linearity was assessed by fitting a regression model with the In transformed pre-dilution factor to the In transformed titers. Samples with titers that were either less than the lowest dilution tested or greater than the highest dilution tested were excluded from the analysis. A MIXED model containing fixed effects for In transformed pre-dilution factor was fit to the In transformed titers for each sample. The overall decrease in titer per 2-fold increase in dilution was determined by fitting a MIXED model containing fixed effects for In transformed pre-dilution factor and for sample and random effects for run and sample by run to the In transformed titers. The overall decrease in titer per 2-fold increase in dilution was calculated as 2b, where b represents the slope from the overall linear regression fit. A MIXED model was used, hence the %GCV of the standard error on the slope was determined.

[00121] The linear model generally represented the data in an acceptable manner as there was a strong correlation between MNT50 and dilution as shown by the slope estimate. The individual slope estimates and dilution bias estimates across all samples are summarized in Table 8.

[00122] Table 8: Fold-decrease in titer per 2-fold increase in dilution across samples

[00123] The overall fold-decrease in titer per 2-fold increase in dilution was between 2.03-fold and 2.16-fold across the four dengue serotypes. The overall slope of the regression line was within -1 ±0.2, hence the assay met the acceptance criteria for linearity.

Claims

1. A method for determining the titer of neutralizing antibodies against each of dengue serotypes 1, 2, 3 and 4 in a blood serum sample, the method comprising the steps of:

(a) seeding cells from a dengue-susceptible cell line on 96-well assay plates and culturing the cells for a culture period;

(b) preparing serial dilutions of the blood serum sample;

(c) separately mixing the serially diluted blood serum samples prepared in step (b) with dengue serotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4 to obtain separate mixtures for each dengue serotype and incubating the separate mixtures;

(d) adding the separate mixtures prepared in step (c) to the cells seeded and cultured in step (a) and incubating the cells with the separate mixtures;

(e) providing an overlay for the cells incubated in step (d) and incubating the cells for an incubation period of 40 to 75 hours;

(f) determining the number of plaques in each well and comparing the number of plaques in each well to an unneutralized control to determine the level of neutralizing antibodies against each of dengue serotypes 1, 2, 3 and 4.

2. Method according to claim 1, wherein in step (e) different incubation periods are used for the mixtures of different dengue serotypes.

3. Method according to claim 1 or 2, wherein in step (e) the incubation period for mixtures of dengue serotype

4 is shorter than the incubation period for mixtures of dengue serotypes 1, 2 and 3.

4. Method according to claim 3, wherein the incubation period for mixtures of dengue serotype 4 is 46±2 hours.

5. Method according to any one of claims 1 to 4, wherein in step (e) the incubation period for mixtures of dengue serotype 2 is longer than the incubation period for mixtures of dengue serotypes 1, 3 and 4.

6. Method according to claim 5, wherein the incubation period for mixtures of dengue serotype 2 is 70±2 hours.

7. Method according to any one of claims 1 to 6, wherein the dengue-susceptible cell line is selected from Vero cells, LLC-MK2 cells and BHK-21 cells.

8. Method according to any one of claims 1 to 7, wherein the culture period in step (a) is 12 to 36 hours.

9. Method according to any one of claims 1 to 8 wherein in step (c) the dengue serotype 1 is DENV-1 strain

16007, dengue serotype 2 is DENV-2 strain 16681, dengue serotype 3 is DENV-3 strain 16562 and dengue serotype 4 is DENV-4 strain 1036.

10. Method according to any one of claims 1 to 9, wherein the separate mixtures in step (c) are incubated overnight at a temperature of 2°C to 8°C.

11. Method according to any one of claims 1 to 10, wherein the overlay in step (e) is selected from the group consisting of methylcellulose, carboxymethylcellulose and agarose.

12. Method according to any one of claims 1 to 11, wherein in step (e) the cells are incubated at a temperature of 33°C to 35°C.

13. Method according to any one of claims 1 to 12, wherein the number of plaques in each well is determined using serotype-specific anti-dengue monoclonal antibodies.

14. Method according to any one of claims 1 to 13, wherein said blood serum sample is obtained from a subject which has not been vaccinated with a dengue virus vaccine.

15. Method according to any one of claims 1 to 13, wherein said blood serum sample is obtained from a subject which has been vaccinated with a dengue virus vaccine.

16. Method according to claim 15, wherein the dengue virus vaccine is a tetravalent dengue virus composition.

17. Method according to claim 15 or 16, wherein the dengue virus vaccine comprises a chimeric dengue serotype 2/1 strain, a dengue serotype 2 strain, a chimeric dengue serotype 2/3 strain, and a chimeric dengue serotype 2/4 strain.

18. Method according to claim 16 or 17, wherein each one of the four live attenuated dengue virus strains has attenuating mutations in the 5'-noncoding region (NCR) at nucleotide 57 from cytosine to thymine, in the NS1 gene at nucleotide 2579 from guanine to adenine resulting in an amino acid change at position 828 from glycine to asparagine, and in the NS3 gene at nucleotide 5270 from adenine to thymine resulting in an amino acid change at position 1725 from glutamine to valine.

19. Method according to claim 15 or 16, wherein the dengue virus vaccine comprises a live attenuated chimeric dengue serotype 1 virus, a live attenuated chimeric dengue serotype 2 virus, a live attenuated chimeric dengue serotype 3 virus and a live attenuated chimeric dengue serotype 4 virus.

20. Method according to claim 19, wherein the live attenuated chimeric dengue serotype 1 virus comprises a genome of an attenuated yellow fever virus whose prM-E sequence has been substituted with the prM-E sequence of dengue serotype 1, the live attenuated chimeric dengue serotype 2 virus comprises a genome of an attenuated yellow fever virus whose prM-E sequence has been substituted with the prM-E sequence of dengue serotype 2, the live attenuated chimeric dengue serotype 3 virus comprises a genome of an attenuated yellow fever virus whose prM-E sequence has been substituted with the prM-E sequence of dengue serotype 3 and the live attenuated chimeric dengue serotype 4 virus comprises a genome of an attenuated yellow fever virus whose prM-E sequence has been substituted with the prM-E sequence of dengue serotype 4.

21. Use of the method according to any one of claims 1 to 14 for determining the dengue serostatus of a subject before vaccination with a dengue virus vaccine.

22. Use of the method according to any one of claims 1 to 13 or 15 to 20 for analyzing a subject's antibody response after vaccination with a dengue virus vaccine.

23. Method for determining whether a subject is dengue-seronegative or dengue-seropositive, comprising the steps of: (a) performing the method according to any one of claims 1 to 20 with a blood sample from said subject;

(b) calculating the MNT50 value based on the level of neutralizing antibodies; and

(c) determining that the subject is dengue-seronegative if the MNT50 value is below the lower limit of detection or determining that the subject is dengue-seropositive if the MNT50 value is above the lower limit of detection of the assay.

24. Method according to claim 23, wherein the lower limit of detection is 10.