Patent Grant
10487120
The present invention relates generally to vaccines, and more specifically to vaccines for dengue viruses.
Dengue virus (DENV), a global disease, is divided into four serotypes (DENV1-4). Cross-reactive and non-neutralizing antibodies against envelope protein (E) of DENV bind to the Fcγ receptors (FcγR) of cells, and thereby exacerbate viral infection by heterologous serotypes via antibody-dependent enhancement (ADE). Identification and modification of enhancing epitopes may mitigate enhancement of DENV infection.
Therefore, identification of B-cell epitopes of DENV E protein, which induce cross-reactive and non-neutralizing antibodies, may provide valuable information for vaccine development. A safe and effective vaccine against DENV is not yet available. Thus, there is a need to identify and substitute the epitopes recognized by poorly neutralizing and highly enhancing antibodies to improve dengue vaccines.
In one aspect, the invention relates to an isolated mutant dengue virus E protein variant comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 1 and having one or more amino acid residue substitutions at position corresponding to Asn8 (N8), Arg9 (R9), Val12 (V12) and/or Glu13 (E13).
In another aspect, the invention relates to an isolated mutant dengue virus E protein variant, wherein the nominated wild-type dengue virus E protein comprises the amino acid sequence of SEQ ID NO: 1, the variant comprising an amino acid sequence that is at least 90% identical to the SEQ ID NO: 1 and lacking an infection-enhancing antibody-binding motif comprising the amino acid sequence of SEQ ID NO: 28 at domain I.
In one embodiment of the invention, the variant comprises one or more mutations selected from the group consisting of: (a) a substitution of the 8th Asn residue; (b) a substitution of the 9th Arg residue; (c) a substitution of the 12th Val residue; and (d) a substitution of the 13th Glu residue.
In one embodiment of the invention, the mutant dengue virus E protein variant has Asparagine at position 8 being substituted. For example, the variant has a substitution of Asn for Ala, Glu, or Arg.
In another aspect, the invention relates to an isolated nucleic acid sequence encoding a mutant dengue virus E protein variant as aforementioned.
In another aspect, the invention relates to a plasmid expressing a mutant dengue virus E protein variant as aforementioned.
In another aspect, the invention relates to a plasmid expressing a virus-like particle, the virus-like particle comprising a mutant dengue virus E protein variant as aforementioned.
In one embodiment of the invention, the aforementioned plasmid comprises (a) an isolated nucleic sequence comprising the nucleotide sequence of SEQ ID NO: 27; and (b) a promoter operably linked to the isolated nucleic sequence.
In another aspect, the invention relates to a DNA vaccine comprising: (a) a recombinant DNA comprising a nucleotide sequence encoding the mutant dengue virus E protein variant as aforementioned and a promoter operably linked to the nucleotide sequence; and (b) gold or tungsten, wherein the recombinant DNA and gold or tungsten forms a complex.
In another aspect, the invention relates to immunized human sera comprising antibodies, the antibodies exhibiting the following characteristics: (a) having specific binding affinity to the wild-type dengue virus E protein and the mutant dengue virus E protein variant as aforementioned; (b) possessing neutralizing and protective activities against dengue virus infection: (c) flee of antibody dependent enhancement of dengue virus infection; (d) the antibodies being elicited by the mutant dengue virus E protein variant; and (e) having no binding activity to dengue virus infection-enhancing epitopes Asn8, Arg9, Val 12 and/or Glu 13 located within domain I of the wild-type dengue virus E protein.
In another aspect, the invention relates to use of immunized sera as aforementioned or use of a mutant dengue virus E protein variant as aforementioned in the manufacture of a medicament for eliciting neutralizing and protecting activity against one or more dengue virus serotypes in a subject in need thereof, and/or in the manufacture of a medicament for neutralizing a dengue virus, reducing, alleviating antibody dependent enhancement of dengue virus infection, and/or increasing survival rate in a subject in need thereof.
In one embodiment of the invention, the medicament is for alleviating the antibody dependent enhancement in a subject whose serum sample exhibits reactivity with an isolated peptide comprising the amino acid sequence of SEQ ID NO: 28.
Alternatively, the invention relates to immunized sera as aforementioned or a mutant dengue virus E protein variant as aforementioned for use in eliciting neutralizing and protecting activity against one or more dengue virus serotypes in a subject in need thereof, or for use in neutralizing a dengue virus, reducing, alleviating antibody dependent enhancement of dengue virus infection, and/or increasing survival rate in a subject in need thereof.
Alternatively, the invention relates to a method of eliciting neutralizing and protecting activity against one or more dengue virus serotypes in a subject in need thereof, or a method of neutralizing a dengue virus, reducing, alleviating antibody dependent enhancement of dengue virus infection, and/or increasing survival rate in a subject in need thereof, comprising administering the immunized sera as aforementioned or a mutant dengue virus E protein variant as aforementioned to the subject in need thereof.
In one embodiment of the invention, immunized sera or a mutant dengue virus E protein variant as aforementioned is for use in alleviating the antibody dependent enhancement in a subject whose serum sample exhibits reactivity with an isolated peptide comprising the amino acid sequence of SEQ ID NO: 28.
In one embodiment of the invention, the isolated peptide is less than 15 amino acid residues in length, and is at least 80%, 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ NOs: 2-12, and 14-26.
Further in another aspect, the invention relates to a method of detecting the presence of a dengue virus in a biological sample, comprising:
Yet in another aspect, the invention relates to a method of detecting the presence of dengue virus infection-enhancing antibodies in a biological sample, comprising:
The biological sample may be selected from the group consisting of: blood, serum, plasma, saliva, cerebrospinal fluid, and urine.
These and other aspects will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings.
The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment.
FIGS. 8A-B show evaluation of neutralizing activity of mAbs against DENV1-4. (A) The neutralizing activity of DB21-6, DB39-2, or 4G2 against DENV1-4 was examined with inhibition assays. DENV1 (MOI=5), DENV2 (MOI=1), DENV3 (MOI=5), or DENV4 (MOI=1) was incubated with mAbs at 4° C. for 1 hour, and then used to infect BHK-21 cells. After 3 days, the cells were fixed and stained with 4G2. Titers are expressed as inhibition percentages. Data shown are from one representative experiment of two independent experiments. (B) Summary of 50% inhibition concentrations of DB21-6, DB39-2, and 4G2 against DENV1-4.
The present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. Various embodiments of the invention are now described in detail. Referring to the drawings, like numbers indicate like components throughout the views. As used in the description herein and throughout the claims that follow, the meaning of “a”, “an”, and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein and throughout the claims that follow, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise. Moreover, titles or subtitles may be used in the specification for the convenience of a reader, which shall have no influence on the scope of the present invention. Additionally, some terms used in this specification are more specifically defined below.
The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term is the same, in the same context, whether or not it is highlighted. It will be appreciated that same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.
As used herein, “around”, “about” or “approximately” shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term “around”, “about” or “approximately” can be inferred if not expressly stated.
The term “treating” or “treatment” refers to administration of an effective amount of a therapeutic agent to a subject in need thereof, who has a disease, or a symptom or predisposition toward such a disease, with the purpose of cure, alleviate, relieve, remedy, ameliorate, or prevent the disease, the symptoms of it, or the predisposition towards it, or reduce incidence of symptoms. Such a subject can be identified by a health care professional based on results from any suitable diagnostic method.
“An effective amount” refers to the amount of an active compound that is required to confer a therapeutic effect on the treated subject. Effective doses will vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment.
The “Guidance for Industry and Reviewers Estimating the Safe Starting Dose in Clinical Trials for Therapeutics in Adult Healthy Volunteers” published by the U.S. Department of Health and Human Services Food and Drug Administration discloses “a human equivalent dose” may be obtained by calculations from the following formula:
HED=animal dose in mg/kg×(animal weight in kg/human weight in kg)0.33,
As used herein, when a number or a range is recited, ordinary skill in the art understand it intends to encompass an appropriate, reasonable range for the particular field related to the invention.
Detection reagent or labeling agent for labeling an antibody has been disclosed in U.S. Pat. No. 8,920,804, which is incorporated herein by reference in its entirety.
The amino acid 1-395 sequence of wild-type DENV2 (16681) E protein (The GenBank accession number: AAB58782) is shown in SEQ ID NO: 1.
Abbreviations: virus-like particles (VLPs); dengue fever (DF); dengue hemorrhagic fever (DHF); E protein domain I (EDI); antibody-dependent enhancement (ADE); glutamic acid (Glu; E); valine (Val; V); asparagine (Asn; N): arginine (Arg; R); pre-membrane (prM); envelope (E) proteins; multiplicity of infection (MOI).
We characterized the cross-reactive DB21-6 and DB39-2 monoclonal antibodies mAbs) against domain I-II of DENV; these antibodies poorly neutralized and potently enhanced DENV infection both in vitro and in vivo. Two enhancing mAbs, DB21-6 and DB39-2, were observed to compete with sera antibodies from patients infected with dengue. The epitopes of these enhancing mAbs were identified. N8, R9, V12, and E13 are the reactive residues DB21-6, while N8. R9, and E13 are the reactive residues of DB39-2. N8 substitution tended to maintain VLP secretion, and decrease the binding activity of DB21-6 and DB39-2. The immunized sera from N8 substitution (N8R) DNA vaccine exerted greater neutralizing and protecting activity than wild-type (WT) immunized sera, both in vitro and in vivo. Treatment with N8R-immunized sera reduced the enhancement of mortality in AG129 mice. These results support identification and substitution of enhancing epitope as a novel strategy for developing safe dengue vaccines.
Exemplary instruments, apparatus, methods and their related results according to the embodiments of the present invention are given below.
Materials and Methods
DENV, Cell Lines, and mAbs
Four dengue virus serotypes, DENV1 Hawaii, DENV2 16681, DENV3 H87, and DENV4 H241, were prepared as described in Wu et al. (2003) (“Identification of a dengue virus type 2 (DEN-2) serotype-specific B-cell epitope and detection of DEN-2-immunized animal serum samples using an epitope-based peptide antigen” J Gen Virol 84, 2771-2779). C6/36 cells were grown in medium having 50% Mitsumashi and Maramorsch insect medium (SIGMA-ALDRICH®) plus 50% Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphoterlein B. The C6/36 cells were infected with DENV at a multiplicity of infection (MOI) of 0.1-1, and incubated at 28° C. for 7 to 9 days. The viruses were harvested from supernatant and titrated in a baby hamster kidney fibroblast cell line (BHK-21) by plaque assay. The aliquots were stored at −80° C. BHK-1K-21 cells were grown in minimal essential medium (MEM) supplemented with 10% FBS, 100 U/ml penicillin. 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B. Human erythroleukaemic K562 and monocytic THP-1 cells were grown in RPMI medium containing 10% FBS. The mouse mAbs, including DB21-6 and DB39-2, were previously generated by immunization of BALB/c mice with DENV2, and produced in hybridoma cells (Li et al., (2012). “Development of a Humanized Antibody with High Therapeutic Potential against Dengue Virus Type 2” PLoS Negl Trop Dis 6, e1636). DB21-6 and DB39-2 were isotyped as IgG1 and purified using PROTEIN G SEPHAROSE™4B gels (GE Healthcare).
In Vitro Measurement of ADE with mAbs
Serial dilutions of mAbs were incubated with DENV1 Hawaii (MOI=1), DENV2 16681 (MOI=1), DENV3 H87 (MOI=5), and DENV4 H241 (MOI=1) for 1 hour at 4° C. The mixtures were used to infect K562 cells for 2 hours at 37° C. After washing, the cells were incubated with 2% FBS in RPMI medium at 37° C. for 3 days. The infected cells were collected and fixed with 3.7% formaldehyde for 10 minutes at 4′C. For staining, the cells were permeabilized with 2% FBS in PBS containing 0.1% saponin (Sigma), followed by staining with 4 μg/ml 4G2 for 0.5 hours at 4° C. The cells were washed and incubated with R-phycoerythrin (RPE)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) for 1 hour at 4° C. The cells were washed, and the percentages of infected cells were determined by flow cytometry. For infection of THP-1 cells, DENV2 16681 (MOI=1 or 10) was incubated with diluted mAbs for 1 hour at 4° C., and then incubated with cells for 2 hours at 37° C. After 3 days, the cells were fixed, permeabilized, and stained with hDB32-6. After washing, the cells were incubated with an RPE-conjugated goat anti-human IgG, and were subsequently analyzed by flow cytometry.
Measurement of In Vivo ADE with mabs in AG129 Mice
Type I and II interferon receptor-deficient mice (AG129; 5- to 6-weeks-old) were purchased from B&K Universal. The AG129 mice were given 5 μg mAbs in 200 μl PBS on days 1 and −1 (i.p.). The mouse IgG1 isotype antibody was used as a negative control. On day 0, mice were inoculated (i.v.) with 1×105 pfu of the mouse-adapted. DENV2 S221 (obtained from S. Shresta) in 100 μl PBS. The survival rates al AG129 mice were recorded for 30 days.
Measurement of Viremia by Quantitative RT-PCR
AG129 mice were infected with 1×105 pfu DENV2 S221 (i.v.) on day 0, and treated with 5 μg mAbs via i.p. on days −1 and 1. Viral RNA was extracted from pooled and infected mice sera using QIAAMP® viral RNA minikit (Qiagen). Quantitative RT-PCR was performed, using LIGHTCYCLER® 480 system (ROCHE™). The standard curve was generated with DENV2 S221 (at concentrations from 101 to 107 pfu/ml). Viremia measurements were expressed as pfu equivalents/ml, which was calculated based on the threshold cycle value (Ct) according to the standard curve for DENV2 S221.
Competitive ELISA of mAbs and Patient Serum Samples
A total of 21 DENV2-infected patient serum samples were collected from 11 DF and 10 DHF patients during an outbreak between 2002 and 2003 in Taiwan. Diagnosis of DENV infection was based on IgM antibody-capture ELISA (MAC-ELISA), reverse-transeriptase PCR (RT-PCR), or virus isolation in cell cultures. These serum samples were collected between days 4 and 22 from the onset of symptoms; such sera contained anti-dengue antibodies. All of these patients were determined to have classical DF ear DHF based on the criteria published by the World Health Organization (WHO) in 2009. Table 2 shows the characteristics of patient serum samples enrolled in this study. Competition ELISA was performed. Briefly, the plates were coated with polyclonal rabbit anti-DENV hyper-immune sera at 4° C. overnight. After blocking, the diluted DENV2 viral supernatants (1×106 pfu) were added for 2 hours at room temperature (R.T.). The diluted mAbs and patient sera (1:100 dilution) were incubated for 2 hours at R.T. After washing, horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmumoResearch Laboratories) was added for 1 hour at R.T. The peroxidase substrate o-phenylenediamine dihydrochloride (OPD, SIGMA-ALDRICH™) was then added, and the reaction stopped with 3N HCl. The optical density (OD) was measured at 490 nm. Normal human serum (NHS) was used as a control. The percentage of competition was calculated as follows: competition (%)=[1−(OD of patient serum-mAb mixture/OD of NHS-mAb mixture)]×100.
Phage Display Biopanning
Phage display biopanning was performed. Briefly, the plate was coated with 100 μg/ml mAbs at 4° C. for 6 hours. After washing and blocking, 4×1010 pfu of phage-displayed peptide library (New England BioLabs, Inc.) were incubated for 50 mins at R.T. After washing, bound phage was eluted with 100 μl 0.2 M glycine/HCl (pH 2.2) and neutralized with 15 μl 1 M Tris/HCl (pH 9.1). The eluted phage was then amplified in ER2738 for subsequent rounds of selection. The phage was titrated onto LB medium plates containing IPTG and X-Gal. The second and third rounds of selection were identical to the first round except for the addition of 2×1011 pfu of amplified phage.
Identification of Immunopositive Phage Clones by ELISA
Plates were coated with 50 μg/ml mAbs. After washing and blocking, the amplified phage was added, and incubated for 1 hour at R.T. After washing, diluted HRP-conjugated anti-M13 antibody (GE Healthcare) was added at RT for 1 hour. The plates were developed and subsequently terminated by 3N HCl. The optical density (OD) was measured at 490 nm.
Identification of Epitopes Using Flow Cytometry-based Binding Assay to Cells Expressing WT and Mutant DENV2 pr/M/E Proteins
The pCBD2-2J-2-9-1 plasmid expressing prM-E proteins of DENV2 has been previously characterized and described (Chang et al., (2003) “Enhancing biosynthesis and secretion of premembrane and envelope proteins by the chimeric plasmid of dengue virus type 2 and Japanese encephalitis virus” Virology 306, 170-180). Site-directed mutagenesis was performed to replace each of the selected amino acid residues. After mutagenesis, the plasmids were sequenced to ensure the absence of any further mutations at non-target sites. BHK-21 cells were transfected with constructs expressing the wild-type (WT) or mutant DENV2 E protein using POLYJET™ in vitro DNA transfection reagent (SignGen Laboratories). After 2 days, the cells were fixed, and permeabilized with 2% FBS in PBS containing 0.1% saponin (SIGMA™). For staining, cells were incubated with DB21-6, DB39-2, 4G2, and mixed mAbs (DB32-6, 3H5, and DB25-2) at a concentration of 1, 1, 1 and 1 μg/ml, respectively, at 4° C. for 0.5 hours. After washing, the cells were incubated with RPE-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. The relative index of a mAb to a mutant E protein was measured using the formula: [intensity of the mutant E/intensity of WT E (recognized by a mAb)]/[intensity of mutant E/intensity of WT E (recognized by mixed mAbs)].
Detection of Secreted VLP's by Capture ELISA
BHK-21 cells were transfected with vectors expressing WT or mutant E protein of DENV2, as described above. At 48 hours post-transfection, culture supernatants were collected. The plates were coated with polyclonal rabbit anti-DENV hyper-immune sera at 4° C. overnight. After blocking, two-fold dilutions of supernatants containing WT or mutant VLP's were added for 2 hour at RT. The wells were then incubated with diluted DB32-6 and 4G2 at RT for 2 hour. After washing, a 1:2000 dilution of HRP-conjugated anti-mouse IgG was added for 1 hour at RT. Finally, the plates were developed, and the reaction was subsequently terminated with 3N HCl. The OD was measured at 490 nm.
Preparation of Plasmids for Immunization
Plasmids expressing WT E protein (SEQ ID NO: 1) of DENV2 or a mutant E protein in which the N8 residue was substituted with R (N8R) were used for immunization (
Immunization of Mice
The abdominal epidermis of 6 week-old female BALB/c mice was injected with a gene gun using a helium pressure setting of 400 lb/inch2. Each mouse was immunized by administering 4 bullets containing 1 μg plasmid DNA. Mice were immunized at 0, 3, and 6 weeks. Serum samples were collected before immunization and 3 weeks after the third immunization (pre-, 1st, 2nd, 3rd immunized sera). The serum samples were pooled from five to six mice for each group and evaluated by ELISA, neutralization assay, and in vivo ADE assay.
Evaluation of Immunized Sera Against DENV2 by ELISA
C6/36 cells infected with DENV2 16681 were used as antigens. C6/36 cells were seeded into each well (2×104 cells/well) of 96-well ELISA plates. After one day. 2×103 pfu of DENV2 16681 (MOI=0.1) was added to infect the cells at 37° C. for 2 hours. The wells were washed with PBS, and then cultured in 2% FBS culture medium at 28° C. for 5 days. The infected cells were fixed with 1:1 methanol/acetone at 4° C. for 10 mins. The plates were blocked with 5% skimmed milk at 4° C. for 24 hours. Diluted immunized sera were then added for incubation at RT for 2 hours. The plates were washed three times with phosphate-buffered saline containing 0.1% (w/v) TWEEN® 20 (PBST0.1), and subsequently incubated with HRP-conjugated anti-mouse IgG. Finally, the plates were developed, and the reaction terminated with 3N HCl. The OD was measured at 490 nm. In vitro and in vivo neutralization assays with immunized sera
DENV2 16681 (MOI=1) was incubated with the 3rd immunized sera for 1 hour at 4° C. The mixtures were used to infect BHK-21 cells for 2 hours at 37° C. After 3 days, the cells were fixed, permeabilized, and stained with 4 μg/ml 4G2. After washing, the cells were incubated with RPE-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Inhibition percentage (%)=[1−(the percentage of infected cells incubated with immunized sera/without immunized sera)]×100.
The ICR mice were purchased from the Laboratory Animal Center, National Taiwan University College of Medicine. Serially-diluted immunized sera were incubated with 1×104 pfu (25-fold lethal dose, 25-fold LD50) of DENV2 16681 for 0.5 hours at 4° C. Two-day-old suckling mice were inoculated with 20 μl of the mixtures through intracranial (i.e.) injection. After challenge, the survival rates were recorded for 28 days.
Studies of In Vivo ADE with Immunized Sera
AG129 mice were given (i.p.) dilutions of immunized sera on days −1 and 1, and inoculated (i.v.) with 1×105 pfu of DENV2 S221 on day 0. The survival rates were recorded for 30 days.
Statistical Analysis
Survival rate was expressed using Kaplan-Meier survival curves, and statistical analyses were performed using GraphPad Prism 5. For competition assays of mAbs and patient sera, Student's t tests were used to identify significant differences and calculate P values (*P<0.05, ***P<0.001, NS not significant). For evaluation of immunized sera against DENV2 by ELISA, two-way ANOVA with Bonferroni post-hoc test was used to determine the significant differences and calculate P values (**P<0.01, NS not significant). GRAPHPAD PRISM® 5 was used to analyze 50% inhibition titers against DENV2, based on inhibition percentages from pooled immunized sera.
Results
Characterization Cross-reactive DB21-6 and DB39-2 Against DENV
To estimate the in vitro neutralizing activity of these mAbs, we infested BHK-21 cells with a mixture of individual mAbs and DENV1-4.
Enhancing Activities of DB21-6 and DB39-2
To investigate in vitro enhancement of DENV infection through ADE, we performed in vitro ADE assays, and detected the increases in the percentage of dengue-infected cells by flow cytometry. The FcγRIIA-bearing K562 cells, which do not express type 1 interferon (IFN), were used to measure the enhancement of infected cells through extrinsic ADE. The serially-diluted mAbs were incubated with DENV1-4, and then used to infect K562 cells. The infection percentage was measured by flow cytometry.
To thriller confirm enhancement of infection, we examined the enhancement of DENV2 16681 infection by DB21-6 and DB39-2 in FcγRI- and FcγRIIA-bearing THP-1 cells. Infection in THP-1 cells was enhanced to a greater extent by DB21-6 and DB39-2 than by 4G2 (
DEN V2 S221 was previously used to study enhancement of mortality via ADE in AG129 mice. To evaluate the in vitro enhancement of DENV2 S221 infection by mAbs, we performed ADE assays using K562 cells and THP-1 cells. As for DENV1-4 infection, high concentrations of DB21-6 and DB39-2 enhanced DENV2 S221 infection in K562 cells (
We confirmed the in vivo enhancing activities in AG129 mice. The AG129 mice treated with 5 μg DB21-6 and infected with DENV2 S221 exhibited increased mortality as compared to control infected mice (
Competition Assay of mAbs and Infected Patient Sera
We performed competition assay to determine whether sera antibodies from dengue patients compete with mAbs for binding to DENV2. Table 2 shows the characteristics of patient serum samples. The results indicate that the sera antibodies from infected patients competed with DB21-6 and DB39-2.
Identification of Enhancing Epitopes of DB21-6 and DB39-2
To identify the enhancing epitopes of DB21-6 and DB39-2, we used a phage-displayed peptide library to screen for the reactive phage clones. After three biopanning rounds, the phage titers were increased to 12,871-fold (DB21-6) and 5,000-fold (DB39-2), respectively, compared to that of the first round (
To further verify the epitopes of DB21-6 and DB39-2, we performed site-directed mutagenesis of the phage-displayed epitope using pCBD2-2J-2-9-1 as template. After confirmation of variants by sequencing, we transfected cells with mutant plasmids, and detected binding activity by flow cytometry. The binding percentages for each transfectant were normalized to those of anti-EDIII mAbs (DB32-6, 3H5, and DB25-2), and relative indices were calculated and as shown in
Examination of Humoral Immune Responses in Mice
The BALB/c mice were immunized with vector, WT, or N8R plasmids at 0, 3, and 6 weeks. After three rounds of immunization, the serum samples were collected and pooled within each group. The immunized sera were examined by ELISA.
Evaluation of Neutralizing Activity of the Immunized Sera
The immunized sera were evaluated for neutralizing activity against DENV2. Both WT- and N8R-immunized sera exhibited high neutralizing activities, while vector-immunized sera did not (
The protective effect of immunized sera against DENV2 16681 in vivo was examined. The survival rates of mice treated with WT-immunized sera at dilutions of 1:100 and 1:200 were significantly higher than that of mice treated with vector-immunized sera at a dilution of 1:100 (
Reduction of In Vivo Enhancing Activity of the Immunized Sera
To study the in vivo enhancement of mortality, we passively transferred different dilutions of WT-, N8R-, or vector-immunized sera into AG129 mice. Following infection with DENV2 S221, the survival rate of mice treated with WT- or N8R-immunized sera (1:25 dilution) was higher than that of mice treated with vector-immunized sera (
To further characterize these enhancing antibodies are produced in immunized sera, we performed competitive ELISA to inhibit the binding of HRP-conjugated DB21-6 or DB39-2 mAbs by immunized sera (
We characterized the ability of DB21-6 and DB39-2 to increase the percentage of dengue virus-infected cells. These mAbs enhance mortality in AG129 mice. The sera antibodies from infected patients compete with these mAbs for binding. We mapped the epitopes of enhancing mAbs DB21-6 and DB39-2 on EDI protein. To investigate how to reduce the enhancing effects while maintaining neutralizing activity, we substituted the N8 residue of E protein, and immunized mice with WT or N8R plasmids with a gene gun delivery system. After three immunizations, N8R-immunized sera produced neutralizing activity against DENV2, and reduced enhancement of mortality as compared to WT-immunized sera. Thus, substitution of enhancing epitope residues can increase the immune response against viral infection while reducing the potential for ADE.
We demonstrated that cross-reactive DB21-6 and DB39-2 against EDI-II had poor neutralizing activities against DENV1-4 (
ADE is regarded as an important mechanism leading to the development of severe dengue disease, including DHF/DSS. Cross-reactive and non-neutralizing antibodies binding to viruses can enhance infection of FcγR-bearing cells by ADE, resulting in increased viral load and/or production of cytokines. High viral load is correlated with dengue disease severity and DHF. Thus, there is a need to be able to confirm the presence of enhancing antibodies in dengue patient sera. Our results indicate that the competition percentages of DB21-6 and DB39-2 were significantly higher in DHF patient sera than those in DF patient sera (
The phage clones selected using DB21-6 and DB39-2 mAbs displayed peptide sequences containing a consensus motif, N—R-x-x-V-E (Table 1). These displayed peptide sequences may be suitable for detecting enhancing antibodies in serum samples from dengue patients, and for providing information on the pathogenesis of dengue. By alignment of displayed peptide sequences and structural modeling, the candidate epitopes were predicted and verified using VLP mutants (
The N8R substitution does not affect DENV2 VLP secretion (
We passively transferred diluted vector-, WT-, or N8R-immunized sera, and then challenged AG129 mice with DENV2 S221. WT- and N8R-immunized sera were protective at a 1:25 dilution, as compared to vector-immunized sera. The mortality of mice was enhanced by treatment with WT-immunized sera at a 1:100 dilution, as compared to treatment with vector-immunized sera. The mortality of mice treated with N8R-immunized sera at a 1:100 dilution was not enhanced (
Substitution of enhancing epitope and preservation of neutralizing epitope in immunized mice provide protective immunity. Such an approach would redirect immunodominance (
Table 1 shows alignment of phage-displayed peptide sequences selected by DB21-6 and DB39-2. Table 2 shows the DENV2-infected patient serum samples used. Table 3 shows comparison of the amino acid sequences of EDI-II proteins of DENV1, 2, 3, and 4a. Table 4 shows the database, gene/protein and accession/ID numbers.
aThe phage-displayed consensus amino acids are indicated by boldface type.
bThe amino acid sequences 8 to 13 in E protein of DENV1-4 were retrieved from GenBank (accession number AIU47321, AAB58782, AAA99437, and AAX48017).
aDHF, dengue hemorrhagic fever; DF, dengue fever.
N
R
V
E
N
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E
N
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E
N
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E
N
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E
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N
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E
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E
N
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N
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N
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N
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V
E
aEDI-II protein of strains of four dengue virus serotypes are aligned. Single letter amino acid abbreviations are shown.
b,cThe critical residues are shown in boldface type and identified by loss of binding for DB21-6 b and DB39-2c, respectively.
We found that the cross-reactive mAbs DB21-6 and DB39-2 exhibit poor neutralizing activity and high capacity for enhancing DENV infection. We identified the epitopes recognized by DB21-6 and DB39-2. To further improve the DNA vaccines against DENV2, in the illustrated example we substituted the N8 residue of wild type (WT) DENV2 E protein with arginine (N8R) in a plasmid for immunization. N8R-immunized sera produced higher neutralizing and protective activity than WT-immunized sera. Treatment of AG129 mice with N8R-immunized sera reduced ADE and mortality, as compared with mice treated with WT-immunized sera. In conclusion, we have identified a novel cross-reactive and infection-enhancing epitope in E protein, and demonstrated that substitution of this enhancing epitope is a promising strategy for development of a safe dengue vaccine.
All references cited, and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.
This application is a national stage application (under 35 U.S.C. 371) of PCT/US2016/015074 filed on 27 Jan. 2016, which claims priority to U.S. provisional application 62/113,811 filed on 9 Feb. 2015, all of which are herein incorporated by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2016/015074 | 1/27/2016 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2016/130321 | 8/18/2016 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20040009469 | Apt | Jan 2004 | A1 |
| Entry |
|---|
| Wahala et al., Recombinant Dengue Type 2 Viruses with Altered E Protein Domain III Epitopes Are Efficiently Neutralized by Human Immune Sera, 2012, Journal of virology, pp. 4019-4023. |
| GenBank Accession # AAB58782, polyprotein [Dengue virus 2 Thailand/16681/84], Jun. 4, 1997. |
| Tang et al., An Epitope-Substituted DNA Vaccine Improves Safety and Immunogenicity against Dengue Virus Type 2, 2015, PLoS Neglected Tropical Diseases, vol. 9, No. 7, pp. 1-23. |
| International Search Report for PCT/US2016/015074, dated May 19, 2016. |
| Written Opinion of International Search Authority for PCT/US2016/015074, dated May 19, 2016. |
| De Alwis et al. Dengue Viruses Are Enhanced by Distinct Populations of Serotype Cross-Reactive Antibodies in Human Immune Sera. PLoS Pathog. 2014, 10(10):e1004386. |
| Leon-Juarez et al. Generation and characterization of a monoclonal antibody that cross-reacts with the envelope protein from the four dengue virus serotypes. APMIS. Sep. 2013; 121(9):848-58. |
| Number | Date | Country | |
|---|---|---|---|
| 20180030097 A1 | Feb 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62113811 | Feb 2015 | US |
