Virus-like Particles displaying envelope domain III of dengue virus type 2 induce virus-specific antibody response in mice

Objective: Currently, dengue represents one of the most significant arboviral disease worldwide, for which a vaccine is not yet available. Persistent challenges in live viral dengue vaccines have sparked a keen interest in exploring non-replicating dengue vaccines. We have examined the feasibility of using the methylotrophic yeast Pichia pastoris to develop a chimeric vaccine candidate displaying the dengue virus type-2 (DENV-2) Envelope Domain III (EDIII), implicated in host receptor binding and in the induction of virus-neutralizing antibodies, on the surface of non-infectious Virus-like Particles (VLP)-based on the Hepatitis B virus core antigen (HBcAg). Methods: We designed a fusion antigen by inserting DENV-2 EDIII into c/e1 loop of HBcAg. A codon-optimized gene encoding this fusion antigen was integrated into the genome of P. pastoris, under the control of the Alcohol Oxidase 1 promoter. The antigen was expressed by methanol induction and purified to near homogeneity by Ni²⁺ affinity chromatography. The purified antigen was characterized physically and functionally to evaluate its ability to assemble into VLPs and elicit DENV-2-specific antibodies in mice. Results: This fusion antigen was expressed successfully to high yields and purified to near homogeneity. Electron microscopy and competitive ELISA analyses showed that it formed VLPs in which the EDIII moiety was accessible to different EDIII-specific antibodies. These VLPs were immunogenic in mice, stimulating the production of antibodies that could specifically recognize DENV-2 and neutralize its infectivity. However, virus-neutralizing antibody titers were modest. Conclusions: Our data show: (i) insertion of EDIII into the c/e1 loop of HBcAg does not compromise particle assembly and (ii) the chimeric VLPs elicit a specific humoral response against DENV-2. The strategy of displaying dengue virus EDIII using a VLP platform will need further optimization before it may be developed into a viable alternative option

Comparative study of risk indicators associated with tooth loss among adult population in urban and rural areas of Muradnagar, Ghaziabad, Uttar Pradesh, India

Background: Oral health objectives prescribed by World Health Organization for the year 2020 have expressed that there ought to be an expansion in the quantity of people with functional dentitions (at least 21 common teeth) at ages of 35–44 and 65–74 years. Aim: The aim of this study is to examine the prevalence of tooth loss and to evaluate and compare the risk indicators associated with tooth loss among adult population in urban and rural areas of Muradnagar, Ghaziabad. Materials and Methods: A cross-sectional study was led among 1200 adults aged 35–74 years in urban and rural areas of Muradnagar, India. Information was assembled by an interview followed by clinical examination (number of missing teeth). Demographic and socioeconomic factors and self-perceived oral health were the independent variables assessed. One-way analysis of variance, post-hoc test (Bonferroni), Chi-square test, Student's t-test, and logistic regression analysis were used for statistical analysis. Results: Low educational status, no dental check-ups, low frequency of brushing, older age, and smoking habit were independent risk factors for tooth loss. The odds of tooth loss in older adults and illiterates were higher; the odds for tooth loss among those who expressed their desire for replacement of missing teeth were 1.3 times lower than their counterparts. Conclusion: The experiences gained up showed that tooth loss was very pervasive in Muradnagar populace and the critical hazard indicators identified were age, education, socioeconomic status, and cigarette smoking

Dengue-specific subviral nanoparticles: design, creation and characterization.

Dengue is today the most significant of arboviral diseases. Novel tools are necessary to effectively address the problem of dengue. Virus-like particles (VLP) offer a versatile nanoscale platform for developing tools with potential biomedical applications. From the perspective of a potentially useful dengue-specific tool, the dengue virus envelope protein domain III (EDIII), endowed with serotype-specificity, host receptor recognition and the capacity to elicit virus-neutralizing antibodies, is an attractive candidate

Dengue-specific subviral nanoparticles: design, creation and characterization

Abstract Background Dengue is today the most significant of arboviral diseases. Novel tools are necessary to effectively address the problem of dengue. Virus-like particles (VLP) offer a versatile nanoscale platform for developing tools with potential biomedical applications. From the perspective of a potentially useful dengue-specific tool, the dengue virus envelope protein domain III (EDIII), endowed with serotype-specificity, host receptor recognition and the capacity to elicit virus-neutralizing antibodies, is an attractive candidate. Methods We have developed a strategy to co-express and co-purify Hepatitis B virus surface (S) antigen in two forms: independently and as a fusion with EDIII. We characterized these physically and functionally. Results The two forms of the S antigen associate into VLPs. The ability of these to display EDIII in a functionally accessible manner is dependent upon the relative levels of the two forms of the S antigen. Mosaic VLPs containing the fused and un-fused components in 1:4 ratio displayed maximal functional competence. Conclusions VLPs armed with EDIII may be potentially useful in diagnostic, therapeutic and prophylactic applications

<i>Pichia pastoris</i>-Expressed Dengue 2 Envelope Forms Virus-Like Particles without Pre-Membrane Protein and Induces High Titer Neutralizing Antibodies

<div><p>Dengue is a mosquito-borne viral disease with a global prevalence. It is caused by four closely-related dengue viruses (DENVs 1–4). A dengue vaccine that can protect against all four viruses is an unmet public health need. Live attenuated vaccine development efforts have encountered unexpected interactions between the vaccine viruses, raising safety concerns. This has emphasized the need to explore non-replicating dengue vaccine options. Virus-like particles (VLPs) which can elicit robust immunity in the absence of infection offer potential promise for the development of non-replicating dengue vaccine alternatives. We have used the methylotrophic yeast <i>Pichia pastoris</i> to develop DENV envelope (E) protein-based VLPs. We designed a synthetic codon-optimized gene, encoding the N-terminal 395 amino acid residues of the DENV-2 E protein. It also included 5’ pre-membrane-derived signal peptide-encoding sequences to ensure proper translational processing, and 3’ 6× His tag-encoding sequences to facilitate purification of the expressed protein. This gene was integrated into the genome of <i>P. pastoris</i> host and expressed under the alcohol oxidase 1 promoter by methanol induction. Recombinant DENV-2 protein, which was present in the insoluble membrane fraction, was extracted and purified using Ni²⁺-affinity chromatography under denaturing conditions. Amino terminal sequencing and detection of glycosylation indicated that DENV-2 E had undergone proper post-translational processing. Electron microscopy revealed the presence of discrete VLPs in the purified protein preparation after dialysis. The E protein present in these VLPs was recognized by two different conformation-sensitive monoclonal antibodies. Low doses of DENV-2 E VLPs formulated in alum were immunogenic in inbred and outbred mice eliciting virus neutralizing titers >1∶1200 in flow cytometry based assays and protected AG129 mice against lethal challenge (<i>p</i><0.05). The formation of immunogenic DENV-2 E VLPs in the absence of pre-membrane protein highlights the potential of <i>P. pastoris</i> in developing non-replicating, safe, efficacious and affordable dengue vaccine.</p></div

Expression of DENV-2 E in <i>P. pastoris</i>.

<p>(A) Map of the DENV-2 expression construct for integration into <i>P. pastoris</i> genome. The <i>DENV-2 E</i> gene is flanked by the <i>AOX1</i> promoter (5’ AOX1) and transcription terminator (TT) at its 5′ and 3′ ends, respectively. The construct contains an <i>E. coli</i> origin of replication (ori) and the selection marker zeocin (Zeo), which is functional in both <i>E. coli</i> as well as <i>P. pastoris</i>. (B) Localization of the recombinant DENV-2 antigen expression in induced <i>P. pastoris</i>. Aliquots of un-induced (UI) and induced (I) cultures were lysed and separated into soluble (S) and membrane-enriched pellet (P) fractions, run on SDS-polyacrylamide gel and subjected to Western blot analysis using mAb 24A12. Pre-stained protein markers were analyzed in lane ‘M’. Their sizes (in kDa) are indicated to the left. The arrow on the right indicates the position of the recombinant DENV-2 E antigen. (C) Ni-NTA His-Sorb ELISA analysis of S and P fractions obtained from UI (blue bars) and I (red bars) cell lysates described in ‘B’.</p

Design of the DENV-2 E antigen.

<p>(A) Schematic representation of the DENV-2 polyprotein, showing the parts of prM and E included in designing the E antigen for expression in <i>P. pastoris</i>. (B) Design of the DENV-2 E antigen consisting of the 395 aa residue E ectodomain, preceded by the C-terminal 34 aa residues of prM. The grey box denotes the pentaglycine linker peptide joining the C-terminus of E ectodomain to the polyhistidine tag (6×H). (C) The predicted aa sequence of the DENV-2 E antigen shown in ‘B’. The color scheme corresponds to that shown in ‘B’. The first two aa residues (MV) were introduced due to the insertion of the initiator codon in a Kozak consensus context. The downward arrows in ‘B’ and ‘C’ denote the signal cleavage site.</p

Evaluation of antibodies elicited by recombinant DENV-2 E antigen VLPs.

<p>(A) Pooled sera from DENV-2 E immunized (solid, blue curve) and mock-immunized (black, dashed curve) Balb/C mice were tested in an indirect ELISA using DENV-2 E protein as the coating antigen. (B) The Balb/C anti-DENV-2 E antiserum (panel A) was tested in ELISAs using recombinant monovalent EDIII-1 (black), EDIII-2 ( blue), EDIII-3 (red) or EDIII-4 (green) antigens. Mock-immunized Balb/C serum was tested against EDIII-1 as coating antigen (black, dashed). (C) Pooled serum from DENV-2 E-immunized Swiss albino mice was tested in ELISAs using either recombinant DENV-2 (solid blue squares) or EDIII-2 (empty blue squares) as the coating antigens. Mock-immunized Swiss albino serum was tested against EDIII-2 as coating antigen (black, dashed). (D) Indirect immunofluorescence analysis of DENV-2-infected BHK cells using mock-immunized serum (i), anti-EDIII-2 antiserum (ii), 4G2 mAb (iii), or anti-DENV-2 E antiserum (iv), as the source of primary antibodies. Bound antibodies were visualized using anti-mouse IgG-FITC conjugate. Antisera in panel (i), (ii) and (iv) were from Balb/C mice.</p

Characterization of DENV-2-specific antibodies elicited by DENV-2 E VLPs.

<p>(A) Analysis of virus-specific antibody titers in anti-DENV-2 E antisera (blue bars) and mock-immune sera (black bars) in indirect ELISAs using infectious DENVs as coating antigen. (B) Determination of virus-neutralizing antibody titers using FACS neutralization assay. Serial dilutions of anti-DENV-2 E antisera were tested for their capacity to neutralize infectivity of all four DENV serotypes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064595#pone.0064595-Kraus1" target="_blank">[30]</a>. The vertical axis denotes the serum dilution corresponding to 50% neutralization (FNT₅₀ titre) of virus infectivity. Murine sera used in experiments shown in panels A and B were from Balb/C mice; the Arabic numerals along the x-axis, in both these panels, indicate DENV serotype. (C) Determination of protective efficacy of DENV-2 E VLP immunization. AG129 mice were either mock-immunized (black curve, n = 4) or immunized with DENV-2 E VLPs (blue curve, n = 6) and challenged with a virulent strain of DENV-2. The mice were monitored daily (up to 18 days post challenge) for mortality and the resultant data plotted as Kaplan-Meir survival curves.</p

Purification and characterization of recombinant DENV-2 E antigen.

<p>(A) Ni²⁺ affinity chromatographic purification of DENV-2 E antigen from the P fraction of induced <i>P. pastoris</i> lysate. The continuous blue and the dashed black curves represent the profiles of UV absorbance (at 280 nm) and the imidazole step gradient, respectively, during chromatography. (B) Coomassie-stained SDS-polyacrylamide gel analysis of the purified protein. (C) Immunoblot analysis of the purified protein using mAb 24A12. (D) Immunoblot analysis of the purified protein using penta-His mAb. (E) Protein blot using Con A-HRPO conjugate. Controls analyzed in parallel include DENV-2 (lane ‘V’), purified EDIII-2 protein (lane ‘III’) and ovalbumin (lane ‘O’). In panels B-E: lanes ‘E’ denote the purified DENV-2 E protein (pooled peak material shown in panel ‘A’). Protein markers (whose sizes, in kDa, are shown to the left of each panel) were run in lanes ‘M’. The arrow to the right of each panel indicates the position of the recombinant DENV-2 E antigen.</p