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Pichia pastoris-expressed dengue 3 envelope-based virus-like particles elicit predominantly domain III-focused high titer neutralizing antibodies.

Tripathi L, Mani S, Raut R, Poddar A, Tyagi P, Arora U, de Silva A, Swaminathan S, Khanna N - Front Microbiol (2015)

Bottom Line: Recently, we used Pichia pastoris, to express recombinant DENV-2 E ectodomain, and found that it assembled into virus-like particles (VLPs), in the absence of prM, implicated in the elicitation of ADE-mediating antibodies.These VLPs elicited predominantly type-specific neutralizing antibodies that conferred significant protection against lethal DENV-2 challenge, in a mouse model.Significantly, they also lack discernible ADE potential toward heterotypic DENVs.

View Article: PubMed Central - PubMed

Affiliation: Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi India.

ABSTRACT
Dengue poses a serious public health risk to nearly half the global population. It causes ~400 million infections annually and is considered to be one of the fastest spreading vector-borne diseases. Four distinct serotypes of dengue viruses (DENV-1, -2, -3, and -4) cause dengue disease, which may be either mild or extremely severe. Antibody-dependent enhancement (ADE), by pre-existing cross-reactive antibodies, is considered to be the major mechanism underlying severe disease. This mandates that a preventive vaccine must confer simultaneous and durable immunity to each of the four prevalent DENV serotypes. Recently, we used Pichia pastoris, to express recombinant DENV-2 E ectodomain, and found that it assembled into virus-like particles (VLPs), in the absence of prM, implicated in the elicitation of ADE-mediating antibodies. These VLPs elicited predominantly type-specific neutralizing antibodies that conferred significant protection against lethal DENV-2 challenge, in a mouse model. The current work is an extension of this approach to develop prM-lacking DENV-3 E VLPs. Our data reveal that P. pastoris-produced DENV-3 E VLPs not only preserve the antigenic integrity of the major neutralizing epitopes, but also elicit potent DENV-3 virus-neutralizing antibodies. Further, these neutralizing antibodies appear to be exclusively directed toward domain III of the DENV-3 E VLPs. Significantly, they also lack discernible ADE potential toward heterotypic DENVs. Taken together with the high productivity of the P. pastoris expression system, this approach could potentially pave the way toward developing a DENV E-based, inexpensive, safe, and efficacious tetravalent sub-unit vaccine, for use in resource-poor dengue endemic countries.

No MeSH data available.


Related in: MedlinePlus

Detection of virus-like particles (VLPs) in the purified DENV-3 E protein preparation. (A) EM analysis of freshly purified DENV-3 E antigen following negative staining with 1% uranyl acetate. (B) Dynamic Light Scattering (DLS) analysis of particle size distribution by intensity. (C) DLS analysis of particle size distribution by volume.
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Figure 1: Detection of virus-like particles (VLPs) in the purified DENV-3 E protein preparation. (A) EM analysis of freshly purified DENV-3 E antigen following negative staining with 1% uranyl acetate. (B) Dynamic Light Scattering (DLS) analysis of particle size distribution by intensity. (C) DLS analysis of particle size distribution by volume.

Mentions: We had observed earlier that the P. pastoris-expressed DENV-2 E protein is able to assemble into VLPs in the absence of prM (Mani et al., 2013). We therefore expected that the DENV-3 E protein produced using this yeast may also manifest this attribute. To verify this, we analyzed the purified recombinant DENV-3 E protein preparation by electron microscopy after negative staining with uranyl acetate, as shown in Figure 1A. Consistent with expectation, the ectodomain of DENV 3 E protein formed discrete VLPs, in the absence of prM. Given that purification was carried out in the presence of urea, VLP formation presumably occurred upon gradual urea removal during dialysis. The observation that P. pastoris-expressed recombinant DENV-3 E protein ectodomain assembles into VLPs is consistent with the behavior of its DENV-2 E counterpart, reported earlier (Mani et al., 2013). The EM data reveal that the DENV-3 VLPs ranged in size from 25 to 50 nm. Further, EM analysis revealed the VLPs to be intact after 2 weeks incubation at 37°C (data not shown). We also analyzed particle size in purified DENV3 E VLP preparation by laser-based DLS. This technique which monitors Brownian movement of the VLPs in native solution can yield information pertaining to average size and frequency distribution of particles. As shown in Figure 1, DLS data represented on the basis of either intensity (Figure 1B) or volume (Figure 1C), resulted in quite similar particle size distribution profiles with average VLP diameter of ~47 nm, which was in fair agreement with EM data.


Pichia pastoris-expressed dengue 3 envelope-based virus-like particles elicit predominantly domain III-focused high titer neutralizing antibodies.

Tripathi L, Mani S, Raut R, Poddar A, Tyagi P, Arora U, de Silva A, Swaminathan S, Khanna N - Front Microbiol (2015)

Detection of virus-like particles (VLPs) in the purified DENV-3 E protein preparation. (A) EM analysis of freshly purified DENV-3 E antigen following negative staining with 1% uranyl acetate. (B) Dynamic Light Scattering (DLS) analysis of particle size distribution by intensity. (C) DLS analysis of particle size distribution by volume.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4585145&req=5

Figure 1: Detection of virus-like particles (VLPs) in the purified DENV-3 E protein preparation. (A) EM analysis of freshly purified DENV-3 E antigen following negative staining with 1% uranyl acetate. (B) Dynamic Light Scattering (DLS) analysis of particle size distribution by intensity. (C) DLS analysis of particle size distribution by volume.
Mentions: We had observed earlier that the P. pastoris-expressed DENV-2 E protein is able to assemble into VLPs in the absence of prM (Mani et al., 2013). We therefore expected that the DENV-3 E protein produced using this yeast may also manifest this attribute. To verify this, we analyzed the purified recombinant DENV-3 E protein preparation by electron microscopy after negative staining with uranyl acetate, as shown in Figure 1A. Consistent with expectation, the ectodomain of DENV 3 E protein formed discrete VLPs, in the absence of prM. Given that purification was carried out in the presence of urea, VLP formation presumably occurred upon gradual urea removal during dialysis. The observation that P. pastoris-expressed recombinant DENV-3 E protein ectodomain assembles into VLPs is consistent with the behavior of its DENV-2 E counterpart, reported earlier (Mani et al., 2013). The EM data reveal that the DENV-3 VLPs ranged in size from 25 to 50 nm. Further, EM analysis revealed the VLPs to be intact after 2 weeks incubation at 37°C (data not shown). We also analyzed particle size in purified DENV3 E VLP preparation by laser-based DLS. This technique which monitors Brownian movement of the VLPs in native solution can yield information pertaining to average size and frequency distribution of particles. As shown in Figure 1, DLS data represented on the basis of either intensity (Figure 1B) or volume (Figure 1C), resulted in quite similar particle size distribution profiles with average VLP diameter of ~47 nm, which was in fair agreement with EM data.

Bottom Line: Recently, we used Pichia pastoris, to express recombinant DENV-2 E ectodomain, and found that it assembled into virus-like particles (VLPs), in the absence of prM, implicated in the elicitation of ADE-mediating antibodies.These VLPs elicited predominantly type-specific neutralizing antibodies that conferred significant protection against lethal DENV-2 challenge, in a mouse model.Significantly, they also lack discernible ADE potential toward heterotypic DENVs.

View Article: PubMed Central - PubMed

Affiliation: Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi India.

ABSTRACT
Dengue poses a serious public health risk to nearly half the global population. It causes ~400 million infections annually and is considered to be one of the fastest spreading vector-borne diseases. Four distinct serotypes of dengue viruses (DENV-1, -2, -3, and -4) cause dengue disease, which may be either mild or extremely severe. Antibody-dependent enhancement (ADE), by pre-existing cross-reactive antibodies, is considered to be the major mechanism underlying severe disease. This mandates that a preventive vaccine must confer simultaneous and durable immunity to each of the four prevalent DENV serotypes. Recently, we used Pichia pastoris, to express recombinant DENV-2 E ectodomain, and found that it assembled into virus-like particles (VLPs), in the absence of prM, implicated in the elicitation of ADE-mediating antibodies. These VLPs elicited predominantly type-specific neutralizing antibodies that conferred significant protection against lethal DENV-2 challenge, in a mouse model. The current work is an extension of this approach to develop prM-lacking DENV-3 E VLPs. Our data reveal that P. pastoris-produced DENV-3 E VLPs not only preserve the antigenic integrity of the major neutralizing epitopes, but also elicit potent DENV-3 virus-neutralizing antibodies. Further, these neutralizing antibodies appear to be exclusively directed toward domain III of the DENV-3 E VLPs. Significantly, they also lack discernible ADE potential toward heterotypic DENVs. Taken together with the high productivity of the P. pastoris expression system, this approach could potentially pave the way toward developing a DENV E-based, inexpensive, safe, and efficacious tetravalent sub-unit vaccine, for use in resource-poor dengue endemic countries.

No MeSH data available.


Related in: MedlinePlus