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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

Characterization of neutralization potency of DENV-3 E VLP induced antibodies. (A) Two-fold serial dilutions of heat inactivated immune serum from DENV-3 E VLP-immunized mice was assessed for its potency to neutralize and inhibit the infectivity of DENV-1 (red), DENV-2 (green), DENV-3 (blue) and DENV-4 (black) using FACS-based neutralization assay. The y-axis corresponds to the observed percentage of virus infection in Vero cells. The dotted horizontal line represents 50% infection. The x-axis corresponds to logarithm of reciprocal serum dilution. (B) Effect of EDIII-specific antibody depletion on the DENV-3 virus neutralization potency of anti-DENV-3 E VLP antiserum. The same experiment as shown in panel A, except that the immune serum was pre-depleted with MBP alone (blue) or MBP-EDIII-3 fusion protein (red) before being assessed for its neutralization potency against DENV-3 infection. Control serum (gray) was analyzed in parallel without any pre-depletion with either MBP or MBP-EDIII-3 fusion protein.
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Figure 3: Characterization of neutralization potency of DENV-3 E VLP induced antibodies. (A) Two-fold serial dilutions of heat inactivated immune serum from DENV-3 E VLP-immunized mice was assessed for its potency to neutralize and inhibit the infectivity of DENV-1 (red), DENV-2 (green), DENV-3 (blue) and DENV-4 (black) using FACS-based neutralization assay. The y-axis corresponds to the observed percentage of virus infection in Vero cells. The dotted horizontal line represents 50% infection. The x-axis corresponds to logarithm of reciprocal serum dilution. (B) Effect of EDIII-specific antibody depletion on the DENV-3 virus neutralization potency of anti-DENV-3 E VLP antiserum. The same experiment as shown in panel A, except that the immune serum was pre-depleted with MBP alone (blue) or MBP-EDIII-3 fusion protein (red) before being assessed for its neutralization potency against DENV-3 infection. Control serum (gray) was analyzed in parallel without any pre-depletion with either MBP or MBP-EDIII-3 fusion protein.

Mentions: Following this preliminary analysis of the antibodies elicited by the DENV-3 E VLPs, we next sought to determine if these could prevent DENV from infecting susceptible cells. To this end, we employed a FACS-based virus neutralization assay, the results of which are depicted in Figure 3A. In this experiment we tested the neutralization capacity of the DENV-3 E VLP immune sera against each one of the four WHO reference DENV strains (Kraus et al., 2007). Our data revealed that the anti-DENV-3 E VLP antibodies neutralized only DENV-3 most effectively (FNT50 titers > 2400). The remaining three serotypes were only weakly neutralized, with neutralization titers against DENV-1, DENV-2, and DENV-4 being 1–5% the titers observed for DENV-3. Heterotypic neutralization titers were very significantly lower compared to homotypic neutralization titers (p < 0.0001).


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)

Characterization of neutralization potency of DENV-3 E VLP induced antibodies. (A) Two-fold serial dilutions of heat inactivated immune serum from DENV-3 E VLP-immunized mice was assessed for its potency to neutralize and inhibit the infectivity of DENV-1 (red), DENV-2 (green), DENV-3 (blue) and DENV-4 (black) using FACS-based neutralization assay. The y-axis corresponds to the observed percentage of virus infection in Vero cells. The dotted horizontal line represents 50% infection. The x-axis corresponds to logarithm of reciprocal serum dilution. (B) Effect of EDIII-specific antibody depletion on the DENV-3 virus neutralization potency of anti-DENV-3 E VLP antiserum. The same experiment as shown in panel A, except that the immune serum was pre-depleted with MBP alone (blue) or MBP-EDIII-3 fusion protein (red) before being assessed for its neutralization potency against DENV-3 infection. Control serum (gray) was analyzed in parallel without any pre-depletion with either MBP or MBP-EDIII-3 fusion protein.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Characterization of neutralization potency of DENV-3 E VLP induced antibodies. (A) Two-fold serial dilutions of heat inactivated immune serum from DENV-3 E VLP-immunized mice was assessed for its potency to neutralize and inhibit the infectivity of DENV-1 (red), DENV-2 (green), DENV-3 (blue) and DENV-4 (black) using FACS-based neutralization assay. The y-axis corresponds to the observed percentage of virus infection in Vero cells. The dotted horizontal line represents 50% infection. The x-axis corresponds to logarithm of reciprocal serum dilution. (B) Effect of EDIII-specific antibody depletion on the DENV-3 virus neutralization potency of anti-DENV-3 E VLP antiserum. The same experiment as shown in panel A, except that the immune serum was pre-depleted with MBP alone (blue) or MBP-EDIII-3 fusion protein (red) before being assessed for its neutralization potency against DENV-3 infection. Control serum (gray) was analyzed in parallel without any pre-depletion with either MBP or MBP-EDIII-3 fusion protein.
Mentions: Following this preliminary analysis of the antibodies elicited by the DENV-3 E VLPs, we next sought to determine if these could prevent DENV from infecting susceptible cells. To this end, we employed a FACS-based virus neutralization assay, the results of which are depicted in Figure 3A. In this experiment we tested the neutralization capacity of the DENV-3 E VLP immune sera against each one of the four WHO reference DENV strains (Kraus et al., 2007). Our data revealed that the anti-DENV-3 E VLP antibodies neutralized only DENV-3 most effectively (FNT50 titers > 2400). The remaining three serotypes were only weakly neutralized, with neutralization titers against DENV-1, DENV-2, and DENV-4 being 1–5% the titers observed for DENV-3. Heterotypic neutralization titers were very significantly lower compared to homotypic neutralization titers (p < 0.0001).

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