Limits...
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

Antibody-dependent enhancement analysis. Serial dilutions (x-axis) of heat-inactivated anti-DENV-3 E VLP immune serum (A) or patient serum (primary DENV-3 infection) were analyzed for their ability to promote uptake (y-axis) of DENV-1 (red), DENV-2 (green), DENV-3 (blue) or DENV-4 (black) into Fcγ-bearing K562 cells, using a FACS-based enhancement assay. Note that y-axis in (B) is at a fivefold bigger scale compared to that in (A). Data from one of two independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4585145&req=5

Figure 4: Antibody-dependent enhancement analysis. Serial dilutions (x-axis) of heat-inactivated anti-DENV-3 E VLP immune serum (A) or patient serum (primary DENV-3 infection) were analyzed for their ability to promote uptake (y-axis) of DENV-1 (red), DENV-2 (green), DENV-3 (blue) or DENV-4 (black) into Fcγ-bearing K562 cells, using a FACS-based enhancement assay. Note that y-axis in (B) is at a fivefold bigger scale compared to that in (A). Data from one of two independent experiments are shown.

Mentions: That the P. pastoris-produced DENV-3 E VLPs contain epitopes recognized by cross-reactive mAbs (Table 1) and the antiserum raised using these VLPs contains cross-reactive antibodies (Figure 2B) is evident from our observations above. Would these cross-reactive antibodies play a role in facilitating uptake of heterotypic DENVs through the Fc receptor pathway? To address this, we examined the potential of the DENV-3 E VLP-induced antibodies to cause enhancement. Fcγ receptor-bearing K562 cells were infected with DENVs in the presence of varying dilutions of the anti-DENV-3 E VLP antiserum, followed by determination of the proportion of cells infected as a function of immune serum dilution. For a comparison, serum from a DENV-3 infected patient was also used in a parallel experiment. From the results (Figure 4) it is evident that the patient serum manifested significant heterotypic enhancement while the DENV-3 E VLP-induced immune serum did not. A 20-fold dilution of the patient serum resulted in ≥35% K-562 cells becoming infected with DENV-1, -2, and -4 (heterotypic enhancement) with virtually no DENV-3 infection. In contrast, a 20-fold dilution of anti-DENV-3 E VLP antiserum manifested virtually insignificant heterotypic enhancement effect on DENV-1, -2, and -4, and a low level of homotypic enhancement of DENV-3 infection.


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)

Antibody-dependent enhancement analysis. Serial dilutions (x-axis) of heat-inactivated anti-DENV-3 E VLP immune serum (A) or patient serum (primary DENV-3 infection) were analyzed for their ability to promote uptake (y-axis) of DENV-1 (red), DENV-2 (green), DENV-3 (blue) or DENV-4 (black) into Fcγ-bearing K562 cells, using a FACS-based enhancement assay. Note that y-axis in (B) is at a fivefold bigger scale compared to that in (A). Data from one of two independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Antibody-dependent enhancement analysis. Serial dilutions (x-axis) of heat-inactivated anti-DENV-3 E VLP immune serum (A) or patient serum (primary DENV-3 infection) were analyzed for their ability to promote uptake (y-axis) of DENV-1 (red), DENV-2 (green), DENV-3 (blue) or DENV-4 (black) into Fcγ-bearing K562 cells, using a FACS-based enhancement assay. Note that y-axis in (B) is at a fivefold bigger scale compared to that in (A). Data from one of two independent experiments are shown.
Mentions: That the P. pastoris-produced DENV-3 E VLPs contain epitopes recognized by cross-reactive mAbs (Table 1) and the antiserum raised using these VLPs contains cross-reactive antibodies (Figure 2B) is evident from our observations above. Would these cross-reactive antibodies play a role in facilitating uptake of heterotypic DENVs through the Fc receptor pathway? To address this, we examined the potential of the DENV-3 E VLP-induced antibodies to cause enhancement. Fcγ receptor-bearing K562 cells were infected with DENVs in the presence of varying dilutions of the anti-DENV-3 E VLP antiserum, followed by determination of the proportion of cells infected as a function of immune serum dilution. For a comparison, serum from a DENV-3 infected patient was also used in a parallel experiment. From the results (Figure 4) it is evident that the patient serum manifested significant heterotypic enhancement while the DENV-3 E VLP-induced immune serum did not. A 20-fold dilution of the patient serum resulted in ≥35% K-562 cells becoming infected with DENV-1, -2, and -4 (heterotypic enhancement) with virtually no DENV-3 infection. In contrast, a 20-fold dilution of anti-DENV-3 E VLP antiserum manifested virtually insignificant heterotypic enhancement effect on DENV-1, -2, and -4, and a low level of homotypic enhancement of DENV-3 infection.

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