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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

Preliminary analysis of anti-DENV-3 E VLP induced antibodies. (A) Pooled sera from DENV-3 E VLP-immunized mice after the first (open blue squares) and second (filled blue squares) boosts and mock-immunized (gray filled squares) BALB/c mice were tested in an indirect ELISA using DENV-3 E protein as the coating antigen. (B) Anti-DENV-3 E antiserum obtained after the second boost was tested in ELISAs using DENV-2 E VLPs (solid green), DENV-3 E VLPs (solid blue), recombinant monovalent EDIII-1 (dashed, red), EDIII-2 (dashed, green), EDIII-3 (dashed, blue) or EDIII-4 (dashed, black) proteins as the coating antigens. The ELISA profile of sera from mock-immunized mice (dashed, gray) is found to be superimposed over that of the immune serum against EDIII-4 as the coating antigen. (C) Indirect immunofluorescence analysis of DENV-3 virus-infected BHK-21 cells using (i) mock-immunized serum, (ii) 4G2 mAb, or (iii) anti-DENV-3 E antiserum, as the source of primary antibodies. Bound antibodies were visualized using anti-mouse IgG-FITC conjugate.
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Figure 2: Preliminary analysis of anti-DENV-3 E VLP induced antibodies. (A) Pooled sera from DENV-3 E VLP-immunized mice after the first (open blue squares) and second (filled blue squares) boosts and mock-immunized (gray filled squares) BALB/c mice were tested in an indirect ELISA using DENV-3 E protein as the coating antigen. (B) Anti-DENV-3 E antiserum obtained after the second boost was tested in ELISAs using DENV-2 E VLPs (solid green), DENV-3 E VLPs (solid blue), recombinant monovalent EDIII-1 (dashed, red), EDIII-2 (dashed, green), EDIII-3 (dashed, blue) or EDIII-4 (dashed, black) proteins as the coating antigens. The ELISA profile of sera from mock-immunized mice (dashed, gray) is found to be superimposed over that of the immune serum against EDIII-4 as the coating antigen. (C) Indirect immunofluorescence analysis of DENV-3 virus-infected BHK-21 cells using (i) mock-immunized serum, (ii) 4G2 mAb, or (iii) anti-DENV-3 E antiserum, as the source of primary antibodies. Bound antibodies were visualized using anti-mouse IgG-FITC conjugate.

Mentions: The immunogenicity of the DENV-3 E VLPs above was evaluated in BALB/c mice using a three-dose regimen as described (Mani et al., 2013). Antibody titers in immune sera collected from these mice were determined using an indirect ELISA, with purified DENV-3 E VLPs as the coating antigen (Figure 2). Consistent with that seen earlier for DENV-2 E VLPs, we found an immune boosting effect with DENV-3 E VLPs as well (Figure 2A). As a result, the next ELISA was done using sera collected after the second boost. In this experiment, shown in Figure 2B, we examined the capacity of DENV-3 E VLP immune sera to cross-react with DENV-2 E VLPs as the coating antigen. This showed that ELISA reactivity toward DENV-2 E VLPs was ~20–25% that seen toward DENV-3 E VLPs. This decrease was statistically significant (p < 0.05). Interestingly, ELISA titers observed when the coating antigen used was the recombinant EDIII protein, revealed that antibodies elicited by DENV-3 E VLPs predominantly recognize EDIII-3 as the coating antigen. The reactivity toward EDIII of serotype 2 was very significantly lower (p < 0.01), and negligible in the case of EDIIIs of serotypes 1 and 4. This suggests that DENV-3 E VLPs tend to induce largely serotype 3-specific antibodies. As a next step, we investigated if these anti-DENV-3 E antibodies would also recognize and bind to infectious DENV-3, using an indirect IFA. The data in Figure 2C show that the DENV-3 E-induced antibodies can efficiently bind to DENV-3 E in infected BHK-21 cells. As in the case of DENV-2 seen earlier (Mani et al., 2013), the immunofluorescence pattern observed using anti-DENV-3 E VLPs antiserum showed DENV-3 replication to be essentially localized to the cytoplasm.


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)

Preliminary analysis of anti-DENV-3 E VLP induced antibodies. (A) Pooled sera from DENV-3 E VLP-immunized mice after the first (open blue squares) and second (filled blue squares) boosts and mock-immunized (gray filled squares) BALB/c mice were tested in an indirect ELISA using DENV-3 E protein as the coating antigen. (B) Anti-DENV-3 E antiserum obtained after the second boost was tested in ELISAs using DENV-2 E VLPs (solid green), DENV-3 E VLPs (solid blue), recombinant monovalent EDIII-1 (dashed, red), EDIII-2 (dashed, green), EDIII-3 (dashed, blue) or EDIII-4 (dashed, black) proteins as the coating antigens. The ELISA profile of sera from mock-immunized mice (dashed, gray) is found to be superimposed over that of the immune serum against EDIII-4 as the coating antigen. (C) Indirect immunofluorescence analysis of DENV-3 virus-infected BHK-21 cells using (i) mock-immunized serum, (ii) 4G2 mAb, or (iii) anti-DENV-3 E antiserum, as the source of primary antibodies. Bound antibodies were visualized using anti-mouse IgG-FITC conjugate.
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: Preliminary analysis of anti-DENV-3 E VLP induced antibodies. (A) Pooled sera from DENV-3 E VLP-immunized mice after the first (open blue squares) and second (filled blue squares) boosts and mock-immunized (gray filled squares) BALB/c mice were tested in an indirect ELISA using DENV-3 E protein as the coating antigen. (B) Anti-DENV-3 E antiserum obtained after the second boost was tested in ELISAs using DENV-2 E VLPs (solid green), DENV-3 E VLPs (solid blue), recombinant monovalent EDIII-1 (dashed, red), EDIII-2 (dashed, green), EDIII-3 (dashed, blue) or EDIII-4 (dashed, black) proteins as the coating antigens. The ELISA profile of sera from mock-immunized mice (dashed, gray) is found to be superimposed over that of the immune serum against EDIII-4 as the coating antigen. (C) Indirect immunofluorescence analysis of DENV-3 virus-infected BHK-21 cells using (i) mock-immunized serum, (ii) 4G2 mAb, or (iii) anti-DENV-3 E antiserum, as the source of primary antibodies. Bound antibodies were visualized using anti-mouse IgG-FITC conjugate.
Mentions: The immunogenicity of the DENV-3 E VLPs above was evaluated in BALB/c mice using a three-dose regimen as described (Mani et al., 2013). Antibody titers in immune sera collected from these mice were determined using an indirect ELISA, with purified DENV-3 E VLPs as the coating antigen (Figure 2). Consistent with that seen earlier for DENV-2 E VLPs, we found an immune boosting effect with DENV-3 E VLPs as well (Figure 2A). As a result, the next ELISA was done using sera collected after the second boost. In this experiment, shown in Figure 2B, we examined the capacity of DENV-3 E VLP immune sera to cross-react with DENV-2 E VLPs as the coating antigen. This showed that ELISA reactivity toward DENV-2 E VLPs was ~20–25% that seen toward DENV-3 E VLPs. This decrease was statistically significant (p < 0.05). Interestingly, ELISA titers observed when the coating antigen used was the recombinant EDIII protein, revealed that antibodies elicited by DENV-3 E VLPs predominantly recognize EDIII-3 as the coating antigen. The reactivity toward EDIII of serotype 2 was very significantly lower (p < 0.01), and negligible in the case of EDIIIs of serotypes 1 and 4. This suggests that DENV-3 E VLPs tend to induce largely serotype 3-specific antibodies. As a next step, we investigated if these anti-DENV-3 E antibodies would also recognize and bind to infectious DENV-3, using an indirect IFA. The data in Figure 2C show that the DENV-3 E-induced antibodies can efficiently bind to DENV-3 E in infected BHK-21 cells. As in the case of DENV-2 seen earlier (Mani et al., 2013), the immunofluorescence pattern observed using anti-DENV-3 E VLPs antiserum showed DENV-3 replication to be essentially localized to the cytoplasm.

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