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Dengue virus infection and virus-specific HLA-A2 restricted immune responses in humanized NOD-scid IL2rgamma mice.

Jaiswal S, Pearson T, Friberg H, Shultz LD, Greiner DL, Rothman AL, Mathew A - PLoS ONE (2009)

Bottom Line: We sought to establish an animal model for dengue virus (DENV) infection and immunity using non-obese diabetic/severe combined immunodeficiency interleukin-2 receptor gamma-chain knockout (NOD-scid IL2rgamma()) mice engrafted with human hematopoietic stem cells.Human CD45(+) cells in the bone marrow of engrafted mice were susceptible to in vitro infection using low passage clinical and established strains of DENV.Human T cells that developed following engraftment of HLA-A2 transgenic NOD-scid IL2rgamma() mice with HLA-A2(+) human cord blood hematopoietic stem cells, were able to secrete IFN-gamma, IL-2 and TNF-alpha in response to stimulation with three previously identified A2 restricted dengue peptides NS4b 2353((111-119)), NS4b 2423((181-189)), and NS4a 2148((56-64)).

View Article: PubMed Central - PubMed

Affiliation: Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT

Background: The lack of a suitable animal model to study viral and immunological mechanisms of human dengue disease has been a deterrent to dengue research.

Methodology/principal findings: We sought to establish an animal model for dengue virus (DENV) infection and immunity using non-obese diabetic/severe combined immunodeficiency interleukin-2 receptor gamma-chain knockout (NOD-scid IL2rgamma()) mice engrafted with human hematopoietic stem cells. Human CD45(+) cells in the bone marrow of engrafted mice were susceptible to in vitro infection using low passage clinical and established strains of DENV. Engrafted mice were infected with DENV type 2 by different routes and at multiple time points post infection, we detected DENV antigen and RNA in the sera, bone marrow, spleen and liver of infected engrafted mice. Anti-dengue IgM antibodies directed against the envelope protein of DENV peaked in the sera of mice at 1 week post infection. Human T cells that developed following engraftment of HLA-A2 transgenic NOD-scid IL2rgamma() mice with HLA-A2(+) human cord blood hematopoietic stem cells, were able to secrete IFN-gamma, IL-2 and TNF-alpha in response to stimulation with three previously identified A2 restricted dengue peptides NS4b 2353((111-119)), NS4b 2423((181-189)), and NS4a 2148((56-64)).

Conclusions/significance: This is the first study to demonstrate infection of human cells and functional DENV-specific T cell responses in DENV-infected humanized mice. Overall, these mice should be a valuable tool to study the role of prior immunity on subsequent DENV infections.

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Related in: MedlinePlus

Expression of viral antigen in target tissues of mice 3–7 days after in vivo infection.Spleen and bone marrow cells were isolated from mice infected by the s.c. or i.p. route (n = 17) with DENV or mock infected (n = 5) mice 3–7 days post infection. Splenocytes and bone marrow cells were assessed for the expression of the envelope protein of DENV-2 using the zenon conjugated 3H5 mAb (A and B) or a non structural protein 1 of DENV-2 using the zenon conjugated 7E11 mAb (C and D). Data shown are frequencies of 3H5+, 7E11+ or ctrl Ab+ cells within the human CD45+ lymphocyte gate.
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pone-0007251-g003: Expression of viral antigen in target tissues of mice 3–7 days after in vivo infection.Spleen and bone marrow cells were isolated from mice infected by the s.c. or i.p. route (n = 17) with DENV or mock infected (n = 5) mice 3–7 days post infection. Splenocytes and bone marrow cells were assessed for the expression of the envelope protein of DENV-2 using the zenon conjugated 3H5 mAb (A and B) or a non structural protein 1 of DENV-2 using the zenon conjugated 7E11 mAb (C and D). Data shown are frequencies of 3H5+, 7E11+ or ctrl Ab+ cells within the human CD45+ lymphocyte gate.

Mentions: To determine whether viral antigen was expressed following in vivo infection, we assessed the distribution of DENV in multiple organs including the spleen and bone marrow by multiparametric flow cytometry. We utilized the DENV-2-specific mAb 3H5 directed against the envelope protein as well as the mAb 7E11 directed against the nonstructural protein NS1 of DENV-2, to detect DENV antigen in NOD-scid IL2rγ mice 3–7 days after in vivo infection. The specificity of staining was confirmed using DENV-specific mAbs on cells from mock infected mice and control isotype antibodies on cells from infected mice (Fig. S1). Data from multiple experiments are summarized in Fig. 3. Splenocytes and bone marrow cells from mice infected 3 days prior with DENV-2 had the highest frequencies of hCD45+ antigen-positive cells using the 3H5 mAb with lower frequencies of hCD45+ antigen-positive cells detected in mice studied 5 or 7 days post infection (Fig. 3A and B). Frequencies of hCD45+ antigen-positive cells were similar using the 7E11 antibody and reflected the results we obtained with the 3H5 antibody in a subset of mice tested (Fig. 3C and D). Frequencies of mCD45+ antigen positive cells in the spleens and bone marrow of infected mice at early time points using the 3H5 antibody ranged from 0.1–1% (n = 9) which were in the range detected in mock infected mice with the exception of 1 animal which had 3.3% mCD45+3H5+ cells. Similarly, frequencies of mCD45+7E11+ cells ranged from 0–0.7% (n = 9) with frequencies in the spleens of 2 animals at 1.7 and 2.6%. Our data indicate that both structural and non-structural DENV proteins could be detected predominantly in hCD45+ cells following in vivo infection of engrafted mice.


Dengue virus infection and virus-specific HLA-A2 restricted immune responses in humanized NOD-scid IL2rgamma mice.

Jaiswal S, Pearson T, Friberg H, Shultz LD, Greiner DL, Rothman AL, Mathew A - PLoS ONE (2009)

Expression of viral antigen in target tissues of mice 3–7 days after in vivo infection.Spleen and bone marrow cells were isolated from mice infected by the s.c. or i.p. route (n = 17) with DENV or mock infected (n = 5) mice 3–7 days post infection. Splenocytes and bone marrow cells were assessed for the expression of the envelope protein of DENV-2 using the zenon conjugated 3H5 mAb (A and B) or a non structural protein 1 of DENV-2 using the zenon conjugated 7E11 mAb (C and D). Data shown are frequencies of 3H5+, 7E11+ or ctrl Ab+ cells within the human CD45+ lymphocyte gate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2749937&req=5

pone-0007251-g003: Expression of viral antigen in target tissues of mice 3–7 days after in vivo infection.Spleen and bone marrow cells were isolated from mice infected by the s.c. or i.p. route (n = 17) with DENV or mock infected (n = 5) mice 3–7 days post infection. Splenocytes and bone marrow cells were assessed for the expression of the envelope protein of DENV-2 using the zenon conjugated 3H5 mAb (A and B) or a non structural protein 1 of DENV-2 using the zenon conjugated 7E11 mAb (C and D). Data shown are frequencies of 3H5+, 7E11+ or ctrl Ab+ cells within the human CD45+ lymphocyte gate.
Mentions: To determine whether viral antigen was expressed following in vivo infection, we assessed the distribution of DENV in multiple organs including the spleen and bone marrow by multiparametric flow cytometry. We utilized the DENV-2-specific mAb 3H5 directed against the envelope protein as well as the mAb 7E11 directed against the nonstructural protein NS1 of DENV-2, to detect DENV antigen in NOD-scid IL2rγ mice 3–7 days after in vivo infection. The specificity of staining was confirmed using DENV-specific mAbs on cells from mock infected mice and control isotype antibodies on cells from infected mice (Fig. S1). Data from multiple experiments are summarized in Fig. 3. Splenocytes and bone marrow cells from mice infected 3 days prior with DENV-2 had the highest frequencies of hCD45+ antigen-positive cells using the 3H5 mAb with lower frequencies of hCD45+ antigen-positive cells detected in mice studied 5 or 7 days post infection (Fig. 3A and B). Frequencies of hCD45+ antigen-positive cells were similar using the 7E11 antibody and reflected the results we obtained with the 3H5 antibody in a subset of mice tested (Fig. 3C and D). Frequencies of mCD45+ antigen positive cells in the spleens and bone marrow of infected mice at early time points using the 3H5 antibody ranged from 0.1–1% (n = 9) which were in the range detected in mock infected mice with the exception of 1 animal which had 3.3% mCD45+3H5+ cells. Similarly, frequencies of mCD45+7E11+ cells ranged from 0–0.7% (n = 9) with frequencies in the spleens of 2 animals at 1.7 and 2.6%. Our data indicate that both structural and non-structural DENV proteins could be detected predominantly in hCD45+ cells following in vivo infection of engrafted mice.

Bottom Line: We sought to establish an animal model for dengue virus (DENV) infection and immunity using non-obese diabetic/severe combined immunodeficiency interleukin-2 receptor gamma-chain knockout (NOD-scid IL2rgamma()) mice engrafted with human hematopoietic stem cells.Human CD45(+) cells in the bone marrow of engrafted mice were susceptible to in vitro infection using low passage clinical and established strains of DENV.Human T cells that developed following engraftment of HLA-A2 transgenic NOD-scid IL2rgamma() mice with HLA-A2(+) human cord blood hematopoietic stem cells, were able to secrete IFN-gamma, IL-2 and TNF-alpha in response to stimulation with three previously identified A2 restricted dengue peptides NS4b 2353((111-119)), NS4b 2423((181-189)), and NS4a 2148((56-64)).

View Article: PubMed Central - PubMed

Affiliation: Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT

Background: The lack of a suitable animal model to study viral and immunological mechanisms of human dengue disease has been a deterrent to dengue research.

Methodology/principal findings: We sought to establish an animal model for dengue virus (DENV) infection and immunity using non-obese diabetic/severe combined immunodeficiency interleukin-2 receptor gamma-chain knockout (NOD-scid IL2rgamma()) mice engrafted with human hematopoietic stem cells. Human CD45(+) cells in the bone marrow of engrafted mice were susceptible to in vitro infection using low passage clinical and established strains of DENV. Engrafted mice were infected with DENV type 2 by different routes and at multiple time points post infection, we detected DENV antigen and RNA in the sera, bone marrow, spleen and liver of infected engrafted mice. Anti-dengue IgM antibodies directed against the envelope protein of DENV peaked in the sera of mice at 1 week post infection. Human T cells that developed following engraftment of HLA-A2 transgenic NOD-scid IL2rgamma() mice with HLA-A2(+) human cord blood hematopoietic stem cells, were able to secrete IFN-gamma, IL-2 and TNF-alpha in response to stimulation with three previously identified A2 restricted dengue peptides NS4b 2353((111-119)), NS4b 2423((181-189)), and NS4a 2148((56-64)).

Conclusions/significance: This is the first study to demonstrate infection of human cells and functional DENV-specific T cell responses in DENV-infected humanized mice. Overall, these mice should be a valuable tool to study the role of prior immunity on subsequent DENV infections.

Show MeSH
Related in: MedlinePlus