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Robust intrapulmonary CD8 T cell responses and protection with an attenuated N1L deleted vaccinia virus.

Mathew A, O'Bryan J, Marshall W, Kotwal GJ, Terajima M, Green S, Rothman AL, Ennis FA - PLoS ONE (2008)

Bottom Line: Vaccinia viruses have been used as a model for viral disease and as a protective live vaccine.Infection by the intranasal, intraperitoneal, and tail scarification routes resulted in the robust induction of cytolytic virus-specific CD8 T cells in the spleens and the lungs.These results indicate that the attenuated vGK5 virus protects against subsequent infection and suggest that the N1L protein limits the strength of the early antiviral CD8 T cell response following respiratory infection.

View Article: PubMed Central - PubMed

Affiliation: Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, Worcester, Massachusetts, USA. anuja.mathew@umassmed.edu

ABSTRACT

Background: Vaccinia viruses have been used as a model for viral disease and as a protective live vaccine.

Methodology and principal findings: We investigated the immunogenicity of an attenuated strain of vaccinia virus engineered to inactivate the N1L gene (vGK5). Using the intranasal route, this recombinant virus was 2 logs less virulent compared to the wildtype VACV-WR. Infection by the intranasal, intraperitoneal, and tail scarification routes resulted in the robust induction of cytolytic virus-specific CD8 T cells in the spleens and the lungs. VACV-specific antibodies were also detected in the sera of mice infected 3-5 months prior with the attenuated vGK5 virus. Finally, mice immunized with vGK5 were significantly protected when challenged with a lethal dose of VACV-WR.

Conclusions: These results indicate that the attenuated vGK5 virus protects against subsequent infection and suggest that the N1L protein limits the strength of the early antiviral CD8 T cell response following respiratory infection.

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Related in: MedlinePlus

Immune responses following infection by the tail scarification and i.p. routes.Mice were infected with 1×106 PFU VACV-WR or vGK5 by the i.p. and t.s. routes. (A) Lung lymphocytes and splenocytes obtained from mice (n = 4 mice/group except for infection with VACV-WR by the t.s. route where splenocytes and lung lymphocytes from 2 mice were pooled together) infected 7 days prior were stained with B8R20–27 tetramer. The data shown represent frequencies of cells that were tetramer positive within the CD3+CD8+ gate. Each symbol represents the frequency of tetramer+ T cells obtained in target organs of individual mice; median values are denoted by horizontal lines. (B) Seven days post infection, splenocytes were isolated and CTL assays were carried out using RMA cells infected with VACV-WR (moi = 5), vGK5 (moi = 5) at different (E/T) ratios. Data shown are representative of 2–3 experiments performed for each condition for the i.p. route. (C) PRNT50 antibody titers were measured in sera of mice immunized 3 months prior with 106 PFU of VACV-WR (n = 3) or vGK5 (n = 4). (D) VACV titers were determined in organs 5 days post infection by the i.p. route and expressed as log10 PFU per gram of lung and spleen tissue and PFU/ovary. – represents median values of titers in respective organs. N.S. = Not significant. P values were determined by Student's t test.
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pone-0003323-g004: Immune responses following infection by the tail scarification and i.p. routes.Mice were infected with 1×106 PFU VACV-WR or vGK5 by the i.p. and t.s. routes. (A) Lung lymphocytes and splenocytes obtained from mice (n = 4 mice/group except for infection with VACV-WR by the t.s. route where splenocytes and lung lymphocytes from 2 mice were pooled together) infected 7 days prior were stained with B8R20–27 tetramer. The data shown represent frequencies of cells that were tetramer positive within the CD3+CD8+ gate. Each symbol represents the frequency of tetramer+ T cells obtained in target organs of individual mice; median values are denoted by horizontal lines. (B) Seven days post infection, splenocytes were isolated and CTL assays were carried out using RMA cells infected with VACV-WR (moi = 5), vGK5 (moi = 5) at different (E/T) ratios. Data shown are representative of 2–3 experiments performed for each condition for the i.p. route. (C) PRNT50 antibody titers were measured in sera of mice immunized 3 months prior with 106 PFU of VACV-WR (n = 3) or vGK5 (n = 4). (D) VACV titers were determined in organs 5 days post infection by the i.p. route and expressed as log10 PFU per gram of lung and spleen tissue and PFU/ovary. – represents median values of titers in respective organs. N.S. = Not significant. P values were determined by Student's t test.

Mentions: Overall our studies thus far showed that mice could tolerate high doses of vGK5 by the i.n. route and these doses elicited robust CD8 T cell responses in the lungs and spleens of acutely infected mice. To determine whether immune responses to the attenuated vGK5 were comparable to wildtype VACV-WR, we administered equivalent doses of both viruses by the i.p. and tail scarification routes (106 PFU) and lower doses by the i.n. route (103.5 PFU) since mice were unable to tolerate 104 or greater doses of wildtype VACV-WR intranasally. Mice that were administered VACV-WR or vGK5 by the i.p. or tail scarification routes did not lose any weight and remained healthy. Seven days post infection, 10% of the CD8+ T cells in the spleens and 13–16% of CD8+ T cells in the lungs of mice infected systemically with VACV-WR or vGK5 were tetramer positive with similar frequencies of B8R20–27 TET+ T cells detected in mice infected by the tail scarification route (Fig. 4A). Splenocytes from mice infected with vGK5 systemically efficiently lysed VACV-infected target cells although VACV-WR elicited slightly higher responses at all E/T ratios tested (Fig. 4B). To compare antibody titers in mice immunized with wildtype or the attenuated vGK5, we collected sera from mice immunized 3 months prior with 106 PFU VACV-WR or vGK5 by the i.p. route. Sera from mice immunized with vGK5 had vaccinia-specific antibody titers ranging from 80–1280 (Geometric Mean Titer = 380) while sera from mice immunized with VACV-WR had PRNT50 titers of 640 (Fig. 4C). There were no statistical differences between the two groups.


Robust intrapulmonary CD8 T cell responses and protection with an attenuated N1L deleted vaccinia virus.

Mathew A, O'Bryan J, Marshall W, Kotwal GJ, Terajima M, Green S, Rothman AL, Ennis FA - PLoS ONE (2008)

Immune responses following infection by the tail scarification and i.p. routes.Mice were infected with 1×106 PFU VACV-WR or vGK5 by the i.p. and t.s. routes. (A) Lung lymphocytes and splenocytes obtained from mice (n = 4 mice/group except for infection with VACV-WR by the t.s. route where splenocytes and lung lymphocytes from 2 mice were pooled together) infected 7 days prior were stained with B8R20–27 tetramer. The data shown represent frequencies of cells that were tetramer positive within the CD3+CD8+ gate. Each symbol represents the frequency of tetramer+ T cells obtained in target organs of individual mice; median values are denoted by horizontal lines. (B) Seven days post infection, splenocytes were isolated and CTL assays were carried out using RMA cells infected with VACV-WR (moi = 5), vGK5 (moi = 5) at different (E/T) ratios. Data shown are representative of 2–3 experiments performed for each condition for the i.p. route. (C) PRNT50 antibody titers were measured in sera of mice immunized 3 months prior with 106 PFU of VACV-WR (n = 3) or vGK5 (n = 4). (D) VACV titers were determined in organs 5 days post infection by the i.p. route and expressed as log10 PFU per gram of lung and spleen tissue and PFU/ovary. – represents median values of titers in respective organs. N.S. = Not significant. P values were determined by Student's t test.
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Related In: Results  -  Collection

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pone-0003323-g004: Immune responses following infection by the tail scarification and i.p. routes.Mice were infected with 1×106 PFU VACV-WR or vGK5 by the i.p. and t.s. routes. (A) Lung lymphocytes and splenocytes obtained from mice (n = 4 mice/group except for infection with VACV-WR by the t.s. route where splenocytes and lung lymphocytes from 2 mice were pooled together) infected 7 days prior were stained with B8R20–27 tetramer. The data shown represent frequencies of cells that were tetramer positive within the CD3+CD8+ gate. Each symbol represents the frequency of tetramer+ T cells obtained in target organs of individual mice; median values are denoted by horizontal lines. (B) Seven days post infection, splenocytes were isolated and CTL assays were carried out using RMA cells infected with VACV-WR (moi = 5), vGK5 (moi = 5) at different (E/T) ratios. Data shown are representative of 2–3 experiments performed for each condition for the i.p. route. (C) PRNT50 antibody titers were measured in sera of mice immunized 3 months prior with 106 PFU of VACV-WR (n = 3) or vGK5 (n = 4). (D) VACV titers were determined in organs 5 days post infection by the i.p. route and expressed as log10 PFU per gram of lung and spleen tissue and PFU/ovary. – represents median values of titers in respective organs. N.S. = Not significant. P values were determined by Student's t test.
Mentions: Overall our studies thus far showed that mice could tolerate high doses of vGK5 by the i.n. route and these doses elicited robust CD8 T cell responses in the lungs and spleens of acutely infected mice. To determine whether immune responses to the attenuated vGK5 were comparable to wildtype VACV-WR, we administered equivalent doses of both viruses by the i.p. and tail scarification routes (106 PFU) and lower doses by the i.n. route (103.5 PFU) since mice were unable to tolerate 104 or greater doses of wildtype VACV-WR intranasally. Mice that were administered VACV-WR or vGK5 by the i.p. or tail scarification routes did not lose any weight and remained healthy. Seven days post infection, 10% of the CD8+ T cells in the spleens and 13–16% of CD8+ T cells in the lungs of mice infected systemically with VACV-WR or vGK5 were tetramer positive with similar frequencies of B8R20–27 TET+ T cells detected in mice infected by the tail scarification route (Fig. 4A). Splenocytes from mice infected with vGK5 systemically efficiently lysed VACV-infected target cells although VACV-WR elicited slightly higher responses at all E/T ratios tested (Fig. 4B). To compare antibody titers in mice immunized with wildtype or the attenuated vGK5, we collected sera from mice immunized 3 months prior with 106 PFU VACV-WR or vGK5 by the i.p. route. Sera from mice immunized with vGK5 had vaccinia-specific antibody titers ranging from 80–1280 (Geometric Mean Titer = 380) while sera from mice immunized with VACV-WR had PRNT50 titers of 640 (Fig. 4C). There were no statistical differences between the two groups.

Bottom Line: Vaccinia viruses have been used as a model for viral disease and as a protective live vaccine.Infection by the intranasal, intraperitoneal, and tail scarification routes resulted in the robust induction of cytolytic virus-specific CD8 T cells in the spleens and the lungs.These results indicate that the attenuated vGK5 virus protects against subsequent infection and suggest that the N1L protein limits the strength of the early antiviral CD8 T cell response following respiratory infection.

View Article: PubMed Central - PubMed

Affiliation: Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, Worcester, Massachusetts, USA. anuja.mathew@umassmed.edu

ABSTRACT

Background: Vaccinia viruses have been used as a model for viral disease and as a protective live vaccine.

Methodology and principal findings: We investigated the immunogenicity of an attenuated strain of vaccinia virus engineered to inactivate the N1L gene (vGK5). Using the intranasal route, this recombinant virus was 2 logs less virulent compared to the wildtype VACV-WR. Infection by the intranasal, intraperitoneal, and tail scarification routes resulted in the robust induction of cytolytic virus-specific CD8 T cells in the spleens and the lungs. VACV-specific antibodies were also detected in the sera of mice infected 3-5 months prior with the attenuated vGK5 virus. Finally, mice immunized with vGK5 were significantly protected when challenged with a lethal dose of VACV-WR.

Conclusions: These results indicate that the attenuated vGK5 virus protects against subsequent infection and suggest that the N1L protein limits the strength of the early antiviral CD8 T cell response following respiratory infection.

Show MeSH
Related in: MedlinePlus