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Sub-nucleocapsid nanoparticles: a nasal vaccine against respiratory syncytial virus.

Roux X, Dubuquoy C, Durand G, Tran-Tolla TL, Castagné N, Bernard J, Petit-Camurdan A, Eléouët JF, Riffault S - PLoS ONE (2008)

Bottom Line: These nanoparticles were named sub-nucleocapsid ring structure (N SRS).Mucosal immunization with N SRS elicited strong local and systemic immunity characterized by high titers of IgG1, IgG2a and IgA anti-N antibodies, antigen-specific CD8(+) T cells and IFN-gamma-producing CD4(+) T cells.This is the first report of using nanoparticles formed by the recombinant nucleocapsid protein as an efficient and safe intra-nasal vaccine against RSV.

View Article: PubMed Central - PubMed

Affiliation: Unité de Virologie et Immunologie Moléculaires (UR892), INRA, Jouy-en-Josas, France.

ABSTRACT

Background: Bronchiolitis caused by the respiratory syncytial virus (RSV) in infants less than two years old is a growing public health concern worldwide, and there is currently no safe and effective vaccine. A major component of RSV nucleocapsid, the nucleoprotein (N), has been so far poorly explored as a potential vaccine antigen, even though it is a target of protective anti-viral T cell responses and is remarkably conserved between human RSV A and B serotypes. We recently reported a method to produce recombinant N assembling in homogenous rings composed of 10-11 N subunits enclosing a bacterial RNA. These nanoparticles were named sub-nucleocapsid ring structure (N SRS).

Methodology and principal findings: The vaccine potential of N SRS was evaluated in a well-characterized and widely acknowledged mouse model of RSV infection. BALB/c adult mice were immunized intranasally with N SRS adjuvanted with the detoxified E. coli enterotoxin LT(R192G). Upon RSV challenge, vaccinated mice were largely protected against virus replication in the lungs, with a mild inflammatory lymphocytic and neutrophilic reaction in their airways. Mucosal immunization with N SRS elicited strong local and systemic immunity characterized by high titers of IgG1, IgG2a and IgA anti-N antibodies, antigen-specific CD8(+) T cells and IFN-gamma-producing CD4(+) T cells.

Conclusions/significance: This is the first report of using nanoparticles formed by the recombinant nucleocapsid protein as an efficient and safe intra-nasal vaccine against RSV.

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Related in: MedlinePlus

T cell-mediated immune response to N SRS.Nasal vaccination with N SRS generated antigen-specific CD8+ T cells and IFN-γ producing CD4+ T cells. (A) Antigen-specific proliferation of CD4+ and CD8+ T splenocytes after 7 days restimulation with RSV-A2 (1 PFU/cell), N (10 µg/ml) or medium. Pooled splenocytes from non immunized (grey lines), LT(R192G) (black lines) or N SRS+LT(R192G) (red lines) immunized groups were stained with CFSE, cultured for 7 days and then labeled with anti-CD8-biot and anti-CD4-PE for flow-cytometry analysis. The data (100,000 events) were acquired with a FACScalibur and analyzed with Cell Quest-Pro. The CD8+ or CD4+ lymphocyte population was gated according to SSC/FCS and FL4 (CD8) or FL2 (CD4) fluorescence criteria and the fluorescence corresponding to CFSE was monitored in FL1. The percentage of proliferating cells (low CFSE staining) is indicated on the plot with the color corresponding to the immunization condition. (Data from one out of two experiments with similar results). (B) Two weeks after the booster immunization with LT(R192G) or N SRS+LT(R192G), spleen (white bars) and draining LN (cervical and sub-maxilliary LN, black bars) were dissected out and cell suspensions prepared. Splenocytes from individual mice and pooled draining LN cells from each group of mice were re-stimulated for 72 hr with N SRS (10 µg/ml) or medium (mock). IFN-γ secretion was measured in cell culture supernatant with a standardized specific sandwich ELISA assay (white bars represent the mean and SEM of 5 individual spleens, black bars represent the pool of LN, data from one out of three experiments). (C) The frequency of IFN-γ secreting splenocytes after 20 hr restimulation with N (10 µg/ml) was monitored by ELISPOT. Spleen cells from LT(R192G) or N SRS+LT(R192G) immunized mice were assayed for each mouse (each bar represents the mean and SEM of 5 mice). Depletion of CD4+ or CD8+ T cells was done by immuno-magnetic separation of pooled splenocytes from either LT(R192G) or N SRS+LT(R192G) groups. (Data from one out of two experiments with similar results).
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pone-0001766-g005: T cell-mediated immune response to N SRS.Nasal vaccination with N SRS generated antigen-specific CD8+ T cells and IFN-γ producing CD4+ T cells. (A) Antigen-specific proliferation of CD4+ and CD8+ T splenocytes after 7 days restimulation with RSV-A2 (1 PFU/cell), N (10 µg/ml) or medium. Pooled splenocytes from non immunized (grey lines), LT(R192G) (black lines) or N SRS+LT(R192G) (red lines) immunized groups were stained with CFSE, cultured for 7 days and then labeled with anti-CD8-biot and anti-CD4-PE for flow-cytometry analysis. The data (100,000 events) were acquired with a FACScalibur and analyzed with Cell Quest-Pro. The CD8+ or CD4+ lymphocyte population was gated according to SSC/FCS and FL4 (CD8) or FL2 (CD4) fluorescence criteria and the fluorescence corresponding to CFSE was monitored in FL1. The percentage of proliferating cells (low CFSE staining) is indicated on the plot with the color corresponding to the immunization condition. (Data from one out of two experiments with similar results). (B) Two weeks after the booster immunization with LT(R192G) or N SRS+LT(R192G), spleen (white bars) and draining LN (cervical and sub-maxilliary LN, black bars) were dissected out and cell suspensions prepared. Splenocytes from individual mice and pooled draining LN cells from each group of mice were re-stimulated for 72 hr with N SRS (10 µg/ml) or medium (mock). IFN-γ secretion was measured in cell culture supernatant with a standardized specific sandwich ELISA assay (white bars represent the mean and SEM of 5 individual spleens, black bars represent the pool of LN, data from one out of three experiments). (C) The frequency of IFN-γ secreting splenocytes after 20 hr restimulation with N (10 µg/ml) was monitored by ELISPOT. Spleen cells from LT(R192G) or N SRS+LT(R192G) immunized mice were assayed for each mouse (each bar represents the mean and SEM of 5 mice). Depletion of CD4+ or CD8+ T cells was done by immuno-magnetic separation of pooled splenocytes from either LT(R192G) or N SRS+LT(R192G) groups. (Data from one out of two experiments with similar results).

Mentions: CFSE-stained splenocytes from non vaccinated, LT(R192G) or N SRS+LT(R192G) immunized mice were co-cultured with either live RSV, N SRS or medium alone and after 7 days the level of CFSE staining on CD4+ and CD8+ cell subsets was quantified by flow cytometry analysis (Fig. 5A). A large percentage of CD4+ and CD8+ splenocytes isolated from N SRS immunized mice and co-cultured with N SRS or RSV displayed low CFSE fluorescence, showing the expansion of CD4+ and CD8+ N-specific T cells (Fig. 5A red lines). To control for the specificity of this response, we showed that CD4+ and CD8+ splenocytes isolated from non immunized mice (grey lines) or LT(R192G) treated mice (black lines) did not proliferate in response to either RSV or N SRS (Fig. 5A).


Sub-nucleocapsid nanoparticles: a nasal vaccine against respiratory syncytial virus.

Roux X, Dubuquoy C, Durand G, Tran-Tolla TL, Castagné N, Bernard J, Petit-Camurdan A, Eléouët JF, Riffault S - PLoS ONE (2008)

T cell-mediated immune response to N SRS.Nasal vaccination with N SRS generated antigen-specific CD8+ T cells and IFN-γ producing CD4+ T cells. (A) Antigen-specific proliferation of CD4+ and CD8+ T splenocytes after 7 days restimulation with RSV-A2 (1 PFU/cell), N (10 µg/ml) or medium. Pooled splenocytes from non immunized (grey lines), LT(R192G) (black lines) or N SRS+LT(R192G) (red lines) immunized groups were stained with CFSE, cultured for 7 days and then labeled with anti-CD8-biot and anti-CD4-PE for flow-cytometry analysis. The data (100,000 events) were acquired with a FACScalibur and analyzed with Cell Quest-Pro. The CD8+ or CD4+ lymphocyte population was gated according to SSC/FCS and FL4 (CD8) or FL2 (CD4) fluorescence criteria and the fluorescence corresponding to CFSE was monitored in FL1. The percentage of proliferating cells (low CFSE staining) is indicated on the plot with the color corresponding to the immunization condition. (Data from one out of two experiments with similar results). (B) Two weeks after the booster immunization with LT(R192G) or N SRS+LT(R192G), spleen (white bars) and draining LN (cervical and sub-maxilliary LN, black bars) were dissected out and cell suspensions prepared. Splenocytes from individual mice and pooled draining LN cells from each group of mice were re-stimulated for 72 hr with N SRS (10 µg/ml) or medium (mock). IFN-γ secretion was measured in cell culture supernatant with a standardized specific sandwich ELISA assay (white bars represent the mean and SEM of 5 individual spleens, black bars represent the pool of LN, data from one out of three experiments). (C) The frequency of IFN-γ secreting splenocytes after 20 hr restimulation with N (10 µg/ml) was monitored by ELISPOT. Spleen cells from LT(R192G) or N SRS+LT(R192G) immunized mice were assayed for each mouse (each bar represents the mean and SEM of 5 mice). Depletion of CD4+ or CD8+ T cells was done by immuno-magnetic separation of pooled splenocytes from either LT(R192G) or N SRS+LT(R192G) groups. (Data from one out of two experiments with similar results).
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Related In: Results  -  Collection

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pone-0001766-g005: T cell-mediated immune response to N SRS.Nasal vaccination with N SRS generated antigen-specific CD8+ T cells and IFN-γ producing CD4+ T cells. (A) Antigen-specific proliferation of CD4+ and CD8+ T splenocytes after 7 days restimulation with RSV-A2 (1 PFU/cell), N (10 µg/ml) or medium. Pooled splenocytes from non immunized (grey lines), LT(R192G) (black lines) or N SRS+LT(R192G) (red lines) immunized groups were stained with CFSE, cultured for 7 days and then labeled with anti-CD8-biot and anti-CD4-PE for flow-cytometry analysis. The data (100,000 events) were acquired with a FACScalibur and analyzed with Cell Quest-Pro. The CD8+ or CD4+ lymphocyte population was gated according to SSC/FCS and FL4 (CD8) or FL2 (CD4) fluorescence criteria and the fluorescence corresponding to CFSE was monitored in FL1. The percentage of proliferating cells (low CFSE staining) is indicated on the plot with the color corresponding to the immunization condition. (Data from one out of two experiments with similar results). (B) Two weeks after the booster immunization with LT(R192G) or N SRS+LT(R192G), spleen (white bars) and draining LN (cervical and sub-maxilliary LN, black bars) were dissected out and cell suspensions prepared. Splenocytes from individual mice and pooled draining LN cells from each group of mice were re-stimulated for 72 hr with N SRS (10 µg/ml) or medium (mock). IFN-γ secretion was measured in cell culture supernatant with a standardized specific sandwich ELISA assay (white bars represent the mean and SEM of 5 individual spleens, black bars represent the pool of LN, data from one out of three experiments). (C) The frequency of IFN-γ secreting splenocytes after 20 hr restimulation with N (10 µg/ml) was monitored by ELISPOT. Spleen cells from LT(R192G) or N SRS+LT(R192G) immunized mice were assayed for each mouse (each bar represents the mean and SEM of 5 mice). Depletion of CD4+ or CD8+ T cells was done by immuno-magnetic separation of pooled splenocytes from either LT(R192G) or N SRS+LT(R192G) groups. (Data from one out of two experiments with similar results).
Mentions: CFSE-stained splenocytes from non vaccinated, LT(R192G) or N SRS+LT(R192G) immunized mice were co-cultured with either live RSV, N SRS or medium alone and after 7 days the level of CFSE staining on CD4+ and CD8+ cell subsets was quantified by flow cytometry analysis (Fig. 5A). A large percentage of CD4+ and CD8+ splenocytes isolated from N SRS immunized mice and co-cultured with N SRS or RSV displayed low CFSE fluorescence, showing the expansion of CD4+ and CD8+ N-specific T cells (Fig. 5A red lines). To control for the specificity of this response, we showed that CD4+ and CD8+ splenocytes isolated from non immunized mice (grey lines) or LT(R192G) treated mice (black lines) did not proliferate in response to either RSV or N SRS (Fig. 5A).

Bottom Line: These nanoparticles were named sub-nucleocapsid ring structure (N SRS).Mucosal immunization with N SRS elicited strong local and systemic immunity characterized by high titers of IgG1, IgG2a and IgA anti-N antibodies, antigen-specific CD8(+) T cells and IFN-gamma-producing CD4(+) T cells.This is the first report of using nanoparticles formed by the recombinant nucleocapsid protein as an efficient and safe intra-nasal vaccine against RSV.

View Article: PubMed Central - PubMed

Affiliation: Unité de Virologie et Immunologie Moléculaires (UR892), INRA, Jouy-en-Josas, France.

ABSTRACT

Background: Bronchiolitis caused by the respiratory syncytial virus (RSV) in infants less than two years old is a growing public health concern worldwide, and there is currently no safe and effective vaccine. A major component of RSV nucleocapsid, the nucleoprotein (N), has been so far poorly explored as a potential vaccine antigen, even though it is a target of protective anti-viral T cell responses and is remarkably conserved between human RSV A and B serotypes. We recently reported a method to produce recombinant N assembling in homogenous rings composed of 10-11 N subunits enclosing a bacterial RNA. These nanoparticles were named sub-nucleocapsid ring structure (N SRS).

Methodology and principal findings: The vaccine potential of N SRS was evaluated in a well-characterized and widely acknowledged mouse model of RSV infection. BALB/c adult mice were immunized intranasally with N SRS adjuvanted with the detoxified E. coli enterotoxin LT(R192G). Upon RSV challenge, vaccinated mice were largely protected against virus replication in the lungs, with a mild inflammatory lymphocytic and neutrophilic reaction in their airways. Mucosal immunization with N SRS elicited strong local and systemic immunity characterized by high titers of IgG1, IgG2a and IgA anti-N antibodies, antigen-specific CD8(+) T cells and IFN-gamma-producing CD4(+) T cells.

Conclusions/significance: This is the first report of using nanoparticles formed by the recombinant nucleocapsid protein as an efficient and safe intra-nasal vaccine against RSV.

Show MeSH
Related in: MedlinePlus