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Induction of pluripotent protective immunity following immunisation with a chimeric vaccine against human cytomegalovirus.

Zhong J, Rist M, Cooper L, Smith C, Khanna R - PLoS ONE (2008)

Bottom Line: In spite of numerous attempts successful licensure of a HCMV vaccine formulation remains elusive.Here we have developed a novel chimeric vaccine strategy based on a replication-deficient adenovirus which encodes the extracellular domain of gB protein and multiple HLA class I & II-restricted CTL epitopes from HCMV as a contiguous polypeptide.These studies demonstrate that the adenovirus chimeric HCMV vaccine provides an excellent platform for reconstituting protective immunity to prevent HCMV diseases in different clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Australian Centre for Vaccine Development, Tumour Immunology Laboratory, Division of Immunology, Queensland Institute of Medical Research, Brisbane, Australia.

ABSTRACT
Based on the life-time cost to the health care system, the Institute of Medicine has assigned the highest priority for a vaccine to control human cytomegalovirus (HCMV) disease in transplant patients and new born babies. In spite of numerous attempts successful licensure of a HCMV vaccine formulation remains elusive. Here we have developed a novel chimeric vaccine strategy based on a replication-deficient adenovirus which encodes the extracellular domain of gB protein and multiple HLA class I & II-restricted CTL epitopes from HCMV as a contiguous polypeptide. Immunisation with this chimeric vaccine consistently generated strong HCMV-specific CD8(+) and CD4(+) T-cells which co-expressed IFN-gamma and TNF-alpha, while the humoral response induced by this vaccine showed strong virus neutralizing capacity. More importantly, immunization with adenoviral chimeric vaccine also afforded protection against challenge with recombinant vaccinia virus encoding HCMV antigens and this protection was associated with the induction of a pluripotent antigen-specific cellular and antibody response. Furthermore, in vitro stimulation with this adenoviral chimeric vaccine rapidly expanded multiple antigen-specific human CD8(+) and CD4(+) T-cells from healthy virus carriers. These studies demonstrate that the adenovirus chimeric HCMV vaccine provides an excellent platform for reconstituting protective immunity to prevent HCMV diseases in different clinical settings.

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Related in: MedlinePlus

Ad-gBCMVpoly induced protection against challenge with recombinant vaccinia expressing gB or IE-1 protein.HHD-2 mice were immunised with Ad-gbCMVpoly vaccine and 21 days following vaccination these mice were challenged (intraperitoneal) with recombinant vaccinia encoding gB (Vacc.gB), IE1 protein (Vacc.IE-1) or control vaccinia (Vacc.TK−) at 107 pfu virus/mouse. Ovaries, splenocytes and peripheral blood samples were collected four days later and used for assessing viral load, antigen-specific T cell response and gB-specific antibody response. A, Virus titres in the ovaries of Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.IE-1, Vacc.gB or Vacc.TK−. B, gB-specific antibody response in Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.gB or Vacc.TK−. C, Ex vivo gB-specific CD3+CD4+ T cell response in Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.gB or Vacc.TK−. Splenocytes from these mice were stimulated with recombinant gB protein (40 µg/ml) overnight and then assessed for IFN-γ production using intracellular cytokine assay. D, Ex vivo IE-1-specific CD3+CD8+ T cell response in Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.IE-1 or Vacc.TK−. Splenocytes from these mice were stimulated with the peptide epitope VLEETSVML (1 µg/ml) overnight and then assessed for IFN-γ production using intracellular cytokine assay. E, Ex vivo expression of IFN-γ and/or TNF-α by antigen-specific CD8+ and CD4+T-cells from Ad-gBCMVPpoly vaccinated mice, challenged with recombinant vaccinia encoding IE-1 or gB. Splenocytes from immunised mice stimulated with either gB protein or IE-1 peptide epitope overnight for intracellular cytokine assay. Data represent the percentage of TNF-α and IFN-γ & TNF-α expressing CD4+ or CD8+ T-cells. All statistical analyses were conducted using GraphPad Prism 4 software.
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pone-0003256-g006: Ad-gBCMVpoly induced protection against challenge with recombinant vaccinia expressing gB or IE-1 protein.HHD-2 mice were immunised with Ad-gbCMVpoly vaccine and 21 days following vaccination these mice were challenged (intraperitoneal) with recombinant vaccinia encoding gB (Vacc.gB), IE1 protein (Vacc.IE-1) or control vaccinia (Vacc.TK−) at 107 pfu virus/mouse. Ovaries, splenocytes and peripheral blood samples were collected four days later and used for assessing viral load, antigen-specific T cell response and gB-specific antibody response. A, Virus titres in the ovaries of Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.IE-1, Vacc.gB or Vacc.TK−. B, gB-specific antibody response in Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.gB or Vacc.TK−. C, Ex vivo gB-specific CD3+CD4+ T cell response in Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.gB or Vacc.TK−. Splenocytes from these mice were stimulated with recombinant gB protein (40 µg/ml) overnight and then assessed for IFN-γ production using intracellular cytokine assay. D, Ex vivo IE-1-specific CD3+CD8+ T cell response in Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.IE-1 or Vacc.TK−. Splenocytes from these mice were stimulated with the peptide epitope VLEETSVML (1 µg/ml) overnight and then assessed for IFN-γ production using intracellular cytokine assay. E, Ex vivo expression of IFN-γ and/or TNF-α by antigen-specific CD8+ and CD4+T-cells from Ad-gBCMVPpoly vaccinated mice, challenged with recombinant vaccinia encoding IE-1 or gB. Splenocytes from immunised mice stimulated with either gB protein or IE-1 peptide epitope overnight for intracellular cytokine assay. Data represent the percentage of TNF-α and IFN-γ & TNF-α expressing CD4+ or CD8+ T-cells. All statistical analyses were conducted using GraphPad Prism 4 software.

Mentions: Having firmly established the immunogenicity of Ad-gBCMVpoly vaccine, the next set of experiments was designed to determine protective efficacy of this vaccine. Due to the species restriction, we challenge immunised HHD-2 mice with recombinant vaccinia encoding HCMV antigens (gB and IE-1) to evaluate the protective efficiency of the Ad-gBCMVpoly vaccine. Data presented in Figure 6A shows that HLA A2 mice immunised with Ad-gBCMVpoly vaccine showed significant reduction in the virus load following challenge with Vacc.gB and Vacc.IE-1. This reduction in the virus load was highly antigen-specific as the vaccinated or naïve animals challenged with Vacc.TK- or Vacc.gB, Vacc.IE-1 respectively showed minimal reduction in the viral load. Although Ad-gBCMVpoly immunized mice showed better protection against Vacc.gB when compared to Vacc.IE-1 (Figure 6A), this better protection was not due to anti-gB antibodies (Figure 6B) as Vacc.gB was not neutralized by serum from immunized animals (data not shown), but due to gB-specific CD4+ T cell responses (Figure 6C). Nevertheless, the anti-gB humoral response should play an important role in human as it induces HCMV neutralizing antibodies. As expected, the reduction in the Vacc.IE-1 virus load in Ad-gBCMVpoly immunised mice was co-incident with the induction of VLE-specific CD8+ T cell responses (Figure 6D). It is important to note that Ad-gBCMVpoly immunised mice challenged with Vacc.gB or Vacc.IE-1 showed significantly higher humoral and T cell responses respectively when compared to mice challenged with Vacc.TK-. We also assessed the level of TNF-α expression by IFN-γ expressing CD4+ and CD8+ T-cells using intracellular cytokine assays. Data presented in Figure 6E shows that following stimulation with gB protein or HCMV IE-1 epitope, a large proportion of CD4+ and CD8+ T cells from these mice showed strong co-expression of IFN-γ and TNF-α.


Induction of pluripotent protective immunity following immunisation with a chimeric vaccine against human cytomegalovirus.

Zhong J, Rist M, Cooper L, Smith C, Khanna R - PLoS ONE (2008)

Ad-gBCMVpoly induced protection against challenge with recombinant vaccinia expressing gB or IE-1 protein.HHD-2 mice were immunised with Ad-gbCMVpoly vaccine and 21 days following vaccination these mice were challenged (intraperitoneal) with recombinant vaccinia encoding gB (Vacc.gB), IE1 protein (Vacc.IE-1) or control vaccinia (Vacc.TK−) at 107 pfu virus/mouse. Ovaries, splenocytes and peripheral blood samples were collected four days later and used for assessing viral load, antigen-specific T cell response and gB-specific antibody response. A, Virus titres in the ovaries of Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.IE-1, Vacc.gB or Vacc.TK−. B, gB-specific antibody response in Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.gB or Vacc.TK−. C, Ex vivo gB-specific CD3+CD4+ T cell response in Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.gB or Vacc.TK−. Splenocytes from these mice were stimulated with recombinant gB protein (40 µg/ml) overnight and then assessed for IFN-γ production using intracellular cytokine assay. D, Ex vivo IE-1-specific CD3+CD8+ T cell response in Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.IE-1 or Vacc.TK−. Splenocytes from these mice were stimulated with the peptide epitope VLEETSVML (1 µg/ml) overnight and then assessed for IFN-γ production using intracellular cytokine assay. E, Ex vivo expression of IFN-γ and/or TNF-α by antigen-specific CD8+ and CD4+T-cells from Ad-gBCMVPpoly vaccinated mice, challenged with recombinant vaccinia encoding IE-1 or gB. Splenocytes from immunised mice stimulated with either gB protein or IE-1 peptide epitope overnight for intracellular cytokine assay. Data represent the percentage of TNF-α and IFN-γ & TNF-α expressing CD4+ or CD8+ T-cells. All statistical analyses were conducted using GraphPad Prism 4 software.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2533118&req=5

pone-0003256-g006: Ad-gBCMVpoly induced protection against challenge with recombinant vaccinia expressing gB or IE-1 protein.HHD-2 mice were immunised with Ad-gbCMVpoly vaccine and 21 days following vaccination these mice were challenged (intraperitoneal) with recombinant vaccinia encoding gB (Vacc.gB), IE1 protein (Vacc.IE-1) or control vaccinia (Vacc.TK−) at 107 pfu virus/mouse. Ovaries, splenocytes and peripheral blood samples were collected four days later and used for assessing viral load, antigen-specific T cell response and gB-specific antibody response. A, Virus titres in the ovaries of Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.IE-1, Vacc.gB or Vacc.TK−. B, gB-specific antibody response in Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.gB or Vacc.TK−. C, Ex vivo gB-specific CD3+CD4+ T cell response in Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.gB or Vacc.TK−. Splenocytes from these mice were stimulated with recombinant gB protein (40 µg/ml) overnight and then assessed for IFN-γ production using intracellular cytokine assay. D, Ex vivo IE-1-specific CD3+CD8+ T cell response in Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.IE-1 or Vacc.TK−. Splenocytes from these mice were stimulated with the peptide epitope VLEETSVML (1 µg/ml) overnight and then assessed for IFN-γ production using intracellular cytokine assay. E, Ex vivo expression of IFN-γ and/or TNF-α by antigen-specific CD8+ and CD4+T-cells from Ad-gBCMVPpoly vaccinated mice, challenged with recombinant vaccinia encoding IE-1 or gB. Splenocytes from immunised mice stimulated with either gB protein or IE-1 peptide epitope overnight for intracellular cytokine assay. Data represent the percentage of TNF-α and IFN-γ & TNF-α expressing CD4+ or CD8+ T-cells. All statistical analyses were conducted using GraphPad Prism 4 software.
Mentions: Having firmly established the immunogenicity of Ad-gBCMVpoly vaccine, the next set of experiments was designed to determine protective efficacy of this vaccine. Due to the species restriction, we challenge immunised HHD-2 mice with recombinant vaccinia encoding HCMV antigens (gB and IE-1) to evaluate the protective efficiency of the Ad-gBCMVpoly vaccine. Data presented in Figure 6A shows that HLA A2 mice immunised with Ad-gBCMVpoly vaccine showed significant reduction in the virus load following challenge with Vacc.gB and Vacc.IE-1. This reduction in the virus load was highly antigen-specific as the vaccinated or naïve animals challenged with Vacc.TK- or Vacc.gB, Vacc.IE-1 respectively showed minimal reduction in the viral load. Although Ad-gBCMVpoly immunized mice showed better protection against Vacc.gB when compared to Vacc.IE-1 (Figure 6A), this better protection was not due to anti-gB antibodies (Figure 6B) as Vacc.gB was not neutralized by serum from immunized animals (data not shown), but due to gB-specific CD4+ T cell responses (Figure 6C). Nevertheless, the anti-gB humoral response should play an important role in human as it induces HCMV neutralizing antibodies. As expected, the reduction in the Vacc.IE-1 virus load in Ad-gBCMVpoly immunised mice was co-incident with the induction of VLE-specific CD8+ T cell responses (Figure 6D). It is important to note that Ad-gBCMVpoly immunised mice challenged with Vacc.gB or Vacc.IE-1 showed significantly higher humoral and T cell responses respectively when compared to mice challenged with Vacc.TK-. We also assessed the level of TNF-α expression by IFN-γ expressing CD4+ and CD8+ T-cells using intracellular cytokine assays. Data presented in Figure 6E shows that following stimulation with gB protein or HCMV IE-1 epitope, a large proportion of CD4+ and CD8+ T cells from these mice showed strong co-expression of IFN-γ and TNF-α.

Bottom Line: In spite of numerous attempts successful licensure of a HCMV vaccine formulation remains elusive.Here we have developed a novel chimeric vaccine strategy based on a replication-deficient adenovirus which encodes the extracellular domain of gB protein and multiple HLA class I & II-restricted CTL epitopes from HCMV as a contiguous polypeptide.These studies demonstrate that the adenovirus chimeric HCMV vaccine provides an excellent platform for reconstituting protective immunity to prevent HCMV diseases in different clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Australian Centre for Vaccine Development, Tumour Immunology Laboratory, Division of Immunology, Queensland Institute of Medical Research, Brisbane, Australia.

ABSTRACT
Based on the life-time cost to the health care system, the Institute of Medicine has assigned the highest priority for a vaccine to control human cytomegalovirus (HCMV) disease in transplant patients and new born babies. In spite of numerous attempts successful licensure of a HCMV vaccine formulation remains elusive. Here we have developed a novel chimeric vaccine strategy based on a replication-deficient adenovirus which encodes the extracellular domain of gB protein and multiple HLA class I & II-restricted CTL epitopes from HCMV as a contiguous polypeptide. Immunisation with this chimeric vaccine consistently generated strong HCMV-specific CD8(+) and CD4(+) T-cells which co-expressed IFN-gamma and TNF-alpha, while the humoral response induced by this vaccine showed strong virus neutralizing capacity. More importantly, immunization with adenoviral chimeric vaccine also afforded protection against challenge with recombinant vaccinia virus encoding HCMV antigens and this protection was associated with the induction of a pluripotent antigen-specific cellular and antibody response. Furthermore, in vitro stimulation with this adenoviral chimeric vaccine rapidly expanded multiple antigen-specific human CD8(+) and CD4(+) T-cells from healthy virus carriers. These studies demonstrate that the adenovirus chimeric HCMV vaccine provides an excellent platform for reconstituting protective immunity to prevent HCMV diseases in different clinical settings.

Show MeSH
Related in: MedlinePlus