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Immunogenic profiling in mice of a HIV/AIDS vaccine candidate (MVA-B) expressing four HIV-1 antigens and potentiation by specific gene deletions.

García-Arriaza J, Nájera JL, Gómez CE, Sorzano CO, Esteban M - PLoS ONE (2010)

Bottom Line: Both vectors were capable of producing similar levels of antibodies against Env.These findings revealed that MVA-B and MVA-B DeltaA41L/DeltaB16R induced in mice robust, polyfunctional and durable T-cell responses to HIV-1 antigens, but the double deletion mutant showed enhanced magnitude and quality of HIV-1 adaptive and memory responses.Our observations are relevant in the immune evaluation of MVA-B and on improvements of MVA vectors as HIV-1 vaccines.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.

ABSTRACT

Background: The immune parameters of HIV/AIDS vaccine candidates that might be relevant in protection against HIV-1 infection are still undefined. The highly attenuated poxvirus strain MVA is one of the most promising vectors to be use as HIV-1 vaccine. We have previously described a recombinant MVA expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (referred as MVA-B), that induced HIV-1-specific immune responses in different animal models and gene signatures in human dendritic cells (DCs) with immunoregulatory function.

Methodology/principal findings: In an effort to characterize in more detail the immunogenic profile of MVA-B and to improve its immunogenicity we have generated a new vector lacking two genes (A41L and B16R), known to counteract host immune responses by blocking the action of CC-chemokines and of interleukin 1beta, respectively (referred as MVA-B DeltaA41L/DeltaB16R). A DNA prime/MVA boost immunization protocol was used to compare the adaptive and memory HIV-1 specific immune responses induced in mice by the parental MVA-B and by the double deletion mutant MVA-B DeltaA41L/DeltaB16R. Flow cytometry analysis revealed that both vectors triggered HIV-1-specific CD4(+) and CD8(+) T cells, with the CD8(+) T-cell compartment responsible for >91.9% of the total HIV-1 responses in both immunization groups. However, MVA-B DeltaA41L/DeltaB16R enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4(+) and CD8(+) T-cell immune responses. HIV-1-specific CD4(+) T-cell responses were polyfunctional and preferentially Env-specific in both immunization groups. Significantly, while MVA-B induced preferentially Env-specific CD8(+) T-cell responses, MVA-B DeltaA41L/DeltaB16R induced more GPN-specific CD8(+) T-cell responses, with an enhanced polyfunctional pattern. Both vectors were capable of producing similar levels of antibodies against Env.

Conclusions/significance: These findings revealed that MVA-B and MVA-B DeltaA41L/DeltaB16R induced in mice robust, polyfunctional and durable T-cell responses to HIV-1 antigens, but the double deletion mutant showed enhanced magnitude and quality of HIV-1 adaptive and memory responses. Our observations are relevant in the immune evaluation of MVA-B and on improvements of MVA vectors as HIV-1 vaccines.

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Related in: MedlinePlus

HIV-1-specific adaptive immune responses induced by MVA-B and MVA-B ΔA41L/ΔB16R.(Upper) Scheme of the DNA prime/MVA-boost immunization protocol used in this study. BALB/c mice were primed i.m with 100µg of DNA-B (50µg of pCMV-BX08gp120+50µg of pCDNA-IIIBGPN), or control DNA and two weeks later infected i.p with 1×107 PFU of MVA-B, MVA-B ΔA41L/ΔB16R or MVA-WT. (A) Vaccine-elicited T-cell responses of splenocytes 11 days after the last immunization with MVA-B, MVA-B ΔA41L/ΔB16R or MVA-WT, in a fresh IFN-γ ELISPOT assay following stimulation with HIV-1 peptide Gag-B. Bars represent the total number of Gag-B-specific IFN-γ secreting cells per 106 splenocytes in each group. Standard deviations from triplicate cultures are shown. *, represent statistically significant differences between groups, p<0.05. A representative experiment out of two is shown. (B) Total HIV-1-specific CD4+ and CD8+ T-cell immune responses induced in mice 11 days after the last immunization with DNA-B/MVA-B or DNA-B/MVA-B ΔA41L/ΔB16R, measured by flow cytometry using ICS assay, following stimulation with different HIV-1 peptide pools that covered the entire HIV-1 sequence present in the poxvirus vector (Env-pool, Gag-pool and GPN-pool). The percentage of HIV-1-specific CD4+ and CD8+ T-cell responses directed against Env-pool, Gag-pool and GPN-pool is indicated by different color codes, and the frequencies were calculated as the addition of single, double and triple positive T cells for the secretion of IFN-γ, TNF-α and IL-2; thus, each responding cell was counted once. The background of the unstimulated controls was subtracted in all cases, and only significant values over the background are represented. Standard deviations are shown. **, represent statistically significant differences between groups, p<0.005. (C) The pie charts summarize the data of panel B, with each set representing the fraction of CD4+ or CD8+ T cells specific for Env-pool, Gag-pool and GPN-pool.
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pone-0012395-g002: HIV-1-specific adaptive immune responses induced by MVA-B and MVA-B ΔA41L/ΔB16R.(Upper) Scheme of the DNA prime/MVA-boost immunization protocol used in this study. BALB/c mice were primed i.m with 100µg of DNA-B (50µg of pCMV-BX08gp120+50µg of pCDNA-IIIBGPN), or control DNA and two weeks later infected i.p with 1×107 PFU of MVA-B, MVA-B ΔA41L/ΔB16R or MVA-WT. (A) Vaccine-elicited T-cell responses of splenocytes 11 days after the last immunization with MVA-B, MVA-B ΔA41L/ΔB16R or MVA-WT, in a fresh IFN-γ ELISPOT assay following stimulation with HIV-1 peptide Gag-B. Bars represent the total number of Gag-B-specific IFN-γ secreting cells per 106 splenocytes in each group. Standard deviations from triplicate cultures are shown. *, represent statistically significant differences between groups, p<0.05. A representative experiment out of two is shown. (B) Total HIV-1-specific CD4+ and CD8+ T-cell immune responses induced in mice 11 days after the last immunization with DNA-B/MVA-B or DNA-B/MVA-B ΔA41L/ΔB16R, measured by flow cytometry using ICS assay, following stimulation with different HIV-1 peptide pools that covered the entire HIV-1 sequence present in the poxvirus vector (Env-pool, Gag-pool and GPN-pool). The percentage of HIV-1-specific CD4+ and CD8+ T-cell responses directed against Env-pool, Gag-pool and GPN-pool is indicated by different color codes, and the frequencies were calculated as the addition of single, double and triple positive T cells for the secretion of IFN-γ, TNF-α and IL-2; thus, each responding cell was counted once. The background of the unstimulated controls was subtracted in all cases, and only significant values over the background are represented. Standard deviations are shown. **, represent statistically significant differences between groups, p<0.005. (C) The pie charts summarize the data of panel B, with each set representing the fraction of CD4+ or CD8+ T cells specific for Env-pool, Gag-pool and GPN-pool.

Mentions: Since DNA prime/MVA boost immunization is an effective protocol to activate T-cell responses to HIV-1 antigens [1], [2], [3], [7], [22], we analyzed the HIV-1-specific immune responses triggered in BALB/c mice by a DNA-B/MVA-B immunization regimen, and compared it with that triggered by the double deletion mutant MVA-B ΔA41L/ΔB16R. For this purpose groups of mice were first primed intramuscularly (i.m.) with 100µg of DNA-B, and two weeks later the animals were boosted by intraperitoneal (i.p.) route with 1×107 PFU/mouse of recombinant viruses MVA-B or MVA-B ΔA41L/ΔB16R. Animals primed with sham DNA (DNA-φ) and boosted with the non-recombinant MVA-WT were used as control group (a diagram is shown on top of Figure 2). Vaccine-elicited adaptive immune responses in splenocytes were measured 11 days after the boost by fresh IFN-γ ELISPOT and ICS assays.


Immunogenic profiling in mice of a HIV/AIDS vaccine candidate (MVA-B) expressing four HIV-1 antigens and potentiation by specific gene deletions.

García-Arriaza J, Nájera JL, Gómez CE, Sorzano CO, Esteban M - PLoS ONE (2010)

HIV-1-specific adaptive immune responses induced by MVA-B and MVA-B ΔA41L/ΔB16R.(Upper) Scheme of the DNA prime/MVA-boost immunization protocol used in this study. BALB/c mice were primed i.m with 100µg of DNA-B (50µg of pCMV-BX08gp120+50µg of pCDNA-IIIBGPN), or control DNA and two weeks later infected i.p with 1×107 PFU of MVA-B, MVA-B ΔA41L/ΔB16R or MVA-WT. (A) Vaccine-elicited T-cell responses of splenocytes 11 days after the last immunization with MVA-B, MVA-B ΔA41L/ΔB16R or MVA-WT, in a fresh IFN-γ ELISPOT assay following stimulation with HIV-1 peptide Gag-B. Bars represent the total number of Gag-B-specific IFN-γ secreting cells per 106 splenocytes in each group. Standard deviations from triplicate cultures are shown. *, represent statistically significant differences between groups, p<0.05. A representative experiment out of two is shown. (B) Total HIV-1-specific CD4+ and CD8+ T-cell immune responses induced in mice 11 days after the last immunization with DNA-B/MVA-B or DNA-B/MVA-B ΔA41L/ΔB16R, measured by flow cytometry using ICS assay, following stimulation with different HIV-1 peptide pools that covered the entire HIV-1 sequence present in the poxvirus vector (Env-pool, Gag-pool and GPN-pool). The percentage of HIV-1-specific CD4+ and CD8+ T-cell responses directed against Env-pool, Gag-pool and GPN-pool is indicated by different color codes, and the frequencies were calculated as the addition of single, double and triple positive T cells for the secretion of IFN-γ, TNF-α and IL-2; thus, each responding cell was counted once. The background of the unstimulated controls was subtracted in all cases, and only significant values over the background are represented. Standard deviations are shown. **, represent statistically significant differences between groups, p<0.005. (C) The pie charts summarize the data of panel B, with each set representing the fraction of CD4+ or CD8+ T cells specific for Env-pool, Gag-pool and GPN-pool.
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Related In: Results  -  Collection

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pone-0012395-g002: HIV-1-specific adaptive immune responses induced by MVA-B and MVA-B ΔA41L/ΔB16R.(Upper) Scheme of the DNA prime/MVA-boost immunization protocol used in this study. BALB/c mice were primed i.m with 100µg of DNA-B (50µg of pCMV-BX08gp120+50µg of pCDNA-IIIBGPN), or control DNA and two weeks later infected i.p with 1×107 PFU of MVA-B, MVA-B ΔA41L/ΔB16R or MVA-WT. (A) Vaccine-elicited T-cell responses of splenocytes 11 days after the last immunization with MVA-B, MVA-B ΔA41L/ΔB16R or MVA-WT, in a fresh IFN-γ ELISPOT assay following stimulation with HIV-1 peptide Gag-B. Bars represent the total number of Gag-B-specific IFN-γ secreting cells per 106 splenocytes in each group. Standard deviations from triplicate cultures are shown. *, represent statistically significant differences between groups, p<0.05. A representative experiment out of two is shown. (B) Total HIV-1-specific CD4+ and CD8+ T-cell immune responses induced in mice 11 days after the last immunization with DNA-B/MVA-B or DNA-B/MVA-B ΔA41L/ΔB16R, measured by flow cytometry using ICS assay, following stimulation with different HIV-1 peptide pools that covered the entire HIV-1 sequence present in the poxvirus vector (Env-pool, Gag-pool and GPN-pool). The percentage of HIV-1-specific CD4+ and CD8+ T-cell responses directed against Env-pool, Gag-pool and GPN-pool is indicated by different color codes, and the frequencies were calculated as the addition of single, double and triple positive T cells for the secretion of IFN-γ, TNF-α and IL-2; thus, each responding cell was counted once. The background of the unstimulated controls was subtracted in all cases, and only significant values over the background are represented. Standard deviations are shown. **, represent statistically significant differences between groups, p<0.005. (C) The pie charts summarize the data of panel B, with each set representing the fraction of CD4+ or CD8+ T cells specific for Env-pool, Gag-pool and GPN-pool.
Mentions: Since DNA prime/MVA boost immunization is an effective protocol to activate T-cell responses to HIV-1 antigens [1], [2], [3], [7], [22], we analyzed the HIV-1-specific immune responses triggered in BALB/c mice by a DNA-B/MVA-B immunization regimen, and compared it with that triggered by the double deletion mutant MVA-B ΔA41L/ΔB16R. For this purpose groups of mice were first primed intramuscularly (i.m.) with 100µg of DNA-B, and two weeks later the animals were boosted by intraperitoneal (i.p.) route with 1×107 PFU/mouse of recombinant viruses MVA-B or MVA-B ΔA41L/ΔB16R. Animals primed with sham DNA (DNA-φ) and boosted with the non-recombinant MVA-WT were used as control group (a diagram is shown on top of Figure 2). Vaccine-elicited adaptive immune responses in splenocytes were measured 11 days after the boost by fresh IFN-γ ELISPOT and ICS assays.

Bottom Line: Both vectors were capable of producing similar levels of antibodies against Env.These findings revealed that MVA-B and MVA-B DeltaA41L/DeltaB16R induced in mice robust, polyfunctional and durable T-cell responses to HIV-1 antigens, but the double deletion mutant showed enhanced magnitude and quality of HIV-1 adaptive and memory responses.Our observations are relevant in the immune evaluation of MVA-B and on improvements of MVA vectors as HIV-1 vaccines.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.

ABSTRACT

Background: The immune parameters of HIV/AIDS vaccine candidates that might be relevant in protection against HIV-1 infection are still undefined. The highly attenuated poxvirus strain MVA is one of the most promising vectors to be use as HIV-1 vaccine. We have previously described a recombinant MVA expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (referred as MVA-B), that induced HIV-1-specific immune responses in different animal models and gene signatures in human dendritic cells (DCs) with immunoregulatory function.

Methodology/principal findings: In an effort to characterize in more detail the immunogenic profile of MVA-B and to improve its immunogenicity we have generated a new vector lacking two genes (A41L and B16R), known to counteract host immune responses by blocking the action of CC-chemokines and of interleukin 1beta, respectively (referred as MVA-B DeltaA41L/DeltaB16R). A DNA prime/MVA boost immunization protocol was used to compare the adaptive and memory HIV-1 specific immune responses induced in mice by the parental MVA-B and by the double deletion mutant MVA-B DeltaA41L/DeltaB16R. Flow cytometry analysis revealed that both vectors triggered HIV-1-specific CD4(+) and CD8(+) T cells, with the CD8(+) T-cell compartment responsible for >91.9% of the total HIV-1 responses in both immunization groups. However, MVA-B DeltaA41L/DeltaB16R enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4(+) and CD8(+) T-cell immune responses. HIV-1-specific CD4(+) T-cell responses were polyfunctional and preferentially Env-specific in both immunization groups. Significantly, while MVA-B induced preferentially Env-specific CD8(+) T-cell responses, MVA-B DeltaA41L/DeltaB16R induced more GPN-specific CD8(+) T-cell responses, with an enhanced polyfunctional pattern. Both vectors were capable of producing similar levels of antibodies against Env.

Conclusions/significance: These findings revealed that MVA-B and MVA-B DeltaA41L/DeltaB16R induced in mice robust, polyfunctional and durable T-cell responses to HIV-1 antigens, but the double deletion mutant showed enhanced magnitude and quality of HIV-1 adaptive and memory responses. Our observations are relevant in the immune evaluation of MVA-B and on improvements of MVA vectors as HIV-1 vaccines.

Show MeSH
Related in: MedlinePlus