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Targeting of HIV-1 antigens for rapid intracellular degradation enhances cytotoxic T lymphocyte (CTL) recognition and the induction of de novo CTL responses in vivo after immunization.

Tobery TW, Siliciano RF - J. Exp. Med. (1997)

Bottom Line: Additionally, a greatly enhanced de novo env-specific CTL response was induced in vivo after immunization of mice with recombinant vaccinia vectors expressing the cytoplasmic env mutant.Similarly, targeting a cytoplasmic protein, HIV-1 nef, to undergo rapid cytoplasmic degradation induced a greatly enhanced de novo nef-specific CD8+ CTL response in vivo after immunization of mice with either recombinant vaccinia vectors or DNA expression plasmids expressing the degradation targeted nef mutant.The targeting of viral antigens for rapid cytoplasmic degradation represents a novel and highly effective vaccine strategy for the induction of enhanced de novo CTL responses in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
CD8+ cytotoxic T lymphocytes (CTLs) have the ability to recognize and eliminate virally infected cells before new virions are produced within that cell. Therefore, a rapid and vigorous CD8+ CTL response, induced by vaccination, can, in principle, prevent disseminated infection in vaccinated individuals who are exposed to the relevant virus. There has thus been interest in novel vaccine strategies that will enhance the induction of CD8+ CTLs. In this study, we have tested the hypothesis that targeting an antigen to undergo more efficient processing by the class I processing pathway will elicit a more vigorous CD8+ CTL response against that antigen. Targeting a type I transmembrane protein, the HIV-1 envelope (env) protein, for expression in the cytoplasm, rather than allowing its normal co-translational translocation into the endoplasmic reticulum, sensitized target cells expressing this mutant more rapidly for lysis by an env-specific CTL clone. Additionally, a greatly enhanced de novo env-specific CTL response was induced in vivo after immunization of mice with recombinant vaccinia vectors expressing the cytoplasmic env mutant. Similarly, targeting a cytoplasmic protein, HIV-1 nef, to undergo rapid cytoplasmic degradation induced a greatly enhanced de novo nef-specific CD8+ CTL response in vivo after immunization of mice with either recombinant vaccinia vectors or DNA expression plasmids expressing the degradation targeted nef mutant. The targeting of viral antigens for rapid cytoplasmic degradation represents a novel and highly effective vaccine strategy for the induction of enhanced de novo CTL responses in vivo.

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Lysis of targets expressing wt or ss− env protein by the envspecific CD8+ CTL clone A42.46. Autologous B-LCL were infected with  the indicated vaccinia expression vectors 2 h before assay (diamonds) or were  infected for 2 h and then incubated overnight at 37°C before assay  (squares). Infected cells were then used as targets for the env-specific CD8+  CTL clone A42.46 in a standard 51Cr release assay at an E/T ratio of 10:1.  One set of targets was infected in the presence of 0.1 mM cycloheximide  (CHX, circles). The experiment was repeated three times with similar results. Data from a representative experiment are shown.
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Figure 1: Lysis of targets expressing wt or ss− env protein by the envspecific CD8+ CTL clone A42.46. Autologous B-LCL were infected with the indicated vaccinia expression vectors 2 h before assay (diamonds) or were infected for 2 h and then incubated overnight at 37°C before assay (squares). Infected cells were then used as targets for the env-specific CD8+ CTL clone A42.46 in a standard 51Cr release assay at an E/T ratio of 10:1. One set of targets was infected in the presence of 0.1 mM cycloheximide (CHX, circles). The experiment was repeated three times with similar results. Data from a representative experiment are shown.

Mentions: To compare the processing of wt and ss− forms of the env protein, we used the env-specific human CD8+ CTL clone A42.46, which recognizes a 9–amino acid env epitope in association with HLA A3.1 (48). As shown in Fig. 1, this clone recognized an autologous B-LCL infected with recombinant vaccinia virus expressing the HIV-1 env protein (vac-env). The clone also lysed target cells infected with a vaccinia vector expressing the vac-ss− env in a standard 51Cr release assay. However, when target cells were assayed at 2 h after infection with vaccinia expression vectors, rather than after the standard overnight incubation, only the vac-ss− env infected cells were recognized by this clone. The recognition of these cells by clone A42.46 was dependent on de novo protein synthesis, as cells infected with vaccinia expression vectors in the presence of cycloheximide were not lysed. This result suggests that targeting of the env protein for expression in the cytoplasm results in more rapid and/or efficient processing and presentation of the antigen by the class I pathway.


Targeting of HIV-1 antigens for rapid intracellular degradation enhances cytotoxic T lymphocyte (CTL) recognition and the induction of de novo CTL responses in vivo after immunization.

Tobery TW, Siliciano RF - J. Exp. Med. (1997)

Lysis of targets expressing wt or ss− env protein by the envspecific CD8+ CTL clone A42.46. Autologous B-LCL were infected with  the indicated vaccinia expression vectors 2 h before assay (diamonds) or were  infected for 2 h and then incubated overnight at 37°C before assay  (squares). Infected cells were then used as targets for the env-specific CD8+  CTL clone A42.46 in a standard 51Cr release assay at an E/T ratio of 10:1.  One set of targets was infected in the presence of 0.1 mM cycloheximide  (CHX, circles). The experiment was repeated three times with similar results. Data from a representative experiment are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196169&req=5

Figure 1: Lysis of targets expressing wt or ss− env protein by the envspecific CD8+ CTL clone A42.46. Autologous B-LCL were infected with the indicated vaccinia expression vectors 2 h before assay (diamonds) or were infected for 2 h and then incubated overnight at 37°C before assay (squares). Infected cells were then used as targets for the env-specific CD8+ CTL clone A42.46 in a standard 51Cr release assay at an E/T ratio of 10:1. One set of targets was infected in the presence of 0.1 mM cycloheximide (CHX, circles). The experiment was repeated three times with similar results. Data from a representative experiment are shown.
Mentions: To compare the processing of wt and ss− forms of the env protein, we used the env-specific human CD8+ CTL clone A42.46, which recognizes a 9–amino acid env epitope in association with HLA A3.1 (48). As shown in Fig. 1, this clone recognized an autologous B-LCL infected with recombinant vaccinia virus expressing the HIV-1 env protein (vac-env). The clone also lysed target cells infected with a vaccinia vector expressing the vac-ss− env in a standard 51Cr release assay. However, when target cells were assayed at 2 h after infection with vaccinia expression vectors, rather than after the standard overnight incubation, only the vac-ss− env infected cells were recognized by this clone. The recognition of these cells by clone A42.46 was dependent on de novo protein synthesis, as cells infected with vaccinia expression vectors in the presence of cycloheximide were not lysed. This result suggests that targeting of the env protein for expression in the cytoplasm results in more rapid and/or efficient processing and presentation of the antigen by the class I pathway.

Bottom Line: Additionally, a greatly enhanced de novo env-specific CTL response was induced in vivo after immunization of mice with recombinant vaccinia vectors expressing the cytoplasmic env mutant.Similarly, targeting a cytoplasmic protein, HIV-1 nef, to undergo rapid cytoplasmic degradation induced a greatly enhanced de novo nef-specific CD8+ CTL response in vivo after immunization of mice with either recombinant vaccinia vectors or DNA expression plasmids expressing the degradation targeted nef mutant.The targeting of viral antigens for rapid cytoplasmic degradation represents a novel and highly effective vaccine strategy for the induction of enhanced de novo CTL responses in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
CD8+ cytotoxic T lymphocytes (CTLs) have the ability to recognize and eliminate virally infected cells before new virions are produced within that cell. Therefore, a rapid and vigorous CD8+ CTL response, induced by vaccination, can, in principle, prevent disseminated infection in vaccinated individuals who are exposed to the relevant virus. There has thus been interest in novel vaccine strategies that will enhance the induction of CD8+ CTLs. In this study, we have tested the hypothesis that targeting an antigen to undergo more efficient processing by the class I processing pathway will elicit a more vigorous CD8+ CTL response against that antigen. Targeting a type I transmembrane protein, the HIV-1 envelope (env) protein, for expression in the cytoplasm, rather than allowing its normal co-translational translocation into the endoplasmic reticulum, sensitized target cells expressing this mutant more rapidly for lysis by an env-specific CTL clone. Additionally, a greatly enhanced de novo env-specific CTL response was induced in vivo after immunization of mice with recombinant vaccinia vectors expressing the cytoplasmic env mutant. Similarly, targeting a cytoplasmic protein, HIV-1 nef, to undergo rapid cytoplasmic degradation induced a greatly enhanced de novo nef-specific CD8+ CTL response in vivo after immunization of mice with either recombinant vaccinia vectors or DNA expression plasmids expressing the degradation targeted nef mutant. The targeting of viral antigens for rapid cytoplasmic degradation represents a novel and highly effective vaccine strategy for the induction of enhanced de novo CTL responses in vivo.

Show MeSH
Related in: MedlinePlus