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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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Enhanced induction of env-specific CTLs by immunization  with vac ss− env. BALB/c mice were immunized with 107 PFU of vac  control, vac-env, or vac-ss− env. Splenocytes from immunized mice were  stimulated for 5 d in IL-2 containing media in the presence of the H-2Ld– restricted env peptide (RGPGGRAFVTI). Stimulated splenocytes were  then assayed for env-specific CTL activity against P815 cells pulsed with  the H-2Ld–restricted env peptide or media alone. The data shown are  representative of three separate experiments.
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Figure 2: Enhanced induction of env-specific CTLs by immunization with vac ss− env. BALB/c mice were immunized with 107 PFU of vac control, vac-env, or vac-ss− env. Splenocytes from immunized mice were stimulated for 5 d in IL-2 containing media in the presence of the H-2Ld– restricted env peptide (RGPGGRAFVTI). Stimulated splenocytes were then assayed for env-specific CTL activity against P815 cells pulsed with the H-2Ld–restricted env peptide or media alone. The data shown are representative of three separate experiments.

Mentions: To determine whether the cytoplasmic targeting of the env protein would result in enhanced generation of de novo CTL responses in vivo, BALB/c mice were immunized with the control vaccinia vector, vac-ss− env, or with vac-env as described in Materials and Methods. Splenocytes from immunized mice were cultured in vitro in the presence or absence of a peptide representing an immunodominant H-2 Ld–restricted epitope within the env protein, RGPGRAFVTI (49). As seen in Fig. 2, stimulated splenocytes from mice immunized with the vac-ss− env vector were able to recognize and efficiently lyse target cells pulsed with the env peptide. Class II negative P815 target cells were used in this assay so that only class I–restricted lysis could be observed. Most importantly, the lysis by CTLs from mice immunized with vacss− env was much greater than the lysis mediated by stimulated splenocytes from mice immunized with the vac-env vector. Splenocytes from mice immunized with a control vaccinia vector did not lyse targets presenting the relevant env epitope. When mice were immunized with a higher dose (108 PFU) of recombinant vaccinia vectors, the immune response to both constructs was blunted (data not shown). However, this dose exceeds the standard dose for immunization with vaccinia virus, and the diminished immune response is most likely a result of other secondary effects. Taken together, these results suggest that targeting the env protein for translation on free ribosomes in the cytoplasm, rather than for co-translational translocation into the endoplasmic reticulum, results in more rapid and efficient processing for presentation to class I–restricted T cells and the more efficient stimulation of de novo CTL responses in vivo.


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)

Enhanced induction of env-specific CTLs by immunization  with vac ss− env. BALB/c mice were immunized with 107 PFU of vac  control, vac-env, or vac-ss− env. Splenocytes from immunized mice were  stimulated for 5 d in IL-2 containing media in the presence of the H-2Ld– restricted env peptide (RGPGGRAFVTI). Stimulated splenocytes were  then assayed for env-specific CTL activity against P815 cells pulsed with  the H-2Ld–restricted env peptide or media alone. The data shown are  representative of three separate experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196169&req=5

Figure 2: Enhanced induction of env-specific CTLs by immunization with vac ss− env. BALB/c mice were immunized with 107 PFU of vac control, vac-env, or vac-ss− env. Splenocytes from immunized mice were stimulated for 5 d in IL-2 containing media in the presence of the H-2Ld– restricted env peptide (RGPGGRAFVTI). Stimulated splenocytes were then assayed for env-specific CTL activity against P815 cells pulsed with the H-2Ld–restricted env peptide or media alone. The data shown are representative of three separate experiments.
Mentions: To determine whether the cytoplasmic targeting of the env protein would result in enhanced generation of de novo CTL responses in vivo, BALB/c mice were immunized with the control vaccinia vector, vac-ss− env, or with vac-env as described in Materials and Methods. Splenocytes from immunized mice were cultured in vitro in the presence or absence of a peptide representing an immunodominant H-2 Ld–restricted epitope within the env protein, RGPGRAFVTI (49). As seen in Fig. 2, stimulated splenocytes from mice immunized with the vac-ss− env vector were able to recognize and efficiently lyse target cells pulsed with the env peptide. Class II negative P815 target cells were used in this assay so that only class I–restricted lysis could be observed. Most importantly, the lysis by CTLs from mice immunized with vacss− env was much greater than the lysis mediated by stimulated splenocytes from mice immunized with the vac-env vector. Splenocytes from mice immunized with a control vaccinia vector did not lyse targets presenting the relevant env epitope. When mice were immunized with a higher dose (108 PFU) of recombinant vaccinia vectors, the immune response to both constructs was blunted (data not shown). However, this dose exceeds the standard dose for immunization with vaccinia virus, and the diminished immune response is most likely a result of other secondary effects. Taken together, these results suggest that targeting the env protein for translation on free ribosomes in the cytoplasm, rather than for co-translational translocation into the endoplasmic reticulum, results in more rapid and efficient processing for presentation to class I–restricted T cells and the more efficient stimulation of de novo CTL responses in vivo.

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