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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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Pulse-chase analysis and immunoprecipitation of nef. P815 cells  infected with vaccinia vectors expressing β-gal (lane 1), nef (lanes 2–5),  UbMNef (lanes 6–9), and UbRNef (lanes 10–13) were pulse labeled with  35S-Met and 35S-Cys for 30 min and then chased with an excess of unlabeled Met and Cys for 0 min (lanes 1, 2, 6, 10), 15 min (lanes 3, 7, 11),  60 min (lanes 4, 8, 12) or 240 min (lanes 5, 9, 13), followed by extraction, immunoprecipitation, and electrophoretic analysis of nef and MHC  class I expression (see Materials and Methods).
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Figure 5: Pulse-chase analysis and immunoprecipitation of nef. P815 cells infected with vaccinia vectors expressing β-gal (lane 1), nef (lanes 2–5), UbMNef (lanes 6–9), and UbRNef (lanes 10–13) were pulse labeled with 35S-Met and 35S-Cys for 30 min and then chased with an excess of unlabeled Met and Cys for 0 min (lanes 1, 2, 6, 10), 15 min (lanes 3, 7, 11), 60 min (lanes 4, 8, 12) or 240 min (lanes 5, 9, 13), followed by extraction, immunoprecipitation, and electrophoretic analysis of nef and MHC class I expression (see Materials and Methods).

Mentions: The env protein was targeted for enhanced class I processing by intentionally inducing the mislocalization of the protein to the cytoplasm. To enhance the class I processing of proteins that are normally localized to the cytoplasm, we made use of the N-end rule that states that the identity of the NH2-terminal residue of a protein determines its half-life (44). We generated fusion protein constructs in which the Ub coding sequence was fused to the codon for the NH2-terminal residue of the HIV-1 nef protein. Constructs with a stabilizing Met residue (UbMNef) or a destabilizing Arg residue (UbRNef) at the NH2-terminus of the nef coding sequence were compared. Preliminary Western blot analysis established that the steady state level of the nef protein in cells expressing the UbRNef construct was much lower than the steady state level of nef in cells expressing the UbMNef or the wt nef, suggesting that the UbRNef construct is subject to rapid degradation (data not shown). The synthesis and degradation rates of the Ub-Nef fusion proteins as well as wt nef were investigated in pulse chase experiments, as described in the Materials and Methods section. Fig. 5 shows that in cells infected with a vaccinia vector carrying the wt nef gene (vVnef), the nef protein was stable, with a slow rate of degradation (t1/2 ∼10 h). In cells infected with a vaccinia vector carrying the Ub-Met-nef fusion construct (vVUbMNef), nef was again slowly degraded. A band at the expected relative molecular mass of a Ub-nef fusion protein, 35 kD, was not observed, indicating the rapid cleavage at the COOH terminus of Ub by the endogenous Ub hydrolases. The resulting nef band derived from the UbMNef construct had a t1/2 slightly less than 10 h. In cells infected with a vaccinia vector carrying the Ub-Arg-nef fusion construct (vVUbRNef), the fusion construct was again rapidly cleaved to give Ub and the nef protein, in this case with an Arg residue at the NH2 terminus. The rate of synthesis of this protein was similar to that of UbMNef, as judged from the intensity of the 0 min chase time bands. However, the UbRNef construct displayed a rapid rate of degradation (t1/2 = 15 min), suggesting that the exposed Arg residue at the NH2 terminus of nef does indeed confer instability and rapid degradation on this protein in mammalian cells.


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)

Pulse-chase analysis and immunoprecipitation of nef. P815 cells  infected with vaccinia vectors expressing β-gal (lane 1), nef (lanes 2–5),  UbMNef (lanes 6–9), and UbRNef (lanes 10–13) were pulse labeled with  35S-Met and 35S-Cys for 30 min and then chased with an excess of unlabeled Met and Cys for 0 min (lanes 1, 2, 6, 10), 15 min (lanes 3, 7, 11),  60 min (lanes 4, 8, 12) or 240 min (lanes 5, 9, 13), followed by extraction, immunoprecipitation, and electrophoretic analysis of nef and MHC  class I expression (see Materials and Methods).
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Figure 5: Pulse-chase analysis and immunoprecipitation of nef. P815 cells infected with vaccinia vectors expressing β-gal (lane 1), nef (lanes 2–5), UbMNef (lanes 6–9), and UbRNef (lanes 10–13) were pulse labeled with 35S-Met and 35S-Cys for 30 min and then chased with an excess of unlabeled Met and Cys for 0 min (lanes 1, 2, 6, 10), 15 min (lanes 3, 7, 11), 60 min (lanes 4, 8, 12) or 240 min (lanes 5, 9, 13), followed by extraction, immunoprecipitation, and electrophoretic analysis of nef and MHC class I expression (see Materials and Methods).
Mentions: The env protein was targeted for enhanced class I processing by intentionally inducing the mislocalization of the protein to the cytoplasm. To enhance the class I processing of proteins that are normally localized to the cytoplasm, we made use of the N-end rule that states that the identity of the NH2-terminal residue of a protein determines its half-life (44). We generated fusion protein constructs in which the Ub coding sequence was fused to the codon for the NH2-terminal residue of the HIV-1 nef protein. Constructs with a stabilizing Met residue (UbMNef) or a destabilizing Arg residue (UbRNef) at the NH2-terminus of the nef coding sequence were compared. Preliminary Western blot analysis established that the steady state level of the nef protein in cells expressing the UbRNef construct was much lower than the steady state level of nef in cells expressing the UbMNef or the wt nef, suggesting that the UbRNef construct is subject to rapid degradation (data not shown). The synthesis and degradation rates of the Ub-Nef fusion proteins as well as wt nef were investigated in pulse chase experiments, as described in the Materials and Methods section. Fig. 5 shows that in cells infected with a vaccinia vector carrying the wt nef gene (vVnef), the nef protein was stable, with a slow rate of degradation (t1/2 ∼10 h). In cells infected with a vaccinia vector carrying the Ub-Met-nef fusion construct (vVUbMNef), nef was again slowly degraded. A band at the expected relative molecular mass of a Ub-nef fusion protein, 35 kD, was not observed, indicating the rapid cleavage at the COOH terminus of Ub by the endogenous Ub hydrolases. The resulting nef band derived from the UbMNef construct had a t1/2 slightly less than 10 h. In cells infected with a vaccinia vector carrying the Ub-Arg-nef fusion construct (vVUbRNef), the fusion construct was again rapidly cleaved to give Ub and the nef protein, in this case with an Arg residue at the NH2 terminus. The rate of synthesis of this protein was similar to that of UbMNef, as judged from the intensity of the 0 min chase time bands. However, the UbRNef construct displayed a rapid rate of degradation (t1/2 = 15 min), suggesting that the exposed Arg residue at the NH2 terminus of nef does indeed confer instability and rapid degradation on this protein in mammalian cells.

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