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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 nef-specific CTLs by immunization with DNA expression vectors encoding UbRNef.  BALB/c mice were immunized  once with 4 × 50 μg pcDNA3  (open), pcDNA3nef (cross  hatched), pcDNA3UbMNef (stippled), or pcDNA3UbRNef  (filled). Splenocytes from immunized mice were stimulated with  psoralen/UV–treated, vVnefinfected CT26 cells at a ratio of  50 responders/stimulator cell for  5 d in the presence of IL-2.  Stimulated splenocytes were assayed for nef-specific CTL activity against CT26 cells infected  with vVnef or with vac control or  against P815 cells infected with  vVnef or vac control at an E/T  ratio of 10:1 or 100:1. The data  shown are from one experiment.  The experiment was repeated a  total of four times, with similar  results.
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Figure 8: Enhanced induction of nef-specific CTLs by immunization with DNA expression vectors encoding UbRNef. BALB/c mice were immunized once with 4 × 50 μg pcDNA3 (open), pcDNA3nef (cross hatched), pcDNA3UbMNef (stippled), or pcDNA3UbRNef (filled). Splenocytes from immunized mice were stimulated with psoralen/UV–treated, vVnefinfected CT26 cells at a ratio of 50 responders/stimulator cell for 5 d in the presence of IL-2. Stimulated splenocytes were assayed for nef-specific CTL activity against CT26 cells infected with vVnef or with vac control or against P815 cells infected with vVnef or vac control at an E/T ratio of 10:1 or 100:1. The data shown are from one experiment. The experiment was repeated a total of four times, with similar results.

Mentions: To confirm the results obtained with vaccinia vectors, we used DNA immunization to express the UbMNef and UbRNef constructs in mice as described in Materials and Methods, and evaluated the responses induced by this form of immunization. As shown in Fig. 8, after antigen-specific stimulation, splenocytes from mice immunized a single time with an expression plasmid encoding UbRNef were able to recognize and lyse nef-expressing CT26 target cells at low E/T ratios. Stimulated splenocytes from mice immunized with plasmid encoding UbMNef, wt nef, or with plasmid alone were unable to recognize and lyse CT26 target cells expressing nef even at E/T ratios as high as 100:1. Nef-expressing P815 target cells were also recognized and lysed by stimulated splenocytes from UbRNef immunized mice at low E/T ratios. Stimulated splenocytes from mice immunized with a plasmid expressing UbMNef or wt nef were also able to recognize and lyse these targets, but only at high E/T ratios and at reduced levels relative to the cells from UbRNef immunized mice. Both target cells used in these experiments are MHC class II−, indicating that the lysis mediated by CTLs from mice immunized with pcDNA3UbRNef was MHC class I restricted. In addition, lysis of nef-expressing targets by antigen simulated splenocytes from immunized mice was completely blocked by antiCD8 antibodies (data not shown). Taken together, these results indicate that when host cells are induced to express the rapidly degraded UbRNef, either by immunization with recombinant vaccinia virus vectors or with a DNA expression plasmid, BALB/c mice mount a strong primary CTL response against nef. However, when immunized with a stable form of nef, either UbMNef or wt nef, the CTL response against nef that they mount is greatly reduced or undetectable.


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 nef-specific CTLs by immunization with DNA expression vectors encoding UbRNef.  BALB/c mice were immunized  once with 4 × 50 μg pcDNA3  (open), pcDNA3nef (cross  hatched), pcDNA3UbMNef (stippled), or pcDNA3UbRNef  (filled). Splenocytes from immunized mice were stimulated with  psoralen/UV–treated, vVnefinfected CT26 cells at a ratio of  50 responders/stimulator cell for  5 d in the presence of IL-2.  Stimulated splenocytes were assayed for nef-specific CTL activity against CT26 cells infected  with vVnef or with vac control or  against P815 cells infected with  vVnef or vac control at an E/T  ratio of 10:1 or 100:1. The data  shown are from one experiment.  The experiment was repeated a  total of four times, with similar  results.
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Related In: Results  -  Collection

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Figure 8: Enhanced induction of nef-specific CTLs by immunization with DNA expression vectors encoding UbRNef. BALB/c mice were immunized once with 4 × 50 μg pcDNA3 (open), pcDNA3nef (cross hatched), pcDNA3UbMNef (stippled), or pcDNA3UbRNef (filled). Splenocytes from immunized mice were stimulated with psoralen/UV–treated, vVnefinfected CT26 cells at a ratio of 50 responders/stimulator cell for 5 d in the presence of IL-2. Stimulated splenocytes were assayed for nef-specific CTL activity against CT26 cells infected with vVnef or with vac control or against P815 cells infected with vVnef or vac control at an E/T ratio of 10:1 or 100:1. The data shown are from one experiment. The experiment was repeated a total of four times, with similar results.
Mentions: To confirm the results obtained with vaccinia vectors, we used DNA immunization to express the UbMNef and UbRNef constructs in mice as described in Materials and Methods, and evaluated the responses induced by this form of immunization. As shown in Fig. 8, after antigen-specific stimulation, splenocytes from mice immunized a single time with an expression plasmid encoding UbRNef were able to recognize and lyse nef-expressing CT26 target cells at low E/T ratios. Stimulated splenocytes from mice immunized with plasmid encoding UbMNef, wt nef, or with plasmid alone were unable to recognize and lyse CT26 target cells expressing nef even at E/T ratios as high as 100:1. Nef-expressing P815 target cells were also recognized and lysed by stimulated splenocytes from UbRNef immunized mice at low E/T ratios. Stimulated splenocytes from mice immunized with a plasmid expressing UbMNef or wt nef were also able to recognize and lyse these targets, but only at high E/T ratios and at reduced levels relative to the cells from UbRNef immunized mice. Both target cells used in these experiments are MHC class II−, indicating that the lysis mediated by CTLs from mice immunized with pcDNA3UbRNef was MHC class I restricted. In addition, lysis of nef-expressing targets by antigen simulated splenocytes from immunized mice was completely blocked by antiCD8 antibodies (data not shown). Taken together, these results indicate that when host cells are induced to express the rapidly degraded UbRNef, either by immunization with recombinant vaccinia virus vectors or with a DNA expression plasmid, BALB/c mice mount a strong primary CTL response against nef. However, when immunized with a stable form of nef, either UbMNef or wt nef, the CTL response against nef that they mount is greatly reduced or undetectable.

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