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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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Stimulation of secondary nef-specific CTLs by UbMNef and UbRNef constructs. PBMCs were isolated from an HIV-1 seropositive donor  and stimulated in the presence of IL-2 with titrating numbers of vVUbMNef (squares) or vVUbRNef (diamonds) infected, psoralen/UV–treated autologous B-LCL. Stimulated PBMCs were assayed for nef-specific CTL activity against vVnef infected (filled) or vac control infected (open) autologous B-LCL  at an E/T ratio of 10.
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Figure 6: Stimulation of secondary nef-specific CTLs by UbMNef and UbRNef constructs. PBMCs were isolated from an HIV-1 seropositive donor and stimulated in the presence of IL-2 with titrating numbers of vVUbMNef (squares) or vVUbRNef (diamonds) infected, psoralen/UV–treated autologous B-LCL. Stimulated PBMCs were assayed for nef-specific CTL activity against vVnef infected (filled) or vac control infected (open) autologous B-LCL at an E/T ratio of 10.

Mentions: Preliminary studies demonstrated that B-LCL infected with vVUbRNef, vVUbMNef, and vVnef were all lysed to comparable extents by the previously described nef-specific human CTL clone 4N225 (50). For other nef-specific human CTL clones, different patterns were observed, with the degradation targeted construct giving preferential lysis in some cases (data not shown). However, the critical test of this strategy is to determine whether cells expressing the degradation targeted construct are better able to stimulate primary and secondary CTL responses. To determine whether the rapid degradation of the UbRNef construct permits enhanced stimulation of secondary human CTL responses, autologous B-LCL infected with recombinant vaccinia vectors expressing either UbMNef or UbRNef were used to stimulate nef-specific memory CTLs present in PBMCs obtained from an HIV-1 seropositive donor. Stimulation of PBMCs with large numbers of either vVUbMNef-infected B-LCL or vVUbRNef-infected B-LCL led to the induction of a nef-specific secondary CTL response, as seen in Fig. 6. However, the number of vVUbMNef-infected stimulating cells required to obtain a positive CTL response was >30-fold higher than the number of vVUbRNef-infected stimulating cells required to induce a comparable response. Thus, on a per cell basis, cells expressing the rapidly degraded UbRNef construct are >30-fold more effective at stimulating a secondary CTL response.


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)

Stimulation of secondary nef-specific CTLs by UbMNef and UbRNef constructs. PBMCs were isolated from an HIV-1 seropositive donor  and stimulated in the presence of IL-2 with titrating numbers of vVUbMNef (squares) or vVUbRNef (diamonds) infected, psoralen/UV–treated autologous B-LCL. Stimulated PBMCs were assayed for nef-specific CTL activity against vVnef infected (filled) or vac control infected (open) autologous B-LCL  at an E/T ratio of 10.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196169&req=5

Figure 6: Stimulation of secondary nef-specific CTLs by UbMNef and UbRNef constructs. PBMCs were isolated from an HIV-1 seropositive donor and stimulated in the presence of IL-2 with titrating numbers of vVUbMNef (squares) or vVUbRNef (diamonds) infected, psoralen/UV–treated autologous B-LCL. Stimulated PBMCs were assayed for nef-specific CTL activity against vVnef infected (filled) or vac control infected (open) autologous B-LCL at an E/T ratio of 10.
Mentions: Preliminary studies demonstrated that B-LCL infected with vVUbRNef, vVUbMNef, and vVnef were all lysed to comparable extents by the previously described nef-specific human CTL clone 4N225 (50). For other nef-specific human CTL clones, different patterns were observed, with the degradation targeted construct giving preferential lysis in some cases (data not shown). However, the critical test of this strategy is to determine whether cells expressing the degradation targeted construct are better able to stimulate primary and secondary CTL responses. To determine whether the rapid degradation of the UbRNef construct permits enhanced stimulation of secondary human CTL responses, autologous B-LCL infected with recombinant vaccinia vectors expressing either UbMNef or UbRNef were used to stimulate nef-specific memory CTLs present in PBMCs obtained from an HIV-1 seropositive donor. Stimulation of PBMCs with large numbers of either vVUbMNef-infected B-LCL or vVUbRNef-infected B-LCL led to the induction of a nef-specific secondary CTL response, as seen in Fig. 6. However, the number of vVUbMNef-infected stimulating cells required to obtain a positive CTL response was >30-fold higher than the number of vVUbRNef-infected stimulating cells required to induce a comparable response. Thus, on a per cell basis, cells expressing the rapidly degraded UbRNef construct are >30-fold more effective at stimulating a secondary CTL response.

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