Limits...
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.

Show MeSH

Related in: MedlinePlus

Enhanced induction of primary nef-specific CTLs by immunization with vVUbRNef. BALB/c mice were immunized with 107 PFU  of vac control (open), vVUbMNef (stippled), or vVUbRNef (filled). Splenocytes from immunized mice were stimulated for 5 d in the presence of  IL-2 with either CT26nef transfectants treated with mitomycin C or with  CT26 cells which had been infected with vVnef and then treated with  psoralen/UV light to inactivate vaccinia virus. The ratio of responders to  stimulators was 50:1. Stimulated splenocytes were then assayed for nefspecific CTL activity against vVnef-infected CT26 cells or vac control– infected CT26 cells. The percentage nef-specific lysis reported is the percentage specific lysis of vVnef-infected targets − the percentage specific  lysis of vac control–infected targets. The data shown are from a single experiment which is representative of three experiments conducted with  similar results.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196169&req=5

Figure 7: Enhanced induction of primary nef-specific CTLs by immunization with vVUbRNef. BALB/c mice were immunized with 107 PFU of vac control (open), vVUbMNef (stippled), or vVUbRNef (filled). Splenocytes from immunized mice were stimulated for 5 d in the presence of IL-2 with either CT26nef transfectants treated with mitomycin C or with CT26 cells which had been infected with vVnef and then treated with psoralen/UV light to inactivate vaccinia virus. The ratio of responders to stimulators was 50:1. Stimulated splenocytes were then assayed for nefspecific CTL activity against vVnef-infected CT26 cells or vac control– infected CT26 cells. The percentage nef-specific lysis reported is the percentage specific lysis of vVnef-infected targets − the percentage specific lysis of vac control–infected targets. The data shown are from a single experiment which is representative of three experiments conducted with similar results.

Mentions: To determine whether the targeting of nef for Ub-dependent degradation could enhance the generation of de novo CTL responses in vivo, BALB/c mice were immunized with vVUbMNef and vVUbRNef as described in Materials and Methods. As shown in Fig. 7, a vigorous nef-specific CTL response was seen after stimulation of splenocytes from mice immunized with vVUbRNef by vVnef-infected CT26 cells. This response was detected using the MHC class I+, MHC class II− cell line, CT26, indicating that the lysis was class I restricted. In contrast, stimulated splenocytes from mice immunized with vVUbMNef exhibited a detectable, but much reduced nefspecific CTL response. Despite this difference in nef-specific CTL activity, splenocytes that were stimulated with vVnef-infected CT26 cells had statistically indistinguishable levels of vaccinia-specific CTL activity, as was seen when vac control–infected CT26 cells were used as targets in the CTL assay (29, 27, and 21% vaccinia-specific lysis by splenocytes from mice immunized with vac control, vVUbMNef, and vVUbRNef, respectively). When splenocytes were stimulated with CT26 cells transfected to express nef, nef-specific CTL activity was detectable in splenocytes from mice immunized with vVUbRNef and was virtually lost in splenocytes from mice immunized with vVUbMNef. This lower level of stimulation is presumably due to the greater than fivefold lower level of expression of nef in the stably transfected CT26 cells than in the vVnef infected CT26 cells as seen by Western blot analysis (data not shown). As expected, stimulated splenocytes from mice immunized with a control vaccinia vector failed to recognize and lyse target cells expressing nef.


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 primary nef-specific CTLs by immunization with vVUbRNef. BALB/c mice were immunized with 107 PFU  of vac control (open), vVUbMNef (stippled), or vVUbRNef (filled). Splenocytes from immunized mice were stimulated for 5 d in the presence of  IL-2 with either CT26nef transfectants treated with mitomycin C or with  CT26 cells which had been infected with vVnef and then treated with  psoralen/UV light to inactivate vaccinia virus. The ratio of responders to  stimulators was 50:1. Stimulated splenocytes were then assayed for nefspecific CTL activity against vVnef-infected CT26 cells or vac control– infected CT26 cells. The percentage nef-specific lysis reported is the percentage specific lysis of vVnef-infected targets − the percentage specific  lysis of vac control–infected targets. The data shown are from a single experiment which is representative of three experiments conducted with  similar results.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Enhanced induction of primary nef-specific CTLs by immunization with vVUbRNef. BALB/c mice were immunized with 107 PFU of vac control (open), vVUbMNef (stippled), or vVUbRNef (filled). Splenocytes from immunized mice were stimulated for 5 d in the presence of IL-2 with either CT26nef transfectants treated with mitomycin C or with CT26 cells which had been infected with vVnef and then treated with psoralen/UV light to inactivate vaccinia virus. The ratio of responders to stimulators was 50:1. Stimulated splenocytes were then assayed for nefspecific CTL activity against vVnef-infected CT26 cells or vac control– infected CT26 cells. The percentage nef-specific lysis reported is the percentage specific lysis of vVnef-infected targets − the percentage specific lysis of vac control–infected targets. The data shown are from a single experiment which is representative of three experiments conducted with similar results.
Mentions: To determine whether the targeting of nef for Ub-dependent degradation could enhance the generation of de novo CTL responses in vivo, BALB/c mice were immunized with vVUbMNef and vVUbRNef as described in Materials and Methods. As shown in Fig. 7, a vigorous nef-specific CTL response was seen after stimulation of splenocytes from mice immunized with vVUbRNef by vVnef-infected CT26 cells. This response was detected using the MHC class I+, MHC class II− cell line, CT26, indicating that the lysis was class I restricted. In contrast, stimulated splenocytes from mice immunized with vVUbMNef exhibited a detectable, but much reduced nefspecific CTL response. Despite this difference in nef-specific CTL activity, splenocytes that were stimulated with vVnef-infected CT26 cells had statistically indistinguishable levels of vaccinia-specific CTL activity, as was seen when vac control–infected CT26 cells were used as targets in the CTL assay (29, 27, and 21% vaccinia-specific lysis by splenocytes from mice immunized with vac control, vVUbMNef, and vVUbRNef, respectively). When splenocytes were stimulated with CT26 cells transfected to express nef, nef-specific CTL activity was detectable in splenocytes from mice immunized with vVUbRNef and was virtually lost in splenocytes from mice immunized with vVUbMNef. This lower level of stimulation is presumably due to the greater than fivefold lower level of expression of nef in the stably transfected CT26 cells than in the vVnef infected CT26 cells as seen by Western blot analysis (data not shown). As expected, stimulated splenocytes from mice immunized with a control vaccinia vector failed to recognize and lyse target cells expressing nef.

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