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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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Precursor CTL (pCTL) frequency analysis. BALB/c mice  were immunized with 107 PFU vac control, vac-env, or vac-ss− env. After 21 d, splenocytes from immunized mice were cultured (48 wells/titration) in the presence of 105 naive splenocytes that had been pulsed with  the H-2Ld–restricted env peptide for 2 h and then irradiated for 7 d. Culture media contained IL-2, T cell growth factor (from Con A–stimulated  rat splenocytes), and methyl-α-d-mannopyranoside. Each well was then  split into two equal parts and assayed for env-specific CTL activity against  P815 targets pulsed with the H-2Ld–restricted env peptide or with media  for 2 h. Positive wells were scored as >3 standard deviations above the  percent specific lysis without effectors. The fraction of negative wells is  plotted on a log scale versus cell number/well and from this line the  pCTL frequency was determined (see Materials and Methods).
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Figure 3: Precursor CTL (pCTL) frequency analysis. BALB/c mice were immunized with 107 PFU vac control, vac-env, or vac-ss− env. After 21 d, splenocytes from immunized mice were cultured (48 wells/titration) in the presence of 105 naive splenocytes that had been pulsed with the H-2Ld–restricted env peptide for 2 h and then irradiated for 7 d. Culture media contained IL-2, T cell growth factor (from Con A–stimulated rat splenocytes), and methyl-α-d-mannopyranoside. Each well was then split into two equal parts and assayed for env-specific CTL activity against P815 targets pulsed with the H-2Ld–restricted env peptide or with media for 2 h. Positive wells were scored as >3 standard deviations above the percent specific lysis without effectors. The fraction of negative wells is plotted on a log scale versus cell number/well and from this line the pCTL frequency was determined (see Materials and Methods).

Mentions: To determine whether the increased CTL response observed in mice immunized with recombinant vaccinia expressing the cytoplasmic ss− env reflects a quantitative increase in the number of env-specific CTL precursors in those mice, we assayed the env-specific CTL precursor frequency in mice immunized with either vac control, vac-env, or vac-ss− env. As shown in Fig. 3, the precursor frequency of CTLs specific for the P18 env peptide was approximately fourfold greater in mice immunized with vac-ss− env (1/69,618) compared to that observed in mice immunized with vac-env (1/267,980). The frequency of env-specific CTL precursors in mice immunized with vac control was 1/1,154,338. Thus, the enhanced CTL response that is seen in mice immunized with vac-ss− env after in vitro stimulation of splenocytes correlates with a fourfold increase in precursor CTL frequency in these mice relative to mice that were immunized with vac-env.


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)

Precursor CTL (pCTL) frequency analysis. BALB/c mice  were immunized with 107 PFU vac control, vac-env, or vac-ss− env. After 21 d, splenocytes from immunized mice were cultured (48 wells/titration) in the presence of 105 naive splenocytes that had been pulsed with  the H-2Ld–restricted env peptide for 2 h and then irradiated for 7 d. Culture media contained IL-2, T cell growth factor (from Con A–stimulated  rat splenocytes), and methyl-α-d-mannopyranoside. Each well was then  split into two equal parts and assayed for env-specific CTL activity against  P815 targets pulsed with the H-2Ld–restricted env peptide or with media  for 2 h. Positive wells were scored as >3 standard deviations above the  percent specific lysis without effectors. The fraction of negative wells is  plotted on a log scale versus cell number/well and from this line the  pCTL frequency was determined (see Materials and Methods).
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Related In: Results  -  Collection

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Figure 3: Precursor CTL (pCTL) frequency analysis. BALB/c mice were immunized with 107 PFU vac control, vac-env, or vac-ss− env. After 21 d, splenocytes from immunized mice were cultured (48 wells/titration) in the presence of 105 naive splenocytes that had been pulsed with the H-2Ld–restricted env peptide for 2 h and then irradiated for 7 d. Culture media contained IL-2, T cell growth factor (from Con A–stimulated rat splenocytes), and methyl-α-d-mannopyranoside. Each well was then split into two equal parts and assayed for env-specific CTL activity against P815 targets pulsed with the H-2Ld–restricted env peptide or with media for 2 h. Positive wells were scored as >3 standard deviations above the percent specific lysis without effectors. The fraction of negative wells is plotted on a log scale versus cell number/well and from this line the pCTL frequency was determined (see Materials and Methods).
Mentions: To determine whether the increased CTL response observed in mice immunized with recombinant vaccinia expressing the cytoplasmic ss− env reflects a quantitative increase in the number of env-specific CTL precursors in those mice, we assayed the env-specific CTL precursor frequency in mice immunized with either vac control, vac-env, or vac-ss− env. As shown in Fig. 3, the precursor frequency of CTLs specific for the P18 env peptide was approximately fourfold greater in mice immunized with vac-ss− env (1/69,618) compared to that observed in mice immunized with vac-env (1/267,980). The frequency of env-specific CTL precursors in mice immunized with vac control was 1/1,154,338. Thus, the enhanced CTL response that is seen in mice immunized with vac-ss− env after in vitro stimulation of splenocytes correlates with a fourfold increase in precursor CTL frequency in these mice relative to mice that were immunized with vac-env.

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