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. 2001 Nov;75(21):10421-30.
doi: 10.1128/JVI.75.21.10421-10430.2001.

CD4(+) T Cells Induced by a DNA Vaccine: Immunological Consequences of Epitope-Specific Lysosomal Targeting

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Free PMC article

CD4(+) T Cells Induced by a DNA Vaccine: Immunological Consequences of Epitope-Specific Lysosomal Targeting

F Rodriguez et al. J Virol. .
Free PMC article

Abstract

Our previous studies have shown that targeting DNA vaccine-encoded major histocompatibility complex class I epitopes to the proteasome enhanced CD8(+) T-cell induction and protection against lymphocytic choriomeningitis virus (LCMV) challenge. Here, we expand these studies to evaluate CD4(+) T-cell responses induced by DNA immunization and describe a system for targeting proteins and minigenes to lysosomes. Full-length proteins can be targeted to the lysosomal compartment by covalent attachment to the 20-amino-acid C-terminal tail of lysosomal integral membrane protein-II (LIMP-II). Using minigenes encoding defined T-helper epitopes from lymphocytic choriomeningitis virus, we show that the CD4(+) T-cell response induced by the NP(309-328) epitope of LCMV was greatly enhanced by addition of the LIMP-II tail. However, the immunological consequence of lysosomal targeting is not invariably positive; the CD4(+) T-cell response induced by the GP(61-80) epitope was almost abolished when attached to the LIMP-II tail. We identify the mechanism which underlies this marked difference in outcome. The GP(61-80) epitope is highly susceptible to cleavage by cathepsin D, an aspartic endopeptidase found almost exclusively in lysosomes. We show, using mass spectrometry, that the GP(61-80) peptide is cleaved between residues F(74) and K(75) and that this destroys its ability to stimulate virus-specific CD4(+) T cells. Thus, the immunological result of lysosomal targeting varies, depending upon the primary sequence of the encoded antigen. We analyze the effects of CD4(+) T-cell priming on the virus-specific antibody and CD8(+) T-cell responses which are mounted after virus infection and show that neither response appears to be accelerated or enhanced. Finally, we evaluate the protective benefits of CD4(+) T-cell vaccination in the LCMV model system; in contrast to DNA vaccine-induced CD8(+) T cells, which can confer solid protection against LCMV challenge, DNA vaccine-mediated priming of CD4(+) T cells does not appear to enhance the vaccinee's ability to combat viral challenge.

Figures

FIG. 1
FIG. 1
Sequences of the LIMP-II plasmids used in this study. The nucleic acid sequence and the related amino acids adjacent to the BglII cloning site are shown above a cartoon of the parental plasmid, pCMV-LIMP-II (pCMV-LII). CMV IE, human cytomegalovirus immediate-early promoter; SVSD/SA and SVpolyA, SV40 splice donor-acceptor and transcription terminator-polyadenylation signal, respectively; LII tail, 20 amino acids from the C terminus of LIMP-II. The stop codon which follows the LII tail is shown as a grey box. Partial or complete amino acid sequences are shown for each of the four LIMP-II constructs used in this study. For HBV core and LCMV NP, the superscripted numerals indicate the position in the full-length protein of the adjacent amino acid. In all cases, the native viral amino acids are shown in regular uppercase type, the 20-residue LIMP-II tail is shown in boldface italic uppercase type, and any additional amino acids inserted as a result of the cloning procedure are shown in regular lowercase type.
FIG. 2
FIG. 2
The LIMP-II tail targets proteins to the lysosomal compartment. Cells were transfected with plasmids encoding the HBV core protein (top row) or the LCMV NP (bottom row), with or without the LIMP-II tail (left and right columns, respectively). After 48 h, cells were stained with FITC-labeled antibodies specific for the HBV core or for the LCMV NP (green signal) and were costained with rhodamine-labeled antibodies specific for the lysosomal protein LAMP-I (red signal). Fluorescence was evaluated using a confocal microscope. A bar representing 10 μm is shown in all panels. The right-hand panels include enlargements of regions showing a dual signal (yellow), which indicates colocalization of the viral antigen and LAMP-I.
FIG. 3
FIG. 3
LIMP-II enhances induction of NP309–328-specific CD4+ T cells. C57BL/6 mice were immunized with pCMV-LII, pCMV-NPTh, or pCMV-NPTh-LII (four mice per group) and 3 weeks later were infected with LCMV. Six days later, spleens were harvested, cells were stimulated with peptide NP309–328, and an ICCS assay was carried out. (A) Representative data, gated on CD4+ T cells, from individual mice are shown. (B) The percentage of CD4+ T cells producing IFN-γ is shown for each group (mean + SEM [error bars]).
FIG. 4
FIG. 4
LIMP-II inhibits induction of GP61–80-specific CD4+ T cells. C57BL/6 mice were immunized with pCMV-LII, pCMV-GPTh, or pCMV-GPTh-LII (four mice per group) and 3 weeks later were infected with LCMV. Six days later, spleens were harvested, cells were stimulated with peptide GP61–80, and an ICCS assay was carried out. (A) Representative data, gated on CD4+ T cells, from individual mice are shown. (B) The percentage of CD4+ T cells producing IFN-γ is shown for each group (mean + SEM [error bars]).
FIG. 5
FIG. 5
Incubation with cathepsin D diminishes the stimulatory activity of GP61–80 but not of NP309–328. Aliquots of the NP309–328 and GP61–80 peptides were pretreated by incubation in acidic medium, either without (−) or with (+) cathepsin D (Cat D) as indicated. After incubation, the stimulatory activities of the resulting materials were determined by incubating them with splenocytes from C57BL/6 mice taken 8 days after LCMV infection. For details, see Materials and Methods.
FIG. 6
FIG. 6
Cathepsin D specifically cleaves GP61–80. Peptide GP61–80 was incubated without or with cathepsin D as described in Materials and Methods, and the resulting products were analyzed by mass spectrometry. The data in the absence of enzyme cleavage appear in the top panel. The MW of the observed peak and the sequence GP61–80 are shown. As shown in the bottom panel, cathepsin D cleavage generated a major peak with an observed MW of 1,585.1 Da; the probable sequence of the peptide forming this peak is shown.
FIG. 7
FIG. 7
Immunization with pCMV-NPTh-LII has no apparent effect on subsequent induction of antibodies or CD8+ T cells. Mice were immunized with one of the three indicated plasmids and, 3 weeks later, were infected with LCMV. At various time points postinfection, mice were sacrificed (three mice per time point, for each vaccine group), and their LCMV-specific antibody titers (A) and CD8+ T-cell responses (B) were determined. (A) Antibody titers (total IgG) at 8 days after infection were determined by ELISA. (B) CD8+ T-cell responses were evaluated by ICCS after stimulating with peptide NP396; the data for days 4 and 6 postinfection are shown for each mouse.
FIG. 8
FIG. 8
Priming of CD4+ T cells alone does not confer protection against LCMV challenge. Groups of mice were immunized with one inoculation of the indicated plasmid DNA vaccine or, as a positive control, with LCMV (2 × 105 PFU, i.p.). Six weeks later, mice were challenged via the i.c. (A) or i.p. (B) routes. (A) Mice (eight per vaccine group) received 20 50% lethal doses of LCMV i.c. and were observed daily for 21 days. For each group, the percentage of mice which survived infection is shown. All deaths occurred between days 7 and 10 postchallenge. (B) Mice (four per vaccine group) were challenged with LCMV (2 × 105 PFU, i.p.), and 4 days postchallenge, spleens were harvested and virus titers were determined by plaque assay. For each vaccine group, the mean + SEM (in PFU per gram of spleen) (error bars) is shown. The vertical lines indicate percentage reductions in titer compared to titers in the negative control (pCMV) animals (dotted line, 99% reduction; dashed line, 99.9% reduction).

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