DNA vaccine encoding human immunodeficiency virus-1 Gag, targeted to the major histocompatibility complex II compartment by lysosomal-associated membrane protein, elicits enhanced long-term memory response

Abstract

Antigen presentation by major histocompatibility complex type II (MHC II) molecules and activation of CD4+ helper T cells are critical for the generation of immunological memory. We previously described a DNA vaccine encoding human immunodeficiency virus-1 p55Gag as a chimera with the lysosome-associated membrane protein (LAMP/gag). The LAMP/gag chimera protein traffics to the MHC II compartment of transfected cells and elicits enhanced immune responses as compared to a DNA vaccine encoding native gag not targeted to the MHC II compartment. We have now investigated the long-term responses of immunized mice and show that the LAMP/gag DNA vaccine promotes long-lasting B cell- and CD4+ and CD8+ T-cell memory responses induced by DNA encoding non-targeted Gag decay rapidly and elicit very low or undetectable levels of gag DNA is sufficient to generate T-cell memory. Following this initial priming immunization with LAMP/gag DNA, booster immunizations with native gag DNA or the LAMP/gag chimera are equally efficient in eliciting B- and T-cell secondary responses, results in accordance with observations that secondary expansion of CD8+ cells in the boost phase does not require additional CD4+ help. These findings underscore the significance of targeting DNA-encoded vaccine antigens to the MHC II processing compartments for induction of long-term immunological memory.

Figure 1

Schematic representation of the plasmids used in this study. The plasmid vectors contain the adeno-associated virus inverted terminal repeats (ITRs) flanking the expression elements. The grey box indicates the open reading frame (ORF) of the lysosome-associated membrane protein (LAMP) luminal domain and the striped box indicates the transmembrane (TM) and cytoplamic (cy) ORFs of LAMP. The black box indicates the gag ORF.

Figure 2

Enhanced and sustained CD4⁺ T-cell response following immunization with lysosome-associated membrane protein (LAMP)/gag DNA. Mice were immunized twice with 50 µg of plasmid DNA encoding LAMP (control), native gag, or the LAMP/gag chimera, as indicated. Ten (a) or 120 (b) days after immunization, splenocytes were prepared and cultured overnight in the presence or absence of p55Gag protein, and the frequency of interferon-γ (IFN-γ)-producing cells was analysed by ELISPOT assay. The data represent the measurements obtained from each mouse group, shown as average and standard deviation.

Figure 3

Enhanced and sustained CD8⁺ T-cell response following immunization with lysosome-associated membrane protein (LAMP)/gag DNA. Mice were immunized as described in the legend to Fig. 2. Ten (a) or 120 (b) days after immunization, splenocytes obtained from these mice were cultured overnight in the presence or absence of the Gag major histocompatibility complex type I (MHC I) epitope, AMQMLKETI, and the frequency of interferon-γ (IFN-γ)-producing cells was analysed by ELISPOT assay. The data represent the measurements obtained from each mouse group, shown as average and standard deviation. SFC, spot-forming cells.

Figure 4

Lysosome-associated membrane protein (LAMP)/gag DNA immunization promotes a potent CD8-recall response to vaccinia-gag challenge. Mice were immunized as described in the legend to Fig. 2. Eleven months later they were challenged with VDK-1 vaccinia/gag. After 5 days, the cells were stimulated in vivo by an intravenous (i.v.) injection of the major histocompatibility complex type I (MHC I)-restricted Gag epitope, AMQMLKETI₆₅₋₇₃. Splenocytes were harvested 2 hr later and (a) stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD8 immunoglobulin and phycoerythrin (PE)-conjugated H-2K^d/AMQMLKETI tetramer, or (b) cultured for 2 hr with Golgi-stop containing monensin, and then stained with FITC-conjugated anti-CD8 immunoglobulin, followed by intracellular staining with PE-conjugated anti-IFN-γ, as described in the Materials and methods. The results indicate the percentage of tetramer- or IFN-γ-positive cells among CD8⁺ cells. The data represent the measurements obtained from each mouse group, shown as average and standard deviation.

Figure 5

A single dose of lysosome-associated membrane protein (LAMP)/gag DNA is sufficient to prime both B- and T-cell responses to a subsequent boost with DNA encoding native Gag. Mice were injected with one dose of DNA plasmids (50 µg) encoding lysosome-associated membrane protein (LAMP) control, native gag, or LAMP/gag chimera (L/g), as indicated. On day 30, some groups of mice received a booster DNA injection of the indicated plasmids. On day 40 after the first immunization, the mice were killed and (a) splenocytes were cultured with medium alone or p55Gag protein for 3 days and supernatant interferon-γ (IFN-γ) levels were analysed by enzyme-linked immunosorbent assay (ELISA); and (b) the amount of anti-Gag serum immunoglobulin G (IgG) of blood samples was analysed by ELISA at a serum dilution of 1 : 300. The data represent the measurements obtained from each mouse group, shown as average and standard deviation.

Figure 6

A single dose of lysosome-associated membrane protein (LAMP)/gag DNA induces the generation of memory T cells. Mice were injected with one dose of DNA plasmids (50 µg) encoding LAMP (control) or LAMP/gag chimera. (a) CD4⁺ response: the mice were killed on day 40 or day 120, as indicated, splenocytes were harvested and cultured with p55Gag for 3 days, and the supernatant interferon-γ (IFN-γ) concentration was analysed by enzyme-linked immunosorbent assay (ELISA). (b) and (c) The CD8⁺ response on day 120 after immunization was analysed as described in Fig. 4 for tetramer binding (b) or intracellular IFN-γ (c). The results indicate the percentage of tetramer- or IFN-γ-positive cells among CD8⁺ cells. The data represent the measurements obtained from each mouse group, shown as average and standard deviation.