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. 1999 Mar;73(3):2280-7.
doi: 10.1128/JVI.73.3.2280-2287.1999.

Dissecting the immune response to moloney murine sarcoma/leukemia virus-induced tumors by means of a DNA vaccination approach

G Milan  1 , A Zambon, M Cavinato, P Zanovello, A Rosato, D Collavo
Affiliations

Dissecting the immune response to moloney murine sarcoma/leukemia virus-induced tumors by means of a DNA vaccination approach

G Milan et al. J Virol. 1999 Mar.

Abstract

The intramuscular inoculation of Moloney murine sarcoma/leukemia (M-MSV/M-MuLV) retroviral complex gives rise to sarcomas that undergo spontaneous regression due to the induction of a strong immune reaction mediated primarily by cytotoxic T lymphocytes (CTL). We used a DNA-based vaccination approach to dissect the CTL response against the Gag and Env proteins of M-MSV/M-MuLV in C57BL/6 (B6) mice and to evaluate whether plasmid DNA-immunized mice would be protected against a subsequent challenge with syngeneic tumor cells expressing the viral antigens. Intramuscular DNA vaccination induced CTL against both Gag and Env proteins. A detailed analysis of epitopes recognized by CTL generated in mice inoculated with the whole virus and with the Gag-expressing plasmid confirmed the presence of an immunodominant peptide in the leader sequence of Gag protein (Gag85-93, CCLCLTVFL) that is identical to that described in B6 mice immunized with Friend MuLV-induced leukemia cells. Moreover, CTL generated by immunization with the Env-encoding plasmid recognized a subdominant Env peptide (Env189-196, SSWDFITV), originally described in the B6.CH-2(bm13) mutant strain. B6 mice immunized with the Gag-expressing plasmid were fully protected against a lethal tumor challenge with M-MuLV-transformed MBL-2 leukemia cells, while vaccination with the Env-expressing plasmid resulted in rejection of the tumor in 44% of the mice and in increased survival of an additional 17% of the animals. Taken together, these results indicate the existence of a hierarchy in the capacity of different structural viral proteins to induce a protective immune response against retrovirus-induced tumors.

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Figures

FIG. 1
FIG. 1
Frequency of CTLp against the Gag85–93 CCLCLTVFL peptide in tumor regressor B6 mice. Minimal frequencies of CTLp specific for MBL-2 cells (●), Gag85–93 peptide-pulsed EL-4− cells (▾), and FMR-MuLV-negative EL-4− cells (▴) were estimated by linear-regression analysis. Limiting numbers of responder spleen cells from virus-induced tumor regressor B6 mice were cultivated as reported in Materials and Methods and divided into three portions for testing in 51Cr release assays.
FIG. 2
FIG. 2
CTL generation in B6 mice immunized with pcDNA3-gag. Plots 1 to 6 show the cytotoxicity of MLTC from splenocytes of six pcDNA3-gag-injected mice after restimulation in vitro with MBL-2 tumor cells. Lytic activity was evaluated with 51Cr-labeled FMR-MuLV-positive MBL-2 cells (●), EL-4+ cells (■), and FMR-MuLV-negative EL-4− cells (▴) as targets. The cytotoxicities of MLTC generated from four tumor regressor mice (plot M-MSV) and from six pcDNA3-injected mice (plot Mock) are shown as mean and SD in the bottom right-hand plots. The lytic activity of CTL from immunized mice was considered positive when it exceeded the mean lysis values of mock-injected animals + 3SD. E/T, effector/target.
FIG. 3
FIG. 3
CTL activity of splenocytes from pcDNA3-gag-injected mice following in vitro restimulation with the Gag85–93 CCLCLTVFL peptide. Plots 1 to 6 show the lytic activity of MLPC from splenocytes of six pcDNA3-gag-injected mice against 51Cr-labelled EL-4+ cells (■), EL-4− cells (▴), and Gag85–93 peptide-pulsed EL-4− cells (▾). The cytotoxicities of MLPC generated from four tumor regressor mice (plot M-MSV) and from six pcDNA3-injected mice (plot Mock) are shown as mean and SD in the bottom right-hand plots. E/T, effector/target.
FIG. 4
FIG. 4
CTL generation in B6 mice immunized with pCMV-ecoenv-bpA. Plots 1 to 7 report the lytic activity of MLTC from splenocytes of seven pCMV-ecoenv-bpA-injected mice, after restimulation in vitro with MBL-2 tumor cells, against 51Cr-labeled FMR-MuLV-positive MBL-2 cells (●), EL-4+ cells (■), and FMR-MuLV-negative EL-4− cells (▴). The cytotoxicities of similar MLTC generated from five tumor regressor mice (plot M-MSV) and from eight pCMV-ecoΔ-injected mice (plot Mock) are depicted as mean and SD in the bottom right-hand plots. E/T, effector/target.
FIG. 5
FIG. 5
Peptide specificity and MHC restriction of CTL induced by immunization of B6 mice with M-MSV/M-MuLV, pcDNA3-gag, or pCMV-ecoenv-bpA. Splenocytes from five tumor regressor mice and five mice vaccinated with either pcDNA3-gag or pCMV-ecoenv-bpA were restimulated in vitro with MBL-2 tumor cells (MLTC) or with Gag85–93 or Env189–196 peptide (MPLC). The lytic activity of the cultures, reported as the mean and SD, was assayed against FMR-MuLV-positive MBL-2 cells (●) Gag85–93 peptide-pulsed 293Db cells (■), and Env189–196 peptide-pulsed 293Kb cells (▴). No lysis was observed against 293Db and 293Kb cells without peptide, 293Db cells plus Env189–196 peptide, or 293Kb cells plus Gag85–93 peptide. E/T, effector/target.
FIG. 6
FIG. 6
Cytotoxic activity of CD8+ CTL clones derived from tumor regressor mice and from mice immunized with pCMV-ecoenv-bpA. The specificity of lysis of two representative CTL clones derived from tumor regressor mice (76GM and 13/1) and two CTL clones derived from pCMV-ecoenv-bpA-injected mice (123 and 167) was evaluated against the indicated target cells at an effector-to-target-cell ratio of 10.
FIG. 7
FIG. 7
Protection of B6 mice vaccinated with pcDNA3-gag from challenge with MBL-2 tumor cells. Shown is a representative survival experiment in which two groups of 10 B6 mice were injected three times with either 100 μg of pcDNA3-gag (▴) or mock plasmid pcDNA3 (■) and then challenged with 2 × 105 MBL-2 leukemia cells s.c. 5 weeks after the last plasmid immunization. Ten nonimmunized mice (●) served as controls.
FIG. 8
FIG. 8
Survival of pCMV-ecoenv-bpA-immunized B6 mice after challenge with MBL-2 tumor cells. B6 mice inoculated three times with 100 μg of pCMV-ecoenv-bpA (n = 18) (▴) or with mock plasmid pCMV-ecoΔ (n = 18) (■) were challenged with 2 × 105 MBL-2 leukemia cells s.c. 5 weeks after the last immunization. Nonimmunized mice (n = 31) (●) were used as controls.
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