Vaccination with a fusion protein that introduces HIV-1 gag antigen into a multitrimer CD40L construct results in enhanced CD8+ T cell responses and protection from viral challenge by vaccinia-gag

Abstract

CD40 ligand (CD40L, CD154) is a membrane protein that is important for the activation of dendritic cells (DCs) and DC-induced CD8(+) T cell responses. To be active, CD40L must cluster CD40 receptors on responding cells. To produce a soluble form of CD40L that clusters CD40 receptors necessitates the use of a multitrimer construct. With this in mind, a tripartite fusion protein was made from surfactant protein D (SPD), HIV-1 Gag as a test antigen, and CD40L, where SPD serves as a scaffold for the multitrimer protein complex. This SPD-Gag-CD40L protein activated CD40-bearing cells and bone marrow-derived DCs in vitro. Compared to a plasmid for Gag antigen alone (pGag), DNA vaccination of mice with pSPD-Gag-CD40L induced an increased number of Gag-specific CD8(+) T cells with increased avidity for major histocompatibility complex class I-restricted Gag peptide and improved vaccine-induced protection from challenge by vaccinia-Gag virus. The importance of the multitrimeric nature of the complex was shown using a plasmid lacking the N terminus of SPD that produced a single trimer fusion protein. This plasmid, pTrimer-Gag-CD40L, was only weakly active on CD40-bearing cells and did not elicit strong CD8(+) T cell responses or improve protection from vaccinia-Gag challenge. An adenovirus 5 (Ad5) vaccine incorporating SPD-Gag-CD40L was much stronger than Ad5 expressing Gag alone (Ad5-Gag) and induced complete protection (i.e., sterilizing immunity) from vaccinia-Gag challenge. Overall, these results show the potential of a new vaccine design in which antigen is introduced into a construct that expresses a multitrimer soluble form of CD40L, leading to strongly protective CD8(+) T cell responses.

FIG 1

Construction of SPD-Gag-CD40L fusion protein. (A) Cloning strategy. For pSPD-Gag-CD40L, a nucleic acid sequence was constructed that fused amino acids 1 to 105 of murine surfactant protein D (SPD) to amino acids 1 to 499 of HIV-1 HXB2 Gag, followed by amino acids 106 to 256 of murine SPD, followed by amino acids 47 to 260 of murine CD40L. For pTrimer-Gag-CD40L, the N-terminal portion of SPD was deleted and replaced with the t-PA signal peptide sequence to direct protein secretion. pSPD-CD40L is a multitrimer CD40L adjuvant molecule that has been previously described (see the text). pGag is an antigen-only construct that contains t-PA signal peptide fused to the HIV-1 Gag sequence. (B) Schematic of proposed four-trimer complex of SPD-Gag-CD40L. CD40L trimers are shown as gray circles, Gag trimers are shown as black circles, and SPD collagen-like domain are shown as black bars. For pTrimer-Gag-CD40L, the N-terminal portion of SPD is absent. Since this N-terminal portion of SPD contains the disulfide-linked “hub” needed for the assembly of the natural four-“arm” SPD structure, the resulting trimer-Gag-CD40L protein only forms a one-trimer molecule that was used as a control for the complete multitrimer SPD-Gag-CD40L structure. (C) 293T cells were transfected with DNA plasmid cytomegalovirus (CMV) promoter expression vectors encoding soluble trimeric CD40L (25), SPD-CD40L, or SPD-Gag-CD40L. After 48 h of culture, supernatant was collected and run on an SDS-PAGE gel in the presence of reducing agent. Western blots were performed with polyclonal antibody to murine CD40L. (D) To confirm the presence of multitrimer complexes, a Western blot was performed on the 293T supernatants as in panel A except using nondenaturing PAGE in the absence of a reducing agent.

FIG 2

Biological activity of SPD-Gag-CD40L in vitro. (A) In vitro activity using a CD40 receptor NF-κB indicator cell line. Equivalent amounts of culture supernatants from 293T cells transfected with pcDNA3.1, pSPD-CD40L, or pSPD-Gag-CD40L were incubated with CD40-293-SEAP reporter cells. NF-κB-driven SEAP production was measured by a colorimetric enzyme assay at OD₆₅₀. In this assay, both the pSPD-CD40L adjuvant protein and the new SPD-Gag-CD40L protein were active as CD40 receptor activators. (B) Stimulating activity on mouse bone marrow-derived DCs (BMDDCs). Equivalent amounts of culture supernatants from 293T cells transfected with pcDNA3.1 or pSPD-Gag-CD40L were incubated with BMDDCs for 18 h. Cells were washed, stained with fluorochrome-conjugated antibodies, and assayed by flow cytometry for the expression of activation and maturation markers. The SPD-Gag-CD40L protein upregulated CD80 and especially CD86 and CCR7. As expected, the CD40 receptor was downregulated by exposure to SPD-Gag-CD40L. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to pcDNA3.1 supernatant). The data represent independent wells in the same experiment.

FIG 3

Vaccine-induced responses to SPD-Gag-CD40L in vivo. (A) Immunization schedule. BALB/c mice (five mice per group) were immunized intramuscularly with either pcDNA3.1 (empty vector), pGag mixed with pcDNA3.1 empty vector, pGag antigen plasmid mixed with either pSPD-CD40L or pIL-12p70 antigen plasmids, or pSPD-Gag-CD40L. Mice were vaccinated on days 0, 14, and 28. Mice received a total of either 100 μg of DNA (pSPD-Gag-CD40L) or 120 μg of DNA (100 μg of pGag plus 20 μg of pcDNA3.1, pSPD-CD40L, or pIL-12p70) by intramuscular injection, half into each hind-limb quadriceps muscle. Two weeks later, mice were sacrificed, and splenocytes were analyzed for Gag-specific immune responses. (B and C) IFN-γ ELISPOT (B) and IL-2 ELISPOT (C) assays for CD8⁺ T cell responses. Splenocytes were collected 2 weeks after vaccination and cultured for 18 h in the presence of 10 μg of HIV-1 Gag CD8⁺ specific peptide AMQMLKETI/ml. SPD-Gag-CD40L significantly increased the number of Gag-specific IFN-γ-secreting cells compared to pGag antigen alone or pGag mixed with separate plasmids for pSPD-CD40L or pIL-12 adjuvants. SPD-Gag-CD40L also significantly increased Gag-specific IL-2 secretion compared to pGag antigen plasmid mixed with separate plasmids for pSPD-CD40L or pIL-12 adjuvants. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) T cell receptor avidity for peptide antigen/MHC-I measured by ELISPOT assay. Splenocytes were cultured with serial dilutions of CD8⁺ T cell specific peptide AMQMLKETI for 18 h. Splenocytes from mice vaccinated with pSPD-Gag-CD40L induced a significant increase in IFN-γ ELISPOT assays after stimulation with Gag peptide AMQMLKETI at concentrations of 1 ng and 10 pg/ml, whereas there was essentially no activity at these doses using splenocytes from mice vaccinated with pGag antigen alone or a mixture of separate plasmids for pGag and ppSPD-CD40L adjuvant. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to pGag alone or pGag plus SPD-CD40L vaccination). (E) IgG antibody responses against Gag antigen. Total IgG specific for Gag was measured by ELISA from mouse serum collected on day 42. Plasmid pGag in combination with pSPD-CD40L or pIL-12 induced higher Gag-specific IgG responses at various dilutions compared to pGag plus pcDNA3.1, but the differences were not statistically significant (P > 0.05 between all groups). IgG antibody levels after vaccination with pSPD-Gag-CD40L were similar to Gag antigen alone.

FIG 4

The multitrimer structure of SPD-Gag-CD40L is necessary for the improved vaccine effect. Vaccinations were performed as detailed in Fig. 3, comparing multitrimer pSPD-Gag-CD40L to a single-trimer Gag-CD40L construct missing the N-terminal disulfide-bonding domain of SPD (pTrimer-Gag-CD40L). IFN-γ (A) and IL-2 (B) ELISPOT assays were performed 2 weeks after the third DNA vaccination. As shown, the number of Gag-specific splenocytes was significantly greater using the multitrimer pSPD-Gag-CD40L vaccine compared to a one-trimer form of an analogous protein.

FIG 5

Protective effects of pSPD-Gag-CD40L vaccination measured by vaccinia-Gag viral challenge. BALB/c mice were immunized intramuscularly with pcDNA3.1 empty vector as a control, pGag mixed with pcDNA3.1 empty vector, pSPD-Gag-CD40L, a mixture of separate plasmids for pGag antigen plus pSPD-CD40L adjuvant, or pTrimer-Gag-CD40L that encodes a one-trimer protein. Immunizations were given on days 0, 14, and 28. At 2 weeks after the final vaccination, the mice were challenged intraperitoneally with vaccinia-Gag (10⁷ PFU). Mice were sacrificed on day 5 after vaccinia virus challenge, and the ovaries were harvested. The vaccinia virus titer was determined on ovary lysates using Vero cells and is expressed as PFU. (A) pSPD-Gag-CD40L vaccination resulted in a significantly lower tissue viral load compared to animals vaccinated with pcDNA3.1 alone (empty vector) or pGag (antigen only). Vaccination with a mixture of pGag antigen plus pSPD-CD40L adjuvant as separate plasmids induced a modest reduction in viral loads that were not significantly reduced compared to pGag antigen alone. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The data represent two independent experiments. (B) Evaluation of single trimer pTrimer-Gag-CD40L construct. Mice were vaccinated with either pcDNA3.1 empty vector (control), pGag antigen only, pTrimer-Gag-CD40L, or pSPD-Gag-CD40L plasmid DNAs. Vaccination with pTrimer-Gag-CD40L did not significantly reduce tissue viral load compared to either or pcDNA3.1 empty vector control or pGag antigen alone.

FIG 6

SPD-Gag-CD40L as part of an Ad5-vectored vaccine. BALB/c mice were immunized intramuscularly with Ad5-GAG or AD5-SPD-Gag-CD40L on days 0 and 14. Two weeks after the final vaccination, mice were challenged intraperitoneally with vaccinia-Gag virus (10⁷ PFU). Mice were sacrificed on day 5 after vaccinia virus challenge, and ovaries were harvested for vaccinia virus PFU determinations using indicator Vero cells. Ad5-SPD-Gag-CD40L vaccination reduced viral load by ∼7 logs after vaccinia-Gag challenge. No detectable virus could be found in the mice that had received this vaccine, indicating complete protection (sterilizing immunity).