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. 2003 Dec;77(23):12742-52.
doi: 10.1128/jvi.77.23.12742-12752.2003.

Codelivery of CCR7 ligands as molecular adjuvants enhances the protective immune response against herpes simplex virus type 1

Felix N Toka  1 , Malgorzata Gierynska, Barry T Rouse
Affiliations

Codelivery of CCR7 ligands as molecular adjuvants enhances the protective immune response against herpes simplex virus type 1

Felix N Toka et al. J Virol. 2003 Dec.

Abstract

Humoral and cellular immunity, associated with long-term protective immunological memory, defines the efficacy of a given vaccine formulation. However, few vaccines achieve this target without the aid of a suitable adjuvant. Molecular adjuvants in vaccination against infectious agents offer a noninvasive means of enhancing the immune response against target antigens. To examine the potency of two beta-chemokines as immunomodulators, plasmid DNA encoding beta-chemokines CCL19 and CCL21 (CCR7L) was codelivered intranasally with plasmid DNA or recombinant vaccinia virus encoding herpes simplex virus (HSV) gB (HSV-gB) in a prime-and-boost vaccination strategy. This vaccination regimen increased serum and vaginal immunoglobulin G (IgG) and IgA, respectively, as well as the numbers of HSV-gB(498-505) peptide-specific gamma interferon-producing CD8(+) T cells. Distinctively, a high number of cytotoxic T lymphocytes was achieved when pCCR7L was applied at both prime and boost as opposed to omission of pCCR7L. A rapid-recall response was induced in the genital tract upon challenge with the HSV McKrae strain, affording a high level of protection and survival of vaccinated mice. Our results demonstrate that high innate immune kinetics and distribution of adaptive response induced in the nasal mucosa appears to be key factors in generating protective memory responses against HSV. Thus CCR7L expressed ectopically may serve as a molecular adjuvant to boost the immune response to a codelivered antigen in mucosal surfaces.

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Figures

FIG. 1.
FIG. 1.
Administration of pCCR7L i.n. increases the numbers of lung DCs. C57BL/6 mice 5 to 6 weeks of age were given i.n. 100 μg of plasmid DNA encoding CCR7L (pCCL19 or pCCL21) with or without rVVgB, vector plasmid DNA encoding β-Gal, or PBS. Lungs and PBLN were removed on days 1 to 5, and DCs were isolated by positive selection with CD11c+ microbeads by MACS, stained with a monoclonal antibody against CD11c FITC, and analyzed by flow cytometry. (A) Comparison of lung CD11c+ DCs from various treatment groups on days 1 to 5; (B) CD11c+ DCs in PBLN following i.n. codelivery of pCCR7L.
FIG. 2.
FIG. 2.
CD11c+ DCs isolated from lungs and PBLN of pCCR7L-treated mice are capable of activating CD8+ T cells from mice previously primed with HSV. Mice were treated with pCCR7L, β-Gal, or PBS i.n., and 3 days later pCCR7L-treated mice were immunized i.n. with rVVgB. One group was infected with rVVgB only. An IFN-γ ELISPOT assay was performed to quantitate the functional capability of CD11c+ DCs originating from lungs and PBLN of pCCR7L-treated and control mice. Seven days following immunization lungs and PBLN were removed and CD11c+ DCs were prepared, purified by MACS, and incubated with splenocytes from mice primed earlier with HSV. CD11c+ DCs (2 × 104) were added to 105 splenocytes per well. (A) Spot-forming cells after stimulation with lung or PBLN CD11c+ DCs; (B) IFN-γ secretion by CD8+ T cells isolated from lungs of pCCR7L-treated mice upon restimulation in vitro with HSV-gB498-505. Intracellular staining for IFN-γ was performed as described in Materials and Methods.
FIG. 3.
FIG. 3.
CD8+ T cells isolated from mice treated with pCCR7L have the capacity to produce more IFN-γ than CD8+ T cells from non-pCCR7L-treated mice. IFN-γ secretion by CD8+ T cells isolated from spleen, MLN, and genital tract was assessed by in vitro stimulation with splenocytes pulsed with gB498-505 in ELISPOT assays. CD8+ T cells were examined at 14 (A; acute phase) and 60 days (B; memory phase) after boost. Data are from a representative experiment of two performed.
FIG. 4.
FIG. 4.
Spleen memory CD8+ T cells from pCCR7L-treated mice secreted IFN-γ upon restimulation ex vivo more rapidly than CD8+ T cells from non-pCCR7L-treated mice. The cells were isolated at 60 days postboost and assayed for IFN-γ production ex vivo by intracellular cytokine staining. Spleen cells (106) were incubated in the presence of 2.5 μg of HSV-gB498-505, 50 U of IL-2, and GolgiPlug for 5 h and subsequently stained with anti-CD8+-FITC and anti-IFN-γ-phycoerythrin (PE) antibodies (except for groups treated with UV-inactivated HSV and PBS, for which IFN-γ-FITC and CD8+-PE were used). FITC-conjugated rat anti-IgG was used for the isotype control (data not shown). Cytometry and data analysis were performed with FACScan and Cell Quest, respectively. Figures show representative data from two independent experiments.
FIG. 5.
FIG. 5.
Induction of SSIEFARL-specific cytolytic activity in mice vaccinated intranasally with rVVgB with or without codelivery of pCCR7L. Splenocytes and MLN were isolated on the 14th (primary phase) and 60th (memory phase) days postboost from vaccinated and control mice and expanded in vitro with syngeneic irradiated splenocytes pulsed with gB498-505 (specific for MHC-I-restricted CD8+ T cells) for 5 days, followed by a 51Cr release assay using MHC-matched MC38 (mouse colon adenocarcinoma) cells pulsed with gB498-505 as the targets. Data were corrected with the formula described in Materials and Methods. E:T ratio, effector-to-target cell ratio.
FIG. 6.
FIG. 6.
Postchallenge cytolytic potential of CD8+ T cells isolated from pCCR7L-immunized mice. The CTL assay was performed as described in Materials and Methods. Cells were isolated from mice at 5 days postchallenge. This is a representative experiment of two performed. E:T ratio, effector-to-target cell ratio.
FIG. 7.
FIG. 7.
Postchallenge IFN-γ secretion by CD8+ T cells. Sixty days after the boosting dose (memory phase) mice were synchronized with 2 mg of medroxyprogesterone/mouse and 5 days later were infected with McKrae at 107 PFU/mouse intravaginally. Five days later mice were sacrificed and CD8+ T cells were isolated from the iliac LN and spleen. To assess IFN-γ production by intracellular cytokine staining, 106 cells were stimulated with 2.5 μg of HSV-gB498-505 for 5 h in the presence of brefeldin A and IL-2 and stained with anti-IFN-γ-FITC and anti-CD8+-phycoerythrin and then analyzed by flow cytometry. Data are representative of two independent experiments performed.
FIG. 8.
FIG. 8.
Survival rates for pCCR7L-vaccinated and non-pCCR7L-vaccinated mice after intravaginal challenge at 60 days after boost immunization. Four mice were used per group and were synchronized by injecting 2 mg of medroxyprogesterone per mouse subcutaneously. Five days later each mouse was anaesthetized with avertin and infected intravaginally with 107 PFU of HSV MacKrae. Each day a specimen of the genital tract wash fluid was collected for virus titration. Mice were monitored daily for clinical illness and pathology, scored according to criteria reported by Gallichan et al. (13) as follows: 0, no apparent infection; 1, mild inflammation of the external genitals and redness and moderate swelling of external genitals; 3, severe redness and inflammation; 4, genital ulceration and severe inflammation; 5, hind limb paralysis and death.
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