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. 2009 Jun;22(3):195-204.
doi: 10.1089/vim.2008.0071.

Listeria monocytogenes delivery of HPV-16 major capsid protein L1 induces systemic and mucosal cell-mediated CD4+ and CD8+ T-cell responses after oral immunization

Waleed Mustafa  1 , Paulo Cesar Maciag, Zhen-kun Pan, Jessica R Weaver, Yuhong Xiao, Stuart N Isaacs, Yvonne Paterson
Affiliations

Listeria monocytogenes delivery of HPV-16 major capsid protein L1 induces systemic and mucosal cell-mediated CD4+ and CD8+ T-cell responses after oral immunization

Waleed Mustafa et al. Viral Immunol. 2009 Jun.

Abstract

Neutralizing antibodies are thought to be required at mucosal surfaces to prevent human papillomavirus (HPV) transmission. However, the potential for cell-mediated immunity in mediating protection against HPV infection has not been well explored. We generated recombinant Listeria monocytogenes (Lm) constructs that secrete listeriolysin O (LLO) fused with overlapping N-terminal (LLO-L1(1-258)) or C-terminal (LLO-L1(238-474)) fragments of HPV type 16 major capsid protein L1 (HPV-16-L1). Oral immunization of mice with either construct induced IFN-gamma-producing CD8+ and CD4+ T cells in the spleen and in the Peyer's patches with the C-terminal construct. Oral immunization with both constructs resulted in diminished viral titers in the cervix and uterus of mice after intravaginal challenge with vaccinia virus expressing HPV-16-L1.

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Figures

FIG. 1.
FIG. 1.
LLO-L11–258 and LLO-L1238–474 secretion were analyzed by Western blots. Lm-PL2-LLO-L1238–474 (lane 1), wild-type Listeria monocytogenes 10403s (lane 2), molecular weight marker (lane 3), HPV-16 VLP (lane 4), and Lm-PL2-LLO-L11–258 (lane 5) were grown on a shaker overnight at 37°C in Lauria-Bertoni broth. Equivalent numbers of bacteria, as determined by OD600-nm absorbance, were pelleted and 18 mL of each supernatant was TCA precipitated. The blots were probed with anti-HPV-16-L1 monoclonal antibody (Neomarker) (A) and anti-LLO polyclonal antibody (B), followed by HRP-conjugated anti-mouse or anti-rabbit secondary antibody (Amersham). The blots were developed using ECL detection reagents (Amersham). Note that in Fig. 1B some lanes have been removed from the original Western blots, but all the lanes shown came from a single Western blot.
FIG. 2.
FIG. 2.
Time course of translocation to the spleen (A) and Peyer's patches (B) after a single oral immunization of C57BL/6 mice with 109 CFU of the recombinant L. monocytogenes strains Lm-PL2-LLO-L11–258 (solid triangles) and Lm-PL2-LLO-L1238–474 (open triangles).. Data are shown as colony-forming units (CFU) per organ homogenate after immunization as described in materials and methods. Individual data are shown for each of four mice per group per time point.
FIG. 3.
FIG. 3.
The induction of L1-specific CD8+ T cells in the spleen after oral immunization and boost with 109 CFU Lm-PL2-LLO-L11–258. Four C57BL/6 mice were immunized and boosted 3 wk after the primary immunization. Spleens were removed on day 7 after the boost, and splenocytes were analyzed for the presence of activated IFN-γ-producing CD8+ T cells. Splenocytes were restimulated ex vivo for 5 h with (A) PMA and ionomycin, (B) L1165–173-specific peptide, or (C) left without stimulation. The plots show CD8+ CD62Llow cells gated using CellQuest software. These data are representative of three different experiments.
FIG. 4.
FIG. 4.
The induction of L1-specific CD8+ T cells in the spleen after oral immunization with 109 colony forming units (CFU) of Lm-PL2-LLO-L11–258 and Lm-PL2-LLO-L1238–274. C57BL/6 mice were immunized and boosted 3 wk after the primary immunization. Spleens were removed on day 7 after the boost, and analyzed for the presence of IFN-γ-secreting CD8+ and CD4+ T cells. Splenocyte suspensions were pooled from four mice from each group and were restimulated ex vivo for 24 h with VLP or left unstimulated. Unstimulated background spots were subtracted from stimulated samples to standardize the results. Whole splenocyte responses are shown in black. CD4+ T-cell enriched splenocytes (i.e., depleted of CD8+ T cells) are shown as white bars, and CD8+ T-cell enriched splenocytes (i.e., depleted of CD4+ cells) are shown as grey bars. Data are presented as mean and standard deviation of three independent experiments.
FIG. 5.
FIG. 5.
Oral immunization with recombinant L. monocytogenes constructs expressing and secreting overlapping HPV16-L1 fragments results in diminished viral titers in the cervix and uterus. Groups of four C57BL/6 female mice were orally immunized and boosted 3 wk later with Lm-PL2-LLO-L11–258, Lm-PL2-LLO-L1238–474, or a control Listeria expressing an irrelevant protein (Lm-NP). The mice were treated with progesterone and then challenged intravaginally with a recombinant vaccinia virus expressing HPV16-L1. The mice were sacrificed 6 d after challenge and the cervices and uteri were harvested. Shown are titers of vaccinia virus in the cervix (A) and uterus (B) from individual mice. The asterisks represent levels below the level of detection (<5 plaques/organ).
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