Sublingual immunization of trivalent human papillomavirus DNA vaccine in baculovirus nanovector for protection against vaginal challenge

Abstract

Here, we report the immunogenicity of a sublingually delivered, trivalent human papillomavirus (HPV) DNA vaccine encapsidated in a human endogenous retrovirus (HERV) envelope-coated, nonreplicable, baculovirus nanovector. The HERV envelope-coated, nonreplicable, baculovirus-based DNA vaccine, encoding HPV16L1, -18L1 and -58L1 (AcHERV-triHPV), was constructed and sublingually administered to mice without adjuvant. Following sublingual (SL) administration, AcHERV-triHPV was absorbed and distributed throughout the body. At 15 minutes and 1 day post-dose, the distribution of AcHERV-triHPV to the lung was higher than that to other tissues. At 30 days post-dose, the levels of AcHERV-triHPV had diminished throughout the body. Six weeks after the first of three doses, 1×10(8) copies of SL AcHERV-triHPV induced HPV type-specific serum IgG and neutralizing antibodies to a degree comparable to that of IM immunization with 1×10(9) copies. AcHERV-triHPV induced HPV type-specific vaginal IgA titers in a dose-dependent manner. SL immunization with 1×10(10) copies of AcHERV-triHPV induced Th1 and Th2 cellular responses comparable to IM immunization with 1×10(9) copies. Molecular imaging revealed that SL AcHERV-triHPV in mice provided complete protection against vaginal challenge with HPV16, HPV18, and HPV58 pseudoviruses. These results support the potential of SL immunization using multivalent DNA vaccine in baculovirus nanovector for induction of mucosal, systemic, and cellular immune responses.

Fig 1. AcHERV-triHPV levels after SL administration.

All mice were given a single dose (1×10⁹ copies) of AcHERV-triHPV via the SL route. Blood and tissue samples were collected 15 minutes, 1 day, and 30 days after immunization. AcHERV-triHPV copy numbers were measured using qRT-PCR. Data are presented as means ± SD (n = 5).

Fig 2. SL and IM immunization and sampling schedules.

All mice were given three immunizations at 2-week intervals via the SL or IM route. Serum and vaginal secretion samples were collected 2, 4, and 6 weeks after the first immunization. Spleens were collected 6 weeks after the first immunization.

Fig 3. HPV type-specific serum IgG antibody titers after SL or IM immunization.

BALB/c mice were administered AcHERV-triHPV or PBS (control) three times over a 2-week interval via the SL or IM route. Mice were sublingually immunized with three different doses of AcHERV-triHPV (1×10⁸, 1×10⁹, and 1×10¹⁰ copies per mouse) or were immunized intramuscularly with 1×10⁹ copies per mouse. Samples were collected 2, 4, and 6 weeks after the first immunization. Antigen-specific serum IgG antibody titers against HPV16L1 (A), HPV18L1 (B), and HPV58L1 (C) were determined by ELISA. *P < 0.05 compared with other groups (ANOVA and Student-Newman-Keuls test).

Fig 4. HPV type-specific vaginal IgA antibody titers after SL or IM immunization.

Mice were administered AcHERV-triHPV or PBS (control) three times over a 2-week interval by the SL or IM route. Mice were sublingually immunized with three different doses of AcHERV-triHPV (1×10⁸, 1×10⁹, and 1×10¹⁰ copies per mouse) or immunized intramuscularly with 1×10⁹ copies per mouse. Vaginal secretion was sampled 2, 4, and 6 weeks after the first immunization. Antigen-specific vaginal IgA antibody titers against HPV16L1 (A), HPV18L1 (B), and HPV58L1 (C) were determined by ELISA. *P < 0.05 compared with other groups (ANOVA and Student-Newman-Keuls test).

Fig 5. Induction of HPV type-specific neutralizing antibodies following SL or IM immunization.

Mice were administered AcHERV-triHPV or PBS (control) three times over a 2-week interval by the SL or IM route. Mice were sublingually immunized with three different doses of AcHERV-triHPV (1×10⁸, 1×10⁹, and 1×10¹⁰ copies per mouse) or immunized intramuscularly with 1×10⁹ copies per mouse. Antigen-specific neutralizing antibody titers against HPV16L1 (A), HPV18L1 (B), and HPV58L1 (C) were determined by SEAP assay. Neutralization assays were performed using serially diluted mouse sera and HPV16, -18, or -58 PVs. Data are expressed as geometric means (log) of reciprocal serum dilutions that yielded a 50% reduction in SEAP (n = 5).

Fig 6. Th1 and Th2 cytokine induction following SL or IM immunization.

Mouse splenocytes were harvested 6 weeks after the first immunization. ELISPOT assays were used to determine the number of (A) IFN-γ-producing HPV16-, HPV18-, or HPV58-specific CD8⁺ T cells, and (B) IL-4-producing HPV16-, HPV18-, or HPV58-specific CD4⁺ T cells. Values represent the number of spots per 10⁶ splenocytes following stimulation with HPV16, -18 or -58 PVs (PV16, PV18, or PV58).

Fig 7. Vaginal challenge of AcHERV-triHPV-immunized mice with PVs.

Mice were administered AcHERV-triHPV or PBS (control) three times over a 2-week interval by the SL or IM route. (A-C) Mice sublingually administered PBS. (D-L) Mice sublingually administered AcHERV-triHPV at a dose of 1×10⁸ copies (D-F), 1×10⁹ copies (G-I), or 1×10¹⁰ copies (J-L). (M-O) Mice intramuscularly immunized with 1×10⁹ copies. Eight weeks after the first immunization, mice received vaginal challenge with luciferase-expressing PV16 (A,D,G,J,M), PV18 (B,E,H,K,N), or PV58 (C,F,I,L,O). Three days after the challenge, mice were anesthetized and injected with luciferin, and the levels of luminescence were detected with an IVIS 200 bioluminescence imaging system.