An intranasally delivered Toll-like receptor 7 agonist elicits robust systemic and mucosal responses to Norwalk virus-like particles - PubMed (original) (raw)

An intranasally delivered Toll-like receptor 7 agonist elicits robust systemic and mucosal responses to Norwalk virus-like particles

Lissette S Velasquez et al. Clin Vaccine Immunol. 2010 Dec.

Abstract

Norwalk virus (NV) is an enteric pathogen from the genus Norovirus and a major cause of nonbacterial gastroenteritis in humans. NV virus-like particles (VLPs) are known to elicit systemic and mucosal immune responses when delivered nasally; however, the correlates of immune protection are unknown, and codelivery with a safe and immunogenic mucosal adjuvant may enhance protective anti-NV immune responses. Resiquimod (R848), an imidazoquinoline-based Toll-like receptor 7 and/or 8 (TLR7/8) agonist, is being evaluated as an adjuvant in FDA-approved clinical vaccine trials. As such, we evaluated the adjuvant activity of two imidazoquinoline-based TLR7 and TLR7/8 agonists when codelivered intranasally with plant-derived NV VLPs. We also compared the activity of these agonists to the gold standard mucosal adjuvant, cholera toxin (CT). Our results indicate that codelivery with the TLR7 agonist, gardiquimod (GARD), induces NV VLP-specific serum IgG and IgG isotype responses and mucosal IgA responses in the gastrointestinal, respiratory, and reproductive tracts that are superior to those induced by R848 and comparable to those induced by the mucosal adjuvant CT. This study supports the continued investigation of GARD as a mucosal adjuvant for NV VLPs and possible use for other VLP-based vaccines for which immune responses at distal mucosal sites (e.g., respiratory and reproductive tracts) are desired.

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Figures

FIG. 1.

Serum Norwalk virus (NV)-specific IgG and isotype production following intranasal immunization with NV virus-like particles (VLPs) codelivered with or without cholera toxin (CT) or Toll-like receptor (TLR) ligands. Female BALB/c mice were immunized intranasally with NV VLPs (5 or 25 μg) on days 0 and 21 with or without CT (1 μg), gardiquimod (GARD) (10 μg), or resiquimod (R848) (10 or 25 μg). Serum samples were collected on days 0, 12, 21, 42, 56, 84, and 119 and analyzed for NV VLP-specific IgG (A), IgG1 (B), and IgG2a (C) by ELISA. By day 42, all mice that received NV VLPs codelivered with or without adjuvant responded with a positive antibody titer (geometric mean titer [GMT] of ≥100) for IgG and IgG1. In addition, all mice responded with a positive anti-NV IgG2a titer except for those receiving R848 (25 μg) plus NV VLP (5 μg) on days 42 (5/8 mice), 56 (6/8 mice), 84 (6/8 mice), and 119 (7/8 mice) or those receiving NV VLP alone on day 42 (7/8 mice). Antigen-specific IgG and IgG2a were not detected (GMT < 100) in all preimmune samples; however, background levels of IgG1 were detected in preimmune samples (data not shown). Error bars represent the standard errors of the means. Values that were significantly different from the values for the PBS control group are shown as follows: ∧, P < 0.05; *, P < 0.01; **, P < 0.001. The horizontal thick broken line indicates the limit of detection for the assay.

FIG. 2.

Fecal NV-specific IgA production following intranasal immunization with NV VLPs codelivered with or without CT or TLR ligands. Fecal pellets were collected on days 0, 12, 21, 42, 56, 84, and 119 and analyzed for NV VLP-specific IgA by ELISAs. By day 42, all mice that received NV VLPs codelivered with or without adjuvant responded with a positive fecal IgA titer (GMT ≥ 2) except for those receiving R848 (25 μg) plus NV VLP (25 μg) (14/16 mice), R848 (25 μg) plus NV VLP (5 μg) (7/8 mice), or NV VLP alone (7/8 mice). Fecal antigen-specific IgA was not detected (GMT < 2) in all preimmune samples (data not shown). Error bars represent the standard errors of the means. Values that were significantly different from the values for the PBS control group are shown as follows: ∧, P < 0.05; *, P < 0.01; **, P < 0.001. The horizontal thick broken line indicates the limit of detection for the assay.

FIG. 3.

NV-specific IgA production in the gastrointestinal tract following intranasal immunization with NV VLPs codelivered with or without CT or TLR ligands. Mice were euthanized on days 56 and 119. Salivary swabs (A) and intestinal lavage samples (B) were collected and analyzed for NV VLP-specific IgA by ELISAs. The number of mice responding with a positive antibody titer (GMT ≥ 2) to the total number of mice is indicated in parentheses above each bar. Error bars represent the standard errors of the mean. Values that were significantly different from the values for the PBS control group are shown as follows: ∧, P < 0.05; *, P < 0.01; **, P < 0.001. There were no significant differences between the groups in panel A. The horizontal thick broken line indicates the limit of detection for the assay.

FIG. 4.

NV-specific IgA production in the respiratory tract following intranasal immunization with NV VLPs codelivered with or without CT or TLR ligands. Following euthanization on days 56 and 119, nasal (A) and bronchoalveolar (B) lavage samples were collected and analyzed for NV VLP-specific IgA by ELISAs. The number of mice responding with a positive antibody titer (GMT ≥ 2) to the total number of mice is indicated in parentheses above each respective bar. Error bars represent the standard errors of the means. Values that were significantly different from the values for the PBS control group are shown as follows: ∧, P < 0.05; *, P < 0.01; **, P < 0.001.There were no significant differences between the groups in panel B. The horizontal thick broken line indicates the limit of detection for the assay.

FIG. 5.

Vaginal NV-specific IgA and IgG production following intranasal immunization with NV VLPs with or without CT or TLR ligands. Vaginal lavage samples were collected on days 0, 12, 21, 42, 56, 84, and 119 and analyzed for NV VLP-specific IgA (A) and IgG (B) by ELISAs. By day 42, all mice that received NV VLPs codelivered with or without adjuvant responded with a positive vaginal IgA antibody titer (GMT ≥ 2). None of the mice responded with a positive vaginal IgG titer (GMT ≥ 2) following a single immunization (days 12 and 21), except for the CT-treated group (1/8 mice). By day 119, 6/8 mice responded with a positive IgG titer in groups receiving NV VLP (25 μg) codelivered with CT, GARD, or R848 (10 μg), whereas groups immunized with NV VLP (25 μg) plus R848 (25 μg), NV VLP (5 μg) plus R848 (25 μg), or NV VLP alone responded with a positive titer in 4/7, 0/8, or 4/8 mice, respectively. Vaginal antigen-specific IgA and IgG were not detected (GMT < 2) in all preimmune samples (data not shown). Error bars represent the standard errors of the means. Values that were significantly different from the values for the PBS control group are shown as follows: ∧, P < 0.05; *, P < 0.01; **, P < 0.001. The horizontal thick broken line indicates the limit of detection for the assay.

FIG. 6.

Frequency of NV-specific IgG-secreting cells in the spleen following intranasal immunization with NV VLPs with or without CT or TLR ligands. Following euthanization on day 56 or 119, splenocytes were isolated and pooled into 3 groups to determine the frequency of NV VLP-specific IgG-secreting cells by an ELISPOT assay. Results are expressed as the average number of antigen-specific IgG-secreting cells per 1 × 107 splenocytes. Error bars represent the standard errors of the means. Values that were significantly different from the values for the PBS control group are shown as follows: ∧, P < 0.05; **, P < 0.001.

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An intranasally delivered Toll-like receptor 7 agonist elicits robust systemic and mucosal responses to Norwalk virus-like particles - PubMed (original) (raw)