TLR7 recognition is dispensable for influenza virus A infection but important for the induction of hemagglutinin-specific antibodies in response to the 2009 pandemic split vaccine in mice

Abstract

Recognition of pathogen-associated molecular patterns by pattern recognition receptors of the innate immune system is crucial for the initiation of innate and adaptive responses and for immunological memory. We investigated the role of TLR7 in the induction of adaptive immunity and long-term memory following influenza virus infection and vaccination in C57BL/6 mice. During infection with influenza A/PR8/34 virus, the absence of either TLR7 or MyD88 leads to reduced virus-specific antibodies in the serum and antibody-secreting cells in their secondary lymphoid organs, particularly in bone marrow. In spite of this, the absence of TLR7/MyD88 signaling did not impair the production of protective antibodies. Following immunization with the 2009 pandemic inactivated split vaccine, TLR7(-/-) mice had significantly lower levels of germinal center formation, antibody-secreting cells, and circulating influenza virus-specific antibodies than control animals. Consequently, TLR7(-/-) mice failed to develop protective immunological memory upon challenge. Furthermore, the immunogenicity of the split vaccine was likely due to TLR7 recognition of virion RNA, as its removal from the split vaccine significantly reduced the levels of influenza virus-specific antibodies and compromised the vaccine protective efficacy in mice. Taken together, our data demonstrate that TLR7 plays an important role in vaccine-induced humoral immune responses to influenza virus through the interaction with viral RNA present in the split vaccine.

Fig 1

TLR7^−/− and MyD88^−/− mice had fewer GC B cells and PR8-specific IgM⁺ ASCs following primary infection. Mice (n ≥ 4) were infected with 25 MID₅₀s of PR8 virus, and spleen, lung, and BM were harvested at the time points indicated. (A) B cells entering GC reactions were identified as CD19⁺ IgD⁻ GL7⁺ CD38⁻ in both lung and spleen. Data are shown as the percentage of GC B cells out of the total number of CD19⁺ cells gated. (B and C) PR8-specific IgG⁺ ASCs (B) or IgM⁺ ASCs (C) from lung, spleen, and BM were measured by ELISpot assay against whole, UV-inactivated PR8 virus. Data are shown as the number of PR8-specific spots per million cells plated.

Fig 2

TLR7^−/− mice had reduced GC reactions and lower frequencies of PR8-specific ASCs in the bone marrow following a lethal IAV challenge. Mice (n ≥ 6) were infected with 25 MID₅₀s of PR8 or mock infected with PBS (naïve) and then challenged at either 1 or 6 months following primary infection with 100 MLD₅₀s of PR8. Lung, spleen, and BM were harvested on day 5 postchallenge. (A) GC B cells (CD19⁺ IgD⁻ GL7⁺ CD38⁻) in either lung or spleen were measured by flow cytometry. Data are shown as the percentage of GC B cells out of the total number of CD19⁺ cells gated. (B and C) PR8-specific IgG⁺ ASCs (B) or IgM⁺ ASCs (C) from lung, spleen, and BM were measured by ELISpot assay against whole, UV-inactivated PR8 virus. Data are shown as the number of PR8-specific spots per million cells plated.

Fig 3

TLR7^−/− and MyD88^−/− mice had comparable levels PR8-specific HI titers, despite reduced levels of IAV-specific IgM and IgG, by 6 months following IAV infection. Mice (n ≥ 15) were infected with 25 MID₅₀s of PR8 virus, and sera were collected at 1, 3, and 6 months following infection. (A to D) The levels of circulating PR8-specfic IgM, IgG, IgG1, and IgG2c were measured by ELISA using whole, UV-inactivated PR8 virus. Each mouse represents one value, and the average is the geometric mean. (E) Sera were treated with RDE, and HI titers were measured by HI assay. Shown is the result for each individual mouse with the geometric mean.

Fig 4

The early-B-cell response to pandemic split vaccine was compromised in TLR7^−/− mice. Mice (n = 5) were vaccinated i.m. with 10 μg pandemic A(H1N1)pdm09 split vaccine. (A) Sera were collected on day 15 (D15) following vaccination, RDE treated, and measured for HI titers against Cal/08 virus. Dashed line, lower limit of assay detection. Shown is the geometric mean with the 95% confidence interval. A representative dot plot of CD19⁺ IgD⁻ GC B cells (GL7⁺ CD38⁻) in spleen (B) and the percentage of splenic GC B cells out of the total number of CD19⁺ B cells from 3 to 5 mice (C) are shown. (D) Representative histogram of CD80 expression pattern from GC B cells (GL7⁺ CD38⁻) or non-GC B cells (CD38⁺). Thin line, isotype control; gray filled line, TLR7^−/− mice; thick black line, B6 mice.

Fig 5

The A(H1N1)pdm09 split vaccine contains viral genomic RNA. Vaccine (150 μg HA) was treated with various concentrations of RNase (10× = 100 μg, 1× = 10 μg) at 37°C for 30 min or left untreated under the same conditions. RNA was then purified and concentrated from vaccine using a Qiagen kit. Samples were run on a 2% ethidium bromide nondenaturing agarose gel.

Fig 6

TLR7 recognition contributed to the B-cell response to split influenza vaccine. B6 and TLR7^−/− mice (n = 10) were immunized i.m. with 10 μg A(H1N1)pdm09 split vaccine, and a control group of B6 mice was immunized with RNase-treated A(H1N1)pdm09 vaccine (RTV) (n = 5). Spleen (A) and BM (B) were harvested at 1 month postvaccination, and Cal/08-specific IgG+ or IgM⁺ ASCs were measured by ELISpot assay against whole, UV-inactivated Cal/08 virus. Data are shown as the number of spots from Cal/08-specific ASCs per million cells plated.

Fig 7

TLR7^−/− mice failed to develop protective immunity following immunization. B6 and TLR7^−/− mice were immunized i.m. with 10 μg Cal/07 split vaccine, and a control group of B6 mice was immunized with RNase-treated Cal/07 vaccine (RTV). (A) Sera were collected at 1 month postvaccination for each group tested (n = 25), RDE treated, and measured for HI titers against Cal/08 virus. Shown are the geometric means of the titers collected with a 95% confidence interval. The HI titers of B6-RTV were under the lower limit of assay detection. (B) At 1 month following immunization, mice (n = 5) from all vaccinated groups were challenged with 2 × 10⁵ MID₅₀s Mex4108. A control group of B6 mice received PBS only and was then challenged. Lungs were harvested at day 4 postchallenge, and lung viral titers were measured in eggs. Shown is each individual titer with the geometric means of the titers collected.