Characterization of humoral immune responses and degree of protection induced by influenza vaccine in cotton rats: Effects of low vaccine dose and single vs booster vaccination

Figures

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  Figure 1. Systemic immune response after a single vaccination. Serum IgG responses were evaluated 30 days after a single immunization with the indicated amounts of WIV. IgG titers are presented as log10 titers for individual cotton rats with means per group (n = 4). LoD is indicated at 2 with a dashed line and significance is represented as *P < .05. IgG, immunoglobulin; LOD, limit of detection; PBS, phosphate‐buffered saline; WIV, whole inactivated virus

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  Figure 2. Effect of a single immunization on clinical symptoms after virus challenge. Cotton rats mock‐immunized with PBS or immunized once with 0.5, 1, or 5 µg WIV and challenged on day 30 with 5 × 107 TCID50 of homologous A/Cal/Gro were followed for 3 days and weight (A‐D), temperature (E‐H), and respiration rate (I‐L) were recorded. Discontinued line with X symbol indicates dead animal. PBS, phosphate‐buffered saline; WIV, whole inactivated virus

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  Figure 3. Effect of a single immunization on lung virus titers. The surviving cotton rats from the experiment described in the legend to Figure 2 were killed 3 days post‐challenge and lung virus titers were determined. Virus titers for individual cotton rats and the mean titers per experimental group are depicted. LoD for the virus titers is indicated at 0.3 with a dashed line and negative samples are assigned a value corresponding to half of the LoD. LOD, limit of detection; PBS, phosphate‐buffered saline

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  Figure 4. IgG responses after prime/boost vaccination and challenge. Serum samples collected on day 0, after the first vaccination (d21), on the day of challenge (d50), and 10 days post‐challenge (d60) were used for the determination of IgG titers. IgG ELISA was performed for sera of (A) vaccinated cotton rats and (B) nonvaccinated cotton rats. Cross‐reactive IgG against A/PR/8 H1N1 (C) and against X‐31 H3N2 (D) was measured in d50 serum samples. Titers are represented as log10 titers with significance *P < .05 and **P < .01. LoD for the IgG titers is indicated at 2 with a dashed line. ELISA, enzyme‐linked immunosorbent assay; IgG, immunoglobulin; LOD, limit of detection; n.d, not determined; PBS, phosphate‐buffered saline; WIV, whole inactivated virus

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  Figure 5. Assessment of the functional potential of systemic antibodies by HI and MN assays. Serum samples were collected at the second vaccination (d21), day of challenge (d50), and 10 days post‐homologous challenge (d60) and functionality of the IgG was measured by HI (A,C) and MN (B,D) assays for vaccinated (A,B) and nonvaccinated cotton rats (C,D). HI and MN antibody titers against NIBRG‐121 virus are represented as log2 titers. Significance as *P < .05, **P < .01. LoD for the MN titers is indicated at 4.32 and LoD for the HI titers is indicated at 2.58, both indicated with a dashed line. HI, hemagglutination inhibition; LOD, limit of detection; MN, microneutralization; n.d, not determined

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  Figure 6. Effects of prime/boost vaccination on clinical symptoms post‐challenge. Cotton rats were injected with PBS, immunized twice with 1 µg WIV, or were left untreated. Thirty days after the second immunization, immunized and mock‐immunized animals were challenged with 1 × 107 TCID50 of homologous virus, nontreated animals were again left untreated. After challenge, animals were followed for weight loss (A‐C), change in temperature (D‐F), and respiration rate (G‐I). Discontinued line with X symbol indicates dead animals. PBS, phosphate‐buffered saline; WIV, whole inactivated virus

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  Figure 7. Effect of prime/boost vaccination on lung virus titers after challenge. Thirty days after the second vaccination, cotton rats were challenged with 107 TCID50 homologous A/Cal/Gro by i.n. administration. One day post‐challenge, lung virus titers were determined by TCID50. Virus titers of individual animals and the mean virus titer per experimental group are depicted. Significance is represented as *P < .05. LoD for the virus titers is indicated at 0.3 with a dashed line. LOD, limit of detection

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  Figure 8. Effect of prime/boost vaccination on gene expression in the lungs upon infection. Cotton rats were (mock‐)immunized, boosted and challenged as previously described. On day 1 and day 10 after virus challenge, expression of cytokine mRNA in lungs of the animals was assessed by qRT‐PCR with gene‐specific primers using the housekeeping gene GAPDH as a reference. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; mRNA, messenger RNA; qRT‐PCR, quantitative real‐time polymerase chain reaction