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. 2011 Nov 8;108(45):18406-11.
doi: 10.1073/pnas.1115348108. Epub 2011 Oct 31.

Intracellular neutralization of viral infection in polarized epithelial cells by neonatal Fc receptor (FcRn)-mediated IgG transport

Yu Bai  1 , Lilin Ye, Devin B Tesar, Haichen Song, Deming Zhao, Pamela J Björkman, Derry C Roopenian, Xiaoping Zhu
Affiliations

Intracellular neutralization of viral infection in polarized epithelial cells by neonatal Fc receptor (FcRn)-mediated IgG transport

Yu Bai et al. Proc Natl Acad Sci U S A. .

Abstract

IgG was traditionally thought to neutralize virions by blocking their attachment to or penetration into mucosal epithelial cells, a common site of exposure to viruses. However, we describe an intracellular neutralizing action for an influenza hemagglutinin-specific monoclonal antibody, Y8-10C2 (Y8), which has neutralizing activity only at an acidic pH. When Y8 was applied to the basolateral surface of Madin-Darby canine kidney cells expressing the rat neonatal Fc receptor for IgG (FcRn), it significantly reduced viral replication following apical exposure of the cell monolayer to influenza virus. Virus neutralization by Y8 mAb was dependent on FcRn expression and its transport of IgG. As both FcRn and Y8 mAb bind their partners only at acidic pH, the Y8 mAb is proposed to carry out its antiviral activity intracellularly. Furthermore, the virus, Y8 mAb, and FcRn colocalized within endosomes, possibly inhibiting the fusion of viral envelopes with endosomal membranes during primary uncoating, and preventing the accumulation of the neutralized viral nucleoprotein antigen in the nucleus. Prophylactic administration of Y8 mAb before viral challenge in WT mice, but not FcRn-KO mice, conferred protection from lethality, prevented weight loss, resulted in a significant reduction in pulmonary virus titers, and largely reduced virus-induced lung pathology. Thus, this study reveals an intracellular mechanism for viral neutralization in polarized epithelial cells that is dependent on FcRn-mediated transport of neutralizing IgG.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Neutralization of influenza PR8 virus in MDCK-FcRn cells by Y8 mAb. Cells (1 × 105/well) were grown in a 0.4-μm transwell insert and allowed to polarize. (A) Neutralization of PR8 virus by Y8 mAb transcytosis. Y8 mAb or IgG2a isotype (400 μg/mL) was added to the basolateral chamber for 2 h at 37 °C; subsequently, PR8 virus (100 pfu/cell) was added to the apical chamber for 1.5 h at 4 °C, then switched to 37 °C for 45 min. Cells in both chambers were completely washed of residual IgG to remove adherent virus particles. Monolayers were then incubated for an additional 24 h at 37 °C. The amount of PR8 virus in the apical medium was analyzed by TCID50 assay. (B) Neutralization of PR8 virus by Y8 mAb is dependent on IgG transcytosis. Y8 mAb (400 μg/mL) was added to the basolateral chamber of MDCK-FcRn, MDCK-FcRn-GFP, or control cells for 2 h at 37 °C. PR8 virus was subsequently added to the apical side for 1.5 h at 4 °C, and then cells were switched to 37 °C for another 45 min to allow for infection. The remaining procedures were performed as in A. *P < 0.05 and **P < 0.01.
Fig. 2.
Fig. 2.
Intracellular colocalizations of Y8 mAb, FcRn, PR8 virus, and endosome. Colocalization of FcRn, EEA1, IgG, and PR8 virus in MDCK-FcRn cells. Cells were grown in 0.4-μm inserts and allowed to polarize. Y8 mAb (400 μg/mL) was added to the basolateral side for 2 h at 37 °C, and subsequently, biotin-PR8 virus (100 pfu/cell) was added to the apical side for 1.5 h at 4 °C. Cells were then incubated for 45 min at 37 °C and thoroughly washed to remove the residual IgG. Cells were fixed and permeabilized. MDCK-FcRn cells were incubated with mouse anti-FcRn (1G3 mAb) or mAb anti-EEA-1 (5 μg/mL) or ZO-1 (5 μg/mL), respectively, followed by Alexa Fluor 488- or Alexa Fluor 555-conjugated IgG of the corresponding species. XY sections are taken at the level of the ZO-1 staining, and the XZ sections are shown (Lower). The ZO-1 staining (blue) indicates the tight junctions at the apical pole of the MDCK-FcRn cells. Colocalization of red and green signals creates a yellow-orange color. (Scale bars: 5 μm.)
Fig. 3.
Fig. 3.
Y8 mAb blocks the entry of PR8 ribonucleoprotein into the nucleus following infection. MDCK-FcRn cells were incubated with Y8 mAb or control IgG2a (400 μg/mL) for 2 h. Cells were then infected with PR8 virus at a multiplicity of infection of 100 pfu/cell at 4 °C for 1.5 h to allow virus attachment. Subsequently, infected cells were shifted to 37 °C to allow virus infection at indicated time points. Cells were fixed, permeabilized, and blocked before staining with mAb anti–EEA-1 (5 μg/mL) (A) or anti–LAMP-2 (5 μg/mL) (B) followed by Alexa Fluor 488- or Alexa Fluor 555-conjugated IgG. NP proteins were stained red by HB-65 mAb for the appearance of PR8 at indicated time. The nucleus is stained with DAPI (blue). (Scale bar: 5 μm.) Pearson colocalization coefficient was calculated to represent the colocalization between NP protein and EEA1 (C), LAMP-2 (D), and DAP1 (E) in PR8-infected MDCK-FcRn cells treated with Y8 mAb or control IgG at different time points following infection. Ten cells were analyzed from at least three different optical regions at each time point by using LSM5 image examiner software (Zeiss).**P < 0.01 and ***P < 0.001.
Fig. 4.
Fig. 4.
PR8 HA-specific Y8 mAb protected mice from virus infection. (A and B) Severity of infection in mice challenged with PR8 virus. Groups of five WT and FcRn-KO mice were intraperitoneally injected with 100 μg Y8 mAb or control IgG. One group of five mice was mock-injected with PBS solution. Four hours later, mice were intranasally challenged with 500 pfu of PR8 virus. The mice were monitored for 10 d. FcRn-KO mice were injected daily with 25–57.5 μg Y8 or control IgG to compensate for IgG catabolism. (A) Survival rate was assessed by recording whether the mice died from the infection. Percentage of mice protected on the indicated days was calculated as the number of mice surviving divided by the number of mice in each group and averaged over three similar experiments (n = 15). The mice were also weighed daily to monitor illness, as defined by percent weight loss (B). For virus titration, lungs were harvested at day 1 (C) or day 5 (D) after infection and homogenized. The amount of PR8 virus in the supernatant was analyzed by TCID50. Data shown are the means of three independent experiments, with five mice per group (**P < 0.01).
Fig. 5.
Fig. 5.
Lung pathologic findings following PR8 virus challenge. (A) Gross pathology of the lung. The lung exhibited edema and a hemorrhagic appearance in the absence of Y8 mAb inoculation or in FcRn-KO mice inoculated with Y8 mAb. H36-4 mAb was used as a positive control. (B) Histopathology in the lung of PR8-infected mice at 6 or 8 d. Inflammation was most severe in infected mice on day 6. After infection with PR8, WT mice treated with Y8 (WT/Y8) or H36 mAb (WT/H36) exhibited few focal areas of epithelial necrosis and peribronchial hemorrhage; however, the WT mice treated with PBS solution or irrelevant IgG, or FcRn-KO mice inoculated with Y8 mAb, showed severe peribronchial inflammation and bronchiolar necrosis with necrotic epithelial cells present in the lumen, and submucosal edema and vascular congestion. A mixed infiltration of inflammatory cells was present throughout at day 6 after infection. All unprotected animals died between days 6 and 7 after infections. Positive control infected mice had similar bronchiolar necrosis as control IgG-inoculated mice. Mock-infected mice had no lesions.
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