Natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity

Abstract

Early control of virus replication by the innate immune response is essential to allow time for the generation of a more effective adaptive immune response. As an important component of innate immunity, complement has been shown to be necessary for protection against numerous microbial infections. This study was undertaken to investigate the role of complement in neutralizing influenza virus. Results demonstrated that the classical pathway of complement mediated serum neutralization of influenza virus. Although nonimmune serum neutralized influenza virus, the mechanism of virus neutralization (VN) required antibody, as sera from RAG1-deficient mice lacked VN activity; moreover, purified natural immunoglobulin M (IgM) restored VN activity to antibody-deficient sera. The mechanism of VN by natural IgM and complement was associated with virion aggregation and coating of the viral hemagglutinin receptor; however, viral lysis did not significantly contribute to VN. Additionally, reconstitution of RAG1-deficient mice with natural IgM resulted in delayed morbidity during influenza virus infection. Collectively, these results provide evidence that natural IgM and the early components of the classical pathway of complement work in concert to neutralize influenza virus and that this interaction may have a significant impact on the course of influenza viral pneumonia.

FIG. 1.

Neutralization of influenza virus is dependent on the classical pathway of complement. (A) PR8 virus was incubated with various concentrations of serum (B6 mice), and the levels of viable virus remaining in the samples were determined by measuring growth, in 10-fold dilutions, on MDCK cells (log10 TCID₅₀). (B) The contribution of complement to virus neutralization was determined by incubating PR8 virus with heat-inactivated (HI) normal serum. (C) The role of classical pathway complements in neutralizing influenza virus was measured by incubating PR8 virus with sera (30%) from B6, C3^−/−, C4^−/−, and C1q^−/− mice. Representative results for two similar experiments are shown. The horizontal line indicates the level of stock PR8 virus used in the assay (0% serum).

FIG. 2.

Complement-dependent neutralization of influenza virus is mediated by natural IgM. (A) Detection of influenza virus-binding nIgM in mouse serum by ELISA. Diluted sera from B6 (▪) or RAG1^−/− (□) mice were added to microtiter wells coated with PR8 virus. IgM binding was detected by goat anti-mouse IgM, and absorbance readings were graphed. Representative results for at least three similar experiments are shown. (B) Influenza virus neutralization by sera (30%) from mice lacking secreted IgM (sIgM^−/−) was measured in a VN assay. Sera from B6 mice were included as a control. (C) Purified IgM (40 μg/ml) from normal mouse sera was used in a VN assay in combination with sera (30%) from RAG1^−/− mice as a source of antibody-free complement. Representative data for at least two similar experiments are shown. The vertical line indicates the level of stock PR8 virus used in the assay.

FIG. 3.

Neutralization of influenza virus occurs by a process that is independent of C5-mediated viral lysis. Sera (30%) from mice with a spontaneous mutation in C5 (−/−) were used in a VN assay. Sera from C5-sufficient mice (+/+) were used as positive controls. C5-deficient serum was also heat inactivated (HI) to demonstrate that the virus-neutralizing activity of this serum was dependent on complement. The horizontal line indicates the level of stock PR8 virus used in the assay.

FIG. 4.

Influenza virus is coated with serum components and forms aggregates in the presence of natural IgM and serum. Electron micrographs of PR8 virus particles incubated with sera (RAG1^−/−, C3^−/−, B6) and nIgM are shown. Samples were stained with uranyl formate before analysis by transmission electron microscopy. Bars represent 100 nm.

FIG. 5.

Complement deposition on influenza virus particles is enhanced by natural IgM and restricts access to the virus receptor, HA. (A and B) PR8 virus-coated microtiter wells were treated with purified nIgM (10 μg/ml) and sera (30%) from B6, C1q^−/−, C4^−/−, C3^−/−, and RAG1^−/− mice. Deposition of C3 (A) and C4 (B) was detected by complement-specific antibodies, and absorbance readings were taken. Background absorbance from non-virus-coated microtiter wells was subtracted. (C) Microtiter wells coated with dilutions of PR8 virus were treated with nIgM (10 μg/ml) or H220-17-4 (H220; 1.0 μg/ml), an HA-specific IgM antibody. Subsequently, sera (30%) from RAG1^−/− mice were added as a source of complement. To determine the level of HA accessibility, biotinylated H36-4-5.2, an HA-specific IgG2a antibody, was added to the wells (2.4 μg/ml), and the level of binding by this antibody was determined by comparison with similarly treated wells that did not receive serum. An irrelevant control IgM antibody allowed binding of H36-4-5.2 similar to that observed with sera (RAG1^−/−) alone (data not shown). None of the antibodies alone blocked H36-4-5.2 in the absence of serum. Results are representative of two similar experiments.

FIG. 6.

Survival of influenza virus-infected mice reconstituted with natural IgM. Groups of RAG1^−/− mice received PBS (▪) or 500 μg of purified nIgM (○) on days −1, 0, and 5. All mice were infected intranasally with 300 TCID₅₀ PR8 virus on day 0.