Complement activation is required for induction of a protective antibody response against West Nile virus infection

Abstract

Infection with West Nile virus (WNV) causes a severe infection of the central nervous system (CNS) with higher levels of morbidity and mortality in the elderly and the immunocompromised. Experiments with mice have begun to define how the innate and adaptive immune responses function to limit infection. Here, we demonstrate that the complement system, a major component of innate immunity, controls WNV infection in vitro primarily in an antibody-dependent manner by neutralizing virus particles in solution and lysing WNV-infected cells. More decisively, mice that genetically lack the third component of complement or complement receptor 1 (CR1) and CR2 developed increased CNS virus burdens and were vulnerable to lethal infection at a low dose of WNV. Both C3-deficient and CR1- and CR2-deficient mice also had significant deficits in their humoral responses after infection with markedly reduced levels of specific anti-WNV immunoglobulin M (IgM) and IgG. Overall, these results suggest that complement controls WNV infection, in part through its ability to induce a protective antibody response.

FIG. 1.

Complement-mediated neutralization of WNV virions and lysis of WNV-infected cells. (A) Neutralization of WNV. Increasing concentrations of rabbit complement was preincubated with WNV virions (30 min at 37°C) in the presence or absence of MAbs against the WNV E (E1, IgG2a; E8, IgG1) or an unrelated viral protein (2E11, IgG2a, and anti-ORF7a of SARS coronavirus) prior to addition to a monolayer of hamster kidney (BHK21-15) epithelial cells. After addition of an agarose overlay and 72-h incubation, plaques were scored visually. The data shown are from one representative experiment of three and was performed in triplicate. In the absence of complement (0%), the number of plaques recorded (means ± standard deviation) was as follows: no antibody, 86 ± 10; anti-ORF7a, 83 ± 8, E1, 98 ± 15; and E8, 89 ± 3. (B) Increasing concentrations of freshly obtained mouse serum were preincubated with WNV in the presence or absence of MAbs against WNV. The experiment and data analysis were performed as described above. (C) Expression of WNV E protein on the surface of infected MC57GL cells. Cells were infected at an MOI of 5 with WNV and were processed 30 h later by flow cytometry as described in Materials and Methods with a MAb against WNV E protein (E1, IgG2a) or the SARS ORF7a protein (negative control; 2E11, IgG2a). The flow cytometric data are expressed as the log of the fluorescence intensity. One representative experiment of four is shown. (D) Lysis of WNV-infected cells. MC57GL mouse fibroblasts were mock infected or infected with WNV and incubated (60 min at 37°C) with increasing concentrations of rabbit complement in the presence or absence of complement-fixing MAbs against WNV (E1 or E16) or an unrelated viral protein (2E11). Subsequently, propidium iodide was added, and cell lysis was determined by flow cytometry. The data shown are from one representative experiment of three and was performed in duplicate.

FIG. 2.

WNV infection in C3-deficient mice. (A) Survival data of wild-type and C3-deficient mice after inoculation with WNV. 129 Sv, C57BL/6, 129 Sv × C57BL/6 F1, and C3-deficient (129 Sv × C57BL/6) mice were inoculated via footpad with 10² PFU of WNV and followed for 28 days. The survival curves were constructed using data from between three and five independent experiments. The number of animals was 10 for 129 Sv, 49 for C57BL/6, 41 for 129 Sv × C57BL/6, and 26 for C3-deficient mice. Survival differences between wild-type and C3-deficient mice were statistically significant (P < 0.0001). (B) Levels of viral RNA in serum. Viral RNA levels were determined from serum of wild-type 129 Sv × C57BL/6 or C3-deficient mice after WNV infection at the indicated days by a real-time fluorogenic RT-PCR assay. Data are expressed as genomic equivalents of WNV RNA per milliliter of serum and reflect the average of at least five independent mice per time point. The dotted line represents the limit of sensitivity of the assay. (C to E) Infectious virus levels in tissues. Virus levels were measured from the spleen (C), spinal cord (D), and brain (E) of wild-type and C3-deficient mice by a viral plaque assay in BHK21 cells after tissues were harvested at the indicated days after inoculation. Data are shown as the average PFU per gram of tissue and reflect 5 to 10 mice per time point for either wild-type or C3-deficient mice. The dotted line represents the limit of sensitivity of the assay.

FIG. 3.

Immunohistochemistry of the brain after WNV infection in wild-type and C3-deficient mice. The brains of infected wild-type (left) and C3-deficient (right) mice were harvested 10 days after infection with WNV, sectioned, and stained with mouse anti-WNV MAbs. Typical sections from the cortex, hippocampus, brain stem, and the amygdala in the base of the brain are shown after several independent brains from either wild-type or C3-deficient mice were reviewed. Red arrows denote infected neurons in wild-type mice, and blue arrows show infected neurons in C3-deficient mice.

FIG. 4.

Development of specific antibodies against WNV in wild-type and C3-deficient mice. Serum was collected from wild-type or C3-deficient mice at the indicated days after infection with 10² PFU of WNV. The development of specific IgM (A) or IgG (B) antibodies against WNV was determined after incubating serum with adsorbed control or purified WNV E protein. Serum was collected from between 5 and 10 wild-type or C3-deficient mice per time point, and individual experiments were performed in duplicate. (C) WNV-specific IgG isotype analysis of wild-type and C3-deficient mice. Serum samples were obtained from five wild-type and four C3-deficient mice at day 10 after WNV infection, diluted 1/50, and analyzed for IgG isotype by enzyme-linked immunosorbent assay. The data are expressed in a logarithmic scale as units of optical density after subtraction of the background (0.05), and the error bars represent standard deviations. For wild-type mice, the levels of IgG2a, IgG2b, and IgG2c were statistically different from levels of IgG1 or IgG3 (P < 0.03). For C3-deficient mice, the levels of IgG2a were statistically different from levels of IgG1 or IgG3 (P = 0.01).

FIG. 5.

WNV infection and humoral response in wild-type and CR1/2-deficient C57BL/6 mice. (A) Survival data of wild-type and CR1/2-deficient mice after infection with WNV. Wild-type and CR1/2-deficient C57BL/6 mice were inoculated via footpad with WNV and followed for 28 days. The survival curves were constructed using data from between three and five independent experiments. Survival differences between wild-type and CR1/2-deficient mice were statistically significant (P < 0.0001). (B) Virus levels were measured by plaque assay from brain and spinal cord tissues of wild-type and CR1/2-deficient mice at day 10 after infection with WNV. The data are shown as a scatter plot with each shaded square (wild-type) or open circle (CR1/2-deficient) corresponding to an individual data point. The horizontal bars reflect the average of the log viral titer, and the P values for statistical significance are shown. The dotted line represents the limit of sensitivity of the assay. (C and D) Development of specific IgM and IgG against WNV in wild-type and CR1/2-deficient mice. Serum samples were collected from wild-type or CR1/2-deficient mice at the indicated days after infection with WNV. The development of specific IgM or IgG antibodies against WNV was determined after incubating serum with adsorbed control or purified WNV E protein. Serum samples from between 5 and 10 wild-type or CR1/2-deficient mice per time point were used, and individual experiments were performed in duplicate. The differences between wild-type and CR1/2-deficient were statistically significant at day 8 (IgM, P < 0.001; IgG, P = 0.01) and day 10 (IgM, P = 0.01; IgG, P = 0.008).