Antibody prophylaxis and therapy against West Nile virus infection in wild-type and immunodeficient mice

Abstract

West Nile virus (WNV) is a mosquito-borne Flavivirus that causes encephalitis in a subset of susceptible humans. Current treatment for WNV infections is supportive, and no specific therapy or vaccine is available. In this study, we directly tested the prophylactic and therapeutic efficacy of polyclonal antibodies against WNV. Passive administration of human gamma globulin or mouse serum prior to WNV infection protected congenic wild-type, B-cell-deficient ( micro MT), and T- and B-cell-deficient (RAG1) C57BL/6J mice. Notably, no increased mortality due to immune enhancement was observed. Although immune antibody completely prevented morbidity and mortality in wild-type mice, its effect was not durable in immunocompromised mice: many micro MT and RAG1 mice eventually succumbed to infection. Thus, antibody by itself did not completely eliminate viral reservoirs in host tissues, consistent with an intact cellular immune response being required for viral clearance. In therapeutic postexposure studies, human gamma globulin partially protected against WNV-induced mortality. In micro MT mice, therapy had to be initiated within 2 days of infection to gain a survival benefit, whereas in the wild-type mice, therapy even 5 days after infection reduced mortality. This time point is significant because between days 4 and 5, WNV was detected in the brains of infected mice. Thus, passive transfer of immune antibody improves clinical outcome even after WNV has disseminated into the central nervous system.

FIG. 1.

(A) Passive administration of mouse serum to μMT mice. Serum was collected from immune (day 28 postinfection; titer ≈1/10,000) wild-type mice and pooled. After heat inactivation, the indicated amounts of serum were administered in a divided dose to μMT mice 1 day prior to and after infection with 10² PFU of WNV. Data reflect between 5 and 10 mice per condition. (B) Passive administration of mouse serum to RAG1 mice. Immune serum (1 ml) or PBS was administered in a divided dose to RAG1 mice 1 day prior to and after infection with 10² PFU of WNV. Data reflect between 5 and 10 mice per condition.

FIG. 2.

Prophylaxis studies with human gamma globulin. (A) Passive administration of human immune gamma globulin to 8-week-old wild-type mice. The indicated amounts of purified immune gamma globulin (lot G12101) were administered as a single dose via an intraperitoneal route immediately prior to administration of 10² PFU of WNV via footpad inoculation. Data reflect at least 20 mice per condition. Statistical differences compared to the PBS control were as follows: 0.2 μg, 2 μg, and 20 μg, P > 0.3; 200 μg, 1,000 μg, 5,000 μg, and 10,000 μg, P ≤ 0.0002. (B) Passive administration of human immune gamma globulin to 8-week-old μMT mice. The indicated amounts of purified immune gamma globulin (lot G12101) were administered as a single dose via an intraperitoneal route immediately prior to administration of 10² PFU of WNV via footpad inoculation. Statistical differences compared to the PBS control were as follows: immune IgG: 200 μg, P > 0.3; 1,000 μg, P = 0.0002; 5,000 μg, P = 0.009; 10,000 μg, P < 0.0001; and nonimmune IgG: 10,000 μg, P > 0.7.

FIG. 3.

WNV burden in serum and the CNS in μMT mice after treatment with immune or nonimmune human gamma globulin. (A) Infectious virus levels in peripheral serum. μMT mice were treated immediately prior to infection with 10 mg of immune (lot G24191) or nonimmune (lot F43312) human gamma globulin. Serum was harvested at the indicated days, and virus levels were measured with a viral plaque assay in BHK21 cells. Data are shown as the average number of PFU per milliliter of serum and reflect at least three mice per time point. The dotted line represents the limit of sensitivity of the assay. (B) Infectious virus levels in the brain. Virus levels were determined in the brain after homogenization by plaque assay and normalized per gram of tissue. (C) Infectious virus levels in the spinal cord. Virus levels were determined from the spinal cord as described above.

FIG. 4.

Therapeutic studies with human gamma globulin in 8-week-old μMT mice. A single 15-mg dose of purified nonimmune (panel A, lot F43312) or immune (panel B, lot G24191) gamma globulin was administered via an intraperitoneal route immediately prior to (day 0) or at the indicated day after (day 1, 2, or 3) administration of 10² PFU of WNV via footpad inoculation. Data reflect between 5 and 10 mice per condition. Statistical differences compared to the PBS control were as follows: immune IgG: day 0, P < 0.005; day 1, P < 0.005; day 2, P = 0.06, day 3, P = 0.12; and nonimmune IgG: days 0, 1, 2, and 3, P > 0.4.

FIG. 5.

Therapeutic studies with human gamma globulin in 5-week-old wild-type mice. (A) Survival curve of 5-week-old wild-type mice. Mice were inoculated via footpad with diluent (mock) or 10² PFU of WNV. The survival curves were constructed with data from two independent experiments. The number of animals for each viral dose ranged from 6 (mock) to 23 (10² PFU). The difference in survival curves was statistically significant: P < 0.001. (B) A single 15-mg dose of purified nonimmune (lot F43312) gamma globulin was administered via an intraperitoneal route to 5-week-old wild-type mice immediately prior to (day 0) or at the indicated day after (day 1, 2, 3, 4, or 5) administration of 10² PFU of WNV via footpad inoculation. Data reflect approximately 20 mice per condition. Significance values for mortality and average survival times are indicated in Table 1. (C) A single dose of 15 mg of purified immune (lot G24191) gamma globulin was administered to 5-week-old wild-type mice via an intraperitoneal route immediately prior to (day 0) or at the indicated day after (day 1, 2, 3, 4, or 5) administration of 10² PFU of WNV via footpad inoculation. Data reflect approximately 20 mice per condition. Significance values for mortality and average survival times are indicated in Table 1. (D) WNV burden in the brain of 5-week-old wild-type mice. Five-week-old mice were treated with a single 15-mg dose of immune (lot G24191) or nonimmune (lot F43312) human gamma globulin immediately prior to infection with 10² PFU of WNV. At days 4 and 5 after infection, brains were harvested and viral burdens were determined by plaque assay after tissue homogenization. The data are expressed as PFU per gram. The following percentage of mice had viral burdens below detection (<20 PFU/g): immune IgG: day 4, 100%; day 5, 100%; nonimmune IgG: day 4, 66%; day 5, 0%.