Recovery of humoral immunity is critical for successful antiviral therapy in disseminated mouse adenovirus type 1 infection

Abstract

Severe adenovirus infections in transplant recipients undergoing immunosuppressive therapy are of increasing concern. Controversy exists on the contribution of antiviral therapy and the host immune response to recovery from these infections. Here, we established a systemic mouse adenovirus type 1 (MAV-1) infection in cyclophosphamide (CyP)-treated BALB/c mice. CyP was administered at 100 mg per kg of body weight every other day for 2, 3, or 4 weeks, thereby inducing general but reversible leukopenia, with a major suppression of the B-cell numbers and functionality that was more pronounced than that seen with T cells. The outcome of MAV-1 infection was dependent on the duration of CyP therapy, as the mice with the most severe immunosuppression were the most vulnerable to MAV-1-induced hemorrhagic enteritis and mortality. The protective effect of concomitant antiviral therapy with cidofovir depended on the level of immunosuppression. The combination of cidofovir treatment with the withdrawal of immunosuppression was the most successful regimen for increasing survival rates. Survival was clearly correlated with the clearance of virus and increased titers of MAV-1-specific antibodies in sera. In addition, the passive transfer of MAV-1-specific immunoglobulin G into MAV-1-infected SCID BALB/c mice caused a marked delay in mortality, the extent of the delay being dependent on the titer of MAV-1-specific antibodies. Based on the critical role of the humoral immune response in the early defense against disseminated adenovirus infection, the concomitant use of adenovirus-specific immunoglobulins and antiviral therapy should be considered for transplant patients at risk for severe adenovirus infections.

FIG. 1.

Schedule of treatment of MAV-1-infected BALB/c mice with CyP and/or cidofovir. CyP (100 mg/kg, injected i.p.) was administered on alternate days. Cidofovir (100 mg/kg, injected subcutaneously) was given for 6 consecutive days and thereafter continued on alternate days.

FIG. 2.

Kinetics of leukocyte populations in spleen and lymph nodes and splenic T- and B-cell functionality in MAV-1-infected BALB/c mice undergoing immunosuppressive treatment with CyP. MAV-1-infected mice were subjected to CyP treatment for 2 or 4 weeks with or without antiviral therapy with cidofovir, as depicted in Fig. 1. (A) Flow cytometric examination of CD4⁺ T cells (black bars), CD8⁺ T cells (gray bars), B220⁺ B cells (white bars), and CD11b⁺ Gr-1⁺ neutrophils (striped bars) in spleens and lymph nodes. (Left panels) Mean number of leukocyte subtypes in spleens or lymph nodes from uninfected, untreated mice (n = 6). (Right panels) On days 3, 14, and 28 p.i., infected mice (n = 2 per treatment condition) were sacrificed and leukocytes from spleens or lymph nodes were pooled for subsequent analysis. These data are expressed as percentages relative to the corresponding values for uninfected, untreated mice. Note that on day 3 p.i., values for CyP-2 and CyP-4 groups were equal, since conditions for these groups were identical and their leukocytes were pooled. (B) With the same spleens, leukocyte functionality was analyzed in a lymphocyte proliferation assay using αCD3, LPS, or no mitogen. The stimulation index was calculated by dividing the response to mitogen (αCD3, gray bars; LPS, white bars) by the response of unstimulated cell populations and is expressed as the percentage of the value obtained for uninfected, untreated mice. †, no surviving mice; *, not determined.

FIG. 3.

(A) Hematoxylin and eosin staining of liver (left panels [panels i, iii, and v]) and intestinal (right panels [panels ii, iv, and vi]) tissue sections taken at 14 and 28 d.p.i. from mice receiving CyP treatment for 2 weeks with or without cidofovir, showing representative MAV-1-induced pathology. The arrows indicate neutrophilic infiltrates. Original magnification, ×200. C, congestion; N, necrosis. (B) Immunohistochemical staining for MAV-1 E3 protein in tissues from moribund MAV-1-infected CyP-3 mice at 14 or 27 d.p.i. receiving, respectively, CyP treatment (upper panels) and CyP-plus-cidofovir therapy (lower panels): (a) liver; (b) small intestine; (c) lung; (d) brain; (e) heart; (f) adrenal; and (g) kidney tissue. Infected cells show dark brown cytoplasmic staining for MAV-1 E3 and are indicated by an arrowhead. Original magnification, ×200 (original magnification of the inset in the upper panel c, ×400). Co, cortical cell; En, endothelial cell; Ep, epithelial cell; H, hepatocyte.

FIG. 4.

Evolution of overall survival, virus replication, and virus-neutralizing antibody titers in MAV-1-infected, immunocompromised BALB/c mice receiving either antiviral or no therapy. BALB/c mice infected with MAV-1 were treated with CyP and/or cidofovir according to various schemes (see Fig. 1 and Materials and Methods for dosing schedules). (A) Survival curve (the total number of mice for each treatment condition is given in the legend box). (B) Viral DNA loads in livers were measured by means of real-time PCR. Data are presented as the numbers of viral DNA copies per nanogram of chromosomal DNA. (C) Titers of MAV-1-neutralizing antibodies in sera were determined in a virus neutralization assay using 1:20 to 1:5,120 serum dilutions. Symbols in panels B and C represent the data obtained from individual mice, with the curves representing trend lines.

FIG. 5.

Suppression of MAV-1 infection in SCID mice receiving virus-specific IgG. Immunocompetent BALB/c mice were challenged with MAV-1, and at 15 and 43 d.p.i., the IgG fractions (designated IgG15 and IgG43, respectively) were isolated from pooled immune sera. Nonimmune IgG was obtained from noninfected BALB/c mice. MAV-1-neutralizing IgG titers were determined by a virus neutralization assay (see inset). Nonimmune IgG, IgG15 (mean titer, 1:130), or IgG43 (mean titer, 1:3,750) was transferred into SCID mice by i.p. injection (in a 200-μl volume) 1 day before and 9 days after infection with MAV-1. Data for each treatment condition were compiled from the results of two independent experiments, each including five mice per group.