Optimal long-term humoral responses to replication-defective herpes simplex virus require CD21/CD35 complement receptor expression on stromal cells

Abstract

Replication-defective herpes simplex virus (HSV) strains elicit durable immune responses and protect against virulent HSV challenge in mice, despite being unable to establish latent infection in neuronal cells. Mechanisms for generating long-lived immunity in the absence of viral persistence remain uncertain. In animals immunized with replication-defective HSV, durable serum immunoglobulin G (IgG) responses were elicited. Surprisingly, Western blot analyses revealed that the specificities of antiviral IgG changed over time, and antibody reactivity to some viral proteins was detected only very late. Thus, some of the durable IgG activity appeared to be contributed by either new or significantly enhanced antibody responses at late times. Following immunization, radiation bone marrow-chimeric mice lacking complement receptors CD21 and CD35 on stromal cells elicited only short-lived serum IgG and failed to mount recall responses to subsequent HSV exposure. Our results suggest that complement-mediated retention of viral antigens by stromal cells, such as follicular dendritic cells, is critical for optimal maintenance of antibody responses and B-cell memory following vaccination with replication-defective HSV.

FIG. 1.

Genomic maps of replication-defective HSV-2 strains used in this study. All viruses were derived from the wild-type HSV-2 186 strain. The dl5 or dl29 replication-defective mutant virus contains a deletion of the entire U_L5 or U_L29 gene, respectively. The 5BlacZ mutant virus expresses a nonfunctional ICP8 protein (U_L29 gene) fused in frame to the E. coli β-galactosidase (βgal) protein. Viruses were propagated on a complementing Vero-derived cell line, V5-29, which expresses the U_L5 and U_L29 products upon HSV infection.

FIG. 2.

Durable IgG responses are elicited following infection with replication-defective HSV-2 strains. The induction and durability of HSV-2 specific IgG were measured using ELISA. Groups of mice were inoculated by the s.c. route at week 0 (black triangle) with 2 × 10⁶ PFU of either strain dl29 (n = 6) (solid line) or strain dl5 (n = 6) (dashed line). At weeks 4 and 8, all mice were boosted by inoculation with 2 × 10⁶ PFU of strain 5BlacZ (white triangles). Sera were collected at the indicated times, and IgG antibody titers were determined. Results are shown as the reciprocals of the mean endpoint dilutions ± SEMs.

FIG. 3.

Specificities of serum IgG responses change at late times following inoculation. Sera were collected as indicated in Fig. 2, and equivalent volumes from each mouse were pooled for use in Western blot assays to determine the breadth of IgG reactivities to HSV-2 proteins. Samples from the dl29 group (A and B) or the dl5 group (C) were used to probe nitrocellulose membranes prepared using cell-free HSV-2 virion protein lysate (A) or HSV-2 infected V5-29 cell lysate (B and C) as described in Materials and Methods. The times of serum collection (in weeks) are indicated at the top. Molecular masses (in kDa) of marker proteins for each blot are shown on the left. Continuous (C), declining (D), and late/enhanced (E) responses, as described in Results, are indicated to the right of each blot.

FIG. 4.

Serum IgG responses to replication-defective HSV infection require stromal cell CD21/CD35. (A) WT mice (n = 7) (black circles), WTBM→Cr2^−/− chimeras (n = 5) (white squares and dashed line), and Cr2^−/− mice (n = 5) (white triangles) were inoculated with 2 × 10⁶ PFU of 5BlacZ by the i.m. route at weeks 0, 4, and 8 (black triangles). Sera were collected at the indicated times, and anti-HSV-2 IgG titers were determined by ELISA. Mean reciprocal IgG titers ± SEMs are shown. (B) Recall responses in these mice were evaluated by i.p. inoculation with 2 × 10⁶ PFU of 5BlacZ at week 32 (white triangle). Serum IgG titers were determined by ELISA, and results are shown as means ± SEMs.