Impaired antibody response causes persistence of prototypic T cell-contained virus

Abstract

CD8 T cells are recognized key players in control of persistent virus infections, but increasing evidence suggests that assistance from other immune mediators is also needed. Here, we investigated whether specific antibody responses contribute to control of lymphocytic choriomeningitis virus (LCMV), a prototypic mouse model of systemic persistent infection. Mice expressing transgenic B cell receptors of LCMV-unrelated specificity, and mice unable to produce soluble immunoglobulin M (IgM) exhibited protracted viremia or failed to resolve LCMV. Virus control depended on immunoglobulin class switch, but neither on complement cascades nor on Fc receptor gamma chain or Fc gamma receptor IIB. Cessation of viremia concurred with the emergence of viral envelope-specific antibodies, rather than with neutralizing serum activity, and even early nonneutralizing IgM impeded viral persistence. This important role for virus-specific antibodies may be similarly underappreciated in other primarily T cell-controlled infections such as HIV and hepatitis C virus, and we suggest this contribution of antibodies be given consideration in future strategies for vaccination and immunotherapy.

Figure 1. Viral Clearance or Persistence Depends on BCR Repertoire Diversity and Correlates with Antiviral Antibody Formation

B cell–deficient (μMT in [A, C, and E]; JHT in [B and D]), BCR-restricted T11μMT and VI10YEN mice, and control C57BL/6 mice were infected i.v. with 10⁶ PFU of LCMV-WE (A, C, and E) or 10⁶ PFU of LCMV Clone 13 (B and D). (A and B) Infectious virus in blood was monitored over time. Numbers next to the VI10YEN curve in (A) indicate viremic VI10YEN mice per number of mice tested at each time point. Comparison of viral clearance kinetics (combined analysis of up to three experiments): LCMV-WE (A) C57BL/6 versus all other groups, VI10YEN versus T11μMT, VI10YEN versus μMT, p < 0.01. LCMV Clone 13 (B) C57BL/6 versus all other groups p < 0.05. All other comparisons p > 0.05. (C and D) nAbs were monitored over time. Double asterisks (**) indicate p < 0.01. (E) LCMV-WE-GP1–specific IgG was measured by ELISA. Symbols represent the mean ± SEM of four mice per group. A single asterisk (*) indicates p < 0.05, and double asterisks (**) indicate p < 0.01. For (A and C), one representative experiment of three similar experiments is shown. (E) displays one of two experiments, with symbols representing the mean ± SEM of three to five mice per group.

Figure 2. Virus Control Requires Secreted IgM and Also AID-Dependent Class-Switch Recombination and Affinity Maturation

(A–E) Mice of the indicated genotypes were infected with 10⁶ PFU of LCMV-WE (A, C, and E) or Clone 13 (B and D) i.v. Viremia (A and B), nAbs (C and D), and LCMV-WE-GP1–binding antibodies (E) were monitored over time. Symbols represent the mean ± SEM of three to four (A, C, and E) or nine to ten (B and D) mice per group. Comparison of viral clearance kinetics: LCMV-WE (A): C57BL/6 versus all other groups, sIgM^−/− versus AID^−/−, sIgM^−/− versus JHT, p < 0.05. LCMV Clone 13 (B): C57BL/6 versus sIgM^−/− p < 0.01, as indicated by double asterisks (**). All other comparisons p > 0.05. n.s., not significant.

Figure 3. Early Adaptive IgM Response Rather Than Natural Antibodies Contributes to Virus Control

(A–C) sIgM^−/− and C57BL/6 control mice were infected with 10⁶ PFU of LCMV-WE i.v. Viremia (A), LCMV-WE-GP1–specific IgM (B), and total serum IgM (C) were determined 8 d later. Bars represent the mean ± SEM of five to ten mice per group. (D) C57BL/6 mice were infected with 10⁶ PFU of LCMV-WE i.v. LCMV-WE-GP1–specific IgM and IgG were measured over time. Symbols represent the mean ± SEM of four mice per group. (E and F) sIgM^−/− mice were substituted i.p. with 2 ml of naive C57BL/6 serum on day −1. Control groups consisted of sIgM^−/− and C57BL/6 mice without serum substitution. All mice were infected with 10⁶ PFU of LCMV-WE i.v. on day 0. Total serum IgM was measured on the day of virus challenge (E), and viremia was determined on day 21 (F). Bars represent the mean ± SEM of five to twelve animals. A single asterisk (*) indicates p < 0.05, and double asterisks (**) indicate p < 0.01. n.s., not significant.

Figure 4. Antibody Therapy Prevents Viral Persistence and Preserves CTL Function in T11μMT Mice

(A and B) Mice of the indicated genotypes were infected with 10⁶ PFU of LCMV WE. On day 4 and day 7 (arrows), they were treated with GP1-specific antibody in normal serum or with normal serum only (control), as outlined in the chart. Viremia was monitored over time. Symbols represent the mean ± SEM of four to five mice per group. The data in (A and B) originate from the same experiment, and the identical C57BL/6 group is shown in both graphs. Virus clearance kinetics in C57BL/6 mice and anti-GP1 antibody-treated T11μMT mice were indistinguishable (p > 0.05), but each one of them differed significantly from the remaining three groups (p < 0.01). All other comparisons p > 0.05. (C–F) C57BL/6 and T11μMT mice were treated as in (A and B). On day 7 (C and D) and day 35 (E and F) after infection, virus-specific cytotoxic activity of CD8⁺ T cells was measured in an in vivo CTL assay (C and E), and virus loads were determined in blood (D and F). Bars represent the mean ± SEM of three to five mice per group. Double asterisks (**) indicate p < 0.01. n.s., not significant.

Figure 5. Efficient Virus Clearance Occurs in the Absence of Either Complement, Fc Receptor γ Chain, or FcγRIIB

Mice of the indicated genotypes were infected with 10⁶ PFU of LCMV-WE, and viremia was monitored over time. Symbols represent the mean ± SEM of three to four mice per group. (A–C) represent independent experiments. C3^−/−C4^−/− mice were tested alongside the experimental groups displayed in Figure 2A, 2B, and 2C, and hence the identical C57BL/6 and JHT control groups are shown in Figure 2A and (A). Comparison of viral clearance kinetics: (A) C57BL/6 versus C3^−/−C4^−/− p > 0.05 (not significant); C57BL/6 versus JHT, C3^−/−C4^−/− versus JHT p < 0.05. (B) C57BL/6 versus FcRγ ^−/−, JHT versus T11μMT p > 0.05 (not significant); C57BL/6 versus JHT, C57BL/6 versus T11μMT, FcRγ^−/− versus T11μMT, FcRγ^−/− versus JHT, p < 0.05. (C) C57BL/6 versus FcγRIIB^−/− p > 0.05 (not significant).