Impairment of CD4(+) T cell responses during chronic virus infection prevents neutralizing antibody responses against virus escape mutants

Abstract

We have shown previously that neutralizing antibodies (nAbs) are important contributors to the long-term immune control of lymphocytic choriomeningitis virus infection, particularly if cytotoxic T cell responses are low or absent. Nevertheless, virus escape from the nAb response due to mutations within the surface glycoprotein gene may subsequently allow the virus to persist. Here we show that most of the antibody-escape viral mutants retain their immunogenicity. We present evidence that the failure of the infected host to mount effective humoral responses against emerging neutralization-escape mutants correlates with the rapid loss of CD4(+) T cell responsiveness during the establishment of viral persistence. Similar mechanisms may contribute to the persistence of some human pathogens such as hepatitis B and C viruses, and human immunodeficiency virus.

Figure 1

Transient control of viremia in CD8^−/− mice. CD8^−/− mice (animals M7 to M11; •) and control B6 mice (○) were infected with 2 × 10⁶ PFU of LCMV-WE-wt intravenously, and sequential blood samples were analyzed for virus titers. Data shown are the mean for five mice ± SEM.

Figure 2

NAb responses against immunizing LCMV-WE-wt and emerging LCMV variants in five individual CD8^−/− mice. (A–E) Five CD8^−/− mice (animals M7–M11) were infected with 2 × 10⁶ PFU of LCMV-WE-wt. Sequential serum samples were tested for neutralizing (Neutr.) activity against LCMV-WE-wt (•) and against the predominant LCMV variant (WE-M7 to WE-M11; ○) isolated from the corresponding mouse after viral reemergence on day 120 after infection (see characterization of variants in Table ). α, anti.

Figure 3

Induction of specific CD4⁺ T cell unresponsiveness in LCMV-infected CD8^−/− mice. B6 mice (triangles) and CD8^−/− mice (circles) were infected with 2 × 10⁶ PFU LCMV-WE intravenously. (A) Splenocytes from the indicated days after infection were stimulated in vitro with the immunodominant LCMV class II–restricted epitope (GP61–80; filled symbols) or with no peptide (open symbols), and the percentage of peptide-specific CD4⁺ T cells expressing intracellular IFN-γ was then assessed. (B) 5 × 10⁵ splenocytes from in vivo CD8-depleted, LCMV-GP61-80–specific CD4⁺ TCR tg SMARTA mice (B6 background) were adoptively transferred intravenously to recipient B6 mice that were either infected with LCMV-WE 1 d later (filled symbols) or were not infected (open symbols). The percentage of tg CD4⁺ T cells (TCR Vα2⁺; Vβ8.3⁺) in the blood was then sequentially determined by FACS^® analysis. Data shown are the mean for four mice ± SEM.

Figure 4

The CD4⁺ T cell population is not depleted during LCMV infection. B6 mice and CD8^−/− mice were infected with 2 × 10⁶ PFU of LCMV-WE. Splenocytes at various time points after infection were stained for CD4. Numbers of CD4⁺ T cells were determined by flow cytometry. Data shown are the mean of three mice per group ± SEM.

Figure 6

In vivo replication of nAb-escape variants. CD8-depleted B6 mice (A) and CD8^−/− mice (B) were infected with 2 × 10⁴ PFU of LCMV-WE-wt or of the indicated nAb-escape LCMV variant (for CD8^−/− mice only variant WE-M7). Sequential blood samples were obtained for quantitation of virus titers. Data shown are the mean of three to four mice per group ± SEM. The experiment shown in A was performed twice with similar results.

Figure 5

Functional VSV-specific T cell help in LCMV-WE infected CD8^−/− mice. B6 mice (triangles) and CD8^−/− mice (circles) infected with 2 × 10⁶ PFU of LCMV-WE 240 d previously (B), as well as naive control animals (A), were immunized with 2 × 10⁶ PFU of VSV-IND. Sequential serum samples were obtained for quantitation of VSV-neutralizing (neutr.) IgM Ab titers (filled symbols) or IgG Ab titers (open symbols), distinguished from IgM by reduction with 0.1 M of β-mercaptoethanol. The results are given as the mean ± SEM of three to four mice per group.

Figure 7

Immunogenicity of LCMV-WE nAb-escape variants. (A–E) Sequential blood samples from CD8-depleted mice infected with 2 × 10⁴ PFU of five nAb-escape variants WE-M7 (A), WE-M8 (B), WE-M9 (C), WE-M10 (D), and WE-M11 (E) were obtained for quantitation of nAb titers against the immunizing virus (•) and of nAb titers against LCMV-WE-wt (○). The dotted lines represent the autologous nAb response after infection of CD8-depleted mice with an equivalent dose of LCMV-WE-wt. The experiment was repeated in CD8^−/− mice for variant WE-M7 (F). Data shown are the mean for three mice per group ± SEM. Neutr., neutralizing.