Nonneutralizing antibodies binding to the surface glycoprotein of lymphocytic choriomeningitis virus reduce early virus spread

Abstract

The biological relevance of nonneutralizing antibodies elicited early after infection with noncytopathic persistence-prone viruses is unclear. We demonstrate that cytotoxic T lymphocyte-deficient TgH(KL25) mice, which are transgenic for the heavy chain of the lymphocytic choriomeningitis virus (LCMV)-neutralizing monoclonal antibody KL25, mount a focused neutralizing antibody response following LCMV infection, and that this results in the emergence of neutralization escape virus variants. Further investigation revealed that some of the escape variants that arose early after infection could still bind to the selecting antibody. In contrast, no antibody binding could be detected for late isolates, indicating that binding, but nonneutralizing, antibodies exerted a selective pressure on the virus. Infection of naive TgH(KL25) mice with distinct escape viruses differing in their antibody-binding properties revealed that nonneutralizing antibodies accelerated clearance of antibody-binding virus variants in a partly complement-dependent manner. Virus variants that did not bind antibodies were not affected. We therefore conclude that nonneutralizing antibodies binding to the same antigenic site as neutralizing antibodies are biologically relevant by limiting early viral spread.

Figure 1.

Rapid selection of antibody escape variants after infection of CTL-deficient TgH(KL25) mice. Blood viremia (top) and neutralizing antibody responses (bottom) in TAP^−/−, TgH(KL25), or TgH(KL25)xTAP^−/− mice after i.v. infection with 2 × 10⁶ or 200 PFU LCMV-WE. The dashed line indicates the detection level for blood virus and neutralizing IgG titers. Error bars represent the mean ± SD (n = 2–3 mice each). One representative out of two independent experiments is shown.

Figure 2.

Characteristics of viruses isolated from TAP^−/− and TgH(KL25)xTAP^−/−. (A) Neutralization sensitivity of single-round subcloned virus isolates obtained from TAP^−/− and TgH(KL25)xTAP^−/− mice. A total of 75 and 55 viral isolates, respectively, from TAP^−/− and TgH(KL25)xTAP^−/− mice were incubated with KL25 at a concentration of 100 μg/ml or mock treated with the same volume of medium, and twofold serially diluted. LCMV-WE was used as a positive control. The difference in viral titers between the antibody and mock-treated preparation are depicted as Δtiter values. Isolates displaying Δtiter values of >1 titer step were considered neutralization sensitive. Solid lines indicate the means. Each point represents an individual isolate. (B) The MFIs of mAb binding to MC57G cells infected with the indicated virus isolates were determined for KL25 and WEN1.3 (which also binds to the GP of KL25 escape variants). Each symbol represents an individual isolate (59 isolates from 8 LCMV-infected TAP^−/− and 55 isolates from 8 TgH(KL25)xTAP^−/− mice are depicted). Values represent the background-corrected MFI of KL25 as a percentile of the background-corrected MFI of WEN1.3. Open circles represent KL25 neutralization–sensitive isolates, and closed circles represent neutralization-resistant isolates. The MFI of LCMV-WE–infected cells is shown for comparison.

Figure 3.

Binding of WEN3.1 and KL25 to recombinantly expressed WT and mutant GP. The extracellular portion of WT or mutant GP was recombinantly expressed as a fusion protein with the Fc portion of human IgG₁. Purified recombinant protein was immobilized on anti–human-Fc–coated plates before titrated amounts of unlabeled WEN3.1 and biotinylated KL25.8 were added, starting at an antibody concentration of 6 μg/ml. After detection using horseradish peroxidase–labeled goat anti–mouse IgG (WEN3.1) or streptavidin (KL25) in combination with ABTS substrate, OD₄₀₅ values were determined and plotted as a function of the dilution. Error bars represent the mean ± SD of duplicate wells. Results are representative of at least two separate experiments.

Figure 4.

Accelerated viral clearance by nonneutralizing antibodies. (A) Neutralizing antibody response of TgH(KL25) mice infected with 200 PFU of the indicated variants of LCMV. (top) Heterologous neutralizing antibody titers induced against WT LCMV-WE. (bottom) Autologous neutralizing antibody titers induced against the infecting viral variant. Error bars represent mean ± SD (n = 3 mice). Complement present in sera was not heat inactivated before the assay. (B) Viral titers in spleen of C57BL/6 and TgH(KL25) mice infected with 200 PFU of LCMV-WE or the indicated variants of LCMV. Each symbol represents the viral titer measured for a single mouse on the indicated day. The solid line indicates the mean of the experimental group, and the dashed line indicates the detection level of the assay.

Figure 5.

Impact of complement on virus clearance of the high-binding isolate cl12.1 in TgH(KL25) mice. (A) Virus titers in spleen of TgH(KL25) mice infected with 200 PFU of the cl12.1 isolate. To deplete activity of the C3 and C5 complement components, animals received 2 U of CVF i.p. before infection and every 24 h thereafter while the control group remained nontreated. Results are representative of at least two separate experiments. (B) Virus titer in spleens of C57BL/6 or C3-deficient mice receiving 3 × 10⁷ TgH(KL25) splenocytes 1 d before infection with 200 PFU cl12.1 i.v. Each symbol represents an individual animal. The solid line indicates the mean of the experimental group, and the dashed line indicates the detection level of the assay. Results are representative of a single experiment.

Figure 6.

Accelerated clearance of cl12.1 is mediated by soluble antibodies alone and does not require the presence of specific B cells. Virus titers in spleen of C57BL/6 mice infected with 200 PFU of the cl12.1 isolate. Mice received either 750 μl of naive TgH(KL25) serum i.v. 20 min before infection to simulate increased natural antibody titers, or 200 μg of purified mAb KL25 1 d after infection to simulate an early nonneutralizing antibody response. As a control, one group of mice did not receive antibodies. Each symbol represents the viral titer measured for a single mouse on the indicated day. The solid line indicates the mean of the experimental group, and the dashed line indicates the detection level of the assay. We estimated a maximal serum concentration of 133 μg/ml KL25 in mice receiving purified mAb, using a conservative assumption of a 1.5-ml blood volume (46) with no diffusion of antibody into the tissue.