Early antibodies specific for the neutralizing epitope on the receptor binding subunit of the lymphocytic choriomeningitis virus glycoprotein fail to neutralize the virus

Abstract

Lymphocytic choriomeningitis virus (LCMV) is a murine arenavirus whose glycoprotein consists of a transmembrane subunit (GP-2) and a receptor-binding subunit (GP-1). LCMV-neutralizing antibodies (nAbs) are directed against a single site on GP-1 and occur 1 month after the infection of cytotoxic-T-lymphocyte (CTL) deficient mice. In wild-type mice, however, CTLs control early infection, and weak nAb titers emerge very late (after 70 to 150 days) if at all. Production of recombinant GP-1 in native conformation enabled us to study the emergence of GP-1-binding antibodies directed against the neutralizing epitope. By combining binding and neutralization assays, we correlated the development of binding antibodies versus nAbs in wild-type and CTL-deficient mice after infection with different LCMV doses. We found that wild-type mice developed GP-1-specific antibodies already by day 8 after exposure to high but not low doses, demonstrating that naive GP-1-specific B cells were infrequent. Furthermore, the induced antibodies bound to the neutralizing GP-1 epitope but failed to neutralize the virus and therefore were of low affinity. In CTL-deficient mice, where massive viremia quickly levels initial differences in viral load, low and high doses induced low-affinity non-neutralizing GP-1-binding antibodies with kinetics similar to high-dose-infected wild-type mice. Only in CTL-deficient mice, however, the GP-1-specific antibodies developed into nAbs within 1 month. We conclude that LCMV uses a dual strategy to evade nAb responses in wild-type mice. First, LCMV exploits a "hole" in the murine B-cell repertoire, which provides only a small and narrow initial pool of low-affinity GP-1-specific B cells. Second, affinity maturation of the available low-affinity non-neutralizing antibodies is impaired.

FIG. 1.

Recombinant expression of GP-1 and of the GP-2 ectodomain of the LCMV-WE glycoprotein. (A) Schematic representation of the full-length wt LCMV-WE glycoprotein. The LCMV-WE glycoprotein is translated as single precursor protein into the lumen of the endoplasmic reticulum and is subsequently cleaved in the Golgi compartment by the cellular protease SKI1/S1P. Cleavage yields the transmembrane subunit GP-2 and the peripheral subunit GP-1. Several features within the GP-2 sequence are indicated: a potential fusion peptide (in black), a trimeric coiled-coil region, a hypothetical disulfide-bonded loop region, and an α-helical region prior to the transmembrane region (TM). At the N terminus the leader sequence (LS) is shown. N-linked glycans are indicated as trees. (B) Sequence of the GP-1-IgG fusion protein expressed in mammalian cells. (C) Sequence of the GP-2 ectodomain expressed in E. coli. (D) SDS-PAGE gel analysis of reduced GP-1-IgG. The sample was either fully glycosylated (lane 2) or PNGase F deglycosylated (lane 1). (E) SDS-PAGE of the reduced and denatured GP-2 ectodomain purified from inclusion bodies. (F) Model of the LCMV glycoprotein spike (19) showing identified surface epitopes (34): the receptor-binding subunit GP-1 contains the only neutralizing epitope (GP-1A). Neutralizing Abs binding to the GP-1A site prevent attachment of the virus to host cells (6). GP-1A partly overlaps with the non-neutralizing epitope GP-1B (not shown). Besides the GP-1A/B epitope there is an additional minor non-neutralizing linear epitope on GP-1 (GP-1C). The GP-2 subunit contains a single non-neutralizing linear epitope, formed by the overlapping epitopes GP-2A, GP-2B and GP-2C. The GP-2A/B/C epitope was shown to be located in a hypothetical disulfide-bonded loop region comprising amino acids 370 to 378 (39).

FIG. 2.

C57BL/6 mice infected with a low dose (200 PFU) of LCMV-WE fail to induce GP-1-specific Abs, whereas high-dose (2 × 10⁶ PFU)-infected mice induce a GP-1-specific Ab response, which fails to neutralize the virus. C57BL/6 mice were i.v. infected with 200 (A to C) or with 2 × 10⁶ PFU (D to F) of LCMV-WE, and blood was collected for a period of about 100 days. Sera were analyzed for their LCMV-WE neutralization potential in a focus-forming assay (A and D) and for virus-specific Ab responses. GP-1-specific IgG titers were detected by ELISA using recombinant GP-1-IgG (A and D), GP-2-specific IgG titers were detected by ELISA using refolded recombinant GP-2 (B and E), and NP-specific IgG titers were detected by ELISA using recombinant NP (C and F). Titers represent twofold above background values. The data are expressed as means ± the standard deviation (SD) of sera from five mice per group and are representative of three separate experiments.

FIG. 3.

GP-1-specific Abs induced in high-dose (2 × 10⁶ PFU) LCMV-WE-infected C57BL/6 mice are predominantly directed against the neutralizing GP-1A epitope. (A) C57BL/6 mice were i.v. infected with 2 × 10⁶ PFU of LCMV-WE, and blood was collected for a period of about 80 days. GP-1-IgG-coated ELISA plates were preincubated for 1 h with the nAb KL 25 (▴), with nonspecific mouse myeloma IgG1 (○), or with buffer (□). Thereafter, serum samples were added, and the plates were incubated for 1 h. Finally, plates were washed, and bound polyclonal serum Abs were detected with a mixture of HRP-coupled α-mouse IgG2a, IgG2b, and IgG3 isotype-specific Abs. HRP-coupled α-mouse IgG1 isotype-specific Ab was excluded, since the competing Ab KL 25 is a mouse monoclonal IgG1. The data are expressed as means ± the SD of sera from five mice per group and are representative of two separate experiments. (B) Average percent binding of polyclonal serum IgG to GP-1-IgG-coated ELISA plates, which were preincubated with KL 25, mouse myeloma IgG1, or buffer (PBS). The signals of the buffer control were defined as 100% binding. The percent binding values represent the average of all time points shown in panel A.

FIG. 4.

CTL-deficient C57BL/6 mice infected with a low or high dose of LCMV-WE alike produce increased GP-1-specific Ab titers and develop early and strong nAb responses. CD8⁺-T-cell-depleted (A and B) or Cd8a^−/− (C and D) C57BL/6 mice were i.v. infected with 200 PFU (A and C) or with 2 × 10⁶ PFU (B and D) of LCMV-WE, and blood was collected over a time period of about 100 days. Sera were analyzed for their neutralization potential and for GP-1-specific Ab titers. GP-1-specific IgG titers were detected by ELISA using recombinant GP-1-IgG. The shaded areas indicate the interval that elapses between the initial detection of GP-1-specific IgG (day 12) and the detection of serum-mediated neutralization (days 30 to 40). The data are expressed as means ± the SD of the sera of five mice per group and are representative of two separate experiments.