Antibody limits in vivo murid herpesvirus-4 replication by IgG Fc receptor-dependent functions

Abstract

Antibody is an important antiviral defence. However, it is considered to do little against human gamma-herpesviruses, which establish predominantly latent infections regulated by T cells. One limitation on analysing these infections has been that latency is already well-established at clinical presentation; early infection may still be accessible to antibody. Here, using murid herpesvirus-4 (MuHV-4), we tested the impact of adoptively transferred antibody on early gamma-herpesvirus infection. Immune sera and neutralizing and non-neutralizing monoclonal antibodies (mAbs) all reduced acute lytic MuHV-4 replication. The reductions, even by neutralizing mAbs, were largely or completely dependent on host IgG Fc receptors. Therefore, passive antibody can blunt acute gamma-herpesvirus lytic infection, and does this principally by IgG Fc-dependent functions rather than by neutralization.

Fig. 1.

Protection against MuHV-4 replication by immune sera. (a) C57BL/6 mice were given 3×10⁴ p.f.u. MuHV-4 intranasally (i.n.) and at the same time treated or not with 200 μl immune serum intraperitoneally (i.p.). Infectious virus in lungs was measured by plaque assay 3 days later. Each point shows the titre for one mouse; × shows mean values. Immune serum reduced lung titres significantly (P<0.0005 by two-tailed Student's t-test). (b) C57BL/6 mice were infected and treated or not with immune serum as in (a). The effects of immune serum on day 3 and day 6 titres were then compared. The reduction in titre was significant at day 6 (P<0.0001), but not at day 3 (P=0.06). (c) BALB/c mice were infected and given immune serum as in (a), or naive serum as an additional control. Virus titres in lungs were determined after 5 days. Immune serum reduced titres significantly compared with either virus only or naive serum (P<0.01). Naive serum had no effect compared with virus only (P=0.5).

Fig. 2.

Immune serum limits MuHV-4 lytic gene expression in lungs and noses. (a) BALB/c mice were infected i.n. with luciferase⁺ MuHV-4 (3×10⁴ p.f.u.) and at the same time given immune serum (200 μl), naive serum (200 μl) or nothing i.p. Virus replication was monitored by luciferin injection and CCD camera scanning at 5 days post-infection. (b) Quantification of signals shown in (a). Each point shows the luciferase signal of one mouse; × shows mean values. Immune serum reduced luciferase expression significantly compared with naive serum (P<0.02 for noses, P<0.004 for lungs) or no-serum controls (P<0.03 for noses, P<0.006 for lungs) using Student's two-tailed t-test. (c) Noses from (a) were titrated for infectious virus by plaque assay. Each point shows the titre for one mouse. Immune serum reduced titres significantly compared with naive serum (P<0.005). Titres with immune serum were also lower than with virus only, although the wide scatter of values made this difference non-significant by t-test (P=0.06).

Fig. 3.

In vitro neutralization of MuHV-4 virions by mAbs. MuHV-4 virions (100 p.f.u.) were incubated with mAbs and then plaque-assayed on BHK-21 cells. The highest amounts of each antibody used for neutralization correspond to the amounts given per mouse. Small changes in infectivity (<3-fold reduction) were considered not significant because of the potential for effects such as virion cross-linking to operate in vitro. Thus, only mAbs MG-2C10, T2C12, 230-5B2 and SC-9A5 were considered to be neutralizing. Consistent results were obtained in at least two experiments for each mAb.

Fig. 4.

Reduction in in vivo MuHV-4 infectivity by glycoprotein-specific mAbs. (a) C57BL/6 mice were infected i.n. with MuHV-4 (3×10⁴ p.f.u.) and at the same time given antibody i.p. (500 μg). Infectious virus titres in lungs were then determined by plaque assay. Each point shows the titre for one mouse; × shows mean values. The gp70-specific mAb LT-6E8 reduced virus titres significantly at day 5 (P<0.005 by Student's two-tailed t-test) compared with the influenza haemagglutinin (flu HA)-specific control, although not at day 3 (P=0.1). (b) In an equivalent experiment, mAb LT-6E8 did not affect the replication of gp70⁻ MuHV-4 (P=0.2). mAb 58-16D2 (50 μg), which recognizes a different domain of gp70, also significantly reduced the 5 day post-infection titres of wild-type (wt; P<0.00002) but not gp70⁻ (P=0.8) MuHV-4. (c) BALB/c mice were infected i.n. with MuHV-4 (3×10⁴ p.f.u.) and at the same time given mAb LT-6E8 i.p. in differing amounts. Infectious virus titres in lungs were determined by plaque assay 5 days later. Each point shows the titre for one mouse; × shows mean values. All LT-6E8 amounts reduced virus titres compared with the no-antibody control (P<0.003); 500 μg was marginally more effective than 56 or 19 μg (P<0.04), but no more effective than 167 μg (P=0.3). (d) BALB/c mice were infected i.n. with MuHV-4 (3×10⁴ p.f.u.) and at the same time given mAb LT-6E8 (500 μg) or mAb 230-4A2 (500 μg) i.p., both together or no antibody. Infectious virus titres in lungs were determined by plaque assay 5 days later. All mAb treatments reduced virus titres significantly compared with the no-antibody control (P<0.02). Both LT-6E8 and 230-4A2 together were more effective than LT-6E8 alone (P<0.03), but not more effective than 230-4A2 alone (P=0.8). (e) C57BL/6 mice were infected i.n. as in (a), and the effect of immune serum (200 μl) was compared with that of the neutralizing gB-specific IgM mAb MG-2C10 (200 μg). Immune serum reduced virus titres significantly (P<0.0001), whereas MG-2C10 had no effect (P=0.7). (f) Mice were infected i.n. as in (a) and at the same time given antibody or not i.p. (results of two separate experiments are shown). Infectious virus in lungs was titrated by plaque assay 5 days later. mAbs 3F7 (IgG_2a, anti-gN, non-neutralizing, 40 μg) and SC-9A5 (IgG₃, anti-gB, neutralizing, 500 μg) reduced virus titres (P<0.002), whereas mAb BN-3A4 (IgG₁, anti-gp150, non-neutralizing, 700 μg) did not (P=0.9).

Fig. 5.

Antibody-mediated protection by non-neutralizing mAbs and by immune serum is IgG Fc receptor-dependent. (a) FcRγ^−/−FcγRII^−/− mice (IgG FcR^−/−) were compared with 129Sv (IgG FcR^+/+) controls for antibody-dependent protection against MuHV-4. Mice were infected i.n. (3×10⁴ p.f.u.) and at the same time given antibody or not i.p. Infectious virus in lungs was titrated 5 days later by plaque assay. Each point shows the titre for one mouse; × shows mean values. mAbs 3F7 (40 μg) and LT-6E8 (500 μg) both reduced virus titres significantly in FcR^+/+ (P<0.02 by Student's two-tailed t-test) but not FcR^−/− (P=0.3) mice. (b) IgG FcR^−/− or C57BL/6 (IgG FcR^+/+) mice were infected i.n. as in (a) and at the same time given i.p. 500 μg LT-6E8 or DW-6F6 (anti-influenza haemagglutinin). Lungs were titrated for infectious virus 5 days later. mAb LT-6E8 reduced titres significantly in IgG FcR^+/+ (P<0.05) but not IgG FcR^−/− (P=0.5) mice compared with the control. (c) IgG FcR^−/− and 129Sv IgG FcR^+/+ mice were compared by lung virus titre 5 days after i.n. MuHV-4 infection and i.p. injection of either nothing (virus only), immune serum (200 μl) or mAb 58-16D2 (50 μg). Immune serum reduced virus titres significantly in IgG FcR^+/+ (P<0.001) but not IgG FcR^−/− (P=0.3) mice. mAb 58-16D2 also reduced virus titres significantly in IgG FcR^+/+ (P<0.02) but not IgG FcR^−/− mice. (d) IgG FcR^−/− and C57BL/6 IgG FcR^+/+ mice were compared by lung virus titre 5 days after i.n. MuHV-4 infection and i.p. injection of either nothing (virus only) or immune serum (200 μl). Immune serum again reduced virus titres significantly in IgG FcR^+/+ (P<0.0002) but not IgG FcR^−/− (P=0.08) mice.

Fig. 6.

Antibody-mediated protection by neutralizing mAbs is also largely IgG Fc receptor-dependent. (a) C57BL/6 (IgG FcR^+/+) or FcRγ^−/−FcγRII^−/− (IgG FcR^−/−) mice were infected with MuHV-4 i.n. (3×10⁴ p.f.u.) and given antibody or not i.p. Five days later, infectious virus in lungs was titrated by plaque assay. Each point shows the titre for one mouse; × shows mean values. IgG FcR^+/+ mice showed a significant reduction in virus replication by mAbs SC-9A5 (500 μg) and 3F7 (40 μg) (P<0.001 by Student's two-tailed t-test), but not by mAb T7F5 (200 μg) (P=0.2). None of the mAbs reduced virus replication significantly in IgG FcR^−/− mice. (b) A repeat experiment again showed a significant reduction in IgG FcR^+/+ lung titres by mAbs 3F7 (40 μg) and SC-9A5 (500 μg) (P<0.0003). This time the reduction in IgG FcR^−/− lung titres by SC-9A5, whilst lower than that of IgG FcR^+/+ (3.7-fold rather than 17-fold), was also significant (P<0.0003), as was that by 3F7 (P<0.02). (c) IgG FcR^+/+ and IgG FcR^−/− mice were further compared for virus titre reductions by the neutralizing gH–gL-specific mAbs T2C12 (500 μg) and 230-5B2 (200 μg), using the same infection protocol as in (a). Both mAbs reduced lung virus titres significantly in IgG FcR^+/+ (P<0.005) but not IgG FcR^−/− (P>0.13) mice.

Fig. 7.

O-Glycosylation may make gp150 a poor therapeutic antibody target. (a) C57BL/6 (IgG FcR^+/+) or FcRγ^−/−FcγRII^−/− (IgG FcR^−/−) mice were infected i.n. with MuHV-4 (3×10⁴ p.f.u.) and given antibody or not i.p. Five days later, infectious virus in lungs was titrated by plaque assay. Each point shows the titre for one mouse; × shows mean values. IgG FcR^+/+ mice showed a significant reduction in virus replication by mAb LT-6E8 (500 μg) (P<0.002), but not by mAb 150-5A10 (200 μg) (P=0.9), using Student's two-tailed t-test. Neither mAb reduced virus replication significantly in IgG FcR^−/− mice. (b) Predicted O-glycosylation sites in gp150 (Julenius et al., 2005). The predicted signal sequence (residues 1–22) and transmembrane domain (residues 459–481) are also shown. (c) Flow-cytometric analysis of BHK-21 cells (solid lines) and NMuMG cells (dashed lines) infected with MuHV-4 (2 p.f.u. per cell, 18 h). Control=secondary antibody only. mAbs 8C1 (anti-gH) and BN-1A7 (anti-gB) provide controls for the level of infection. None of the mAbs stained uninfected cells. Staining with mAb 150-5A10 was equivalent to that of BN-3A4. (d) Immunoblot of virions derived from NMuMG cells (NMu) or BHK-21 cells (BHK). Each blot shows two independently grown virus stocks from each cell type. mAb 150-7D1 recognizes the ORF17 capsid component and provides a loading control. (e) Virions derived from NMuMG cells or BHK-21 cells were untreated (nil), digested with PNGase F to remove N-linked glycans (N−) or digested with sialidase+O-glycanase to remove O-linked glycans (O−). Samples were then immunoblotted for gp150. mAb 150-7D1 (ORF17) provides a loading control; mAb 58-16D2 demonstrates O-linked glycan removal from gp70 in the same samples.