Protective capacity of neutralizing and non-neutralizing antibodies against glycoprotein B of cytomegalovirus

Abstract

Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals, such as organ transplant recipients and infants infected in utero. Antiviral chemotherapy remains problematic due to toxicity of the available compounds and the emergence of viruses resistant to available antiviral therapies. Antiviral antibodies could represent a valuable alternative strategy to limit the clinical consequences of viral disease in patients. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus neutralizing antibodies. However, the role of anti-gB antibodies in the course of the infection in-vivo remains unknown. We have used a murine CMV (MCMV) model to generate and study a number of anti-gB monoclonal antibodies (mAbs) with differing virus-neutralizing capacities. The mAbs were found to bind to similar antigenic structures on MCMV gB that are represented in HCMV gB. When mAbs were used in immunodeficient RAG-/- hosts to limit an ongoing infection we observed a reduction in viral load both with mAbs having potent neutralizing capacity in-vitro as well as mAbs classified as non-neutralizing. In a therapeutic setting, neutralizing mAbs showed a greater capacity to reduce the viral burden compared to non-neutralizing antibodies. Efficacy was correlated with sustained concentration of virus neutralizing mAbs in-vivo rather than their in-vitro neutralizing capacity. Combinations of neutralizing mAbs further augmented the antiviral effect and were found to be as potent in protection as polyvalent serum from immune animals. Prophylactic administration of mAbs before infection was also protective and both neutralizing and non-neutralizing mAbs were equally effective in preventing lethal infection of immunodeficient mice. In summary, our data argue that therapeutic application of potently neutralizing mAbs against gB represent a strategy to modify the outcome of CMV infection in immunodeficient hosts. When present before infection, both neutralizing and non-neutralizing anti-gB exhibited protective capacity.

Fig 1. MCMV gB and monoclonal antibodies against MCMV gB.

(A) Linear representation of MCMV gB and its structural domains. The regions representing individual structural domains are displayed in different colors in analogy to the HCMV gB crystal structure [36]. TM: transmembrane region. Numbers indicate the beginning of the domains. The antigenic regions (AD) corresponding to identified domains on HCMV gB are indicated. (B) 3D ribbon model of the domain architecture of monomeric gB colored according to (A). (C) Accessible surface representation of a trimer of the MCMV gB model with two protomers coloured in light and dark grey, respectively, and one monomer colored as in A. (D) List of anti-gB mAbs and their binding region on gB. Neutralizing antibodies are shown in bold.

Fig 2. Viral load of RAG^-/- mice after therapy with neutralizing antibodies.

Mice were infected with 10⁵ pfu MCMV157luc and treated with the indicated mAbs three days after infection. A total of 250 μg IgG or 200 μl serum per animal was injected. Viral load in aliquots of organ homogenates containing 30 μg protein was determined ten days after infection by a luciferase based assay. RLU: relative light units. Statistics: One way ANOVA using Bonferroni´s multiple comparison test *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001. Dotted line: detection limit. Representative data from 3 independent experiments. Apart from the statistics shown in the figure there were also statistical differences between M11/97.3 vs 27.7/1F11 in liver (p<0.05), kidney (p<0.01) and salivary gland (p<0.05). Differences in lung were not statistically significant.

Fig 3. Neutralization titer of sera after adoptive transfer of mAbs.

Serum was obtained from infected mice one and four days after injection of mAbs or immune serum. Neutralization titer was determined in-vitro on murine embryonic fibroblasts using MCMV157luc. Individual mice receiving the respective mAb are indicated by number and color. Dotted line: 50% neutralization.

Fig 4. Viral load of RAG^-/- mice after therapy with non-neutralizing antibodies.

Mice were infected with 10⁵ pfu MCMV157luc and treated with the indicated antibodies three days after infection. A total of 250 μg IgG per animal was injected. Viral load in aliquots of organ homogenates containing 30 μg protein was determined ten days after infection by a luciferase based assay. RLU: relative light units. Statistics: One way ANOVA using Bonferroni´s multiple comparison test *: p<0.05, **: p<0.01, ***:p<0.001, ****:p<0.0001. Dotted line: detection limit. Representative data from 2 independent experiments.

Fig 5. Viral load of RAG^-/- mice after therapy with antibody combinations.

(A) Mice were infected with 10⁵ pfu MCMV157luc and treated with the indicated combination of mAbs three days after infection. (B) Mice were infected with 10⁵ pfu MCMV-lucMCK2 and treated with the indicated combination of mAbs one day after infection. A total of 250 μg IgG per animal was injected. Viral load in aliquots of organ homogenates containing 30 μg protein was determined ten days after infection by a luciferase based assay. RLU: relative light units. nt: M11+97.3; nnt: 18A5+20H7; mix: M11+97.3+18A5+20H7. MCK-: MCMV containing MCK2 mutation, MCK+: MCMV carrying intact MCK2 gene. Statistics: One way ANOVA using Bonferroni´s multiple comparison test *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001. Dotted line: detection limit. Representative data from 3 independent experiments.

Fig 6. Plaque development in the presence of antibodies.

Murine embryonic fibroblasts were infected in 96well plates with 2000 pfu/well of MCMV C3X-gfp. The inoculum was removed 4 h after infection and the indicated antibodies were added. Development of plaques was monitored in live cells on days 3, 5 and 7 after infection. Concentration of mAbs: 20μg/ml, serum dilution 1:100. Numbers of infected cells were counted from 5–7 plaques on day 7 and were found to be (mean and range): No antibody: 25.5 (17–38); immune serum: 4.6 (3–7); non-immune serum: 10.9 (4–18); M11: 1.4 (1–2); 97.3: 7.0 (4–11); 20H7: 14.0 (5–29); 18A5 (10–18). Statistics: (One way ANOVA using Bonferroni´s multiple comparison test) M11 vs no antibody: p<0.001; M11 vs 20H7: p<0.01; M11 vs 18A5: p>0.01; M11 vs serum immune: n.s.; M11 vs serum naïve: n.s.

Fig 7. Protective capacity of mAb combinations following prophylactic application.

A total of 250 μg IgG (M11+97.3 or 18A5+20H7) per mouse or 200 μl serum/PBS was injected one day before infection with 10⁴ pfu of MCMV157luc (A) or MCMVlucMCK2 (B). Blood was taken at the indicated time points and qPCR performed. n = 4 in antibody treated groups and n = 3 in the PBS treated group. Values represent mean (SEM) of all mice within one group and duplicate determinations per sample. MCMV genome copy number is given per 1μg total DNA. Detection limit: 1 copy/50ng total DNA.