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. Author manuscript; available in PMC: 2021 Apr 23.
Published in final edited form as: Antiviral Res. 2018 Apr 17;154:149–157. doi: 10.1016/j.antiviral.2018.04.014

Anti-respiratory syncytial virus (RSV) G monoclonal antibodies reduce lung inflammation and viral lung titers when delivered therapeutically in a BALB/c mouse model

Hayat Caidi a, Congrong Miao a, Natalie J Thornburg a,*, Ralph A Tripp b, Larry J Anderson c, Lia M Haynes a
PMCID: PMC8063470  NIHMSID: NIHMS1011719  PMID: 29678551

Abstract

RSV continues to be a high priority for vaccine and antiviral drug development. Unfortunately, no safe and effective RSV vaccine is available and treatment options are limited. Over the past decade, several studies have focused on the role of RSV G protein on viral entry, viral neutralization, and RSV-mediated pathology. Anti-G murine monoclonal antibody (mAb) 131–2G treatment has been previously shown to reduce weight loss, bronchoalveolar lavage (BAL) cell number, airway reactivity, and Th2-type cytokine production in RSV-infected mice more rapidly than a commercial humanized monoclonal antibody (mAb) against RSV F protein (Palivizumab). In this study, we have tested two human anti-RSV G mAbs, 2B11 and 3D3, by both prophylactic and therapeutic treatment for RSV in the BALB/c mouse model. Both anti-G mAbs reduced viral load, leukocyte infiltration and IFN-γ and IL-4 expression in cell-free BAL supernatants emphasizing the potential of anti-G mAbs as anti-inflammatory and antiviral strategies.

Keywords: RSV, G protein, CX3C, CX3CR1, Virus, Pathogenesis, Monoclonal antibody

1. Introduction

Respiratory syncytial virus (RSV) is a negative-sense, single—stranded RNA virus of the family Pneumoviridae that causes acute respiratory tract infections (Palivizumab and a humanized, 1998; Reducing transmission of, 2007; Alvaro and Zuccotti, 2000; Anderson et al., 2013; Manohar Lal Choudhary et al., 2013; Rima et al., 2017) often leading to lower respiratory tract disease such as pneumonia and bronchiolitis. RSV is a major cause of pediatric lower respiratory tract hospitalizations globally (DeVincenzo, 2000, (DeVincenzo, 2008); Empey et al., 2010; Hall et al., 1991). The RSV genome encodes 11 proteins, two of which, the F and G surface glycoproteins, induce protective immunity in small animal models (Collins and Malero, 2011; Graham and Anderson, 2013). For decades, substantial efforts have been made toward producing a safe and effective vaccine, however none have been successful in achieving the correct balance of safety and efficacy (Statement on the recommen, 2003; Anderson et al., 2013; BS., 2011; Buckingham et al., 2002; Choi et al., 2012; Crowe et al., 1999; de Waal et al., 2004). A humanized anti-F monoclonal antibody (mAb), Palivizumab, has been shown effective in preventing serious lower respiratory tract infection and hospitalization in high-risk patients when delivered prophylactically, but less effective for treating active infection (Fitzgerald, 2009; Gill and Welliver, 2009; Gonzalez et al., 2000; Gorman et al., 2001; Mejías et al., 2004).

The F protein induces high titers of neutralizing antibodies and a level of cross-protection against different strains of RSV (Connors et al., 1991; Sullender, 1995; Sullender et al., 1998), while the G protein also has an important role in inducing and modulating the host immune responses to infection. The RSV G protein is approximately 50% conserved among circulating RSV strains with two conserved regions, the cytoplasmic/transmembrane region (amino acids [aa] 1 to 63), and the central conserved region (CCR) (Sullender, 1995). Within the CCR, there is a region with 100% conservation (aa 164–176) and a larger region that is relatively conserved (aa 153–207). Furthermore, there is a CX3C chemokine motif (aa 182 –186) that binds to the CX3C chemokine receptor CX3CR1, and mimics several activities of the only known CX3C chemokine, fractalkine (FKN) (Chirkova et al., 2013; Tripp et al., 2001).

Several recent studies have indicated that mAbs directed against G neutralize RSV in primary cell culture (Cortjens et al., 2017; Johnson et al., 2015). Treatment of mice with mouse anti-G mAb, 131–2G after inoculation with RSV reduced infection through a molecular mechanism that may involve blocking G protein binding to CX3CR1 (Caidi et al., 2012; Haynes et al., 2009; Miao et al., 2009; Radu et al., 2010). Treatment with other human anti-G mAbs 3G12 and 3D3, which bind G at residues 167–176 and 164–172, respectively, within the CCR were shown to be effective at decreasing inflammation and reactive airway disease in mice (Han et al., 2014). Based on these observations, we hypothesized that other human anti-RSV G mAbs may also be effective treatments for RSV infections.

In this study, we tested two human mAbs, the previously characterized 3D3, and a novel human mAb, 2B11, that binds the same antigenic region as 131–2G (Collarini et al., 2009). Consistent with earlier findings, our results show that both anti-RSV G mAbs were effective in reducing viral lung titers and inflammation when delivered prior to viral inoculation or one day after viral inoculation in the BALB/c challenge model. Taken together, these results suggest that high affinity human mAbs may be promising antiviral candidates.

2. Materials and methods

2.1. Animals

This study was performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Centers for Disease Control and Prevention (CDC) Institutional Animal Care and Use Committee (Protocol Number: 2415HAYMOUC). No surgeries were performed. All efforts were made to minimize animal suffering during all procedures performed. Four-to-six week old, specific-pathogen-free, female BALB/c (The Jackson Laboratories) mice were used in all experiments. Only females were used for consistency and comparability to previously published studies. The mice were housed in microisolator cages and were fed sterilized water and food ad libitum and maintained in accordance with the guidelines of the CDC Institutional Animal Care and Use Committee.

2.2. Antibodies

Anti-RSV G mAbs (2B11, 3D3), targeting the region CCR of RSV G protein and control human IgG were obtained from Trellis Bioscience (South San Francisco, CA). The 2B11 and 3D3 binding sites were mapped to residues 162–172 and 164–172, respectively (Collarini et al., 2009 (Fig. 1). Anti-RSV F mAb (Palivizumab) was purchased from MedImmune (Gaithersburg, MD) and provided by Trellis Bioscience. Murine monoclonal antibody 131–2G that reacts within the 13 aa central conserved 164–176 aa, was purified from mouse ascites fluid or hybridoma supernatant on a protein G column. Antibodies were delivered via the intraperitoneal route at 300 μg in a 2 ml volume diluted in sterile, endotoxin free PBS.

Fig. 1. Cartoon of RSV G protein and peptides bound by mAbs.

Fig. 1.

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Linear representation of the 298 aa RSV G protein, with the cytoplasmic domain (CD), the transmembrane domain (TM), the conserved central region (CCR), and CXC3 binding region labeled. The amino acid sequence of residues 162-186 are shown with the four cysteine residues participating in two disulfide bonds. Peptide reactivities of 2B11, 3D3, 3G12, and 131-2G mAbs are shown above the cartoon representation of G.

2.3. Virus infection and tissue collection

The A2 strain of RSV was used in all experiments and propagated in Vero cells (ATCC CCL 881) as previously described (Tripp et al., 1999) and was partially purified by limiting dilution. Mice were anesthetized by intraperitoneal (i.p.) administration of Avertin (2% 2, 2, 2-tri-bromoethanol, 2% tert-amyl-alcohol, 180–250 mg/kg), and intranasally (i.n.) challenged with 106 plaques forming units (PFU) of RSV in serum free DMEM (50 μL volume) or mock-infected Vero cells with serum-free DMEM. At days 3, 5, 7, 10 and 14 after challenge, mice were anesthetized with Avertin and exsanguinated by severing the right auxiliary artery. Bronchoalveolar leukocyte (BAL) cells were collected by lavaging the lungs three times with 1 ml sterile Dulbecco’s PBS. The mean number of BAL cells per ml ( ± standard error, SE) was determined. Lungs were harvested and stored at −80°C until use. No fewer than three mice per treatment per time point were examined.

2.4. Treatments regimens

2.4.1. Prophylactic treatment

One day prior to i.n. virus infection, at least four mice per group were i.p. treated with either 5 mg/kg, 1.5 mg/kg, 0.15 mg/kg, 0.015 mg/kg or 0.0015 mg/kg with anti-RSV G protein mAbs, 2B11, 3D3, anti-RSV F mAb Palivizumab or normal human IgG (nIg). Endotoxin concentrations in the mAbs were less than 5 endotoxin units per mg in accordance with the manufacturer’s instructions (Pierce).

2.4.2. Therapeutic treatment

On day 3 p.i., at least 4 mice per group were i.p. treated with 5 mg/kg, body weight with anti-RSV G protein mAb 2B11, mAb 3D3, anti-RSV F mAb Palivizumab or control normal mouse immunoglobulin G (IgG), nIg (Trellis Bioscience). Endotoxin in the mAb preparations was detected by an endotoxin quantitation kit (Pierce) and was less than 5 endotoxin units per mg for all preparations.

2.5. Flow cytometry

The procedure used for extracellular staining of BAL cells was modified for microculture staining as described (Tripp et al., 1999). Briefly, BAL cells from four mice per group were blocked in 10% normal mouse serum in staining buffer (D-PBS with 1% bovine serum albumin) and then stained with the appropriate combinations of fluorescein isothiocyanate-, allophycocyanin-, or phycoerythrin-labeled anti-CD4, anti-CD8, anti-CD3ε, anti-CD45R/B220, anti-CD1lb, anti-PMN cell (RB6-8C5), anti-NK cell (DX5), and mouse isotype antibody controls (Trellis Bioscience). The distribution of cell surface markers was analyzed on a BD LSRII flow cytometer using FACSDiva software (Becton Dickinson, Mountain View, CA) from 10,000 lymphocyte-gated events.

2.6. Virus titers

Viral titers in the lungs of RSV-infected mice were determined as previously described (Haynes et al., 2002). Briefly, lungs were weighed, homogenized in 1 ml/lung of sterile Dulbecco PBS containing 25% sucrose, and 10-fold serial dilutions (in serum-free DMEM) of the lung homogenates were added to confluent Vero cell monolayers in 24-well plates. After adsorption for 2 h at 37 °C, cells monolayers were overlaid with tissue culture DMEM (Life Technologies) containing 10% fetal bovine serum, incubated at 37 °C for 3–5 days, and then enumerated by immunostaining with mAbs against the G and F proteins (232-1F, 130-6D, and anti-F, 131-2A).

2.7. Real-time RT-PCR

The level of RSV replication in lung tissue was also determined by quantitative real-time reverse transcription PCR (qRT-PCR) of the RSV matrix (M) gene. Briefly, total RNA was extracted from homogenized lung tissue using a viral RNA purification kit (QIAGEN, Valencia, CA) per manufacturer’s protocol and stored at −80 °C until use. AgPath-ID One Step qRT-PCR Kit (Ambion, Applied Biosystems) was used for analysis of expression of viral message with RSV matrix M primers (forward, position 3257–3282) 5′-GGC AAA TAT GGA AAC ATA GCT GAA-3′ and (reverse, position 3312–3340) 5′-TCT TTT TCT AGG ACA TTG TAY TGA ACA G-3′ (Caidi et al., 2012), Since the concentration of RSV in the control sample was given as PFU/ml, the concentrations in test samples are given as PFU equivalents/ml (PFUe/ml).

2.8. Cytokine analysis

Cell-free BAL supernatants were assayed for IFN-γ and IL-4 by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (eBioscience). All samples were examined in duplicate and the assays were repeated three independent times.

2.9. Lung histopathalogy

The lungs of infected and treated mice were removed 5 days after challenge, fixed and stained with hemotoxylin and eosin (H&E). Lung histopathological examination was performed for each group of treated mice where lung tissues were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with H&E prior to light microscopy observation using methods previously described (Zhang et al., 2010). Perivasculitis was defined as inflammatory cells of various types that accumulate around the periphery of small airways. Each parameter was scored separately under blinded conditions and was assigned a score based on a scale of 0 (no inflammation) to 4 (maximum inflammation) (Zhang et al., 2010).

2.10. Statistical analysis

Data were analyzed for statistical significance using a Student’s t-test, where a P value of < 0.05 was considered statistically significant, and are marked with asterisks *. Data are expressed as the mean ± standard error of the mean (SEM).

3. Results

3.1. Reduction in pulmonary inflammation is mediated by prophylactic antibody treatment

Human anti-G mAbs 2B11 and 3D3 have been shown to bind residues 162–172 and 164–172, respectively in the region that mouse mAb 131–2G also binds (Collarini et al., 2009) (Fig. 1). 131–2G also requires Cysl86 for binding (Johnson et al., 2015). To assess the prophylactic effectiveness of these mAbs, mice were delivered 5 mg/kg, 1.5 mg/kg, 0.15 mg/kg, 0.015 mg/kg, or 0.0015 mg/kg of each G mAb, anti-F mAb, or an isotype control one day prior to challenge with 106 PFU/ml of RSV A2 per mouse. RSV was reduced in the lungs of mice that had received 5 mg/kg and 1.5 mg/kg doses of anti-G mAbs on day 5 post-inoculation as compared to isotype control treated mice as assayed by plaque assay (Fig. 2A) or qRT-PCR (Fig. 2B). Similarly, there were fewer total BAL cells in the mice that had received prophylactic treatment with either 5 mg/kg anti-RSV G mAbs or Palivizumab in comparison to isotype controls (Fig. 2C). However there were no detectable reductions in the total lung BAL cells when mice were delivered 1.5 mg/kg in any prophylactic groups (Fig. 2C). Prophylactic treatment of the mice with mAbs at 0.15 mg/kg, 0.015 mg/kg, and 0.0015 mg/kg did not reduce either lung viral titers or cells detected in BAL in any group (data not shown). Specific cell types in the BAL were examined by flow cytometry. The decrease in total BAL cell number was associated with significant decreases in CD3+ T, B220 + B, CD11b + myeloid, and DX5 + NK cells in anti-G mAb treated mice (Table 1). Specifically, BAL CD3+ cells were only significantly reduced in mice treated with 2B11, but not 3D3 or Palivizumab at 5 mg/kg. BAL CD1lb+, B220+, and DX5 + NK cells were significantly reduced in mice treated with 5 mg/kg of either 2B11 or 3D3 mAbs, While Palivizumab prophylaxis reduced percentages of B220 + B, CD11b + myeloid, RB86 + neutrophilic, and DX5+ NK cells, the reductions were not statistically significant. Reductions in BAL specific cell types were not significant with 1.5 mg/kg prophylactic dose of any mAb (Table 1).

Fig. 2. Effects of mAbs 2B11 and 3D3 on RSV titers and pulmonary leukocyte trafficking when administered one day prior to inoculation.

Fig. 2.

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RSV infected mice were prophylactically treated with 5 or 1.5 mg/kg isotype control, anti-F (Palivizumab), or anti-G mAbs (2B11 or 3D3) one day prior to inoculation with RSV A2. Lungs were collected at day 5 and used to test for (A) Virus titers (PFU/g of lung tissue) and (B) Level of viral replication as determined by reverse qRT-PCR for M gene expression (relative genome level equivalent PFU/g of lung tissue). BAL were also collected at day 5 pi and used for (C) Mean number of cells (cells per ml) recovered from BAL fluid, (D) amount of IFN-γ in BAL cell-free fluid, (pg/ml) and (E) amount of IL-4 levels in BAL cell-free fluid (pg/ml). Asterisks indicate a significant difference (p < 0.05) between nIg-treated mice and mAbs treated mice as calculated with student’s T test ND, not detectable (levels of virus were below the limit of detection). Results are representative of three independent experiments with three to four mice per time point.

Table 1.

Enumeration of cell types present in BAL as characterized by flow cytometry staining in mice treated prophylactically with isotype control, anti-F, and anti-G mAbs prior to inoculation with RSV A2.

Dose (mg/kg) Phenotype nIg Isotype (Mean number cells (103) ± SE) mAb treatment
Anti-F 2B11 3D3
Mean number cells (103) ± SE % Reductiona Mean number cells (103) ± SE % Reductiona Mean number cells (103) ± SE % Reductiona
5 CD3 24.54 ± 0.96 34.88 ± 1.33 0.0 8.25 ± 0.57 63.3b 9.30 ± 0.40 62.1
CD4 25.13 ± 0.98 28.84 ± 0.98 0.0 21.20 ± 0.96 16.4b 20.96 ± 0.92 16.5
CD8 15.72 ± 0.61 20.38 ± 0.77 0.0 15.47 ± 0.70 0.0 13.75 ± 0.60 12.5
B 24.25 ± 0.95 16.30 ± 0.62 33.0 7.62 ± 0.24 68.5b 8.51 ± 0.37 65.0b
CD11b 102.31 ± 4.01 92.26 ± 3.52 9.0 44.70 ± 2.01 56.3c 48.47 ± 2.13 52.6b
PMN 17.05 ± 0.66 15.70 ± 0.60 7.9 18.54 ± 0.84 0.0 16.63 ± 0.73 2.0
NK 31.01 ± 1.21 23.10 ± 0.88 25.5 9.77 ± 0.44 68.4b 10.61 ± 0.46 65.7b
1.5 CD3 24.88 ± 1.36 31.53 ± 1.78 0.0 14.96 ± 0.89 39.8 18.98 ± 1.06 23.7
CD4 23.69 ± 1.29 28.65 ± 1.62 0.0 24.44 ± 1.49 0.0 26.28 ± 1.46 0.0
CD8 10.13 ± 0.55 15.40 ± 0.87 0.0 16.45 ± 0.98 0.0 14.30 ± 0.79 0.0
B 23.84 ± 1.30 34.27 ± 1.94 0.0 16.45 ± 0.98 31.0 15.33 ± 0.85 36.0
CD11b 84.78 ± 4.64 77.04 ± 4036 9.1 63.78 ± 3.82 25.0 66.43 ± 3.71 22.0
PMN 12.96 ± 0.70 11.66 ± 0.66 10.0 14.28 ± 0.85 0.0 19.71 ± 1.10 0.0
NK 21.75 ± 1.19 14.54 ± 0.82 33.1 12.51 ± 0.75 42.5 14.74 ± 0.82 32.2
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RSV A2-infected mice were treated prophylactically with normal immunoglobulin G (nIg), anti-RSV G mAb 2B11 or 3D3 or mAb anti-F on day 1 prior infection. Bronchoalveolar lavage (BAL) samples from 4 mice per group were examined on day 5 post-infection. Data are the total number of BAL cells expressing CD4, CD8, B220 (B cells), NK (natural killer cells, DX5 +), CD11b (macrophages and/or monocytes), or RB6-8C5 (neutrophils [PMNs]) per lung on day 5 post-challenge shown as mean with standard error of the mean.

a

Percent reduction is the change in total cell type after anti-RSV mAbs treatment relative to the total cell type after treatment with nIg.

b

p < 0.001 between nIg and mAbs treated mice.

c

p < 0.05 between nIg and mAbs treated mice.

Consistent with decreased cell infiltration, we observed a decrease in IFNγ in Palivizumab and anti-G treated mice at the 5 and 1.5 mg/kg doses in cell-free BAL supernatants relative to the isotype control treated mice (Fig. 2D). Treatment with anti-G mAbs also reduced IL-4 in comparison to isotype control, while treatment of mice with Palivizumab did not reduce IL-4, but did reduce IFNγ in comparison to isotype control treatment (Fig. 2D and E). At 0.15 mg/kg, 0.015 mg/kg, and 0.0015 mg/kg the anti-G mAbs did not demonstrate substantial effects on cell infiltration and RSV genome expression (data not shown). Since significant differences were detected for all parameters at 5 mg/kg, this dose was used for the therapeutic study.

3.2. Effects of early therapeutic treatment with mAbs 2B11 and 3D3 on pulmonary inflammation

To assess the therapeutic effectiveness of the human anti-G mAbs 2B11 and 3D3, the previously characterized mouse anti-G mAb 131–2G, and the mouse anti-RSV F mAb 131–2A (Caidi et al., 2012; Haynes et al., 2009; Miao et al., 2009; Radu et al., 2010), mice were delivered 5 mg/kg antibodies at day 3 post-challenge with RSV A2. No change in body weight was observed at any time-point with any treatment (data not shown). Therapeutic treatment with anti-F, 2B11, 3D3, and 131–2G anti-G mAbs decreased viral lung titers at day 5 post-inoculation (Fig. 3A). Anti-G mAb treatment was associated with a reduction in total lung leukocytes (Fig. 3B) in comparison to isotype control treated mice at day 5 post-inoculation, while treatment with anti-F mAb did not reduce total BAL cells. IFNγ was reduced at days 5 and 7 (Fig. 3C) and IL-4 at day 5 (Fig. 3D) in cell-free BAL supernatants when mice were treated with anti-F or anti-G mAbs. Our results are consistent with previous studies where treatment with murine anti-F mAb, 143–6C, did not reduce BAL cellularity, but did marginally decrease pro-inflammatory and Th2-type cytokines (Boyoglu-Barnum et al., 2015a,b; Han et al., 2014; Haynes et al., 2009).

Fig. 3. Reduction in RSV-associated pulmonary inflammation after therapeutic treatment with mAbs 2B11 and 3D3.

Fig. 3.

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RSV-infected mice were treated with 5 mg/kg with anti-G protein mAbs 2B11, 3D3, anti-F mAb Palivizumab and isotype control on Day 3 post-inoculation. Lungs were collected on days 3, 5, 7, 10 and day 14 pi and used for (A) Virus titers (PFU/g of lung tissue) BAL were collected on days 3,5,7,10 and day 14 pi. and used for (B) Mean number of cells (cells per ml) recovered from BAL fluid, (C) amount of IFN-γ in BAL cell free fluid (pg/ml), and (D) amount ofIL-4 levels in BAL cell-free fluid (pg/ml). Asterisks indicate a significant difference (p < 0.05) between nlg-treated mice and mAbs treated mice as calculated by Student’s T test. The arrows indicate the time of treatment. ND, not detectable (levels of virus were below the limit of detection). Results are representative of three independent experiments with four mice per time point.

To determine the alveolar inflammatory cell types affected by anti-RSV G mAb treatment, the BAL cell subsets were characterized at various time points post-treatment by flow cytometry. At 5 mg/kg, treatments with mAbs 2B11 and 3D3 were associated with a significant reduction in B220+B cells (Fig. 4A), DX5+NK cells (Fig. 4B), CD3+T cells (Fig. 4C), and CDS+T cells (Fig. 4D at 5 days post-challenge, which corresponds to the peak of infection in this model. At day 5, B220 + B (Fig. 4A), CD3 + T (Fig. 4B), DX5 + NK (Fig. 4C), and GD8 + T (Fig. 4D) were not reduced in mice treated with anti-F mAbs. We also enumerated neutrophils, which were not reduced with any treatment (data not shown).

Fig. 4. Treatment with 3D3 and 2B11 mAbs reduces the total number of B, NK, and T cells.

Fig. 4.

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BAL were collected on days 3, 5, 7, 10, and 14 days post RSV A2 inoculation in mice treated with 5 mg/kg isotype control, anti-F protein (Palivizumab), human anti-G protein mAbs (2B11 or 3D3), or mouse anti-G 131-2G mAbs at 3 days post-inoculation. BAL cells were stained and used for flow cytometry characterization and enumeration with antibodies against (A) B220+ cells (B) DX5+ cells, (C) CD3+ cells, and (D) CD8+ cells. The data are expressed as the number of BAL/lung ( ± SEM). A representative experiment from three independent experiments is shown. Asterisks indicate a significant difference (P < 0.05) between isotype control-treated and antibody-treated mice as calculated with a Student’s T test. The arrows indicate the time of treatment.

Lung histopathology following therapeutic treatment was also evaluated in the different experimental groups at day 5 post-challenge and scored as indicated in Materials and Methods and shown in Table 2. Pathological abnormalities were described by perivascular inflammatory infiltrates composed of lymphocytes, histiocytes, and/or plasma cells. No perivascular inflammatory infiltration was observed in mock-infected mice with a score of 0.02 (Fig. 5A). In mice treated with nIg after RSV inoculation, a high magnification photomicrograph of a small pulmonary vein (V) in the lungs showed extensive and severe perivascular infiltrate (Fig. 5B). Additionally, there was a focal inflammatory infiltrate underneath the venular endothelium. The inflammatory infiltrate was mononuclear (lymphocytes, plasma cells, and lesser numbers of histiocytes). In mice treated with anti-RSV G protein mAbs 3D3 and 2B11 after inoculation with RSV A2, milder infiltration was observed (Fig. 5C and D, respectively). Despite the neutralizing activity of Palivizumab, treatment of mice after inoculation with RSV A2 did not significantly reduce the inflammation in the lungs (score = 0.45) (Fig. 5E) (Table 2). These mice exhibited and severe perivascular infiltrate, similar to nIg treated mice. Similar to human anti-RSV G mAbs treatment, high magnification photomicrograph of a small pulmonary vein (V) in mice treated with murine mAb, 131–2G, showed a mild perivascular infiltrate (Fig. 5F).

Table 2.

Lung histopathology score (LHS) at 5 days post infection.

Treatment Number of anatomical structures evaluated Cumulative pathological abnormalities Pathological score
Mock-infected 50 1 0.02
Isotype 77 30 0.38
3D3 58 10 0.17
2B11 59 6 0.10
Anti-F mAb 77 35 0.45
131-2G 77 8 0.11
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BALB/c mice (4–5 mice/group) were challenged with RSV A2 strain virus, and treated on day 3 post challenge with infected with normal immunoglobulin G (nIg), anti-RSV G 3D3 or 2B11 mAb, or mAb anti-RSV F (Palivizumab) as described in methods section. Number of anatomical structures defined by perivascular inflammatory infiltrates (composed of lymphocytes, and/or histiocytes, and/or plasma cells) were evaluated on day 5 post challenged, and cumulative pathological abnormalities were scored. The pathologic score was determined by dividing the cumulative pathological abnormalities by the total number of anatomical structures evaluated. The results are representative of two independent experiments.

Fig. 5. Treatment with 3D3 & 2B11 mAbs reduces inflammation in the lungs of infected mice.

Fig. 5.

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Lung histopathology was examined by H&E staining in (A) mock-infected mice or mice infected with RSV A2 and then treated with (B) isotype control antibody, (C) anti-G mAb 3D3, (D) anti-G mAb 2B11 (E) anti-F mAb Palivizumab, or (F) anti-G mAb 131-2G. The arrows indicate areas of inflammatory infiltrates. There are lymphocytes and plasma cells (solid black arrow) around a venule (V). 200× original magnification, scale bar = 50 μm.

4. Discussion

Of the two major surface proteins of RSV, the F protein has most often been the focus of both vaccine and antiviral drug development. The F protein is more effective at inducing neutralizing antibodies and cross-protective immunity than the G protein. Recent studies have shown that some anti-G mAbs neutralize RSV in primary cell cultures in addition to reducing lung inflammation in small animal models (Boyoglu-Barnum et al., 2015a,b; Chirkova et al., 2015; Cortjens et al., 2017; Johnson et al., 2015). The potential added value of an anti-inflammatory effect suggests that anti-RSV G protein mAb may be more efficient in treating established infection than just preventing virus replication as suggested by anti-RSV F protein mAbs (Palivizumab and Motavizumab).

In this study, we examined the in vivo effectiveness of two novel human anti-G mAbs in reducing the inflammatory response to RSV infection prophylactically and therapeutically. These anti-RSV G protein mAbs were derived from B cells of RSV-infected adults using a high-throughput single-cell phenotyping technology, CellSpot. These two mAbs react, at least partially, within the 13 a.a sequences of G protein that are conserved among all RSV strains. There still may be portions of the epitopes either linear or conformational that lie outside the conserved peptide. The affinity of the selected Abs was comparable or superior to the best murine hybridoma mAbs recognizing a similar target epitope (Collarini et al., 2009).

The results of this study and two other studies (Boyoglu-Barnum et al., 2015a,b; Han et al., 2014) support the added value of the anti-inflammatory and neutralizing effects of these anti-G protein mAbs. This study with human mAb 2B11 and earlier studies, with mouse mAb 131–2G and human mAb 3D3, show that all three effectively treated active infection as indicated by decreased lung viral titers, BAL cell number, airway reactivity, and Th2-type cytokine production. In contrast, a Palivizumab-like mouse mAb (143–6C) or Palivizumab itself did not decrease disease when delivered post-inoculation. These findings are consistent with the clinical studies that Palivizumab is not effective for treating active human RSV infection (Mejías et al., 2005; Mejías and Ramilo, 2008; Piedimonte et al., 2000).

Previous studies have demonstrated that when administered three days post-inoculation, mAb 3D3 was more potent in reducing lower respiratory tract infection as well as virus lung titers than anti-F mAbs (Collarini et al., 2009; Han et al., 2014). Our results here confirm that a new human-derived anti-G mAb, 2B11, also reduces lower respiratory tract infection and viral lung inflammation, even when delivered three days post-inoculation. Both 3D3 and 2B11 mAbs bind similar epitopes in the CCR of G protein, adjacent the CX3CR1 binding region, and have been shown to neutralize virus in primary cell-culture systems (Collarini et al., 2009). Blocking RSV G - CX3CR1 interactions inhibits RSV infection of differentiated human airway epithelial cells (Johnson et al., 2015). In mice, treatment with either anti- G or anti-CX3CRl mAbs reduced G-mediated lung inflammation, indicating neutralization may be contributing to the anti-inflammatory nature of anti-G mAbs (Haynes et al., 2009; Tripp et al., 2003). These combined anti-inflammatory activities, and effectiveness when delivered post-inoculation suggest anti-G mAbs may be strong candidates for development as anti-RSV therapies.

Acknowledgements

The authors would like to thank Dr. Tamas Nagy (University of Georgia) for histopathological evaluation and Dr Azaibi Tamin for his critical revision of the manuscript. Financial support was provided by Trellis Biosciences through the CDC Foundation, in collaboration with the Centers for Disease Control and Prevention.

Funding statement

The funding was supported by a grant from Trellis Biosciences through the CDC Foundation.

Footnotes

Potential conflicts of interest

HC, CM, LH, and NJT do not have potential conflicts of interest.

LA has done paid consultancies on RSV vaccines for MedImmune, Novartis Vaccines and Therapeutics, Crucell, and AVC.

LA is on scientific advisory boards for Moderna Therapeutics and Bavarian Nordic.

LA’s laboratory has received funding for a study of RSV treatment in mice through a grant to Emory University from Trellis RSV Holdings.

LA and RT are co-inventors on patents held by the CDC on RSV vaccines and treatment that include mutations in the CX3C motif in the RSV G proteins for live RSV vaccines.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.antiviral.2018.04.014.

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