Mutating the CX3C Motif in the G Protein Should Make a Live Respiratory Syncytial Virus Vaccine Safer and More Effective

Abstract

Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. Through a CX3C chemokine motif ( 182 CWAIC 186 ) in the G protein, RSV binds to the corresponding chemokine receptor, CX3CR1. Since RSV binding to CX3CR1 contributes to disease pathogenesis, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A 186 , within the CX3C motif, mutating it to CX4C ( 182 CWAIAC 187 ), which is known to block binding to CX3CR1, might decrease disease. We studied the effect of the CX4C mutation in two strains of RSV (A2 and r19F) in a mouse challenge model. We included RSV r19F because it induces mucus production and airway resistance, two manifestations of RSV infection in humans, in mice. Compared to wild-type (wt) virus, mice infected with CX4C had a 0.7 to 1.2 log 10 -fold lower virus titer in the lung at 5 days postinfection (p.i.) and had markedly reduced weight loss, pulmonary inflammatory cell infiltration, mucus production, and airway resistance after challenge. This decrease in disease was not dependent on decrease in virus replication but did correspond to a decrease in pulmonary Th2 and inflammatory cytokines. Mice infected with CX4C viruses also had higher antibody titers and a Th1-biased T cell memory response at 75 days p.i. These results suggest that the CX4C mutation in the G protein could improve the safety and efficacy of a live attenuated RSV vaccine.IMPORTANCE RSV binds to the corresponding chemokine receptor, CX3CR1, through a CX3C chemokine motif ( 182 CWAIC 186 ) in the G protein. RSV binding to CX3CR1 contributes to disease pathogenesis; therefore, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A 186 , within the CX3C motif, mutating it to CX4C ( 182 CWAIAC 187 ), known to block binding to CX3CR1, might decrease disease. The effect of this mutation and treatment with the F(ab=) 2 form of the anti-RSV G 131-2G monoclonal antibody (MAb) show that mutating the CX3C motif to CX4C blocks much of the disease and immune modulation associated with the G protein and should improve the safety and efficacy of a live attenuated RSV vaccine.

Effects of Alterations to the CX3C Motif and Secreted Form of Human Respiratory Syncytial Virus (RSV) G Protein on Immune Responses to a Parainfluenza Virus Vector Expressing the RSV G Protein

“…1C) (39), and G_wCX4C has the insertion of an alanine residue between positions 185 and 186 that disrupts the spacing of the motif (Fig. 1C) (30). Constructs viii and ix (G_dCX3C_B3CT and G_dCX3C_B3TMCT) have the C186R mutation in combination with the B3CT and B3TMCT substitutions.…”

“…The presumed CT, TM, and ectodomains are demarcated with dashed lines. Sequences from RSV G and BPIV3 (Continued on next page) ablation of the CX3C domain and/or the expression of sG would be expected to reduce the chemotactic influx of immune cells and might reduce disease, and this is supported by several studies with mutant RSVs (30,31), although there also are contradictory data (32). Other effects of G also have been described.…”

Structures of respiratory syncytial virus G antigen bound to broadly neutralizing antibodies

“…RSV with the G gene deleted is highly attenuated in vivo (19). Moreover, RSV with an insertion in the CX3C motif of G (CX4C) that prevents CX3CR1 binding has markedly reduced disease severity in vivo (20). In a recent study, elevated concentrations of both anti-G and anti-pre-fusion-F antibodies were associated with lower clinical disease severity scores, despite the substantially lower absolute abundance of anti-G antibodies compared to anti-F antibodies (7).…”

Conformational Flexibility in Respiratory Syncytial Virus G Neutralizing Epitopes

“…In addition, treatment with anti-RSV G mAbs reduces bronchoalveolar lavage (BAL) fluid cell influx, including RSV G protein-induced leukocyte migration and eosinophilic inflammatory responses, resulting in decreased airway congestion (15,33,42). Anti-G mAbs have also been shown to reduce mucus production and to restore beneficial antiviral alpha interferon (IFN-␣) (18,(42)(43)(44). Most of the anti-G bnmAbs that have been studied to date bind with high affinity to RSV G (K D [binding dissociation constant] ϭ 1.1 pM to 3.3 nM) and bind to linear epitopes within the RSV G CCD as determined by linear epitope mapping techniques (17,21,40,45).…”