To study the humoral response against the central unglycosylated region of the RSV G protein, we screened sera from RSV-infected adults for reactogenicity against recombinant GST-RSV G fusion proteins. Our approach utilized rational partitioning of the RSV G unglycosylated central residues into the PCC, DCC, and the entire CC region; in contrast, previous reports have utilized overlapping peptides or bacterially derived hypervariable G domains with or without the unglycosylated core as screening reagents to determine RSV G domain-specific serological responses [4
For each of the six RSV G derivatives (PCC, DCC, and CC encoded by RSV A2 and B1 strains), we observed robust IgG titers in acute sera and in a homo- and heterosubtypic-specific manner. This is not unexpected since adults in this study would have experienced a number of RSV subtype A and B infections during their lifetimes [2
]. In convalescent sera, we noted statistically significant titer increases against the homosubtypic and heterosubtypic RSV G PCC and CC domains. The subtype-independent reactogenicity against the PCC and CC regions may primarily be due to the canonical aa sequence HFEVFNFVPCSIC (residues 164–176; ) that overlaps most of the PCC residues and is embedded within the CC [3
]. Our observations are also consistent with the fact that anti-RSV G MAbs with cognate epitopes that overlap residues 164–176 typically recognize RSV G in a subtype-independent manner [13
Interestingly, convalescent sera from subtype A-infected adults responded weakly to B1-derived DCC and subtype B-infected adults responded minimally to A2-derived DCC, i.e. there is homosubtype-specific serum reactogenicity against the RSV G DCC subdomain. It has previously been reported that peptides bearing RSV A2 or B1 strain-derived G residues 158–189 were recognized in a subtype-specific manner in paired sera from pediatric patients with primary RSV infection [7
]. Our results with adult convalescent sera confirm and extend these results by defining the potential residues that may be responsible for such subtype-specific targets of immune response to those within the DCC.
Between the RSV A2- and B1-derived DCC, we note the following six residue differences: A2: CSICSNNPTCWAICKRIP vs. B1: CSICG
IP, in which differences at positions 177, 180, 181, 183, 184, and 188 are underlined and in bold. Based on comparative analysis of reference RSV G sequences, the RSV A2 and B1 DCC are likely to be representative of subtype A and B-specific residues 173–190 of the RSV G protein ([19
]Murata, et al., unpublished observations). The existence of subtype-specific differences within the DCC subdomain may be evolutionarily conserved since bovine RSV (BRSV) G residues 180, 182, and 184 of strain 391-2 (subtype A) (CSTCEGNLACLSLCHIET) differ from those of BOV-X (subtype B) (CSTCEGNP
; differences underlined and in bold) [7
]. We conclude that ≥ 1 of the six residue differences between subtype A and B viral strains confers homosubtype specificity of acute to convalescent serum anti-RSV G DCC reactogenicity increases in adult RSV infections. Furthermore, our analysis of corresponding BRSV DCC residues implies the existence of similar DCC homosubtype-specific humoral response in BRSV infections.
Our results are relevant to the structural and functional aspects of the RSV G DCC region, which is not essential for RSV replication in vitro or in vivo but elicits robust immunomodulatory effects, e.g. CX3C motif-mediated cellular trafficking [14
]. Our results suggest that the presence of cysteines is required for the antigenic/structural integrity of DCC-embedded epitopes since the substitution of all four cysteine residues with serine rendered the resulting DCCmut moiety non-reactogenic. Particularly notable is our observation that for both RSV and BRSV, the subtype-specific residue differences at residues 183 and 184, i.e. between C182 and C186, involve the CX3C motif; for BRSV G protein, mutations of residues 183 and 184 are predicted to have major structural consequences of the antigenic surface of the G “loop” region [25
]. Taken together, these observations raise the possibility of viral subtype-specific, evolutionarily conserved immunomodulatory effects of the RSV DCC subdomain, including the CX3C homology region.
Our data also have implications for clinical course and severity of disease among RSV-infected adults. For subtype A-infected adults, there were greater and statistically significant increases in A2 PCC- and DCC-specific convalescent titers among hospitalized patients as compared to those of subjects diagnosed with RSV as outpatients; a similar but non-statistically significant (p = 0.08) trend was also observed for A2 CC domain (). Among subtype B-infected adults, outpatients bore higher anti-B1 PCC titers than did inpatients at the time of diagnosis (i.e. acute sera) and hospitalized adults bore higher anti- A2 and –B1 CC convalescent titers than did outpatients. Thus for RSV-infected adults, hospitalization may be correlated with greater burden of disease, i.e. elevated viral loads, and subsequent IgG increases in convalescent sera titers against RSV G CC and its subdomains [27
]. For RSV subtype B-infected adults, elevated acute titers against the RSV G PCC may correlate with severity of illness at diagnosis and may potentially be utilized as serological correlates of disease severity. We acknowledge the limitation that the number of non-hospitalized subtype-B infected adults was relatively small (n=8) and thus the clinical utility of such serological correlates requires additional studies.
Lastly, our results have implications for immunological function of RSV G and potential epitopes for host adaptive humoral response and vaccine development. The RSV G CC domain contains immunologically important structural and functional motifs, including: 1) aa 184–198, thought to be involved in CD4+ T cell-mediated eosinophila; 2) canonically conserved residues + G “loop” that collectively have been associated with broad anti-inflammatory effects; and 3) the presence of a human HLA DP4-restricted CD4+ T-cell epitope (minimum epitope residues defined as either 163–171 or 162–175) [28
]. Adult and pediatric subjects who were either naturally infected with RSV or received live attenuated RSV vaccine candidate generated IgG response that inhibited RSV G-mediated leukocyte chemotaxis and RSV G-CXC3 receptor interactions [14
]. To enhance such RSV G-specific immune response, particularly against potential subtype-specific immunomodulatory effects, it may be necessary to engineer the RSV G “loop” region as DCC or the entire CC domain from both subtype A and B viruses for potential vaccine epitopes. As an alternative immunogen, the RSV G PCC from either subtype A or B virus strain may be sufficient to elicit viral subtype-independent antibody production and protective efficacy. Such possibilities require empirical testing in animal immunogenicity studies.