Abstract
Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in infants. RSV repeatedly reinfects individuals: this may be due in part to the variability of the attachment (G) glycoprotein and changes in this protein have been shown to be under positive selection. Infants experiencing their primary infection show a genotype-specific antibody response with respect to the variable regions of the G protein. A prospective study of RSV infections in a birth cohort in rural Kenya identified infants experiencing repeat infections with RSV. The serum antibody responses of these infants were investigated with respect to their anti-RSV reactions in an enzyme-linked immunosorbent assay (ELISA) and the specificity of the response to a variable region of the G protein by ELISA and immuno-blotting using bacterially expressed polypeptides representative of the currently circulating strains of RSV. The results presented here confirm that the primary antibody response to the variable regions of the G protein is generally genotype-specific, but show that the response may become cross-reactive (at least within group A viruses) during secondary infections even where the secondary infection is of the same genotype as the initial infection. Also, some infants who did not mount a detectable antibody response to whole RSV antigens during their primary infection nevertheless showed genotype-specific responses to the G protein. In conclusion, the strain-specific nature of the serum antibody response to the variable regions of the G protein of RSV observed in primary infections can become cross-reactive in subsequent reinfections.
Keywords: respiratory syncytial virus, reinfection, antibody responses
Introduction
Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract disease particularly in infants and young children, but also in the elderly. An unusual characteristic of the virus is that it can repeatedly reinfect individuals although second and subsequent infections are usually less severe than the primary infection [Henderson et al., 1979; Glezen et al., 1986]. The ability of the virus to reinfect may be due to an inadequate immune response and/or to strain variability allowing evasion of the immune response. The observation that there appears to be positive selection acting on the attachment (G) glycoprotein resulting in antigenic drift implies that strain variability at least contributes to the ability of the virus to survive at the community level [Cane and Pringle, 1995; Melero et al., 1997].
Since infants are seldom hospitalised during their second and subsequent infections, there have been few studies analysing the nature of the virus in each infection. It has been reported that RSV reinfection in infants is more likely to occur with a different group of RSV, although the numbers examined were small [Mufson et al., 1987]. However, it has also been shown that reinfections can occur with the same group of RSV as that of the previous infection [Mufson et al., 1987; Sullender et al., 1998; Sato et al., 2005; Scott et al., 2006; Broor et al., 2007]. Hall et al. [1991] showed that it was possible to repeatedly reinfect some, but not all, adult volunteers with the same strain of virus.
More recently, detailed molecular analyses have been carried out on the viruses found to be causing reinfections in infants. Parveen et al. [2006] looked at seven children and found that in five children their reinfections were caused by a heterologous group or genotype of the virus. In contrast, two children were reinfected with very similar viruses implying that reinfection with closely related viruses was possible. Likewise it has been shown that in eight children with clear reinfections, they were reinfected with either very similar viruses or viruses of different groups [Scott et al., 2006]. These studies are complicated by the well-described phenomenon of replacement of the predominant strain of circulating RSV in successive epidemics (reviewed in Cane [2007]), thus limiting the potential for reinfection with an identical strain of the virus in subsequent years.
A study of a cohort of children following their primary and subsequent infections with RSV has been previously described [Nokes et al., 2004; Scott et al., 2004, 2006]. It has been demonstrated by several groups that the antibody response in primary infections to the variable regions of the G protein is often specific to the infecting strain [Cane et al., 1996; Cane, 1997; Palomo et al., 2000; Jones et al., 2002; McGill et al., 2004]. This report extends these observations with analyses of the strain specificity of the serum antibody responses to a variable region of the G protein observed in the children experiencing reinfections with RSV.
Materials and Methods
Study Population and Samples
The patients studied in this report have been previously described [Nokes et al., 2004; Scott et al., 2006]. Briefly, a cohort of 338 babies was recruited at or close to birth and then intensively monitored for RSV infections. Ethical approval for this study was provided by the Kenya Medical Research Institute, the National Review Committee in Kenya, and the Coventry, United Kingdom, Research Ethics Committee. Serum samples were obtained at regular intervals of approximately 3 months, and also in the acute and convalescent phases following diagnosis of RSV by immunofluorescence testing of nasal washings. RSV infections were characterised with respect to their G gene sequences as previously described [Scott et al., 2004]. Ten out of 12 patients described in Scott et al. [2006] provided at least two serum samples which are analysed further here. Table I summarises the molecular characterisation of the infecting viruses of these reinfections.
Table I. Summary of Primary and Secondary RSV infections Observed in Patients Included in This Study [Adapted From Scott et al., 2006].
| Patient designation | Primary infection group and genotypea | Secondary infection group and genotypea | Comment |
|---|---|---|---|
| Ken01 | A1 | A1 | Probable persistent infection |
| Ken02 | A2 | A2 | Similar virus in two epidemics |
| Ken03 | A2 | A2 | Similar virus in two epidemics |
| Ken04 | A2 | A1 | Reinfected in same epidemic |
| Ken05 | A1 | B1 | Different group infection in two epidemics |
| Ken07 | A2 | A2 | Probable persistent infection |
| Ken09 | A1 | A2 | Reinfected in same epidemic |
| Ken10 | A2 | A2 | Probable persistent infection |
| Ken11 | A1 | B1 | Different group infection in two epidemics |
| Ken12 | A2 | B2 with G gene duplication | Different group infection in two epidemics |
Accession numbers and relatedness of strains are described in Scott et al. [2006]. Genotype designations are as in Scott et al. [2004].
Detection of Anti-RSV Antibodies
The enzyme-linked immunosorbent assay (ELISA) used for the detection of RSV-specific IgG in oral fluid was adapted from Wilson et al. [2000] with some modifications. RSV laboratory strain A2 was used to infect Hep 2 cells. Following harvesting of lysate from both mock and infected cells, the resulting supernatant was sonicated (Sonics & Materials, Inc., Newton, CT) at 70% amplitude, 3 x 1 min cycles with 1 sec pulse and 1 sec rest. The lysates were then vortexed before coating triplicate wells of Nunc-Immuno™ 96-well MaxiSorp plates (Fisher Scientific, Leicestershire, UK) with 25 μl of lysate diluted 1/32 in PBS coating buffer. The plates were dried overnight at 37°C in a rotating incubator. They were then blocked with 200 μl/well of 5% dried milk (Marvel) in phosphate buffered saline (PBS) and incubated for 1 hr at 37°C. Serum samples and controls were diluted 1/100 in dried milk in PBS. Resultant test optical density (OD) readings were adjusted for mock antigen results. The rest of the assay was carried out as previously described by Wilson et al. [2000].
Generation of Fusion Proteins Expressing the Carboxy-Terminal Region of the G Protein with Glutathione-S-Transferase (GST)
Groups A and B and RSV strains representative of the viruses detected in the epidemics under study were selected for expression of the carboxy-terminal region of the G protein as GST fusion proteins. The appropriate region was amplified by PCR from the BG10-F1 RT-PCR products (nucleotide 154 of the G gene to nucleotide 22 of the F gene) as described in Scott et al. [2004, 2006]. Amplification of group A samples was undertaken using Primer 1 [Cane et al., 1996] and F1GST, 5′-(GGAATTCGTCGAC)AACTCCATTGTTATTTGC3′, corresponding to the end of the intergenic region between G and F genes and the beginning of the F gene. Amplification of group B strains was done using the primers BG8, 5′-(GCGGATCCG)AGACCCCAAAACAC-CAGCC-3′, and F1GST. Amplification of a B strain with the 60 nucleotide duplication detected in one patient [Scott et al., 2006] was undertaken using primers BG8(alt), 5′-(GCGGATCCG)AGACCCAAAAACGC-TAACC-3′, corresponding to the equivalent region of the G gene to BG8 above, but specific for these new strains, and F1GST. The sequences in parentheses indicate restriction site sequences added to the end of the primers to facilitate cloning.
The PCR products were cloned into Bluescribe (Vector Cloning Systems, San Diego, CA) and subcloned into pGEX-5X-1 (Amersham Pharmacia, Amersham, UK) using BamHI and SalI restriction sites as previously described [Cane et al., 1996]. Sequencing of the pGEX-5X-1-RSV constructs was done to confirm that no errors had been introduced. Large-scale expression of the fusion proteins was undertaken in BL21 Escherichia coli cells by growing 500 ml cultures for 2 hr followed by induction of protein expression with IPTG for 3 hr. The GST-fusion proteins from the cultures were then extracted, using B-PER Bacterial Protein Extraction Reagent (in phosphate buffer; Pierce Biosciences, Cranlington, UK) according to the manufacturer’s instructions. The extracted fusion proteins were then purified by affinity chromatography with glutathione sepharose (Amersham Pharmacia) and expression of the G gene products checked as previously described [Cane et al., 1996] with the addition that for GST fusion proteins derived from group B viruses, the primary antibody used for detection was an anti-GST antiserum (Amersham Biosciences). The designations and derivations of the GST fusion proteins are shown in Table II.
Table II. Designations and Derivations of G Gene-GST Fusion Proteins.
| Designation | Strain number (accession number) | Group and genotypea |
|---|---|---|
| A | Ken7/02 (AY524655) | A1 |
| B | Ken16/01 (AY524602) | A1 |
| C | Ken163/02 (AY524607) | A1 |
| D | Ken6/02 (AY524650) | A2 |
| E | Ken8/01 (AY524658) | A2 |
| F | Ken164/02 (AY524608) | A2 |
| G | Ken2/03 (AY660684) | B1 |
| H | Ken29/03 (AY660681) | B2 with duplication |
| J | Ken1/02 (AY524580) | A2 |
Genotype designations are as in Scott et al. [2004].
Immunoblotting
The purified GST fusion proteins were separated on 12% SDS–polyacrylamide gels to determine the appropriate dilution resulting in approximately equal amounts of protein for use in immunoblot analysis, using anti-GST antibody (Amersham Pharmacia) as primary antibody followed by protein G horseradish peroxidase (HRP) conjugate. Group A fusion proteins were also tested with a monoclonal antibody (021/9G) kindly provided by Jose Melero (Institut de Salud Carlos III, Madrid). The proteins were then diluted to give equivalent concentrations and used in immunoblots as previously described [Cane et al., 1996] for reaction with the infant sera.
Enzyme-Linked Immunosorbent Assay (ELISA)
Purified fusion proteins were tested using anti-GST antibody and protein G HRP conjugate to determine the optimum dilution levels for each of them prior to use. Subsequently, the sera were tested against the GST-fusion proteins by ELISA as previously described [Cane et al., 1996].
Results
Adequate serum samples were available from 10 of the 12 patients previously described [Scott et al., 2006]. These serum samples were tested for their reactions with whole RSV A2 antigens by ELISA, and with the GST fusion proteins expressing the carboxy-terminal region of the G proteins from a range of strains representing the circulating RSV variants during the epidemics under study by both ELISA and immunoblotting. For the strain-specific immunoassays, ELISA usefully provides a continuous numerical readout, but non-specific reactions can be a major problem particularly with sera from older infants (Cane, unpublished observations), while the immunoblotting allows confidence in the specificity of the reactions but interpretation is subjective. Results from the ELISAs using recombinant RSV G-GST fusion proteins are shown in Figure 1 and results from immunoblotting are given in Table III.
Fig. 1.
Antibody responses showed as optical densities determined by ELISA to recombinant RSV G-GST fusion proteins. Panels a–j show results from Ken01 to Ken12, respectively (omitting Ken06 and Ken08). The designations (A–H) on the x-axis indicate the derivation of the G-GST fusion protein as summarised in Table II. The individual bars are from serum samples at different ages (in days).
Table III. Reactions of Infant Sera in Immunoblots With Carboxy Terminal G Proteins Expressed as GST Fusion Proteins.
| Infant | Age (days) when serum sample taken | Days post-diagnosis of infections | Infecting virus genotype (age at diagnosis) | Reaction with recombinant fusion proteinsa | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| A1 | A2 | B | |||||||||
| A | B | C | D | J | G | H | GST | ||||
| KenOl | 92 | 2 | A1 (90) | – | – | – | – | – | – | – | – |
| 125 | 7, 35b | A1 (118) | +++ | +++ | +++ | – | – | – | – | – | |
| Ken02 | 126 | 7 | A2 (119) | + | ++ | + | +++ | ++ | – | – | – |
| 150 | 31 | + | + | + | +++ | ++ | – | – | – | ||
| 345 | 1, 226 | A2 (344) | + | + | + | ++ | + | – | – | – | |
| 381 | 37, 262 | +++ | +++ | +++ | +++ | +++ | – | – | – | ||
| Ken03 | 55 | 3 | A2 (52) | – | – | – | – | – | – | – | – |
| 84 | 32 | +/− | +/− | +/− | ++ | + | – | – | – | ||
| 331 | 3, 279 | A2 (328) | ++ | ++ | +++ | +++ | ++ | – | – | – | |
| 363 | 35, 311 | ++ | ++ | +++ | +++ | +++ | – | – | – | ||
| Ken04 | 83 | 3 | A2 (80) | +/− | +/− | + | +++ | + | – | – | – |
| 124 | 2, 44 | A1 (122) | + | + | +/− | +++ | ++ | – | – | – | |
| 152 | 30, 72 | ++ | ++ | + | +++ | ++ | – | – | – | ||
| Ken05 | 54 | 2 | A1 (52) | +/− | +/− | +/− | – | – | – | – | – |
| 97 | 45 | ++ | ++ | ++ | – | – | – | – | – | ||
| 312 | 0, 260 | B1 (312) | – | +/− | +/− | – | – | – | – | – | |
| 544 | 228, 492 | – | – | – | – | – | – | – | |||
| Ken07 | 69 | 1 | A2 (68) | +/− | +/− | +/− | +/− | – | – | – | – |
| 97 | 5, 29 | A2 (92) | +/− | +/− | +/− | ++ | + | – | – | – | |
| 128 | 36, 60 | ++ | ++ | ++ | +++ | ++ | – | – | – | ||
| Ken09 | 32 | 7 | A1 (25) | ++ | ++ | ++ | – | – | – | – | – |
| 141 | 4, 116 | A2 (137) | ++ | ++ | ++ | + | + | – | – | – | |
| Ken 10 | 81 | 6 | A2 (75) | – | – | – | + | – | – | – | – |
| 97 | 3, 23 | A2 (94) | + | + | + | ++ | + | – | – | – | |
| 107 | 13, 32 | ++ | ++ | + | +++ | ++ | – | – | – | ||
| 124 | 30, 49 | ++ | ++ | + | +++ | ++ | – | – | – | ||
| Ken 11 | 88 | 4 | A1 (84) | +/− | +/− | – | – | – | – | – | – |
| 124 | 40 | +++ | +++ | +++ | + | + | – | – | – | ||
| 322 | 1, 238 | B1 (321) | + | + | + | – | – | – | – | – | |
| 472 | 151, 388 | +/− | +/− | +/− | +/− | +/− | – | +++ | – | ||
| Ken 12 | 40 | 1 | A2 (39) | + | + | + | – | – | – | – | – |
| 72 | 33 | + | + | + | +++ | +++ | – | – | – | ||
| 331 | 5, 292 | B-red (326) | ++ | + | + | +++ | + | + | ++ | – | |
| 359 | 33, 320 | – | – | – | + | – | – | ++ | – | ||
Group and genotype designations as shown in Table II.
Where two values are given, these are days after the second and first diagnosis of RSV infection, respectively.
Genotype-Specific Responses
Patients Ken01, Ken07 and Ken10 appeared to have persistent infections, as previously reported [Scott et al., 2006]. Ken01 was infected with genotype A1 virus and Ken07 and Ken10 with genotype A2 type virus. In the case of Ken01, sera up to day 35 post-primary infection only were available for strain-specific antibody determinations. By immunoblotting, the convalescent serum showed a clear strong response to the homologous genotype (Table III), with no reaction to the heterologous group A or group B derived proteins. By ELISA, the greatest change in reaction was with the homologous protein but there was some cross-reaction with the other proteins (Fig. 1a). Ken07 and Ken10 (infected with genotype A2 virus) showed similar reactions, with specific reactions to the genotype A2 fusion proteins initially. However, with time, this reaction became broader showing reactivity with other group A fusion proteins, though not with the group B derived proteins. This initial specific reaction with subsequent broadening was also shown in the ELISA determinations (Fig. 1f,h).
Two patients (Ken02 and Ken03) were infected with genotype A2 type virus in two separate epidemics. By immunoblotting both patients showed a genotype-specific response initially, but this then broadened to the other group A genotype, while no reaction was seen with the B group fusion proteins (Table III). This was also reflected in the ELISA results (Fig. 1b,c).
Two patients showed repeat infections within the same epidemic, both with group A strains but different genotypes. Patient Ken04 was infected with genotype A2 virus then A1, while patient Ken09 was infected with genotype A1 then A2. By immunoblotting patient Ken04 showed a strong antibody reaction with genotype A2 derived protein even in the acute serum, and then developed a cross-reactive response with respect to all of the group A derived proteins following the second infection. These observations were less clear in the ELISAs due to background reactions with the antigens in this assay (Fig. 1d), illustrating the value of the more specific immunoblot assay for some patient samples. Patient Ken09 showed reaction mainly with genotype A1 derived protein and then this broadened to include genotype A2 following the second infection. The ELISAs for this patient gave a similar result to the immunoblotting for the group A derived antigens but also some cross-reactivity with the group B derived antigens which was not seen in the immunoblots (Fig. 1g).
The remaining three patients (Ken05, Ken11 and Ken12) were infected with a group A virus in the first epidemic and then group B in the second. Patient Ken05 was initially infected with A1 genotype and showed a specific response by immunoblotting to the homologous antigen. Unfortunately, there was no suitable convalescent serum available for the second infection and a serum sample drawn 7 months after the second infection showed no reaction with any of the test antigens. Patient Ken11 was also initially infected with genotype A1 virus and showed a strong specific reaction in the immuno-blots to antigen derived from this genotype. Following infection with a group B virus, the patient’s antiserum reacted strongly with a group B derived antigen, but showed little reaction with the group A antigens. Both these patients showed cross-reactive, probably nonspecific reactions with most of the antigens in the ELISA tests, with little change between the samples. Patient Ken12 was infected first with genotype A2 virus followed by a group B virus containing a duplication in the region of the G gene examined in these assays. As observed with the other patients infected with genotype A2 virus, this patient showed a strong specific reaction to the homologous antigens following both infections as seen in both the immunoblots and ELISAs. This patient like-wise showed a reaction with the group B derived protein following the second infection. Of note, the second infection convalescent sera from patients Ken11 and Ken12 did not show increased reaction to the group A antigens previously reacted with following their infection with the group B viruses, unlike the apparent recall reactions seen when the second infection was with a distinct group A genotype (patients Ken04 and Ken09; Table III and Fig. 1e,i,j).
Overall Anti-RSV Antibody Responses
The antibody response to whole RSV A2 antigen was determined by ELISA. The decay of maternal antibody and baby anti-RSV antibody responses following infection will be described elsewhere for the entire birth cohort (Ochola et al., in preparation). In most cases for the infants described here there was insufficient serum for antibody tests at all time points. However, interesting data were obtained for two of the reinfected patients (Fig. 2). Ken03 (infected with genotype A2 in two epidemics) showed a steady decline in anti-RSV antibody levels from age 55 to 252 days, although a specific response to the G protein fusion proteins was observed with a convalescent serum taken 32 days post-infection. When this patient experienced a second infection at age 328 days, there was an immediate high increase in reaction to both anti-RSV and genotype-specific antigens. In fact this secondary response was so rapid that the first serum taken 3 days after the reinfection diagnosis, already showed high levels of reaction (Fig. 2a).
Fig. 2. Antibody responses with time (age in days) determined by ELISA to whole RSV antigens derived from laboratory strain A2 for patients Ken03 (panel a) and Ken12 (panel b).
Ken12 (infected with genotype A2 virus followed by a B group virus in the next epidemic) showed only a slight increase in anti-RSV levels in the first convalescent sample taken at 33 days but a decline by 46 days following the first infection, despite a very strong genotype-specific response. As for Ken03, there was a rapid increase in the response following the second infection (Fig. 2b).
Discussion
This article describes the strain specificity of anti-G protein antibody responses in 10 infant patients who experienced primary and secondary infections with RSV. As previously described for group A infections, there were some strong genotype-specific antibody responses after primary infections with the variable regions of the G protein [Cane et al., 1996; Cane, 1997; Jones et al., 2002]. This report extends these observations to include infections with group B viruses, although only secondary infections with group B viruses were observed in this study. Where acute sera from primary infections in some patients (Ken02, Ken04 and Ken09) already showed some strain-specific antibody reactions, it may be that these were derived from persisting maternal antibodies.
With respect to the group A infections, both persistent and secondary, the initially specific antibody response appeared to broaden with time to include reactions with recombinant proteins derived from both of the genotypes of group A RSV examined in this study. This extension of the reactivity of the infant sera occurred even where the reinfection was with the same genotype as the primary infection. It is not clear whether this broadening of the spectrum of epitopes recognised by the antibody response is a maturation of the primary response and whether the mechanism in prolonged infections is the same as in reinfection with the same strain. The recall of antibodies observed in the primary infection during a secondary infection with a different strain may in part be reminiscent of the phenomenon of “antigenic sin” which has been described for influenza virus, namely, the strain-specific serological response to earlier influenza strains after infection or vaccination with later variants, irrespective of whether one or more antigenic shifts had occurred during the observation period of the study [Haaheim, 2003]. Whether the development of cross-reacting antisera described here is due to the appearance of further genotype-specific antibodies or due to the development of antibodies cross-reactive between the genotypes, as has been described for influenza virus [Masurel and Drescher, 1976], remains to be determined. However, the broadening of the antibody response observed here was confined to group A variants and did not extend to the group B derived recombinant proteins.
The repeated demonstration that the variable regions of the G protein are under positive selection [Cane and Pringle, 1995; Melero et al., 1997; Martinez et al., 1999; Woelk and Holmes, 2001; Zlateva et al., 2004, 2005] poses the question as to the selective pressure for such evolution. If specific antibodies are seen only in primary infections, then this would suggest that the selective pressure for change of the G protein acts during such primary infections as has also been suggested for influenza virus [Lambkin et al., 1994]. Alternatively, the selective pressure may act early during secondary infection enabling virus escape before the cross-reacting response is evoked. It is also possible that antibodies to different epitopes vary in their ability to inhibit virus replication. The antibodies to variable domains may act to limit virus infection and drive virus evolution in both primary and secondary infections although in the latter poorly inhibitory antibodies to conserved domains may also be present giving the appearance of a cross-reactive response in binding assays. Also, this study analysed the reactions only of strain-specific antibodies that reacted with epitopes shown on non-glycosylated bacterially expressed polypeptides. It has previously been shown that carbohydrate side chains can influence the expression of these epitopes [Palomo et al., 2000], and thus the assays reported here have examined only part of the antibody repertoire. A further additional factor could be that serum antibodies are not entirely reflective of mucosal antibodies which are the front line of defence against infection [McIntosh et al., 1978]. It has been proposed that the greater susceptibility of the upper respiratory tract compared with the lungs may be related to antibody specificity or avidity differences between the sites [Taylor, 2007].
It is known that there is sometimes a poor antibody response to RSV during primary infection in young infants and this may in part be due to the presence of maternal antibody inhibiting antibody responses, but not T cell responses, as has been shown in animal models [Siegrist et al., 1998; Crowe et al., 2001]. It is interesting to note that two patients in this study (Ken03 and Ken 12) showed little effect in their anti-RSV antibody response but nevertheless demonstrated some (Ken03) or considerable (Ken12) response to the G derived recombinant proteins. However, this needs to be interpreted in the context of the antibodies reacting with the whole RSV antigen (laboratory strain A2) would be those recognising conserved antigens only, not strain-specific epitopes.
In conclusion, this study confirms that infants mount genotype-specific serum antibody responses to a variable region of the RSV G protein during primary infection, but subsequent infections induce a response with wider reactivity, at least within the group A viruses. The role of these antibody responses in modulating subsequent infections in the individual and at the population level remains to be determined.
Acknowledgments
We thank the study volunteers for their generous participation. We are indebted to the outpatient clinic field workers and clinical officers, paediatric ward, maternity and MCHC staff, and senior hospital staff from Kilifi District Hospital for support and cooperation in the running of this study. The manuscript is published with permission of the Director of the Kenya Medical Research Institute.
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