The study presents results on RSV molecular epidemiology in a birth cohort of 635 children closely monitored over 4 RSV epidemic seasons to identify patterns of infection in relation to previous exposure. Previously, a number of population-level studies have observed that the RSV groups that predominate epidemics regularly oscillate [9
], whereas the predominant genotypes are often replaced in consecutive seasons [13
]. These observations have led to the hypothesis that RSV groups/genotypes create homologous herd immunity after an epidemic, favoring heterologous variants to circulate in a subsequent season [9
This study investigated whether individuals previously infected with a particular RSV variant are more likely to be reinfected with a heterologous than with a homologous variant, despite the prevailing group or genotype prevalence. We characterized 53 repeat infections from an intensively monitored cohort and determined that 28 of these were of the heterologous group and 25 were of the homologous group. Among the homologous group reinfections, only 10 were with a homologous genotype, 5 of which showed limited amino acid changes in the fragment of the G-gene that we sequenced.
Therefore, overall, 80% of the reinfections that we characterized from the cohort were heterologous at group or genotype level. To understand the epidemiological context in which these reinfections occurred, we genotyped virus strains from children in whom we identified only one antigen-confirmed infection during the study period. Comparing this sample of background circulating variants with the reinfecting variants by epidemic, we found no difference in the genotype distributions (Table ). This observation raises the question of whether the observed heterologous group/genotype reinfections were influenced by the children's previous exposures or reflected the prevailing distribution of genotypes in the community.
After examining the reinfections patterns by epidemic, the hypothesis of stronger homologous group immunity, compared with heterologous group immunity, was supported by data from the 2004 epidemic but not from the 2004–2005 epidemic (Table ). Of note, the 2004–2005 epidemic was the first epidemic in Kilifi that was predominated by the newly recognized group B genotype virus strains (BA) that possess a 60-nucleotide duplication in the G-gene and that have replaced previously circulating group B strains worldwide [37
]. Paradoxically, immunological studies in our laboratory that focused on strain-specific immunity found that infant convalescent serum samples from children infected with the SAB1 strains neutralize to the same level as the strains with the 60-nucleotide duplication [44
Ten (~20%) of 53 of the reinfections that we observed were due to a homologous genotype. Five of these possessed amino acid sequences differences between the pairs, which were few in number (<3), except for one that had 12 amino acid changes. The immunological significance of such limited sequence differences is uncertain, particularly in understanding whether this contributed to the reinfection process. However, some of the substitutions occurred in known antigenic epitope domains. For instance, changes affecting N-glycosylation pattern (Ken/4123) and protein length (Ken 3912) are both known to have a profound effect on antigenicity [38
]. However, in animal immunization studies, Sullender et al showed that recombinant G proteins that are up to 15% different in amino acid sequence give similar protection against homologous and heterologous isolates in intra-group challenge [25
This observation of homologous group or genotype reinfection raises the question of the completeness and durability of RSV strain-specific immune responses [4
]. The interval between the infections for the group B homologous genotype reinfections (Ken/3757 and Ken/3912) exceeded a year. Using data derived from this cohort, we have recently shown that, after infection, immunity to reinfection appears to last up to 6 months and is only 60%–70% effective [40
]. Thus, it is possible that previous-exposure immune responses in some of these children waned to below protective levels. There was also a significant interval between the infections in Ken/4123 (21.7 months) and Ken/3793 (9.4 months) homologous genotype group A reinfections. Individual Ken/3793 provided a clear example of a reinfection with an identical virus in the G-gene occurring in a subsequent epidemic.
For the repeat positive samples of the same group (A) and genotype, separated by a short interval (<2 months; some, 21–24 days) and with limited or no genetic differences, it was not possible to be definitive on whether these were reinfections or persistent single infections. Although we did not determine whole genome sequences, the portion that we sequenced is the most variable in RSV genomes and the protein product of the G-gene is of most immunological significance together with the F protein. Partial sequencing of the F gene for some of these pairs did not find any differences (Supplementary Figure 3
). Previous studies have shown that the mean duration of RSV shedding in infants and young children is 7 days (range, 1–21 days; reviewed in [41
]), and prolonged shedding occurs mostly in individuals with impaired immunity [42
] or cardiopulmonary disease [43
]. Our examination of the records on the baseline characteristics of these children did not find presence of a predisposing condition to prolonged shedding, such as very low birth weight, malnutrition, or congenital disease.
With regard to disease severity, pneumonia occurred in 32% of the reinfections. We did not observe a statistically significant higher risk of pneumonia after heterologous group reinfection, compared with homologous group reinfection.
The sampling and testing methods of the present study undoubtedly missed some infections and reinfections in the cohort, thus presenting a possible bias [40
]. Ideally, sampling should have been more frequent, irrespective of symptoms, to capture possible subclinical cases, and screening could have been done using the more sensitive molecular methods. Although our study presents data from a considerably increased sample size, compared with previous studies on RSV reinfection, the observed numbers in the individual epidemics were insufficient for conclusive interpretation.
In conclusion, our study shows that the vast majority of RSV reinfections in nature are with genetically distinct strains and that repeat infections with variants identical in the amino acid sequence of the G protein to the primary infecting variant can occur. The direct role of strain-specific immunity at individual level in reinfections remains unclear, because we observed that the distribution of the genotypes in the reinfection cases mirrored the distribution of genotypes observed at the same time in the wider study population.