Longitudinal changes in viral genetic variation, immune responses, and disease progression have rarely been investigated in HIV-1 subtype C infected children. We have previously characterized the evolution of the Env C2-V4 region of subtype C HIV-1 and the humoral immune response from one infected infant. In the present study, we expanded our study by correlating the changes of the Env longitudinally with disease outcome, in seven children, divided into two groups based on rapid or slow disease progression. In addition, with these two groups, we were able to examine the contribution of Env length and glycosylation in disease progression, and the role of humoral immunity, both passively acquired and developed de novo, to clinical outcomes.
Phylogenetic analyses show that maternal and infant viruses were epidemiologically linked in each of the seven pairs, and support the concept that selective transmission occurred [33, 59–61]. Rapid progressors, those who died in the first 12 months, received and maintained a genetically homogeneous viral population throughout the short disease course. Slow progressors initially also exhibited low levels of variation, but attained higher levels of diversity over time. These findings are consistent with previous studies that showed higher genetic diversity associated with slow disease progression in children [33, 53, 54].
In both groups of children, a large number of unique, but closely related haplotypes were sampled, matching predictions for a population that was exponentially growing in size from a homogeneous starting point. Estimates of dN/dS can be used to determine whether selective pressure, in addition to expanding population size, played a role in the diversification of the infant viral populations. Our data show that dN/dS values were high in all 7 individuals, exceeding 1.0 in 7 of 24 populations sampled. Values of dN/dS greater than 1.0 provide evidence of positive Dawinian selection .
One of the primary selective pressures acting on Env is neutralizing antibody. The earliest infant Nab responses are largely due to passive transfer from the mother. Passively acquired maternal immunity can play a critical role in protecting infants from infections; however, the specific contribution of maternal or passively-acquired neutralizing antibodies in limiting HIV-1 transmission or disease progression in children is not well understood. Our observations indicate that the neutralizing activity in maternal and infant baseline plasma varied in its effectiveness for the initial infant virus but did not differentiate rapid from slow progressors. Since our assays for Nab activity relied on co-cultured virus, and selection during co-culture may bias the results away from the main phenotype of virus in the original population, the lack of difference between groups should be taken as a tentative result. Nevertheless, consistent with other findings [33, 63, 64], all children developed de novo neutralizing responses within the first 6 months post-infection regardless of the disease course. But our results show that even when children develop effective de novo neutralization responses, they may still progress rapidly (Figure 4A). In contrast, we also observed children who failed to mount high neutralizing responses to later virus, yet have remained clinically asymptomatic throughout the study (Figure 4B). These findings indicate that the development of effective neutralizing responses in children fails to protect them from disease progression, but surprisingly, failure to develop effective responses is not predictive of rapid progression. Moreover, there is no association between the replication kinetics and disease progression, since viral isolates isolated from similar time points (4–8 month) from both rapid and slow progressors replicated with similar pattern (Figure 5A and 5B), even though the late viruses from slow progressors replicated slightly faster than early viruses from the same hosts (Figure 5B). Similarly, our study did not reveal any differences in cytopathicity of the viruses from either progressors or non-progressors from different time points, suggesting a lack of correlation between viral cytopathicity and disease progression among the viruses that were analyzed.
The genotypic and phenotypic parameters leading to preferential transmission of particular virus variants from donor to recipient remain unclear. In heterosexual transmission between discordant couples, it was found that subtype C viruses with shorter V1–V4 regions and fewer putative glycans were preferentially transmitted and were neutralization sensitive [57, 58]. In addition, another study of heterosexually acquired subtype A viruses suggested that transmitted viruses have shorter V1–V2 length and few N-linked glycosylation sites . An extension of these findings is that evolution in the newly infected individual would lead to longer and more glycosylated Env proteins with time. These patterns have not been confirmed in subtype B sexual transmission [65–67]. The genotypic and phenotypic parameters leading to preferential transmission of particular virus variants were also evaluated in mother to child transmission. An investigation of subtype A mother to child transmission has revealed that the transmitted viruses were more resistant to neutralization by maternal plasma although the viruses harbored fewer putative glycosylation sites . In our study, we have observed that both neutralization sensitive and resistant viruses were transmitted to both slow and rapid progressors. It is worth noting that contrasting results between sexual transmission and vertical transmission studies could be due to fundamental differences between these processes, since vertical transmission occurs in the presence of neutralizing antibodies, but in sexual transmission there are presumed to be no baseline antibodies present.
It has been hypothesized that the extensive glycosylation of the HIV-1 Env shields the protein from immunological recognition, or conversely, targets recognition to less functionally constrained domains where hypervariability can be tolerated . Interestingly, neither pattern was confirmed with later viruses in our infant samples, suggesting that lengthening of the V1–V5 domain and acquisition of glycosylation sites were not always a component of glycoprotein evolution in newly infected individuals (Figure 3 and Table 1). Only in one case (infant 1084), a pattern consistent with this hypothesis was obtained, with increasing V1–V5 length and number of PNGS (Figures 3). Collectively, our results highlight the necessity to refine our understanding of the relationships between viral genotype, viral phenotype and different routes of transmission. Our observations and those of others also stress the need to further explore genetic and immunologic correlates of mother to child transmission in non-B subtypes.
Comparison of the rates of non-synonymous and synonymous substitutions has been used as an index of selective pressure exerted by the immune system [20, 55, 69]. There are reports that higher dN/dS ratios are linked with long-term survival [20, 55]; however, we found that the highest dN/dS value was estimated for envelopes from a rapid progressor child at the final timepoint prior to death (Table 1, 8-month sample from infant 1449). In addition, dN/dS values were highly variable in both groups and not statistically different. Despite the variation in dN/dS values, the estimates were high in all cases, suggesting that natural selection is a strong determinant of the diversification and evolution in the Env glycoprotein. Further evidence of this selective pressure comes from the observation that amino acid replacements are not evenly distributed in the protein sequence, but occur in 'hot-spots' in particular domains (Figure 2). We can predict two broad mechanistic explanations for these changes; (1) they modulate glycoprotein function thus enhancing viral fitness (currently under investigation), (2) they modulate immune recognition of the viral glycoprotein by altering epitopes. Despite differences in timing of sampling, or in ultimate disease outcome, some hot spots are shared among all children, and no hot spot differentiates the rapid from the slow progressors. One example of these common hot-spots is the region in C3 just carboxy-terminal to the V3 loop. Structurally this domain corresponds to alpha helix 2 from the alignment of HXBC2 to the intact SIV atomic structure . This sequence, which is perpetually changing, is located on the silent face of the trimeric structure as determined for subtype B. The clustering of polymorphisms as well as the differential binding of antibodies from subtype B versus C infected individuals to this region points to this as a good candidate for a subtype C neutralizing epitope.
Genetic assessment of variation, as indicated by dN/dS, shows that the protein is undergoing selective changes in a non-random fashion. Nevertheless, the magnitude and distribution of these changes do not segregate slow and rapid progressors. In addition, the apparent clustering of positively selected residues in particular regions of the subtype C glycoprotein that are normally not subject to high levels of mutation in subtype B implies significant structural differences between the glycoprotein of the two subtypes.
The sole parameter that discriminates rapid and slow progressors appears to be time to death. We found that neither genetic variation in env, nor maternal neutralizing activity at parturition, nor the level of passively acquired neutralizing antibody, nor the level of the de novo neutralization response was linked to differences in disease progression in the children studied. However, characteristics of the transmitted viruses other than Nab and the genetic make up of viral populations could be linked to disease progression. Such factors might include, but are not limited to, the fitness of the transmitted viruses, binding affinity to CD4 and/or co-receptors, and cell mediated immunity. The potential role of these factors in predicting disease outcome in subtype C HIV-1 infected children will be the focus of further investigations.