We generated full-length viable env
clones from 5 mother-infant pairs and extensively characterized their V1-V5 genotypes and phylogeny. Phylogenetic analyses showed that infant sequences were more homogeneous than maternal viral sequences. The highest sequence diversity seen in the infants, 0.3%, fits well with the model of Keele et al
], which indicates that the maximum diversity expected within an individual shortly after infection with a single virus is 0.6%. Of the 8 transmitted/founder variants identified in the infants of our cohort, seven represented minor variants of the maternal quasispecies at the time of sampling, which was within a few weeks of transmission. These data support previous findings [13
] suggesting a selective bottleneck during MTCT.
Consensus gp160 endpoint dilution sequences from two randomly selected infants were identical to those obtained by SGA. These results are compatible with a recent report that standard PCR and SGA provide similar measures of viral diversity when sufficient templates are analyzed [30
Several groups have reported shorter hypervariable regions and fewer N-linked glycosylation sites in Clade C sexually (reviewed [31
]) or maternally [32
] transmitted viruses. Our data on Clade B viruses are compatible with others' work that did not find altered env
length or glycosylation site number in transmitted Clade B viruses [33
All infant clones were R5 tropic, consistent with numerous prior reports [34
transmission of HIV-1 is hypothesized to occur across the mucosa, although the exact mechanisms have not been determined (reviewed [37
]). Efficient HIV-1 infection usually requires the expression of relatively high levels of the CD4 receptor and CCR5 co-receptor on the surface of target cells [21
]. However, levels of CD4 and CCR5 on mucosal and submucosal cell subsets can be much lower than on CD4+
memory T cells [40
]. Titration on cell lines expressing different levels of CD4 and CCR5 demonstrated efficient infection of cells with variable levels of these molecules. This finding is supported by our failure to observe any systematic differences in the sensitivity of maternal and infant env
to inhibition by sCD4 or CCR5 inhibitors. It is also in agreement with a recent report that sexual HIV-1 transmission does not appear to select for viruses that can preferentially utilize lower levels of CD4 or CCR5 [39
CCR5 co-receptor usage has traditionally been equated with macrophage-tropism. Peters et al
. have recently clarified that not all R5 viruses are macrophage-tropic (reviewed [41
]). Only 1 of 35 plasma-derived env
clones achieved greater than 1% of their TZMbl titers on MDM. Our results are in agreement with prior data [42
] demonstrating that peripheral blood viruses frequently exhibit low levels of macrophage infectivity, and that sexually transmitted R5 tropic variants replicate poorly in macrophages [43
]. Finally, these findings support recent models of HIV-1 transmission, which suggest that cell subtypes other than macrophages are the first to encounter HIV-1 during mucosal transmission [44
We screened our clones to determine their sensitivity to neutralization by a panel of well-characterized monoclonal Nabs. Sensitivity to these NAbs varied both between and within mother-infant pairs. Clones were uniformly sensitive to 2F5. Only one clone (from infant P1046) was resistant to 4E10; this clone exhibited the F673L natural polymorphism associated with resistance to this Nab [24
]. All clones with 2G12 resistance correlated with loss of one of five PNGS that make up the 2G12 epitope.
At least two groups have reported that infant env
clones are relatively resistant to neutralization by autologous maternal plasma [15
]. Relatively high levels (IC50
≥ 100 μg/ml) of autologous maternal plasma IgG were required to neutralize maternal and infant viruses; however, all infant viruses were neutralized by pooled sera from HIV-1 infected individuals, implying that they were not inherently neutralization-resistant.
CCR5 antagonists are a potent new class of entry inhibitors. Since only R5 variants are vertically transmitted, CCR5 antagonists may be highly relevant to blocking MTCT; however their effectiveness against infant isolates has not been well characterized, and partial resistance to CCR5 antagonists in a treatment-naïve individual has been reported [28
]. All env
clones in our panel were sensitive to Maraviroc. All infant clones were also sensitive to T20 and sCD4, and no significant differences in sensitivity were seen between maternal and infant viruses. The latter is in contrast with data from Keele et al
], who demonstrated significantly higher IC50
values for T1249, a fusion inhibitor with a mechanism of action similar to T20, among viruses from acutely infected as compared to chronically infected subjects.