Restriction of HIV-1 genotypes in breast milk does not account for the population transmission genetic bottleneck that occurs following transmission

Abstract

Background: Breast milk transmission of HIV-1 remains a major route of pediatric infection. Defining the characteristics of viral variants to which breastfeeding infants are exposed is important for understanding the genetic bottleneck that occurs in the majority of mother-to-child transmissions. The blood-milk epithelial barrier markedly restricts the quantity of HIV-1 in breast milk, even in the absence of antiretroviral drugs. The basis of this restriction and the genetic relationship between breast milk and blood variants are not well established.

Methodology/principal findings: We compared 356 HIV-1 subtype C gp160 envelope (env) gene sequences from the plasma and breast milk of 13 breastfeeding women. A trend towards lower viral population diversity and divergence in breast milk was observed, potentially indicative of clonal expansion within the breast. No differences in potential N-linked glycosylation site numbers or in gp160 variable loop amino acid lengths were identified. Genetic compartmentalization was evident in only one out of six subjects in whom contemporaneously obtained samples were studied. However, in samples that were collected 10 or more days apart, six of seven subjects were classified as having compartmentalized viral populations, highlighting the necessity of contemporaneous sampling for genetic compartmentalization studies. We found evidence of CXCR4 co-receptor using viruses in breast milk and blood in nine out of the thirteen subjects, but no evidence of preferential localization of these variants in either tissue.

Conclusions/significance: Despite marked restriction of HIV-1 quantities in milk, our data indicate intermixing of virus between blood and breast milk. Thus, we found no evidence that a restriction in viral genotype diversity in breast milk accounts for the genetic bottleneck observed following transmission. In addition, our results highlight the rapidity of HIV-1 env evolution and the importance of sample timing in analyses of gene flow.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Plasma and breast milk viral load.

Viral loads determined by Roche Amplicor (PL) and Roche Amplicor Ultrasensitive (BM) assays. Gray lines are means.

Figure 2. Maximum likelihood trees of region gp160, PL and BM samples obtained contemporaneously.

All trees were calculated under the GTR+G+I model, rooted with 4 subtype C reference sequences obtained from LANL sequence database. In all subjects, HIV-1 RNA sequences from the left breast (white circles) and from the right breast (gray circles) were intermixed. The scale at the bottom left of each tree corresponds to the number of substitutions per site (for example, 0.01 = 1 substitution per 100 sites).

Figure 3. Maximum likelihood trees of region gp160, PL samples obtained previous to BM samples.

All trees were calculated under the GTR+G+I model, rooted with 4 subtype C reference sequences obtained from LANL sequence database. In all subjects, HIV-1 RNA sequences from the left breast (white circles) and from the right breast (gray circles) were intermixed. The scale at the bottom left of each tree corresponds to the number of substitutions per site (for example, 0.01 = 1 substitution per 100 sites).

Figure 4. Correlation between sampling interval and measures of compartmentalization.

(A) Snn score vs. sampling interval; Snn scores close to 1 indicate segregated populations, while scores close to 0.5 indicate mixed populations. (B) Simmonds Associative Index (AI) vs. sampling interval; the AI is based on a grouping score (weighted by position in the tree) in which higher AI  =  less grouping of sequences from same tissue in the tree. (C and D) Generalized correlation coefficient rb and r vs. sampling interval; rb and r offer a way to correlate the number of internal nodes (rb) or branch length (r) between two sequences in a tree with the information about whether or not they were isolated from the same compartment. P-values from Spearman's Rho tests indicate significant linear correlations in A, B, and C.

Figure 5. Diversity and divergence in breast milk and plasma.

(A) Comparing mean diversity of virus in breast milk and plasma HIV-1 RNA gp160 sequences of contemporaneously sampled subjects. Triangles  =  plasma, circles  =  breast milk; star  =  significant difference between plasma and breast milk diversity, p-values from Wilcoxon rank sums test. (B) Comparing diversity in breast milk and plasma in all contemporaneously sampled patients in aggregate, using Gilbert, Rossini, and Shankarappa's method for comparing intra-individual genetic sequence diversity between populations. Black lines are means. (C) Comparison of mean divergence of virus in BM and PL within contemporaneously sampled subjects. P-values from Wilcoxon rank sums test. (D) Comparison of viral divergence between the BM and PL of all contemporaneously sampled subjects in aggregate. P-value obtained using a GEE model. Black lines are means.

Figure 6. Percentage of sequences in plasma and breast milk predicted to have a syncytium-inducing (SI) phenotype.

Prediction made by the Web PSSM from the V3 amino acid sequence. No SI sequences were predicted from the V3 loops in Subjects 31, 32, 17, and 3.