Target cell availability, rather than breast milk factors, dictates mother-to-infant transmission of SIV in sooty mangabeys and rhesus macaques

Abstract

Mother-to-infant transmission (MTIT) of HIV is a serious global health concern, with over 300,000 children newly infected in 2011. SIV infection of rhesus macaques (RMs) results in similar rates of MTIT to that of HIV in humans. In contrast, SIV infection of sooty mangabeys (SMs) rarely results in MTIT. The mechanisms underlying protection from MTIT in SMs are unknown. In this study we tested the hypotheses that breast milk factors and/or target cell availability dictate the rate of MTIT in RMs (transmitters) and SMs (non-transmitters). We measured viral loads (cell-free and cell-associated), levels of immune mediators, and the ability to inhibit SIV infection in vitro in milk obtained from lactating RMs and SMs. In addition, we assessed the levels of target cells (CD4+CCR5+ T cells) in gastrointestinal and lymphoid tissues, including those relevant to breastfeeding transmission, as well as peripheral blood from uninfected RM and SM infants. We found that frequently-transmitting RMs did not have higher levels of cell-free or cell-associated viral loads in milk compared to rarely-transmitting SMs. Milk from both RMs and SMs moderately inhibited in vitro SIV infection, and presence of the examined immune mediators in these two species did not readily explain the differential rates of transmission. Importantly, we found that the percentage of CD4+CCR5+ T cells was significantly lower in all tissues in infant SMs as compared to infant RMs despite robust levels of CD4+ T cell proliferation in both species. The difference between the frequently-transmitting RMs and rarely-transmitting SMs was most pronounced in CD4+ memory T cells in the spleen, jejunum, and colon as well as in central and effector memory CD4+ T cells in the peripheral blood. We propose that limited availability of SIV target cells in infant SMs represents a key evolutionary adaptation to reduce the risk of MTIT in SIV-infected SMs.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. Similar cell-free and cell-associated SIV levels in blood and milk from RMs and SMs following pharmacologic induction of lactation.

Lactation was induced using intramuscular injections of estradiol and medroxyprogesterone along with oral haloperidol followed by oxytocin injections to stimulate the milk ejection reflex. Over the 20 weeks of this study, plasma viral loads were measured via SIV RNA real time RT-PCR every 2 weeks (A). Breast milk was collected from RMs and SMs and cell-free SIV RNA was quantified in the de-fatted milk fraction by RT-PCR (B). Cell-associated SIV DNA was then measured in both PBMCs (C) and breast milk cells (D) with simultaneous albumin detection to determine cell number per reaction. Dashed lines represented the lower limit of detection for the assay. Undetectable values were plotted as half the lower limit of detection. Area under the curve analyses followed by nonparametric Mann-Whitney test was used to assess significance.

Figure 2. Memory and effector CD4+ and CD8+ T cell subsets in SM milk.

The cellular fraction of breast milk was stained with monoclonal antibodies to identify the relative proportions of T cell subsets. Gating strategy was as follows: CD45 vs CD3 to identify hematopoietic T cells, then CD20 vs CD14 to exclude B cells and monocytes/macrophages, respectively, then CD4 vs CD8. The CD4+ and CD8+ populations were stained with CD28 and CD95 to identify naïve (CD28+CD95−) and memory (CD28+/−CD95+) subsets. The memory population was further differentiated into effector (CCR7−) and central (CCR7+) memory subsets.

Figure 3. Inhibition of SIV infection by milk from RMs and SMs.

The inhibitory capacity of serial dilutions of de-fatted breast milk from eight RMs and seven SMs against SIVsmm subtype 1 (FFv18NOV04PLENV2.1), SIVsmm subtype 2 (FWk12AUG04ENV2.1), SIVmac251.6 (neutralizing antibody-sensitive), or SIVmac251.CS (neutralizing antibody-resistant) viral envelopes was determined in the Tzm-bl neutralization assay. Comparisons between RMs and SMs were made at the dilutions where inhibition was seen. Adjusted against wells including no milk, percent viral infectivity in the presence of 1∶10 diluted (A) or 1∶50 diluted (B) milk is depicted on the vertical axis. Horizontal lines depict the median of each group. A nonparametric Mann-Whitney test was used to determine significance.

Figure 4. T cell differentiation and proliferation in infant SM and RM tissues.

(A) The proportion of naïve (CD28+CD95−) and memory (CD28+/−CD95+) CD4+ T cells in lymphoid and gastrointestinal tissues of infant SMs and RMs was measured at necropsy. For each tissue site, the total may not add to 100% because of minor populations excluded in gating. (B) The level of cell activation/proliferation was measured in CD4+ T cells isolated from multiple tissues of infant RMs and SMs using the marker Ki-67. Mann-Whitney test was used to determine significance. Comparisons without p value shown were non-significant.

Figure 5. Few CD4+CCR5+ SIV target cells present in infant SMs.

(A) The proportion of CD4+CCR5+ SIV target cells was measured by flow cytometry in total and memory CD4+ T cells isolated from lymphoid and gastrointestinal tissues of infant SMs (red) and RMs (black) at necropsy. Representative flow plots showing CCR5 expression on effector memory (CD28+CD95+CCR7−) and central memory (CD28+CD95+CCR7+) CD4+ T cells from the spleen, mesenteric LN, and jejunum from infant SMs (B) and RMs (C). (D) The percentage of CD4+CCR5+ T cells among the naïve (CD28+CD95−CCR7+), central memory (CD28+/−CD95+CCR7+), and effector memory (CD28+/−CD95+CCR7−) subsets in the peripheral blood of infant SMs (red) and RMs (black). Mann-Whitney test was used to determine significance. * p

Figure 6. RM, but not SM, infants upregulate CCR5 expression on oral and upper gastrointestinal tract tissues early in life.

The level of proliferation (Ki-67+) and SIV coreceptor expression (CCR5+) on CD4+ T cells isolated from the buccal mucosa, esophagus, and tonsils of representative SM (A) and RM (B) infants. CCR5 levels on CD4+ T cells remain low at these sites in SMs beyond three months of age, but are elevated before one month of age in RMs. DOL  =  day of life.

Figure 7. Alternative coreceptor expression in infants SMs and RMs.

RNA expression of the alternative coreceptors GPR15 and CXCR6 was measured in sorted CD4+ T cells from infant SMs and RMs by real time PCR. CD4+ T cells were negatively selected by MACS microbeads and purity was assessed by flow cytometry (>80% for all animals). Mann Whitney test was used to assess significance.