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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Infect Dis. Author manuscript; available in PMC Jun 27, 2012.
Published in final edited form as:
PMCID: PMC3384735
NIHMSID: NIHMS359241

Association of Levels of HIV-1–Infected Breast Milk Cells and Risk of Mother-to-Child Transmission

Abstract

Understanding how the level of human immunodeficiency virus type 1 (HIV-1)–infected breast milk cells (BMCs) affects HIV transmission via breast-feeding can shed light on the mechanism of infection and aid in establishing effective interventions. The proportion of infected cells to total cells was measured in serial breast milk samples collected from 291 HIV-1–infected women in Nairobi, Kenya, by use of real-time DNA polymerase chain reaction amplification of BMCs. The number of infected BMCs per million cells was associated with levels of cell-free viral RNA in breast milk (R = .144; P = .032), levels of cell-free virus in blood plasma (R = .365; P < .001), and the detection of proviral DNA in cervical and vaginal secretions (P < .001 and P = .030, respectively). The number of infected BMCs per million cells was lower in colostrum or early milk than in mature milk (P < .001). Previous studies demonstrated that the concentration of BMCs varies throughout lactation, and we used these data to transform infected BMCs per million cells to infected BMCs per milliliter. The estimated concentration of infected BMCs per milliliter was higher in colostrum or early milk than in mature milk (P < .001). Each log10 increase in infected BMCs per milliliter was associated with a 3.19-fold–increased risk of transmission (P = 002), after adjustment for cell-free virus in plasma (hazard ratio [HR], 2.09; P = 03) and breast milk (HR, 1.01; P = 1.00). This suggests that infected BMCs may play a more important role in transmission of HIV via breast-feeding than does cell-free virus.

More than one-third of all mother-to-child transmission of HIV-1 in breast-feeding populations is estimated to occur via breast milk [1]. The exact mechanisms of this transmission are unknown. Although both cell-free virus and infected cells have been identified in breast milk [26], their respective roles in infant infection remain undefined. In vitro, studies suggest that infection of gastrointestinal cells and mucosal cells can be initiated by both cell-free virus and infected cells (reviewed in [7]). For example, several reports indicate that epithelial cells from the upper gastrointestinal tract mucosa can selectively take up and transfer both cell-free virus and cell-associated virus to target cells in vitro [812]. In rhesus macaque models, it has been demonstrated that both cell-free and cell-associated virus can be transmitted via both oral and genital mucosa [1316]. It is difficult to know how well these model systems mimic the transmission process in humans and whether the results are applicable to transmission of HIV-1 in humans via breast-feeding.

Breast milk contains many inhibitors to viral replication, such as secretory leukocyte protease inhibitor, chemokines, and lactoferrin [1719]. Secretory leukocyte protease inhibitor has been shown to inhibit infection of lymphocytes by cell-free virus in culture but not to inhibit cell-associated transcytosis of HIV-1 [20]. Chemokines and lactoferrin have been postulated to prevent viral infection by blocking virus-to-cell binding (reviewed in [21, 22]). These data suggest that these factors may inactivate some of the cell-free virions present in breast milk. Thus, infected cells may play a more significant role than does cell-free virus in transmission via breast-feeding. However, to date there have been few published data reporting the levels of HIV-1–infected breast milk cells (BMCs) and their association with HIV-1 transmission relative to the levels of cell-free virus in breast milk.

To develop effective interventions to prevent transmission of HIV-1 via breast-feeding, the relationship of the level of virus in breast milk to transmission needs to be clearly defined. Studies of cell-free virus in breast milk have demonstrated that the concentration of cell-free virus is associated with HIV-1 transmission, the level of virus in maternal plasma, cell-associated virus in genital secretions, and breast disease [3, 5, 23]. Also, cell-free HIV-1 RNA concentrations in breast milk have been found to be highest early after delivery, a time when transmission via breast milk may be high [1, 3, 2427]. Understanding how the level of cell-associated virus in breast milk correlates with the level of cell-free virus in breast milk, with virus levels throughout the body, and with mother-to-child transmission may shed light on the mechanism of transmission via breast-feeding and the relationship between virus levels in different body compartments.

To address the above questions, infected BMCs and total cells in breast milk were quantified in women enrolled in a randomized clinical trial of transmission of HIV-1 via breast-feeding in Nairobi, Kenya, by means of real-time polymerase chain reaction (PCR) amplification methods. The relationship between these measurements and the risk of mother-to-child transmission, concentrations of cell-free virus in breast milk, virus levels in blood and genital secretions, and CD4 T cell count was assessed.

SUBJECTS, MATERIALS, AND METHODS

Subjects

The subjects for the present study participated in a randomized clinical trial conducted between 1992 and 1998, which investigated HIV-1 transmission by infected mothers, both breast-feeding and formula-feeding, in Nairobi, Kenya [1]. The methods for enrollment, counseling, randomization, and sample collection (including blood, breast milk, and cervical and vaginal swabs) have been described elsewhere [1, 2, 28, 29]. Breast milk samples were scheduled to be collected within the first week after delivery and then at 6 and 14 weeks, 6 months, and quarterly thereafter up to 2 years or to the cessation of breast-feeding, whichever occurred first [1]. Data on maternal plasma virus level (HIV-1 RNA copies per milliliter), CD4 and CD8 T cell counts (per microliter), the detection of infected cells in maternal genital secretions, and HIV-1 infection in the infant were available [1, 2830]. Infants with a positive result for HIV-1 by PCR from a sample collected at birth (either cord blood or peripheral blood mononuclear cells) were presumed to be infected in utero. These infants were excluded from analyses related to transmission via breast milk. Although it is difficult to discriminate transmission during delivery from transmission during the early breast-feeding window, all infants with a positive result for HIV-1 DNA by PCR at any subsequent visit were included in the analyses of transmission, because our previous studies showed that approximately one-half of infant infection during the first 6 weeks after delivery was attributable to transmission via breast-feeding in this cohort [1].

Informed consent was obtained from all study participants, and the human-experimentation guidelines of the US Department of Health and Human Services were followed. The present study was approved by the institutional review boards of the University of Washington, the Fred Hutchinson Cancer Research Center, the University of Nairobi, and the Ministry of Health of Kenya.

Real-time PCR

BMC pellets were prepared as described elsewhere [2]. Cellular DNA was extracted from the BMC pellets by use of Qiagen Midi kits. DNA was similarly extracted from ACH2 cell controls. ACH2 is a cell line that was initially infected with HIV-1 at an MOI of 1 infectious unit/cell [31]. This cell line has very low levels of expression of HIV-1 and is thought to maintain a single provirus per cell. For this reason, ACH2 cells were used as controls for quantification of virus.

HIV-1 provirus copies were quantified by real-time PCR am-plification [32], with primers and a probe specific for regions of the p31 integrase domain of polymerase (pol), a region conserved among Kenyan isolates [33]. The forward primer (5′-TACAGTGCAGGGGAAAGAATA-3′) corresponded to nt 4809–4829 of HXB2, the reverse primer (5′-CTGCCCCTTCACCTT-TCC-3′) to nt 4957–4974, and the probe sequence (5′-TTTCGGGTTTATTACAGGGACAGCAG-3′) to nt 4896–4922. Tenfold serial dilutions of a full-length HIV-1 clone, Q23-17 [34], ranging from 1 to 106 copies/reaction, were used to define a standard curve to quantify HIV-1 provirus. To keep the total DNA concentration constant in each standard, plasmid DNA was diluted in 1 ng/mL herring sperm DNA. Both herring sperm DNA alone and water were used as negative controls. Three cloned plasmids of HIV-1 subtypes A, D, and B (both A and D are frequent in this Kenyan population [29]) were amplified by this method. Each of these templates amplified with similar kinetics (data not shown), indicating that this assay is likely to amplify these subtypes equally. Twenty microliters of cellular DNA template was added to each PCR amplification in a total volume of 100 μL. This assay cannot distinguish between a single cell with many copies of HIV-1 provirus and several cells infected with 1 provirus each. For simplicity of calculation, each HIV-1 provirus copy detected with this assay was assumed to represent a single infected cell.

β-Actin was quantified with primers and probe from Taqman β-actin Detection Reagents (Perkin-Elmer). Tenfold serial dilutions of human genomic DNA (β-actin detection reagent; Perkin-Elmer), ranging from a total of 100 to 0.01 ng per reaction (equivalent to 16,650–1.67 cellular genomes), were used to generate a standard curve to quantify the number of copies of the β-actin gene. One to ten microliters of genomic DNA template was added to each β-actin amplification in a total reaction volume of 50 μL.

The amplification reactions included 150 μmol/L MgCl2, 200 μmol/L dNTPs, 1 μmol/L each primer, 1 μmol/L probe, 2.5 U of Surestart Taq, and recommended quantities of core buffer and dye (Brilliant Quantitative PCR Core Reagent Kit; Strategene). The cycling parameters (50°C for 2 min, 95°C for 10 min, 1 cycle; 95°C for 10 s, 60°C for 1 min, 40 cycles) were established on the basis of recommendations by the ABI Prism 7700 Sequence Detection System (Perkin-Elmer). All amplifications were performed in duplicate.

Real-time assay validation

Several control experiments were conducted to determine the inter- and intra-assay variability. Two dilutions of ACH2 cellular DNA were amplified 36 times each within a single assay plate with the β-actin primers and probe. This set of amplifications was repeated in a total of 6 replicate assays. The maximum inter-assay variation among the resulting β-actin concentrations was 1.5-fold. The maximum intra-assay variation was 2.2-fold. Any duplicate sample whose β-actin concentration varied by more than the maximum-fold-observed intra-assay variation (rounded up to the next whole integer, or 3-fold) was reanalyzed in duplicate. If the repeated assays were within the intra-assay variation, these values were used in the analysis. Otherwise, the assay was repeated until the duplicate values were within the intra-assay variation, and only the final set of duplicate values was used in the analysis.

ACH2 cells were combined with uninfected cells (CEMx174) to create a panel of samples with the same total cell concentration (6 × 103/μL) and a range of HIV-1 copies, from 1 to 100 copies/μL [35, 36]. From this panel, the samples with 10 and 100 copies of HIV-1 were amplified 36 times in 1 HIV-1 pol amplification assay plate, and this set of amplifications was repeated in 6 separate assays. The maximum inter-assay and intra-assay variation was 5-fold and 3.35-fold, respectively. Any duplicate sample whose HIV-1 concentration was found to be higher than the maximum intra-assay variation (5-fold) was repeat assayed in duplicate. If the repeated assays were within the intra-assay variation, these values were used in the analysis. Otherwise, the assay was repeated until the duplicate values were within the intra-assay variation, and only the final set of duplicate values was used in the analysis.

The same ACH2 samples used for the β-actin amplification and the HIV-1 pol amplifications were run as internal controls with every assay. If the control samples were not within the range of values previously obtained in the control experiments (3-fold β-actin, 5-fold pol), the entire assay was repeated until the duplicates were within the appropriate range, and only the last duplicate values were used in the analysis.

Statistical analysis

All samples with < 1 × 104 cells tested and no detectable HIV-1 copies per assay were excluded from the analyses (n = 20). For all additional samples with no detectable HIV-1 copies, the value of 0.5 copies per number of BMCs tested was assigned and was converted to number of BMCs per million cells. Because the units used in this report were BMCs per million cells, these values were <1 (and thus the log of these values was negative) for all samples for which >1 million cells were analyzed and no HIV-1 was detected or when >1 million cells were analyzed and <1 copy of HIV-1/million cells was detected.

First BMC samples were defined as those collected closest to the delivery date. Sixty-three percent of the first BMC samples were collected from colostrum or early milk (0–10 days after delivery), 21% at 10 days to 2 months, 8% at 2–4 months, and the remainder ≥4 months after delivery. First BMC samples were used for statistical analyses involving blood plasma and genital samples, because the first BMC samples were collected closest to the others in time.

Spearman’s correlation coefficient was used to compare levels of infected cells from first BMC samples to levels of cell-free virus, CD4 cell count, and virus levels in maternal plasma. The t test was used to compare levels of infected cells in first BMC samples among women with and without HIV-1–infected cells detected in their genital secretions. Univariate and multivariate Cox proportional hazards regression models were used to assess the association between levels of infected BMCs or levels of cell-free virus in breast milk and transmission among women who breast-fed, did not transmit in utero, and had data beyond delivery (n = 134). These included 30 noncompliant women randomized to formula feed, who had a median duration of breast-feeding of 12 months and whose infants received a median of 92% of their nutritional intake via breast milk. Variables in the multivariate analyses were assessed for collinearity by use of variance inflation factors before inclusion in the models. The largest variance inflation factor was 1.2, indicating that all variables could be included in the model together.

For the analysis of changes in levels of infected BMCs over time, intervals were established on the basis of the scheduled visit dates of the women in the trial (birth to 10 days, 6 weeks, 14 weeks, 6 months, 9 months, and ≥12 months, ±4 weeks for each visit after the first), as described elsewhere [3]. The paired t test was used to compare the samples from colostrum or early milk (birth to 10 days after delivery) with those from later time intervals.

Counts of infected BMCs per million cells from colostrum or early milk were converted to concentrations of infected BMCs per milliliter by multiplying by a previously defined colostrum cell concentration, 3.8 × 106 BMCs/mL [37]. Similarly, infected BMC counts per million cells at 6 and 14 weeks (±1 month) were multiplied by 0.08 × 106 BMCs/mL and counts at 6 months and later (±1 month) were multiplied by 0.05 × 106 BMCs/mL, on the basis of previously defined cell concentrations specific to those time points [37]. Only samples from breast-feeding women who transmitted during or after delivery were considered in analyses of changes in levels of virus in breast milk over time.

RESULTS

Study subjects and levels of infected cells in breast milk

Two hundred ninety-one women contributed 630 BMC samples to this study. The demographic and clinical characteristics of this cohort were similar to the original cohort and the subset previously analyzed (data not shown) [1, 3].

HIV-1 provirus was detected in 70% of the samples. The maximum number of infected cells was 4.25 log10 BMCs/million cells (17,857 infected BMCs/million cells), and the minimum was <0 log10 BMCs/million cells (<1 infected BMCs/million cells). The mean log10 number of infected BMCs per million cells for each woman was calculated, and the mean of these means equaled 1.56 log10 infected BMCs/million cells (36 infected BMCs/million cells).

Estimates of the concentration of infected BMCs per milliliter were calculated from counts of BMCs per million cells by use of previously reported information about uninfected BMCs per milliliter concentrations at various time points [37]. The maximum concentration was 3.90 log10 infected BMCs/mL (7943 infected BMCs/mL), and the minimum concentration was <0 log10 infected BMCs/mL. The mean log10 infected BMCs per milliliter for each woman was calculated, and the mean of these means equaled 1.19 log10 infected BMCs/mL (15 infected BMCs/mL).

Correlates with levels of virus in other compartments and CD4 T cell count

The number of HIV-1–infected BMCs per million cells was positively correlated with the concentration of cell-free virus per milliliter of breast milk (R = .144; P = .032; figure 1A). It was also positively correlated with cell-free virus per milliliter of maternal plasma (R = .365; P < .001; figure 1B) and negatively correlated with CD4 T cell count (R =−.231; P < .001). Similar results were obtained by comparing the concentration of infected BMCs per milliliter with the concentration of cell-free virus per milliliter of breast milk (R = .281; P < .001), cell-free virus per milliliter of maternal plasma (R = .291; P < .001), and CD4 T cell count (R =−.237; P < .001). In addition, the number of infected BMCs per million cells in first breast milk was higher on average in women who had HIV-1 DNA detected in third-trimester cervical secretions (1.76 vs. 1.28 log10 infected cells/million cells; P < .001) or vaginal secretions (1.71 vs. 1.40 log10 infected cells/million cells; P = .030) than in women who did not have HIV-1 detected in these secretions. The same associations were found by comparing the concentration of infected BMCs per milliliter with DNA detection in third-trimester cervical or vaginal secretions (1.80 vs. 1.36 [P < .001] and 1.81 vs. 1.44 [P = .04], respectively).

Figure 1
Correlation of the no. of HIV-1–infected breast milk cells (BMCs) per million cells with the concentration of cell-free virus per milliliter of breast milk (A) and cell-free virus per milliliter of maternal blood plasma (B) among breast-feeding ...

Mother-to-child transmission

For each time interval around scheduled visits, the numbers of infected BMCs in mothers who transmitted virus and who breast-fed were compared with the numbers of infected BMCs in mothers who did not transmit virus. The mothers who transmitted the virus during or after delivery had higher numbers of infected BMCs per million cells on average than did the nontransmitting mothers (figure 2A), except at the first and last time intervals. These results were similar to those found when the concentration of infected BMCs per milliliter and HIV-1 transmission were examined (figure 2B).

Figure 2
Distribution of levels of HIV-1 in breast milk from transmitting and nontransmitting mothers, by time interval. The no. of infected breast milk cells (BMCs) per million cells (A) and the no. of infected BMCs per milliliter (B) were compared between transmitting ...

In univariate analyses, each log10 increase in number of infected BMCs per million cells, concentration of infected BMCs per milliliter of breast milk, or concentration of cell-free virus per milliliter of breast milk was associated with an increase in risk of transmission during or after delivery (hazard ratio [HR], 2.79 [95% confidence interval {CI}, 1.59–4.88; P < .001]; HR, 3.42 [95% CI, 2.08–5.65; P < .001]; and HR, 1.8 [95% CI, 1.12–2.67; P = .004], respectively).

After adjustment for cell-free virus level in maternal plasma and in breast milk, each log10 increase in concentration of infected BMCs per milliliter was associated with a 3.19-fold increased risk in hazard of transmission during or after delivery (95% CI, 1.54–6.60; P = .002). In the same multivariate analysis, there was no significant association with cell-free virus per milliliter of breast milk and transmission during or after delivery (table 1). In another similar analysis that replaced the concentration of infected BMCs per milliliter with the number of infected BMCs per million cells, each log10 increase in infected BMCs per million cells was marginally associated with an increased risk of transmission during or after delivery (HR, 2.16; 95% CI, 0.87–5.36; P = .10), after adjustments for cell-free virus per milliliter of maternal plasma and per milliliter of breast milk (table 1).

Table 1
Multivariate Cox regression models for HIV-1 transmission by breast-feeding mothers.

When the above analysis was restricted to only those infants who were known to be uninfected at 4 weeks of life (which included only 4 infants) and adjustments were made for cell-free virus level in maternal plasma and in breast milk, the number of infected BMCs per milliliter was marginally associated with transmission at ≥4 weeks after delivery (HR, 12.93; 95% CI, 0.44–376.71;P = .10). The number of infected BMCs per million cells was similarly associated with transmission at ≥4 weeks after delivery (HR, 12.93; 95% CI, 0.44–376.71; P = .10).

Changes in levels of infected cells over time

The mean number of infected BMCs per million cells was significantly lower in colostrum or early milk than in samples collected after 10 days (table 2). Conversely, the estimated concentration of infected BMCs per milliliter in colostrum or early milk was significantly higher than at all other time points (table 2). The number of cell-free virus particles present per milliliter of breast milk was significantly higher than the estimated number of infected BMCs per milliliter for each time point (P < .001 for each comparison; figure 3).

Figure 3
Changes in levels of HIV-1 in breast milk over time. The mean no. of infected breast milk cells (BMCs) per million cells, the mean concentration of infected BMCs per milliliter, and the mean no. of cell-free virus particles per milliliter of breast milk ...
Table 2
Comparison of levels of HIV-1 provirus in colostrum or early milk with those in breast milk at later time intervals.

DISCUSSION

In the present study, we have found that the concentration of infected BMCs per milliliter was associated with a significant increase in risk of mother-to-child transmission among breast-feeding women, independent of the level of cell-free viral RNA in breast milk or the level of virus in maternal plasma, 2 factors previously shown to be associated with mother-to-child transmission [5, 23, 30, 38, 39]. This trend remained even after the analysis was restricted only to infants uninfected at 4 weeks of age, despite the small number of infections among these infants (n = 4). Conversely, the concentration of cell-free virus was not significantly associated with an increase in transmission via breast-feeding, after adjustment for the number of infected BMCs per milliliter and virus load in maternal plasma. This suggests that infected BMCs play an important role in transmission and may be more likely to mediate HIV-1 transmission via breast-feeding than are cell-free virus particles in breast milk.

Several antiviral compounds are present in breast milk (e.g., secretory leukocyte protease inhibitor, lactoferrin, chemokines, and antibodies). These antiviral factors may have less influence on infected cells than on cell-free virus and, thus, may be less effective at preventing infant infection via BMCs. Because levels of both cell-free and cell-associated virus are associated with transmission, the effect of antiviral factors in breast milk on transmission is likely to depend on the levels of cell-free and cell-associated virus. However, these factors can be correlated, making evaluation of their effect complex. For example, mastitis, which is also a risk factor for infant infection, is associated with an increase in both HIV-1 and several antiviral factors (secretory leukocyte protease inhibitor, lactoferrin, and chemokines) [23, 30, 40]. Perhaps studies examining the role of levels of cell-free and cell-associated virus in breast milk that can adequately adjust for the levels of potential antiviral compounds in breast milk will more clearly define the role of these factors in transmission via breast-feeding.

Our findings have revealed a higher concentration of infected BMCs per milliliter in colostrum and early milk than in mature milk. Thus, infants are exposed to a higher concentration of infected cells soon after delivery. This is also the time when infants are exposed to the greatest concentration of cell-free virus [3]. However, our data have shown that, during the first 10 days after delivery (the period of colostrum or early milk), there was not a significant difference in the concentration of infected BMCs among transmitting and nontransmitting mothers. This may be because a large fraction of the infections detected during this time period may be due to exposure to virus during delivery rather than exposure to HIV-1 in breast milk. However, it is not possible to distinguish transmission during delivery from transmission due to early breast-feeding, and therefore the effect of virus levels in breast milk on transmission via breast-feeding shortly after delivery is difficult to assess.

Levels of infected BMCs at later time points were associated with significantly increased transmission risk. Thus, children continue to be at risk for infection as long as they breast-feed. This suggests that interventions aimed at reducing mother-to-child transmission should be designed to reduce infant exposure to infected BMCs throughout lactation.

The ratio of infected cells to total number of cells in breast milk quantified in the present study is similar to the ratios of infected cells to total cells in other bodily secretions described in previous studies. The average ratio of infected cells to total cells in breast milk, 28 infected BMCs/million cells (range, 0–3600 infected BMCs/million cells), is slightly higher than the ratio reported in vaginal secretions (average, 19 infected cells/million cells; range, 0–333 infected cells/million cells [41]) and semen (median, 6 infected cells/million cells; range, <6 to 2171 infected cells/million cells [42]) but lower than that reported in cervical secretions (average, 43 infected cells/million cells; range, 0–953 infected cells/million cells [41]). It is considerably lower than in blood (average, 1199 infected cells/million cells; range, 25–6410 infected cells/million cells [43]). The values given in each of these studies, including our own, were limited to analysis of infected cells in relation to total cells, irrespective of whether the cells were susceptible targets of HIV-1 infection, namely lymphocytes and macrophages. Nonetheless, our study indicates that the levels of cell-associated virus in various bodily fluids (genital secretions, blood plasma, and breast milk) are interrelated. This is also true for cell-free virus, indicating that similar mechanisms may regulate the migration of cell-free and cell-associated virus in these bodily compartments.

The observed correlation in our study between cell-free virus and levels of infected cells in breast milk is most consistent with a model in which cell-free virus originates from infected cells. However, from these data we cannot exclude the possibility that some of the cell-free virus in breast milk originates from another site, especially given the correlation between cell-free virus in breast milk and virus levels in plasma [3]. In situ studies would be useful for determining how commonly HIV-1 provirus–positive BMCs express cell-free virus and which cell types in breast milk are the main reservoirs of virus.

In summary, infected BMCs have a significant association with the risk of mother-to-child transmission that is independent of the levels of cell-free virus and plasma virus load. In contrast, the strong association between the risk of mother-to-child transmission and the level of cell-free virus in breast milk disappeared once the level of infected BMCs was taken into account, suggesting that cell-associated virus may play a more important role in transmission via breast milk. The highest concentration of cell-associated virus per milliliter of breast milk was found in colostrum or early milk. It may be important to design interventions that include the reduction of levels of cell-associated virus in breast milk to reduce transmission of HIV-1 via breast-feeding.

Acknowledgments

We thank the Nairobi City Council and the Kenyatta National Hospital, for permission to use the facilities; the clinical and research staff involved in the project; the women and children who participated in our study; and Gretchen L. Strauch, for editorial assistance.

Financial support: National Institutes of Health (NIH; grant HD-23412 to J.K.K.); Elizabeth Glaser Pediatric AIDS Foundation Scientist Award (to J.O.); Virology Training grant (NIH National Institute grant T32 CA09229 to C.M.R.).

Footnotes

Presented in part: 12th annual meeting of the International Genetic Epidemiology Society, Los Angeles, 3–4 November 2003 (abstract 107).

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