Mother-to-infant transmission is the leading cause of HIV infection in children younger than 15 years of age.1 Transmission from mother to infant can occur in utero, during delivery, and as a result of breast-feeding. The risk of breast milk transmission has been estimated at 14% to 29% depending on infection status at the time of delivery.2 Abstinence from breast-feeding eliminates transmission by this route and, therefore, is recommended for many women. Unfortunately, in the developing world, where the HIV pandemic is at its worst, abstinence is often not attainable. Attempts to provide women with formula as a replacement to breast milk have not all been successful.3 Water is often scarce or unsafe for infant consumption, infants who do not breast-feed are more susceptible to parasitic and bacterial diseases that can cause diarrhea and dehydration, and mixed feeding of formula and breast milk has been associated with an increased risk of transmission compared with breast-feeding alone.3 The use of antiretrovirals has been evaluated and shown to reduce mother-to-infant transmission by as much as 50%4-6; however, drugs are often not available to women in the developing world, and there is mounting evidence of cytotoxic side effects.7-10 The lack of practical intervention strategies precipitates the need for an understanding of the mechanisms of transmission and the correlates of protection in breast milk transmission of HIV.
High plasma viral load and low CD4+ T-cell count in the mother have been identified as the general correlates of mother-to-infant transmission.11,12 In addition, breast milk transmission has been associated with increased milk viral loads and persistent shedding of virus in milk.13 Unfortunately, the correlates of protection are not as clear. There is conflicting evidence regarding the role that the humoral immune response plays in preventing transmission. Humoral immune responses targeting the envelope glycoproteins of HIV have been observed in the milk of HIV-1-infected women, and several in vitro experiments have shown that antibodies to HIV should be able to prevent binding and translocation of virus across epithelial cells.14-18 In addition, passive immunization of infant macaques provided partial protection against challenge with SHIV, indicating that antibodies are capable of protecting infants against retrovirus transmission.19 However, studies of breast-feeding cohorts suggest that the antiviral humoral immune response does not provide protection against breast milk transmission. Several studies of HIV-positive women and their nursing infants have indicated that transmitting and nontransmitting women have similar levels of HIV-specific antibody in their milk.14-18 Further studies using the macaque model can address these conflicting results and clarify the importance of quality and quantity of antibody required for infant protection.
The SIV-infected rhesus macaque has successfully been used as a model for studying HIV transmission and pathogenesis. Although disease progression is more rapid in the SIV-infected macaque than in HIV-infected humans, infected macaques have a similar disease course and die of opportunistic infections much the same as infected humans.20 We have used the SIV-infected macaque to model breast milk transmission of HIV.21,22 In this study, 14 dams were inoculated with SIV after delivery and housed with their infants to allow for natural breast-feeding. We observed varying rates of disease progression among the mothers and a high rate of mother-to-infant SIV transmission that occurred at time points throughout the disease course. Rapid transmission to the infant during the period of acute maternal viremia was correlated with rapid disease progression in the mother. Transmission during the chronic phase of the disease course was correlated with higher average milk viral loads and more persistent viral expression in milk. Transmission at later time points was not associated with disease progression as measured by plasma viral load or peripheral CD4+ T-cell counts.
As an extension of these studies, we evaluated the humoral immune responses in the lactating macaques to determine if the levels of immunoglobulin in plasma or milk were associated with breast milk transmission. Total immunoglobulin and SIV envelope-specific IgG and IgA concentrations were measured in milk and plasma, and the avidity of envelope-specific IgG was determined. In addition, the association between humoral immune response and transmission was assessed. These studies describe the antibody response to SIV in paired plasma and milk samples and validate the relevance of the SIV-infected macaque as a model for deciphering the mechanisms associated with milk transmission of HIV.
MATERIALS AND METHODS
Blood and milk samples were collected from 14 female rhesus macaques before and after intravenous inoculation with SIV/DeltaB670. As described previously,21,22 females were inoculated within 7-54 days after vaginal delivery of normal infants. Blood specimens were collected in EDTA anticoagulant, and milk samples were collected by manual expression at several time points over the course of the study. Milk samples were stored on ice and processed within 2 hours of collection. Blood and milk samples were separated into cellular and aqueous fractions and stored at −70°C. Aliquots of paired plasma and milk samples were thawed and kept at 4°C for use in this study. Infants were considered SIV infected when peripheral blood mononuclear cells were positive for proviral DNA by polymerase chain reaction assay.21
Detection of Total IgG and IgA Levels
Total levels of IgG and IgA were determined by enzyme linked immunosorbent assays (ELISAs) as described by Tryphonas et al23 with the following modifications. Microtiter plates were coated with 5 μg/mL goat antibody to monkey IgG or IgA (Rockland Immunochemicals, Inc., Gilbertsville, PA) in phosphate-buffered saline (PBS). Blocking was done at 4°C overnight with 4% whey and 10% goat serum in PBS. Samples were diluted 1:100,000 for quantitation of IgG in plasma, 1:10,000 for IgG in milk, and 1:1000 for IgA in milk. Peroxidase- or biotin-conjugated rhesus antisera (0.5 μg/mL) (Rockland Immunochemicals, Inc.) was added after washing, and the samples were incubated for 1 hour at room temperature. TMB developing solution (KPL, Gaithersburg, MD) was added, and the samples were incubated for 3 minutes followed by addition of 1 M H3PO4 to stop the reaction. The absorbance was then read at 450 nm. The concentration was extrapolated from a standard curve generated from a rhesus IgG standard (kindly provided by Dr. James Robinson, Tulane University) or human IgA standard (ICN, Costa Mesa, CA).
Detection of SIV-Specific Antibodies
SIV-specific antibodies were determined by an ELISA similar to that used to detect total antibody, except that plates were coated with 3 μg/mL recombinant SIVmac239 gp130 (AIDS Research and Reference Reagent Program, Quality Biologicals, Gaithersburg, MD) diluted in PBS. To run multiple samples from several time points in a single assay, serial 10-fold dilutions of plasma and milk were used to determine the dilution at which most samples tested in the linear range of a standard curve created from plasma from an SIV-infected macaque. The dilution at which most samples tested in the linear range of the standard curve was then chosen, and all samples were run at a single dilution. Plasma samples obtained during the first 35 days of infection were compared at a dilution of 1:300; otherwise, plasma was diluted 1:10,000 for IgG for comparisons, and milk was diluted 1:50 for IgG and 1:10 for IgA. Peroxidase-conjugated antibody to rhesus IgG or IgA was then added to the wells to detect bound antibodies, as described above. Samples were considered positive if their values were greater than twice the background value observed in PBS-treated wells.
Affinity of IgG antibodies in plasma and breast milk for rgp130 was determined using a protocol adapted from Cole et al.24 The standard ELISA technique described above was used with the following modifications. After coating the plate with rgp130 in PBS and blocking nonspecific binding sites with blocking buffer, samples were added at a 1:8000 dilution and incubated for 1 hour at room temperature. This dilution produced an absorbance of 450 nm in the linear range of the SIV-specific ELISA described above and allowed multiple samples to be tested at once; 8 M urea in PBS was then added to duplicate sample wells in half of the plate, while PBS was added to the other half as a control. The urea was allowed to disrupt low-affinity interactions for 20 minutes. Horseradish peroxidase-conjugated goat antibody to monkey IgG was added, and the sample was incubated for 1 hour at room temperature; the plate was developed, and the absorbance was read at 450 nm as described previously. The affinity index was calculated by dividing the absorbance of wells treated with urea by the absorbance of wells treated with PBS alone. The affinity index was determined only for IgG due to limited sample volume.
The two-tailed Mann-Whitney U test was used to analyze all data. P < 0.05 was considered significant.
Breast Milk Transmission
To evaluate the role of humoral immune responses in breast milk transmission of HIV, we used a collection of milk and plasma samples obtained from 14 rhesus macaques inoculated with the pathogenic quasispecies SIV/DeltaB670 after delivery of their infants. As summarized in Table 1, SIV transmission via breast milk occurred in 10 of 14 mother-infant pairs at different stages in the maternal disease course. Two females rapidly transmitted SIV to their infants during the period of peak viremia and were labeled as early transmitters. Eight additional infants became infected during the chronic phase of disease after the viral set point had been reached in the mother. Two mothers transmitted by 81 and 84 days after inoculation (intermediate transmitters), and 6 females transmitted at time points ranging from 235 to 360 days after inoculation (late transmitters). Four infants remained SIV negative despite maternal disease progression and continued lactation (nontransmitters). Because transmission occurred at various time points throughout the disease course, we hypothesized that different mechanisms may be responsible for transmission at different stages of infection. Characteristics of disease progression were therefore compared between animal groups based on the timing of transmission as designated in Table 1. As reported previously, transmitting and nontransmitting females had similar viral loads, averaging 107 copies/mL at 14 days after inoculation and 106 copies/mL at 98 days after inoculation. Milk viral loads averaged 104 copies/mL at 14 days after inoculation and 102 copies/mL at 98 days after inoculation and were not significantly different between the 4 transmission groups. However, late transmitters had at least 1 time point when viral load in milk was >500 copies/mL and had significantly higher average milk viral loads than nontransmitters (P = 0.028). Milk viral loads in nontransmitting females never reached 500 copies/mL after the set point. CD4+ T-cell counts fell during the primary phase of infection to levels ranging from 212 to 829/μL and remained low in the early transmitters but rebounded to varying levels in all others. No significant differences were found in CD4 cell counts in the nontransmitting and transmitting females.21,22
Total IgG and IgA Concentrations
Total immunoglobulin levels in milk and plasma were measured over the first 4 months of infection to evaluate associations between immunoglobulin levels and transmission groups (Table 2). Immunoglobulin concentrations varied considerably for the 14 animals; however, values were similar to ranges reported for normal macaques and HIV-seropositive humans.23,25,26 Total plasma IgG levels did not change considerably from the day of inoculation through 4 months after inoculation in all female macaques. The concentration of IgG in milk, however, was higher at each of 4 monthly time points after inoculation, as compared with levels in seronegative macaques (P < 0.01 at all time points). In contrast, the levels of IgA in milk decreased after inoculation as compared with those in seronegative controls (P < 0.01 at all time points). Although differences in milk immunoglobulin levels were observed between all SIV-infected animals and seronegative controls, no differences were observed between nontransmitting females and early, intermediate, or late transmitters.
SIV Envelope-Specific Antibody
Because transmission of SIV occurred rapidly in 2 mother-baby pairs, we monitored the development of SIV envelope-specific antibodies in plasma of females after inoculation during the time frame that early transmission occurred. ELISAs were performed using a recombinant SIVmac239 gp130 as the capture antigen to evaluate responses specifically directed to critical envelope antigens. Tenfold serial dilutions of plasma obtained at 7 and 14 days after inoculation indicated that seroconversion to envelope antigens occurred in all animals by 14 days (Fig. 1). At 21 and 28 days after inoculation, antibody levels continued to increase in all animals. It is interesting that the early transmitters tended to have the lowest levels of SIV envelope-specific IgG during these early time points. To compare all the animals longitudinally in a single assay, a single dilution was chosen such that most samples tested in the linear range of a standard curve created from plasma from an SIV-infected macaque. Early transmitters had SIV env-specific IgG titers that were barely detectable at this dilution. Further experiments indicated that these animals had titers that were below 1:5000. SIV-specific IgG responses in plasma continued to develop in the other 12 animals and plateaued between 3 and 4 months after inoculation (Fig. 2). Additional assays indicated that at 98 days after inoculation, the range of specific antibody titers in these animals was 20,000 to 320,000. There were animals in each transmission group with low and high values within this range.22 Because there were only 2 early transmitters and 2 intermediate transmitters, we were unable to determine the statistical significance of the difference in antibody level at each of the 4 time points tested; however, when the values at all time points were pooled, early transmitters had significantly lower IgG responses in plasma than did intermediate transmitters (P ≤ 0.001), late transmitters (P ≤ 0.001), and nontransmitting females (P ≤ 0.001). There was no significant difference between the other 3 groups.
The concentration of SIV env-specific IgG in milk was ∼200-fold lower than that observed in plasma. Specific IgG increased over the first 4 months of infection similar to observations made in plasma. When values were pooled over the 4 months, early transmitters had significantly lower IgG levels in milk than did intermediate transmitters (P ≤ 0.01), late transmitters (P ≤ 0.001), and nontransmitters (P ≤ 0.001). In contrast, intermediate and late transmitters had higher levels of IgG in milk than did nontransmitters (intermediate transmitters, P < 0.05; late transmitters, P < 0.05). SIV-specific IgA levels in milk were much lower than specific IgG levels in all animals, despite normal levels of total IgG and IgA in milk. Similar results have been reported for HIV-infected mothers.14,17,18,27 SIV envelope-specific IgA concentrations in milk increased over the first 4 months of infection, paralleling the development of IgG responses in milk. Early transmitters had barely detectable levels of IgG and IgA in milk at 1:50 and 1:10 dilutions, respectively, which were significantly different than those for intermediate transmitters (P ≤ 0.01), late transmitters (P ≤ 0.001), and nontransmitting females (P ≤ 0.01) when all values were pooled. There was no significant difference between the other 3 groups. These observations indicate that rapid transmission is associated with a lack of development of antibody responses in milk and plasma. However, a quantitative difference in specific antibody was not likely responsible for protection from transmission during the chronic phase of disease. In addition, because plasma and milk viral loads were similar in all the transmission groups at both 14 and 98 days after inoculation, it is unlikely that differences in specific antibody were attributable to antigen concentration.
Recognizing that the specificity of the antibody response may differ in transmitting and nontransmitting females, we used a modified ELISA to assess the avidity of SIV-specific antibody. After incubation of the sample, 8 M urea was used to disrupt low-avidity interactions. An avidity index was calculated by dividing the absorbance of urea-treated wells by the absorbance of wells treated with PBS alone.24 The avidity of envelope-specific IgG was measured in the plasma and milk of 12 animals. The 2 early transmitters were not evaluated in this assay due to their low titer of IgG. As shown in Figure 3, the IgG avidity index was initially low in both milk and plasma but similar for all 12 animals. An average avidity index of 16.5% was observed in plasma 1 month after inoculation, while a slightly higher avidity index of 30% was observed in milk. The avidity levels increased over the first 4 months of disease, peaking at a mean avidity index of 72% for transmitters and 46% for nontransmitters in plasma. Although the avidity index in plasma of late and intermediate transmitters was slightly higher than that of nontransmitters, the difference was not statistically significant (P = 0.17 at 3 months; P = 0.16 at 4 months). The IgG avidity index in milk at 4 months after inoculation was lower than that observed in plasma, peaking at 48% for transmitters and 38% for nontransmitters.
Lactating rhesus macaques infected with SIV were used as a model to evaluate the role of antibodies in breast milk transmission of HIV. In this study, total immunoglobulin levels in plasma and milk were measured and found not to be predictive of infant infection through breast-feeding. Transmitting and nontransmitting dams had similar levels of total IgG in plasma and IgG and IgA in milk that were within ranges reported by other investigators for normal macaques and HIV-infected women. After infection of the dams, no change in total plasma IgG levels was observed. In milk, however, total IgG concentrations were higher and total IgA concentrations were lower at all time points tested in infected dams as compared with seronegative controls. This has not been observed for humans; however, HIV-positive women have been shown to have higher levels of total IgG in plasma as compared with seronegative controls.27 These observations for the macaques were likely due to the small number of macaque samples evaluated, the variability of immunoglobulin concentrations in outbred animals, and the differences in immunoglobulin concentrations in colostrum and mature milk.26
The predominate SIV-specific immunoglobulin in macaque milk was IgG. The lack of a robust SIV-specific IgA response in other mucosal compartments of macaques has been reported previously.28 In addition, the predominance of an HIV-specific IgG response in human milk has been well documented.14,17,18,27 Although the lack of IgA responses to SIV may contribute to transmission, they are not directly responsible, because 4 infants remained SIV uninfected despite the lack of maternal envelope-specific IgA and similar levels of SIV-specific IgG.
In this model, breast milk transmission of SIV occurred rapidly in 2 of 14 mother-baby pairs. Early transmission during the acute viremic phase of disease could not be correlated with a significant difference in viral or antibody levels in plasma or milk at 14 days after inoculation. However, early transmitters developed significantly lower levels of specific antibody than did the other dams and had lower viral set points at 56 days after inoculation. A small percentage of macaques infected with several different strains of SIV have been characterized as rapid progressors based on a lack of SIV-specific humoral immune responses, high plasma viral loads, and a rapid progression to simian AIDS.29,30 It is notable that the rapid progressing animals in this study were the only females that transmitted virus to their infant within the first few weeks of infection. Because viral levels in plasma and milk were not statistically different from those of other dams at 14 days and the specific humoral immune responses were just developing and similar in all animals, it is likely that innate immune responses and/or host genetic factors were more directly responsible for transmission.
SIV envelope-specific antibody levels in milk and plasma were comparable in nontransmitting macaque females and those that transmitted during the chronic stage of infection. Similarly, the levels of HIV-specific antibodies in milk and plasma of HIV-infected women were comparable, regardless of transmission status.14-18 Although quantitative differences in the humoral immune responses between the transmitting and nontransmitting dams were not observed, qualitative differences may exist between the groups. We were able to measure the avidity of the viral specific IgG and observed it to be similar in milk and plasma of transmitters and nontransmitters. This has also been observed in HIV-infected women.27 Unfortunately, we were unable to assay any additional functional differences in the antibody responses due to the limited volume of milk that could be collected from each dam. Future studies with this model can be designed to examine the differences in neutralizing antibody and the specificity of binding over the course of disease in transmitting and nontransmitting females.
Passive immunotherapy has been used to protect infant macaques from oral challenge of SIV, demonstrating that SIV-specific antibody can prevent infant infection.19,31,32 Van Rompay et al31 described protection in 6 of 6 neonates orally challenged with SIVmac251 after the administration of SIV hyperimmune serum just after birth. The hyperimmune serum given to the infant macaques was shown to contain 24.3 mg/mL total IgG and had a titer of antibody to SIV env of 102,400, a 53% avidity index, and a half-life in the infant of ∼2.5 weeks. Although the avidity was similar, this concentration of antibody is higher than that observed in the milk of both transmitting and nontransmitting females and likely contributed to the protection seen in the neonates. Ruprecht et al19,32 showed protection of neonatal macaques from oral SHIV challenge after administration of monoclonal antibodies targeting neutralizing epitopes of the HIV envelope. This targeted specificity is likely responsible for the protection observed. On the basis of these studies, passive administration of antibody is a potential therapy for prevention of breast milk transmission because it provides infants with a high concentration of specific antibodies that the mother is unable to provide. Passive immunotherapy for the infant may also prevent natural breast milk transmission by augmenting the specific humoral response expressed in the milk. However, natural transmission of SIV via breast milk occurs in the presence of high titers of envelope-specific antibody, suggesting that continued evolution of virus in the mother might allow evasion of the humoral immune response resulting in infant infection. Passive administration of antibodies to the infant may only be protective if the antibodies are broadly neutralizing and capable of targeting transmissible viral variants.
These and previous studies21,22 demonstrate that the SIV-infected lactating rhesus macaque provides a relevant model for HIV transmission via breast milk. We have shown that the same correlates of transmission identified in HIV-infected mother-infant pairs-namely, consistent expression of virus in milk-are observed in the monkey model. In this study, we characterized the IgG responses in plasma and the IgG and IgA responses in milk of SIV-infected monkeys and found, as was observed in humans, that no associations could be made between antibody levels and transmission status. Future studies, using this natural transmission model, can lead to the development and improvement of antibody-specific interventions.
The authors thank James Robinson and Nedra Lacour for helpful discussions and technical advice.
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Keywords:© 2005 Lippincott Williams & Wilkins, Inc.
HIV/SIV; mother-to-infant transmission; antibody; breast milk; rhesus macaque