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Vertical transmission of human immunodeficiency virus type 1: Autologous neutralizing antibody, virus load, and virus phenotype
ORIGINAL ARTICLES
Vertical transmission of human immunodeficiency virus type 1: Autologous neutralizing antibody, virus load, and virus phenotype Robert N. Husson, MD, Yan Lan, MD, Eiji Kojima, MS, David Venzon, PhD, Hiroaki Mitsuya, MD, PhD, and Kenneth Mclntosh, MD From the Divisionof infectious Diseases, Children's Hospital, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts,and the Experimental RetrovirologySection, Medicine Branch, and the Biostatisticsand Data Management Section, National Cancer Institute, Bethesda, Maryland
Objective: To evaluate immunologic and virologic correlates of vertical transmission of human immunodeficiency virus type I (HIV-I). Design: Case-control study. Patients: Women who were prospectively enrolled in a natural history study of HIV-I infection in women and infants. Sixteen HIV-1-infected women whose infants became infected were matched by CD4 ÷ cell percentage and use of zidovudine during pregnancy with women whose infants did not become infected. Measurements: Maternal autologous neutralizing antibody, virus load determined by RNA-polymerase chain reaction (RNA-PCR), and virus phenotype. Results: Most women in both groups had low titers of autologous neutralizing antibody, and no difference in neutralizing titers was observed (range, <4 to 181 in both groups). The HIV-I copy number in maternal plasma was not significantly different in the two groups but was inversely correlated with maternal CD4 ÷ cell percentage (p <0.005). Five women in the transmitting group and four in the nontransmitting group had syncytium-inducing (SI) phenotype virus. Two infected infants had SI phenotype virus. The SI phenotype virus was associated with a greater HIV-I copy number in maternal plasma (p <0.05) and an increase in the mortality rate for the infants (p <0.01). Conclusions: In women matched for CD4 ÷ cell percentage, low titers of autologous neutralizing antibody, high virus load, and SI phenotype virus were not associated with an increased risk of transmission of HIV-I to their infants. (J PEDIATR 1995; 126:865-71 ) Supported by grants from the Pediatric AIDS Foundation (5029114-PG), the Charles H. Hood Foundation, and the Milton Fund of Harvard University, and by Women and Infants Transmission Study contract NO1 AI-82507 from the National Institutes of Health. Dr. Husson was the recipient of a Markey Scholar Award. Presented in part at the 33rd Interscience Conference on Antimicrobial Agents and Chemotherapy, October 1993, Anaheim, Calif. (Abstract No. 1138). Reprint requests: Robert N. Husson, MD, Division of Infectious Diseases, Children's Hospital, 300 Longwood Ave., Boston, MA 02115. 9/20/'64351
Although reported rates of vertical transmission of in~nmnodeficiency virus type 1 have varied widely, only a small proportion of infants born to infected women become infected. Two large prospective studies of infants born to HIV-l-infected women in Europe found vertical transmission rates of 14.4% and 18.3%, respectively.],2 In the ongoing Women and Infants Transmission Study in the United States, the overall transmission rate is less than 20%. 3 In Africa, reported rates of transmission are higher, although infection occurs in substantially fewer than half of the infants.4, 5 The recently demonstrated efficacy of zidovudine in
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HIV-1 NSI RNA-PCR SI TCIDs0
Human immunodeficiency virus type 1 Non-syncytium-inducing Polymerase chain reaction of reverse transcribed fibonucleic acid Syncytium-inducing Fifty percent tissue culture infectivity dose
reducing the rate of vertical transmission is likely to decrease transmission in populations where this drug is available and the HIV-I status of women of childbearing age is known. 6 Despite the importance of the pathogenesis of vertical transmission of HIV-1 and the variables that determine why some infants born to HIV-l-infected women become infected and others do not, relatively little is known about these phenomena. Among the variables that have been consistently associated with an increased likelihood of transmission are advanced maternal clinical disease or markers of advanced disease such as low CD4 + cell counts and high CD8 + cell counts, and markers of high maternal virus burden, such as p24 antigenemia or I-IIV-1 culture positivity.1.5, 7-9 Other variables that may play a role are premature birth, placental inflammation, mode of delivery, and breastfeeding 1,2,5,7,10-14 The role of maternal HIV-l-specific immunity in vertical transmission remains unclear, with conflicting results regarding the association of maternal anti-HIV-1 antibody and transmission, l°, 15-20 A better understanding of the correlates of mother-to-infant transmission of HIV-1 would provide information highly relevant to the prevention of both vertical and horizontal transmission. We therefore conducted a case-control study to assess the role of immunologic and virologic factors in relation to mother-to-infant transmission of HIV-1.
METHODS Study population. The HIV-1-infected women enrolled in the Boston centers for the Women and Infants Transmission Study between January 1990 and November 1992, who had given bi~h to infants known to be infected with HIV- 1, were identified by study number through a review of the study records. Infants were defined as infected if they had two or more positive HIV-1 culture results, excluding cord blood, from samples obtained at different times in the first year of life. 3 Of the 20 mother-infant pairs identified, 16 were found to have appropriate virus isolates and plasma or serum samples available for study. From the same study population, women whose infants were known not to be infected were identified, and control mother-infant pairs were selected by matching for maternal CD4 + cell percentage, for use of zidovudine during pregnancy, and, if possible, for mode of delivery. Infants were defined as being uninfected if at least three cultures, including at least two when an infant was more than 1 month of age, were negative for HIV-1. 3 Addi-
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tional data on maternal age, prior pregnancies, infant birth weight, gestational age, and breast-feeding were obtained. The Women and Infants Transmission Study research protocols were approved by the human experimentation review boards of the participating institutions. Written informed consent was obtained from all women enrolled in the study, and from the parent or legal guardian of all infants. Virus isolates and neutralization assay. Each patient's primary HIV-1 isolate was amplified once in phytohemagglutinin-stimulated normal donor peripheral blood mononuclear cell cocultnre, the supematant was harvested, and aliquots were frozen at - 7 0 ° C. Each virus isolate was titered, and a fresh aliquot of titered frozen virus stock was used in each neutralization experiment. For the mothers, the virus isolate obtained from the delivery blood sample, or from the sample obtained as close as possible to the time of delivery, was used. For the infected infants, the first isolate containing virus, excluding cord blood isolates, was used. Neutralization by maternal plasma or serum of maternal and infant HIV- 1 isolates was determined with modifications of reported methods. 21-24Maternal plasma or serum samples from the time of delivery were used except in five cases for which samples obtained during the second or third trimester were used. Plasma or serum was separated from whole blood on the day the blood was drawn (usually within 4 hours), and frozen at -70 ° C until use. Samples were heat inactivated at 56 ° C for 30 minutes, and fourfold serial dilutions from 1:4 to 1:1024 were prepared in a final volume of 50 ml. These dilutions were then incubated in 96-welt plates at 37 ° C for 60 minutes with 50 ml of virus sup~natant containing an lnoculum of 100 TCIDs0, followed by the addition of 2 x 105 phytohemagglutinin-stimulated peripheral blood mononuclear cells in 100 ml of medium. The medium was changed on days 1 and 4, and the p24 antigen concentration in the supematant was determined by enzyme-linked immunosorbent assay (DuPont, Wilmington, Del.) on day 7. Neutralization assays were performed in quadruplicate. The neutralizing titer was defined as the dilution of plasma or serum that resulted in a 50% reduction in p24 :oncentration compared with the no-serum control, and was calculated by the Karber method. 25 The assay was validated by means of a polyclonal HIV-1 immune globulin of defined titer from the National Institutes of Health AIDS Research and Reference Reagent Program (catalog No. 192). This antibody was used as a positive control, and HIV-l-seronegative plasma served as a negative control in each assay. Syncytium-inducing phenotype determination. Virus phenotype was determined as described.26 Briefly, 100 TCIDs0 of each virus stock was used to infect 5 x 104 MT-2 cells and the syncydum-inducing-non-syncytium-inducing phenotype was determined by visual inspection, with syncytium formation defined as more than three syncytia per
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well. The cultures were continued for 14 days, after which time the virus was scored NSI if syncytia were not observed.
Quantitative RNA polymerase chain reaction, virus culture, and p24 antigen determination. Viral RNA quantification in plasma was performe d by reverse transcription of viral RNA followed by amplification by polymerase chain reaction of viral pol sequences as previously described.27 The HIV-1 cultures of maternal and infant cells were grown in phytohemagglutinin-stimulated peripheral blood mononuclear cells as described) Immune complexdissociated p24 antigen concentration in plasma was determined with a commercial enzyme-linked immunosorbent assay kit (DuPont) according to the manufacturer's directions. Statistical analysis• Maternal age, CD4 + cell percentages, CD8 ÷ cell percentages, number of prior pregnancies, birth weights, gestational ages, viral copy number determined by ribonucleic acid-PCR, and p24 concentration in plasma were compared between the two groups by means of the Wilcoxon signed rank test. The RNA-PCR data were log-transformed before analysis. Neutralizing titers, mode of delivery, ethnicity, virus phenotype, and the maternal mortality rate were compared by means of the McNemar test. The relation between virus phenotype and neutralizing titers, and between virus phenotype and mortality rate for the infected infants, was analyzed with the Fisher Exact Test. The Mann-Whitney U test was used to compare viral copy number of SI versus NSI phenotype virus. The Spearman rank correlation was computed to determine the intersample correlation in the RNA-PCR assay and to assess the relation between neutralizing antibody titers against maternal and infant virus, between maternal CD4 ÷ cell percentage and viral copy number, and between neutralizing antibody titer and viral copy number.
RESULTS
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infants, two had positive culture results at birth, another had positive results at day 6 of life, and another at day 14. The one very low birth weight infant (745 gin) was in the infected group.
Virus isolation and neutralization of autologous and infant isolates. Human immunodeficiency virus type 1 was grown and titered from the 32 infected women and from the 16 infected infants. For four isolates, two from women in the transmitting group and two from the infants born to these two women, a titer of at least 100 TCIDsd50 gl (2 x 103/ml) was not obtained, so a lower titer was used in the neutralization assay (69 TCIDs0 for the two maternal isolates and 24 and 41 TCIDs0 for the infant isolates). The neutralizing titer of each woman's serum or plasma against her own (autologous) virus for the transmitting and nontransmitting women is shown in the Table. In both groups most neutralizing titers were low. The median for the transmitting group was 8 (range, <4 to 181), and the median for the nontransmitting group was 6 (range, <4 to i81); six titers in the transmitting group and seven in the nontransmitting group were below the limit of detection of the assay. There was no statistically significant difference in autologous neutralization between the two groups (p = 1.0), and no significant difference if the case patient and the control subject who were not matched for use of zidovudine during pregnancy were excluded. No difference was observed between the neutralizing titers obtained against the maternal isolates versus the titers against the infant isolates (Table). The neutralizing titer of maternal antibody against maternal virus was highly correlated with the titer of the mother's antibody against her infant's virus (p = 0.84; p = 0.001), with one striking exception, motherinfant pair T-12, for whom the titer against the infant isolate was repeatedly higher than the titer against the maternal isolate.
Study population. The study population, consisting of 16
Quantitative RNA-PCR, plasma p24 antigen concentration, and viral culture. The median quantitative RNA-
HIV-1-infected women whose infants also became infected, was matched for CD4 + cell percentage and use of zidovudine during pregnancy with a control population, consisting of 16 HIV-l-infected women whose infants did not become infected. As expected from the matching, there was no significant difference in the two groups in maternal CD4 + cell percentage, use of zidovudine, or mode of delivery of the infant. One case-control pair was discordant for zidovudine use because the case mother was enrolled in a masked trial of zidovudine versus placebo during pregnancy. In addition to the matched variables, there was no significant difference between the groups in maternal CD8 + cell count, maternal age at the time of delivery, ethnicity, number of prior pregnancies, infant birth weight, or infant gestational age. None of the infants in either group was breast fed. Of 16 infected
PCR-detem~ined viral copy number per milliliter of plasma was 785 (range, <100 to 24,000) in the transmitting group and 1240 (range, <100 to 122,500) in the nontransmitting group (p = 0.08). The results obtained from the duplicate samples for each individual were highly correlated (p = 0.98; p <0.001), indicating excellent intersample reproducibility of this assay. Significant negative correlations were observed between the RNA-PCR-determined plasma viral load and maternal CD4 + cell percentage (p =-0.53; p <0.005) and between plasma viral load and maternal autologous neutralizing titer (9 = - 0.37; p <0.05). The p24 antigen concentration in plasma was determined after acid dissociation of immune complexes for 15 of the 16 pairs of women. The concentration was below the limit of detection in the majority of samples from both groups (11 of
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Table. Neutralization of maternal and infant virus isolates Neutralizing titer Transmitting mother
CD4 ÷ cell percentage
Autologous virus
Infant virus
Nontransmitting mother
CD4 ÷ cell percentage
Neutralizing titer of autologous virus
T-l* T-2 T-3 T-4 T-5 T-6* T-7* T-8 T-9 T-t0 T-11 T-12 T-13 T-14 T-15 T-t6*
3 4 9 15 16 19 22 26 27 29 29 35 35 37 41 46
<4? 181 4 <4 128 32 8 <4 <4? 11 128 8 23 128 <4 <4
<45 512 11 <4 181 64 <4 6 <4§ 45 128 >1024 23 128 <4 <4
NT-1 NT-2 NT-3 NT-4 NT-5 NT-6 NT-7 NT-8 NT-9 NT-10 NT-1I NT-12 NT-13 NT-14 NT-15 NT-16
7 11 12 19 21 15 17 22 27 24 28 31 36 37 44 43
<4 <4 32 <4 <4 <4 <4 45 8 8 <4 181 32 45 16 4
*Infantculturepositivefor HIV-1 at 14 days of age or younger. "~Virusinoculumof 69 TCIDs0. SVirus inoculumof 41 TCIDs0. §Virus inoculumof 24 TCIDs0. 15 in the transmitting group and 9 of 15 in the nontransmitting group; p value not significant). No significant difference between the groups was observed. Samples for nonquantitative HIV- 1 culture were obtained from women enrolled in the Women and Infants Transmission Study at up to four visits during pregnancy: first, second, and third trimesters and delivery. Most women in both groups had at least two cultures. There was no difference in the rate of culture positivity between the transmitting and the nontransmitting women (29 of 34 vs 26 of 32 cultures grew HIV-1). Virus phenotype. Five of sixteen isolates from the transmitring group of women and 4 of 16 from the nontransmitting group of women had the SI phenotype. Virus isolates from two of the five infected infants whose mothers had SI phenotype virus were SI and three were NSI. The SI phenotype virus was less likely than the phenotype virus to be neutralizable by maternal antibody; autologous maternal antibody had a neutralizing 6ter of 4 or less against 7 of 9 SI isolates, in comparison with 8 of 23 NSI isolates (p <0.05). The SI virus was also associated with greater replication in vivo. The median number of HIV- 1 particles per milliliter of plasma in the nine women with SI virus was 8650 (range, 980 to 122,500), compared with 410 (range, <100 to 66,000) in the 23 women with NSI virus (p <0.05). Maternal and infant mortality rates. The median duration of follow-up of the women enrolled in this study from the time of delivery was 1.7 years for the mothers whose infants became infected and 2.4 years for the women whose
infants did not become infected. Despite the longer follow-up in the nontransmitting women, all the women who died during the follow-up period were those whose infants became infected (4/16 vs 0/16; p = 0.13). There were no deaths in the HIV- 1-uninfected infants during the follow-up period. Among the 16 infected infants, two cried during the follow-up period. One very low birth weight infant died at 2 weeks of age, and the other child died at 2 years of age. Both infants who died had SI phenotype virus; all 14 infants who survived had NSI virus (p <0.01).
DISCUSSION In this study of mother-to-infant transmission of HIV-1, in which the transmitting and nontransmittingmothers were matched by maternal CD4 + cell count and use of zidovudine, we did not find an association between HIV-I transmission and the titer of maternal autologous neutralizing antibody. The lack of association between titers of maternal autologous neutralizing antibody and transmission is consistent with the findings of a small European study in which there was no association between maternal autologous neutralizing antibody and vertical transmission.2s That study did suggest that mothers whose infants did not become infected were more likely to have neutralizing antibody against one or more heterologous primary isolates. In that study, however, there was a significant difference in CD4 + cell counts and p24 antigen positivity between the transmitting and nontransmitting mothers, which suggests that the observed difference in het-
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erologous neutralizing titers may not have been independent of disease stage and viral load. This interpretation is supported by the inverse correlation between maternal plasma virus load and autologous neutralizing antibody observed in this study, and by previous studies of children and adults that correiate advanced disease with low titers of neutralizing antibody. 29' 30 Another study found a trend toward lower titers of autologous neutralizing antibody in transmitting versus nontransmitring women. 31 The reason for the difference between the results of that study and our results may be related to differences in the neutralization assays, or to differences in virus phenotype or virus load in the two groups of women in that study. In addition, although we examined a larger number of transmitting and nontransmitting women than were examined in previous studies, the relatively small sample size, the low titers, and the large proportion of titers below the limit of detection of the neutralization assay reduce the power of our study to detect small differences in autologous neutralization. Other studies of functional maternal anti-HIV-1 antibody in vertical HIV-1 transmission have also failed to identify differences in titer between transmitting and nontransmitting women. A study of neutralizing antibody against the IIIB strain of HIV-1 found no difference in titer between transmitting and nontransmitting women. 1° A study of maternal anti-SI antibody, an antibody function that correlates with neutralization, and a study of maternal antibodydependent cellular cytotoxicity both failed to observe differences in titers between transmitting and nontransmitting mothers.32, 33 Although the low autologous neutralizing titers in both groups limit the power of this study to detect differences between the groups, the finding of low titers in both groups is itself striking. The low titers of autologous anti-HIV-1 neutralizing antibody observed in this and other studies 2a, 34 suggest that passive immunization with immunoglobulin preparations containing high titers of neutralizing antibodies against a broad spectrum of HIV-1 isolates has the potential to increase endogenous humoral immunity. This rationale is the basis of an ongoing AIDS Clinical Trials Group study to prevent vertical transmission. The titer of maternal autologous neutralizing antibody and the titer of maternal neutralizing antibody against the infant' s first virus isolate were highly correlated in all but one mother-infant pair. This result indicates that the virus transmitted to the infant is not an unusual variant of the maternal vires population. This interpretation is consistent with the observation of Kliks et al.31 that the replication kinetics of maternal and infant viruses were highly similar. We also did not observe an association of maternal virus load and vertical
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transmission as measured by quantitative RNA-PCR, immune-complex-disrupted p24 antigen concentration in plasma, or HIV-1 culture positivity. This result contrasts with recent studies of unmatched transmitting and nontransmitting women that demonstrated a correlation between maternal virus load and vertical HIV-I transmission. 8, 9 The absence of association between viral load and transmission in our study is likely the result of matching for CD4 + cell percentage between the two groups. This interpretation is supported by the highly significant inverse correlation between CD4 + cell percentage and RNA-PCR-determined viral load observed in this study, a finding that is consistent with known associations between high viral load, low CD4 ÷ cell counts and advanced disease stage. 3537 We also did not observe a difference in virus phenotype between the transmitting and nontransmitting women. The SI phenotype has been associated with more advanced disease, but its role in transmission is not known. The phenotype discordance between women with SI virus and infants with NSI virus indicates that the SI phenotype is not transmitted preferentially. This discordance of transmitted phenotype has also been observed in horizontal transmission.%. 39 This finding suggests that there is selection against expression of this phenotype in newly infected infants, either at the level of transmission or by suppression of SI phenotype virus replication by the infant immune system. As has been observed in adults, SI phenotype virus in the infant appears to be associated with a poor prognosis. In older children, SI phenotype has been associated with lower CD4 + cell counts, although this association was not independent of age. 4° Consistent with the in vitro phenotype, we found that SI phenotype virus was associated with increased viral load in vivo, and this phenotype occurred predominantly in women with 10w CD4 + cell counts. The low titers of autologous neutralizing antibody in this and other studies, and the absence of a difference in titers between the groups in our matched case-control study, suggest that neutralizing antibody against autologous virus is unlikely to play a major role in determining the probability of vertical transmission. The results of several studies, together with the efficacy of zidovudine in decreasing the rate of vertical transmission of HIV-1, suggest that maternal virus load is a major determinant of the probability of transmission. Our study and others, however, demonstrate that infants of women with low viral loads may become infected, and infants of women with high viral burdens may escape infection. Thus the pathogenesis of vertical HIV-1 transmission is likely to be multifactorial, and other viral and immune variables, systemically and at the mucosal surface of the infant and the mother, are likely to play a role in determining
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whether an infant born to an HIV-infected woman becomes infected. 16. We thank the Women and Infants Transmission Study investigators, research nurses, and laboratory personnel, including Ruth Tuomola, Arlene Buck, Rosemary Galvin, Patrick Donegan, Steven Pelton, Ellen Cooper, Ethel Hudson, Kathy Khoury, Elizabeth Jones, Martha Muehlmann, and Jenifer Davis. REFERENCES
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Vertical transmission of human immunodeficiency virus type 1: Autologous neutralizing antibody, virus load, and virus phenotype Robert N. Husson, MD, Yan Lan, MD, Eiji Kojima, MS, David Venzon, PhD, Hiroaki Mitsuya, MD, PhD, and Kenneth Mclntosh, MD From the Divisionof infectious Diseases, Children's Hospital, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts,and the Experimental RetrovirologySection, Medicine Branch, and the Biostatisticsand Data Management Section, National Cancer Institute, Bethesda, Maryland
Objective: To evaluate immunologic and virologic correlates of vertical transmission of human immunodeficiency virus type I (HIV-I). Design: Case-control study. Patients: Women who were prospectively enrolled in a natural history study of HIV-I infection in women and infants. Sixteen HIV-1-infected women whose infants became infected were matched by CD4 ÷ cell percentage and use of zidovudine during pregnancy with women whose infants did not become infected. Measurements: Maternal autologous neutralizing antibody, virus load determined by RNA-polymerase chain reaction (RNA-PCR), and virus phenotype. Results: Most women in both groups had low titers of autologous neutralizing antibody, and no difference in neutralizing titers was observed (range, <4 to 181 in both groups). The HIV-I copy number in maternal plasma was not significantly different in the two groups but was inversely correlated with maternal CD4 ÷ cell percentage (p <0.005). Five women in the transmitting group and four in the nontransmitting group had syncytium-inducing (SI) phenotype virus. Two infected infants had SI phenotype virus. The SI phenotype virus was associated with a greater HIV-I copy number in maternal plasma (p <0.05) and an increase in the mortality rate for the infants (p <0.01). Conclusions: In women matched for CD4 ÷ cell percentage, low titers of autologous neutralizing antibody, high virus load, and SI phenotype virus were not associated with an increased risk of transmission of HIV-I to their infants. (J PEDIATR 1995; 126:865-71 ) Supported by grants from the Pediatric AIDS Foundation (5029114-PG), the Charles H. Hood Foundation, and the Milton Fund of Harvard University, and by Women and Infants Transmission Study contract NO1 AI-82507 from the National Institutes of Health. Dr. Husson was the recipient of a Markey Scholar Award. Presented in part at the 33rd Interscience Conference on Antimicrobial Agents and Chemotherapy, October 1993, Anaheim, Calif. (Abstract No. 1138). Reprint requests: Robert N. Husson, MD, Division of Infectious Diseases, Children's Hospital, 300 Longwood Ave., Boston, MA 02115. 9/20/'64351
Although reported rates of vertical transmission of in~nmnodeficiency virus type 1 have varied widely, only a small proportion of infants born to infected women become infected. Two large prospective studies of infants born to HIV-l-infected women in Europe found vertical transmission rates of 14.4% and 18.3%, respectively.],2 In the ongoing Women and Infants Transmission Study in the United States, the overall transmission rate is less than 20%. 3 In Africa, reported rates of transmission are higher, although infection occurs in substantially fewer than half of the infants.4, 5 The recently demonstrated efficacy of zidovudine in
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HIV-1 NSI RNA-PCR SI TCIDs0
Human immunodeficiency virus type 1 Non-syncytium-inducing Polymerase chain reaction of reverse transcribed fibonucleic acid Syncytium-inducing Fifty percent tissue culture infectivity dose
reducing the rate of vertical transmission is likely to decrease transmission in populations where this drug is available and the HIV-I status of women of childbearing age is known. 6 Despite the importance of the pathogenesis of vertical transmission of HIV-1 and the variables that determine why some infants born to HIV-l-infected women become infected and others do not, relatively little is known about these phenomena. Among the variables that have been consistently associated with an increased likelihood of transmission are advanced maternal clinical disease or markers of advanced disease such as low CD4 + cell counts and high CD8 + cell counts, and markers of high maternal virus burden, such as p24 antigenemia or I-IIV-1 culture positivity.1.5, 7-9 Other variables that may play a role are premature birth, placental inflammation, mode of delivery, and breastfeeding 1,2,5,7,10-14 The role of maternal HIV-l-specific immunity in vertical transmission remains unclear, with conflicting results regarding the association of maternal anti-HIV-1 antibody and transmission, l°, 15-20 A better understanding of the correlates of mother-to-infant transmission of HIV-1 would provide information highly relevant to the prevention of both vertical and horizontal transmission. We therefore conducted a case-control study to assess the role of immunologic and virologic factors in relation to mother-to-infant transmission of HIV-1.
METHODS Study population. The HIV-1-infected women enrolled in the Boston centers for the Women and Infants Transmission Study between January 1990 and November 1992, who had given bi~h to infants known to be infected with HIV- 1, were identified by study number through a review of the study records. Infants were defined as infected if they had two or more positive HIV-1 culture results, excluding cord blood, from samples obtained at different times in the first year of life. 3 Of the 20 mother-infant pairs identified, 16 were found to have appropriate virus isolates and plasma or serum samples available for study. From the same study population, women whose infants were known not to be infected were identified, and control mother-infant pairs were selected by matching for maternal CD4 + cell percentage, for use of zidovudine during pregnancy, and, if possible, for mode of delivery. Infants were defined as being uninfected if at least three cultures, including at least two when an infant was more than 1 month of age, were negative for HIV-1. 3 Addi-
The Journal of Pediatrics June 1995
tional data on maternal age, prior pregnancies, infant birth weight, gestational age, and breast-feeding were obtained. The Women and Infants Transmission Study research protocols were approved by the human experimentation review boards of the participating institutions. Written informed consent was obtained from all women enrolled in the study, and from the parent or legal guardian of all infants. Virus isolates and neutralization assay. Each patient's primary HIV-1 isolate was amplified once in phytohemagglutinin-stimulated normal donor peripheral blood mononuclear cell cocultnre, the supematant was harvested, and aliquots were frozen at - 7 0 ° C. Each virus isolate was titered, and a fresh aliquot of titered frozen virus stock was used in each neutralization experiment. For the mothers, the virus isolate obtained from the delivery blood sample, or from the sample obtained as close as possible to the time of delivery, was used. For the infected infants, the first isolate containing virus, excluding cord blood isolates, was used. Neutralization by maternal plasma or serum of maternal and infant HIV- 1 isolates was determined with modifications of reported methods. 21-24Maternal plasma or serum samples from the time of delivery were used except in five cases for which samples obtained during the second or third trimester were used. Plasma or serum was separated from whole blood on the day the blood was drawn (usually within 4 hours), and frozen at -70 ° C until use. Samples were heat inactivated at 56 ° C for 30 minutes, and fourfold serial dilutions from 1:4 to 1:1024 were prepared in a final volume of 50 ml. These dilutions were then incubated in 96-welt plates at 37 ° C for 60 minutes with 50 ml of virus sup~natant containing an lnoculum of 100 TCIDs0, followed by the addition of 2 x 105 phytohemagglutinin-stimulated peripheral blood mononuclear cells in 100 ml of medium. The medium was changed on days 1 and 4, and the p24 antigen concentration in the supematant was determined by enzyme-linked immunosorbent assay (DuPont, Wilmington, Del.) on day 7. Neutralization assays were performed in quadruplicate. The neutralizing titer was defined as the dilution of plasma or serum that resulted in a 50% reduction in p24 :oncentration compared with the no-serum control, and was calculated by the Karber method. 25 The assay was validated by means of a polyclonal HIV-1 immune globulin of defined titer from the National Institutes of Health AIDS Research and Reference Reagent Program (catalog No. 192). This antibody was used as a positive control, and HIV-l-seronegative plasma served as a negative control in each assay. Syncytium-inducing phenotype determination. Virus phenotype was determined as described.26 Briefly, 100 TCIDs0 of each virus stock was used to infect 5 x 104 MT-2 cells and the syncydum-inducing-non-syncytium-inducing phenotype was determined by visual inspection, with syncytium formation defined as more than three syncytia per
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well. The cultures were continued for 14 days, after which time the virus was scored NSI if syncytia were not observed.
Quantitative RNA polymerase chain reaction, virus culture, and p24 antigen determination. Viral RNA quantification in plasma was performe d by reverse transcription of viral RNA followed by amplification by polymerase chain reaction of viral pol sequences as previously described.27 The HIV-1 cultures of maternal and infant cells were grown in phytohemagglutinin-stimulated peripheral blood mononuclear cells as described) Immune complexdissociated p24 antigen concentration in plasma was determined with a commercial enzyme-linked immunosorbent assay kit (DuPont) according to the manufacturer's directions. Statistical analysis• Maternal age, CD4 + cell percentages, CD8 ÷ cell percentages, number of prior pregnancies, birth weights, gestational ages, viral copy number determined by ribonucleic acid-PCR, and p24 concentration in plasma were compared between the two groups by means of the Wilcoxon signed rank test. The RNA-PCR data were log-transformed before analysis. Neutralizing titers, mode of delivery, ethnicity, virus phenotype, and the maternal mortality rate were compared by means of the McNemar test. The relation between virus phenotype and neutralizing titers, and between virus phenotype and mortality rate for the infected infants, was analyzed with the Fisher Exact Test. The Mann-Whitney U test was used to compare viral copy number of SI versus NSI phenotype virus. The Spearman rank correlation was computed to determine the intersample correlation in the RNA-PCR assay and to assess the relation between neutralizing antibody titers against maternal and infant virus, between maternal CD4 ÷ cell percentage and viral copy number, and between neutralizing antibody titer and viral copy number.
RESULTS
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infants, two had positive culture results at birth, another had positive results at day 6 of life, and another at day 14. The one very low birth weight infant (745 gin) was in the infected group.
Virus isolation and neutralization of autologous and infant isolates. Human immunodeficiency virus type 1 was grown and titered from the 32 infected women and from the 16 infected infants. For four isolates, two from women in the transmitting group and two from the infants born to these two women, a titer of at least 100 TCIDsd50 gl (2 x 103/ml) was not obtained, so a lower titer was used in the neutralization assay (69 TCIDs0 for the two maternal isolates and 24 and 41 TCIDs0 for the infant isolates). The neutralizing titer of each woman's serum or plasma against her own (autologous) virus for the transmitting and nontransmitting women is shown in the Table. In both groups most neutralizing titers were low. The median for the transmitting group was 8 (range, <4 to 181), and the median for the nontransmitting group was 6 (range, <4 to i81); six titers in the transmitting group and seven in the nontransmitting group were below the limit of detection of the assay. There was no statistically significant difference in autologous neutralization between the two groups (p = 1.0), and no significant difference if the case patient and the control subject who were not matched for use of zidovudine during pregnancy were excluded. No difference was observed between the neutralizing titers obtained against the maternal isolates versus the titers against the infant isolates (Table). The neutralizing titer of maternal antibody against maternal virus was highly correlated with the titer of the mother's antibody against her infant's virus (p = 0.84; p = 0.001), with one striking exception, motherinfant pair T-12, for whom the titer against the infant isolate was repeatedly higher than the titer against the maternal isolate.
Study population. The study population, consisting of 16
Quantitative RNA-PCR, plasma p24 antigen concentration, and viral culture. The median quantitative RNA-
HIV-1-infected women whose infants also became infected, was matched for CD4 + cell percentage and use of zidovudine during pregnancy with a control population, consisting of 16 HIV-l-infected women whose infants did not become infected. As expected from the matching, there was no significant difference in the two groups in maternal CD4 + cell percentage, use of zidovudine, or mode of delivery of the infant. One case-control pair was discordant for zidovudine use because the case mother was enrolled in a masked trial of zidovudine versus placebo during pregnancy. In addition to the matched variables, there was no significant difference between the groups in maternal CD8 + cell count, maternal age at the time of delivery, ethnicity, number of prior pregnancies, infant birth weight, or infant gestational age. None of the infants in either group was breast fed. Of 16 infected
PCR-detem~ined viral copy number per milliliter of plasma was 785 (range, <100 to 24,000) in the transmitting group and 1240 (range, <100 to 122,500) in the nontransmitting group (p = 0.08). The results obtained from the duplicate samples for each individual were highly correlated (p = 0.98; p <0.001), indicating excellent intersample reproducibility of this assay. Significant negative correlations were observed between the RNA-PCR-determined plasma viral load and maternal CD4 + cell percentage (p =-0.53; p <0.005) and between plasma viral load and maternal autologous neutralizing titer (9 = - 0.37; p <0.05). The p24 antigen concentration in plasma was determined after acid dissociation of immune complexes for 15 of the 16 pairs of women. The concentration was below the limit of detection in the majority of samples from both groups (11 of
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Table. Neutralization of maternal and infant virus isolates Neutralizing titer Transmitting mother
CD4 ÷ cell percentage
Autologous virus
Infant virus
Nontransmitting mother
CD4 ÷ cell percentage
Neutralizing titer of autologous virus
T-l* T-2 T-3 T-4 T-5 T-6* T-7* T-8 T-9 T-t0 T-11 T-12 T-13 T-14 T-15 T-t6*
3 4 9 15 16 19 22 26 27 29 29 35 35 37 41 46
<4? 181 4 <4 128 32 8 <4 <4? 11 128 8 23 128 <4 <4
<45 512 11 <4 181 64 <4 6 <4§ 45 128 >1024 23 128 <4 <4
NT-1 NT-2 NT-3 NT-4 NT-5 NT-6 NT-7 NT-8 NT-9 NT-10 NT-1I NT-12 NT-13 NT-14 NT-15 NT-16
7 11 12 19 21 15 17 22 27 24 28 31 36 37 44 43
<4 <4 32 <4 <4 <4 <4 45 8 8 <4 181 32 45 16 4
*Infantculturepositivefor HIV-1 at 14 days of age or younger. "~Virusinoculumof 69 TCIDs0. SVirus inoculumof 41 TCIDs0. §Virus inoculumof 24 TCIDs0. 15 in the transmitting group and 9 of 15 in the nontransmitting group; p value not significant). No significant difference between the groups was observed. Samples for nonquantitative HIV- 1 culture were obtained from women enrolled in the Women and Infants Transmission Study at up to four visits during pregnancy: first, second, and third trimesters and delivery. Most women in both groups had at least two cultures. There was no difference in the rate of culture positivity between the transmitting and the nontransmitting women (29 of 34 vs 26 of 32 cultures grew HIV-1). Virus phenotype. Five of sixteen isolates from the transmitring group of women and 4 of 16 from the nontransmitting group of women had the SI phenotype. Virus isolates from two of the five infected infants whose mothers had SI phenotype virus were SI and three were NSI. The SI phenotype virus was less likely than the phenotype virus to be neutralizable by maternal antibody; autologous maternal antibody had a neutralizing 6ter of 4 or less against 7 of 9 SI isolates, in comparison with 8 of 23 NSI isolates (p <0.05). The SI virus was also associated with greater replication in vivo. The median number of HIV- 1 particles per milliliter of plasma in the nine women with SI virus was 8650 (range, 980 to 122,500), compared with 410 (range, <100 to 66,000) in the 23 women with NSI virus (p <0.05). Maternal and infant mortality rates. The median duration of follow-up of the women enrolled in this study from the time of delivery was 1.7 years for the mothers whose infants became infected and 2.4 years for the women whose
infants did not become infected. Despite the longer follow-up in the nontransmitting women, all the women who died during the follow-up period were those whose infants became infected (4/16 vs 0/16; p = 0.13). There were no deaths in the HIV- 1-uninfected infants during the follow-up period. Among the 16 infected infants, two cried during the follow-up period. One very low birth weight infant died at 2 weeks of age, and the other child died at 2 years of age. Both infants who died had SI phenotype virus; all 14 infants who survived had NSI virus (p <0.01).
DISCUSSION In this study of mother-to-infant transmission of HIV-1, in which the transmitting and nontransmittingmothers were matched by maternal CD4 + cell count and use of zidovudine, we did not find an association between HIV-I transmission and the titer of maternal autologous neutralizing antibody. The lack of association between titers of maternal autologous neutralizing antibody and transmission is consistent with the findings of a small European study in which there was no association between maternal autologous neutralizing antibody and vertical transmission.2s That study did suggest that mothers whose infants did not become infected were more likely to have neutralizing antibody against one or more heterologous primary isolates. In that study, however, there was a significant difference in CD4 + cell counts and p24 antigen positivity between the transmitting and nontransmitting mothers, which suggests that the observed difference in het-
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erologous neutralizing titers may not have been independent of disease stage and viral load. This interpretation is supported by the inverse correlation between maternal plasma virus load and autologous neutralizing antibody observed in this study, and by previous studies of children and adults that correiate advanced disease with low titers of neutralizing antibody. 29' 30 Another study found a trend toward lower titers of autologous neutralizing antibody in transmitting versus nontransmitring women. 31 The reason for the difference between the results of that study and our results may be related to differences in the neutralization assays, or to differences in virus phenotype or virus load in the two groups of women in that study. In addition, although we examined a larger number of transmitting and nontransmitting women than were examined in previous studies, the relatively small sample size, the low titers, and the large proportion of titers below the limit of detection of the neutralization assay reduce the power of our study to detect small differences in autologous neutralization. Other studies of functional maternal anti-HIV-1 antibody in vertical HIV-1 transmission have also failed to identify differences in titer between transmitting and nontransmitting women. A study of neutralizing antibody against the IIIB strain of HIV-1 found no difference in titer between transmitting and nontransmitting women. 1° A study of maternal anti-SI antibody, an antibody function that correlates with neutralization, and a study of maternal antibodydependent cellular cytotoxicity both failed to observe differences in titers between transmitting and nontransmitting mothers.32, 33 Although the low autologous neutralizing titers in both groups limit the power of this study to detect differences between the groups, the finding of low titers in both groups is itself striking. The low titers of autologous anti-HIV-1 neutralizing antibody observed in this and other studies 2a, 34 suggest that passive immunization with immunoglobulin preparations containing high titers of neutralizing antibodies against a broad spectrum of HIV-1 isolates has the potential to increase endogenous humoral immunity. This rationale is the basis of an ongoing AIDS Clinical Trials Group study to prevent vertical transmission. The titer of maternal autologous neutralizing antibody and the titer of maternal neutralizing antibody against the infant' s first virus isolate were highly correlated in all but one mother-infant pair. This result indicates that the virus transmitted to the infant is not an unusual variant of the maternal vires population. This interpretation is consistent with the observation of Kliks et al.31 that the replication kinetics of maternal and infant viruses were highly similar. We also did not observe an association of maternal virus load and vertical
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transmission as measured by quantitative RNA-PCR, immune-complex-disrupted p24 antigen concentration in plasma, or HIV-1 culture positivity. This result contrasts with recent studies of unmatched transmitting and nontransmitting women that demonstrated a correlation between maternal virus load and vertical HIV-I transmission. 8, 9 The absence of association between viral load and transmission in our study is likely the result of matching for CD4 + cell percentage between the two groups. This interpretation is supported by the highly significant inverse correlation between CD4 + cell percentage and RNA-PCR-determined viral load observed in this study, a finding that is consistent with known associations between high viral load, low CD4 ÷ cell counts and advanced disease stage. 3537 We also did not observe a difference in virus phenotype between the transmitting and nontransmitting women. The SI phenotype has been associated with more advanced disease, but its role in transmission is not known. The phenotype discordance between women with SI virus and infants with NSI virus indicates that the SI phenotype is not transmitted preferentially. This discordance of transmitted phenotype has also been observed in horizontal transmission.%. 39 This finding suggests that there is selection against expression of this phenotype in newly infected infants, either at the level of transmission or by suppression of SI phenotype virus replication by the infant immune system. As has been observed in adults, SI phenotype virus in the infant appears to be associated with a poor prognosis. In older children, SI phenotype has been associated with lower CD4 + cell counts, although this association was not independent of age. 4° Consistent with the in vitro phenotype, we found that SI phenotype virus was associated with increased viral load in vivo, and this phenotype occurred predominantly in women with 10w CD4 + cell counts. The low titers of autologous neutralizing antibody in this and other studies, and the absence of a difference in titers between the groups in our matched case-control study, suggest that neutralizing antibody against autologous virus is unlikely to play a major role in determining the probability of vertical transmission. The results of several studies, together with the efficacy of zidovudine in decreasing the rate of vertical transmission of HIV-1, suggest that maternal virus load is a major determinant of the probability of transmission. Our study and others, however, demonstrate that infants of women with low viral loads may become infected, and infants of women with high viral burdens may escape infection. Thus the pathogenesis of vertical HIV-1 transmission is likely to be multifactorial, and other viral and immune variables, systemically and at the mucosal surface of the infant and the mother, are likely to play a role in determining
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whether an infant born to an HIV-infected woman becomes infected. 16. We thank the Women and Infants Transmission Study investigators, research nurses, and laboratory personnel, including Ruth Tuomola, Arlene Buck, Rosemary Galvin, Patrick Donegan, Steven Pelton, Ellen Cooper, Ethel Hudson, Kathy Khoury, Elizabeth Jones, Martha Muehlmann, and Jenifer Davis. REFERENCES
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