PERINATAL TRANSMISSION OF HIV AND PEDIATRIC AIDS IN THAILAND

Perinatal Transmission of HIV and Pediatric AIDS in Thailand

Usa Thisyakorn,* Siriraj Paupanwatana,^ Varit Kanchanamayul, Tawee Chotpitayasunondh,¤ Tim Brown,# and Werasit Sittitrai**

*Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; ^Chiang Rai Prachanukroh Hospital, Chiang Rai, Thailand; Rayong Hospital, Rayong, Thailand; ¤Children's Hospital, Bangkok, Thailand; #Program on Population, East-West Center, Hawaii, USA; and **Program on AIDS, Thai Red Cross Society, Bangkok, Thailand

A collaborative study group of researchers from four hospitals in Thailand was formed to collect information on children born to HIV-infected mothers since the beginning of the first hospital case until the end of December 1992. The study, which was done to verify the status of perinatal transmission of HIV infection in Thailand, has shown a rapid increase in HIV seropositivity among Thai mothers. The rate of vertical transmission varied between 25% and 42%. The majority of children with symptomatic HIV infection present with delayed growth and development, pneumonia, diarrhea, oral candidiasis, lymphadenopathy, or hepatosplenomegaly. At Chiang Rai Prachanukroh Hospital, two cases of Pe nicillium marneffei infection have been confirmed. The problems to be addressed are the size of the epidemic and the future management of the infants born to HIV-infected mothers.

The global AIDS epidemic is volatile, dynamic, and unstable, and its major impacts are yet to come. By early 1992, 12.9 million people around the world--including 4.7 million women, 7.1 million men, and 1.1 million children--had been infected with HIV. About one-fifth (2.7 million, or 21%) have thus far developed AIDS; of these, over 90 percent (n early 2.5 million) have died. HIV infection has recently exploded in Southeast Asia, especially in Thailand, Myanmar, and India, where within only a few years more than 1 million people have become infected. The global lesson is that HIV will reach most, if not all, communities.

The epidemic becomes ever more complex as it matures. Most important, the direct and indirect impact of the pandemic on women is increasing dramatically. In recent years, the rate of infection among women has increased more rapidly than that of men. In Mexico, for example, the ratio of HIV-infected men to women decreased from 25:1 in 1984 to 4:1 i n 1990. In the United States, HIV infection among women is spreading more rapidly than among men. By the year 2000, it is estimated that the number of women and men infected will be equal globally.

In Thailand, heterosexual transmission is becoming the major mode of HIV spread. With increasing evidence of heterosexual transmission, the number of infected women and consequently their children is increasing. Most HIV-infected women are of childbearing age and, because more than 80% of HIV-infected children acquire the infection from their moth ers through vertical transmission, the expected increase in the number of infected women will directly affect the number of pediatric AIDS cases. The remaining 20% of HIV-infected children acquired infection parenterally, from unscreened blood transfusions, or through exposure to blood-contaminated needles and syringes. Sexual transmission of HIV in children could occur in the cases of child prostitution and unsafe sexual practices among adolescents.1

In Thailand, the first case of AIDS was reported in a Thai man in September 1984. Three years later, the disease was seen in Thai women. The first documented case of a Thai baby who was born to an HIV-infected mother was seen at Chulalongkorn Hospital in July 1988 and the first Thai baby with AIDS was reported in the same year.2,3

Patients and Methods

The four hospitals involved in this study were selected because of their extensive experience with the study of HIV in Thai children, thus providing a window for such a study. The four hospitals were: (1) Chulalongkorn Hospital, whose staff members have been treating HIV disease since the earliest days of the Thai AIDS epidemic in 1984; (2) Chiang Rai Prachanukroh Hospital, a provincial hospital in a northern city with one of the highest HIV prevalences in the country; (3) Rayong Hospital, a provincial hospital in an eastern city where HIV prevalence in pregnant women has been rising rapidly; and (4) Bangkok Children's Hospital, the hospital with the highest prevalence of children being bo rn to HIV-positive mothers in Bangkok and the largest number of HIV-infected children currently under care in Bangkok. The researchers collected information on children born to HIV-infected mothers from the first case in their hospitals until the end of December 1992.

Results

In September 1991 Chulalongkorn Hospital began routine screening for HIV antibodies in pregnant mothers during the first visit to the antenatal care clinic. At Chiang Rai Prachanukroh Hospital, antibody testing was begun in May 1989, both at the first visit to the antenatal care clinic and immediately before or after delivery. Rayong Hospital bega n this testing at the first visit to the antenatal care clinic in October 1990. At Bangkok Children's Hospital, antenatal screening was begun in February 1989. At all hospitals pre- and post-test counseling is done. This screening has detected a large number of HIV-positive mothers. The numbers of children born to HIV-positive mothers at each hosp ital from the start of testing through December 1992 are listed in Table 1. The annual data on children born to HIV-positive mothers at Chiang Rai Prachanukroh and Bangkok Children's Hospitals are shown in Tables 2 and 3.

At Chulalongkorn Hospital, the vertical transmission rate is 33.3%. A study was done of the first 15 babies who were born to HIV-infected mothers. All had been followed through 18 months after birth. Of these, 2 had clinical AIDS and 3 had asymptomatic infection. Another 10 infants were seronegative and free of symptoms. Of these 15, 10 of the mot hers were injecting drug users, 3 were prostitutes (2 of whom were sexual partners of injecting drug users), and 2 were housewives, 1 of whom had multiple sexual partners even though her husband himself was HIV negative. At Bangkok Children's Hospital from 1989 through March 1992, 151 children were born to HIV-positive mothers. Of these, 22 were f ollowed through the age of 15 months. Six were found to be infected with HIV, yielding a vertical transmission rate of 27.3%. The mix of risk factors for the mothers changed over the years, with a decreasing proportion of injecting drug users and female prostitutes and an increasing proportion of heterosexual transmission cases. The percentage of those who were injecting drug users or female prostitutes diminished from 46% in 1989 to 18% in 1992, while women reporting heterosexual transmission from spouses or boyfriends increased from 54% to 82%. At Rayong Hospital, of 24 babies born to HIV-infected mothers from 1990 to 1991, evaluations at 18 months after birth showed 6 (25%) to be infect ed. Results from Chiang Rai are showing a higher rate of 42%. A study was done during the period of 1989 to 1991, when 145 babies were born to HIV-infected mothers. Of 121 babies who returned for follow-up and were evaluated at 15 months after birth, 42% were found to be infected. All of the mothers in Chiang Rai Prachanukroh Hospital reported het erosexual transmission of HIV as a risk factor.

The number of children with clinical AIDS who were seen at each of the hospitals through December 1992 is presented in Table 4. In terms of progression to symptomatic HIV infection, the 22 cases followed at Chulalongkorn Hospital mainly progressed within the first year, with a range of 1 month to 18 months. The age at diagnosis for these 22 childr en is presented in Figure 1. At Bangkok Children's Hospital, ages at diagnosis varied from 1 month to 24 months. Those progressing to symptomatic HIV infection in Chiang Rai mostly did so within the first 6 months. Figure 2 shows the age distribution of AIDS cases in children in Chiang Rai Prachanukroh Hospital. The doctors at Rayong Hospital have only recently begun to see AIDS in children and are not comfortable enough to diagnose the cases.

After progression to AIDS, death generally follows quickly for infants, and the babies in this study followed this pattern. At Chulalongkorn Hospital, the time from diagnosis of AIDS to death varied between 1 month and 13 months. In Chiang Rai Prachanukroh Hospital, the time from diagnosis to death varied between 2 and 4 months and at Bangkok Chil dren's Hospital survival times were between 10 days and 4 months.

The first 22 pediatric AIDS cases at Chulalongkorn Hospital were followed closely. The clinical features observed in these cases are shown in Table 5. Related data are also presented for the 69 pediatric AIDS cases at Bangkok Children's Hospital. In Chiang Rai Prachanukroh Hospital, most of the symptomatic cases presented with delayed growth and d evelopment. The common clinical features were oral candidiasis, pneumonia, and diarrhea. Two cases of Penicillium marneffei infection were confirmed.

Discussion

This study has demonstrated the rapid growth of HIV seropositivity in Thai mothers, which is in accordance with the sentinel surveillance figures of the past three years. The sentinel surveillance has shown that HIV seropositivity in Thai women presenting at antenatal clinics has been increasing steadily.4 The increase in the number of infected wo men will have a direct impact on the number of pediatric AIDS cases. This study has shown that the majority of pregnant women were infected through heterosexual transmission. The rapid spread of the disease by heterosexual contact has led to the increase in identifying HIV-infected children whose mothers are completely unaware that they have been infected by their sexual partners. The eventual magnitude of the problems faced by children depends critically on the reproductive decisions of HIV-infected women. The problems may be reduced through advanced planning and skilled counseling to help these women cope with the consequence of HIV infection for themselves and their children before, dur ing, and after birth.

The exact timing of transmission is under extensive study because of its implications for interruption of the transmission process. Studies in the United States and Europe have shown vertical transmission rates ranging from 7% to 42%, while studies in Africa have tended to show higher transmission rates--closer to 30% or 40%. Some maternal factors that may affect the efficiency of transmission include viral loads, stage of disease, immune status, antibody levels, and coinfection with other diseases.

Variation in ability of divergent HIV strains to attach to different tissues may also play a part in determining the probability of vertical transmission. The variation in vertical transmission rates from different hospitals in this study could be explained by the different study designs used by each hospital. More detailed comparisons of these da ta may provide important clues about the factors involved in maternal-child transmission in Thailand. For example, the epidemic in the north began earlier than that in the central regions (Bangkok and Rayong), as reflected by the higher seroprevalences in the north. One possible explanation for these higher transmission rates could be that the mot hers in the north have been infected longer and are thus more immunocompromised. The difference may also be attributed to subtype variations. Since there are two major HIV subtypes circulating in Thailand, future molecular epidemiology studies may be useful in addressing the importance of these viral variations.2,4

It is well documented that children progress more rapidly from HIV infection to AIDS than adults do. The European Collaborative Study found that 26% of perinatally HIV-infected children developed AIDS and 17% died within the first year.5 A study in Zambia found that 44% of HIV-infected newborns died within two years of follow-up.6 In our study, th e preliminary results show that the progression rates from HIV infection to AIDS appear to be lower than those in Africa and more in keeping with the values in Europe. In Chiang Rai, for example, 21.2% of those who were followed had progressed to AIDS by 15 months of age. The initial clinical presentations are in keeping with the studies done in o ther sites. After progression to AIDS, death generally follows quickly for the infants in this study, probably due to the lack of antiretroviral agents. At Chulalongkorn Hospital, several pediatric AIDS cases have now received antiretroviral agents and have shown a marked improvement in their condition.

The majority of children with AIDS in this study present with lymphadenopathy, delayed growth and development, oral candidiasis, pneumonia, diarrhea, or hepatosplenomegaly. At Chiang Rai Prachanukroh Hospital, in addition to the previously mentioned presentations, two cases of Penicillium marneffei infection have been confirmed in children. Infection from this organism has proven to be particularly prevalent in Chiang Mai, a province in northern Thailand adjacent to Chiang Rai.7

The increase in the number of HIV-infected children through perinatal transmission is alarming. Primary prevention involves prevention of HIV infection in women. A plan for the management of infants born to HIV-infected mothers should be developed.

Selected Papers

1. Mann J, Tarantola DJM, Netter TW. AIDS in the World. Cambridge, MA: Harvard University Press, 1992.
2. Thisyakorn U. Pediatric AIDS. In: Thisyakorn U, Thisyakorn C, eds. Tropical Pediatrics. Bangkok: Desire. 1993:99-117.
3. Thisyakorn U, Chumdermpadetsuk S, Thaithumyanon P, et al. Perinatal HIV transmission in Bangkok, Thailand. Presented at the 7th Congress of the Federation of the Asia-Oceania Perinatal Societies. October 25-29, 1992, Bangkok, Thailand.
4. Brown T, Sittitrai W, Thisyakorn U, Paupunwatana S, Kanchanamayul V, Chotpitayasunondh T. Medical consultants and the impact of HIV on children--a situation assessment (submitted 1993).
5. European Collaborative Study. Children born to women with HIV-1 infection: natural history and risk of transmission. Lancet 1991;337:253-60.
6. Hira SK, Kamanga J, Bhat GJ, et al. Perinatal transmission of HIV-1 in Zambia. Br Med J 1989;299:1250-2.
7. Supparatpinyo K, Chiewchanvit S, Hirunsri P, Uthammachai C, Nelson KE, Sirisanthana T. Penicillium marneffei infection in patients infected with human immunodeficiency virus. Clin Infect Dis 1992;14:871-4.

Pediatric AIDS in Thailand

Somsak Lolekha and Piyaporn Bowonkiratikachorn

Department of Pediatrics, Ramathibodi Hospital and Charoenkrung Pracharak Hospital, Thailand

The first pediatric AIDS case attributed to perinatal transmission of HIV in Thailand was reported in 1988. The number has increased rapidly over the past three years. On December 31, 1993, 430 pediatric AIDS cases (225 male, 205 female) from perinatal transmission were reported to the Ministry of Public Health. The actual number may be much highe r. The rate of vertical transmission varies from 21% to 33% in Bangkok to 42% to 46% in the north. Almost all mothers other than commercial sex workers contracted HIV from their husbands or steady sexual partners.

A prospective study of 95 HIV-positive mothers from April 1991 to May 1993 was done at Charoenkrung Pracharak Hospital. Five infants (5%) had positive polymerase chain reaction (PCR) for HIV at birth. There was no difference in the birth weight, maternal age, or method of delivery between infected and uninfected infants. Eighty percent of uninfect ed infants were seronegative at 10 months of age (range from 5 to 13 months, median 9 months).

Presenting symptoms of 82 pediatric AIDS cases at Chiang Mai Hospital were fever, respiratory symptoms, and diarrhea. Signs and symptoms at the time of the first admission in 151 cases--82 cases at Chiang Mai Hospital and 69 cases at the Children's Hospital in Bangkok--were hepatomegaly (93%), failure to thrive (75%), splenomegaly (72%), chronic o r recurrent pneumonia (69%), oral candidiasis (64%), chronic or recurrent diarrhea (52%), generalized lymphadenopathy (50%), prolonged fever for more than 1 month (48%), and generalized dermatitis (17%). Thrombocytopenia (platelet<100,000/mm³) Penicillium marneffei infection were not as common as in adults.

Risk and Predictors of Perinatal Transmission of HIV-1

Marc Lallemant,*,^ Sophie Lallemant-Le Coeur,* Samuel Nzingoula, D. Cheynier, Lea Samba, A. Baillou,¤ Maurice Mampaka,# Pierre M'Pel,# Francis Barin,¤ Max Essex,* and the Congolese Research Group on Mother-to-Child Transmission of HIV-1

*Department of Cancer Biology, Harvard School of Public Health, Boston, USA; ^ORSTOM / Urbanization and Health, Brazzaville, Congo, and Paris, France; Department of Maternal and Child Health, Ministry of Health, Brazzaville, Congo; ¤Laboratory of Virology, Bretonneau Hospital, Tours, France; and #National Program on AIDS, Ministry of Health, Bra zzaville, Congo

Perinatal transmission of HIV-1 is the primary source of pediatric AIDS. The number of infants at risk worldwide is increasing steadily. At present, about two-thirds of all HIV-1-infected women live in Africa. From 1987 to 1991, a prospective study was conducted in Brazzaville, Congo, to evaluate the overall risk of perinatal transmission of HIV-1 , to determine the factors involved, and to study the clinical presentation of the disease in infants.

Pregnant women who agreed to have themselves and their infants tested were recruited at two mother-child clinics and one maternity hospital. Infants born to HIV-1-positive mothers were followed from birth for at least two years together with control infants born to HIV-1-negative mothers and matched by age, parity, and place of residence. Clinical examination and blood sampling were performed soon after birth, at one month, at three months, and every three months thereafter. The mothers' sera were tested by enzyme-linked immunosorbent assay (Diagnostics Pasteur), and positive results were confirmed by Western Blot (Du Pont de Nemours). The HIV-1 status of infants born to seropositive mothe rs was assessed serologically at fifteen months, when they had lost their passively transferred maternal antibodies. Infants who died before serology was interpretable were classified as infected if they had AIDS (WHO case definition) or if their symptoms and the circumstances of their deaths met the EEC/WHO criteria for HIV-1-attributable infant death. The mothers' socio-demographic characteristics, clinical status, and antibody response to HIV-1 were studied in relation to transmission.

Enrolled in the study were 118 infants born to HIV-1-positive mothers and 208 controls. Newborns of HIV-1-positive mothers had a significantly lower mean birth weight (2,873g vs. 3,083g, p=0.0004) and a higher prematurity rate (17.9% vs. 7.7%, p=0.005). After exclusion of twins, the lower mean birth weight remained the only significant difference (2,938g vs. 3,083g, p=0.01). Survival, however, differed substantially between the two groups at as early as three months. While the probability of survival among control infants was 0.99 (95% CI: 0.96-1.0) at three months, 0.96 (95% CI: 0.92-0.98) at twelve months, and 0.95 (95% CI: 0.91-0.97) at fifteen months, the corresponding survival probabi lities for infants born to HIV-1-positive mothers were 0.91 (95% CI: 0.85-0.95), 0.74 (95% CI: 0.66-0.82), and 0.71 (95% CI: 0.61-0.79), respectively.

Causes of death in infants born to HIV-1-positive mothers were prematurity (n=5, including three twins); pulmonary infection (n=9); severe sepsis (n=6); persistent diarrhea (n=3); AIDS (n=8); and unknown (n=2). By fifteen months, 59 infants had seroreverted and 17 were seropositive. Of the 33 children who had died before fifteen months, 23 were in fected with HIV-1 (AIDS=8; HIV-1-related deaths=15), and 10, who did not meet the criteria of HIV-1-related death, had a serology that remained indeterminate. Nine infants were lost to follow-up. Hepatosplenomegaly, failure to thrive, and upper and lower respiratory infections were common and twice as frequent in infected infants as in uninfected and control infants. These nonspecific manifestations were clearly associated with the infectious, parasitic, and nutritional environment in which these African infants live. By contrast, minor neurologic signs such as diminished alertness and active and passive tonus were highly specific manifestations found in infected infants. Remarkably, uninf ected infants born to HIV-1-positive mothers were clinically indistinguishable from controls.

The overall transmission rate of HIV-1-infected infants who met the criteria of HIV-1-attributable death and those who were seropositive at fifteen months was 40.4% (95% CI: 30.7%-50.1%). When the difference in mortality rates between HIV-1-exposed and control infants was used as an indirect estimate of HIV-1-related deaths, the calculated rate of transmission was 42.7% (95% CI: 32.7% - 52.7%).

Women showing symptoms during pregnancy also had an increased risk of transmitting the virus to their infants. Moreover, maternal antibody titers to peptides corresponding to the third variable domain of gp120 (V3) and antibody titers to the immunodominant domain of gp41 had a dose response-like relationship to the risk of perinatal transmission. These responses were specific and did not reflect an overall heightened immune response to the virus as indicated by the weak correlation between antibody titers to V3 and gp41 and the lack of association between transmission and antibody response to p24 or to the whole virus. Antibody response to V3, to gp41, and to symptoms were independently as sociated with transmission.

Using women's individual data in the logistic regression model allowed the determination of their predicted risk of transmission. Asymptomatic women whose antibody titer to V3 was below 1:500 and antibody titer to gp41 was below 1:50 had a risk of 0.02 of transmitting HIV-1 to their infants. Conversely, the predicted risk was 0.88 for symptomatic women whose antibody titers to V3 and to gp41 were highest.

While HIV-1 infection has little clinical impact at birth, HIV-1 disease progressed much more rapidly in our study than in similar cohorts in Europe and North America. The transmission rate was also higher than that observed in industrialized countries, although it falls within the range reported in Africa. The large proportion of women carrying t heir pregnancy to term at an advanced stage of disease, the rarity of Cesarean section, the high frequency of poorly controlled co-infections, and the universal practice of breastfeeding may contribute to the increased perinatal transmission rates.

Although the actual timing of perinatal transmission and factors that influence it are not well understood, drug and immune therapy trials to interrupt transmission are under way. While the association we have observed may give new insight into our understanding of the actual mechanisms of perinatal transmission, measurement of the antibody respon se in infected women with or without clinical symptoms may also provide a powerful tool for identifying women who would most benefit from such interventions.

References

1. Lallemant M, Lallemant-Le Coeur S, Nzingoula S. Perinatal transmission of HIV-1 in Africa. In: Essex M, et al., eds., AIDS in Africa. New York: Raven Press, 1994;211-236.
2. Lallemant M, Lallemant-Le Coeur S, Cheynier D, et al. Mother-child transmission of HIV-1 and infant survival in Brazzaville, Congo. AIDS 1989;3:643-646.
3. La Rosa GJ, Davide JP, Weinhold K, et al. Conserved sequence and structural elements in the HIV-1 principal neutralizing determinant. Science 1990;249:932-935.
4. Baillou A, Janvier B, Leonard G, et al. Fine serotyping of human immunodeficiency virus serotype 1 (HIV-1) and HIV-2 infections by using synthetic oligopeptides representing an immunodominant domain of HIV-1 and HIV-2/simian immunodeficiency virus. J Clin Microbiol 1991;29:1387-1391.
5. Nowak MA, Anderson RM, McLean AR, et al. Antigenic diversity thresholds and the development of AIDS. Science 1991;254:963-969.

Viral and Immunologic Factors Influencing Vertical Transmission of HIV-1

Srisakul C. Kliks, Diane W. Wara,* Daniel V. Landers,^ and Jay A. Levy

Division of Pediatric Immunology* and the Cancer Research Institute, School of Medicine, University of California, San Francisco, USA ^Department of Obstetrics and Gynecology, San Francisco General Hospital, San Francisco, USA

The majority (80%) of pediatric AIDS cases are the result of HIV-1 transmission from the infected mother to the child. This transmission occurs at a rate ranging from 12% to 45%, depending on the geographic, demographic, and epidemiologic features of the studied cohorts. Three modes of transmission have been implicated: intrauterine, intrapartum, and postpartum via breastfeeding. Because of the presumed different routes of transmission, a variety of intrinsic and extrinsic factors may be involved in HIV-1 transmission from mothers to children. Defining these parameters could help in preventing this mode of HIV-1 transmission.

The maternal factors considered to increase risk for mother-to-child transmission include: late stage of the disease,1 low CD4 T cell count,2 high viral load,1 low anti-gp120 antibodies in the serum,3 and the absence of antibodies against specific domains of gp120.4 These parameters place an emphasis on viral and immunologic factors in the transmission. Considering the high degree of genetic and biologic variation of HIV-1, virus growth capabilities may affect the extent of viral load in mothers and thus influence the transmission to their infants. In addition, circulating HIV-1 antibodies in mothers could affect the viral level via viral neutralization resulting in protection. Alternative ly, the antibodies can also exert various selective pressures on the type of virus that would be transmitted to the infants. To assess the relationship of the viral and immunologic factors in mother-to-child transmission, the biologic and serologic properties of HIV-1 isolates recovered from non-transmitting mothers, transmitting mothers, and their corresponding infants were evaluated. Further, since an HIV-1 isolate exists as "quasispecies"5 with varying degrees of genetic diversity, sequence variation on the viral envelope of the paired mother/infant isolates were analyzed to gain insight into the relationship between the HIV-1 phenotype and genotype transmitted to the infants.

Study Population

The clinical samples used in our study came from HIV-1-infected women and infants in a prospective study on HIV-1 transmission by the Bay Area Perinatal AIDS Center in San Francisco, USA. The vertical transmission rate during the studied period was approximately 15.1%. The infection status in infants was determined by the detection of proviral HIV-1 genome in the peripheral blood mononuclear cells (PBMCs) of subjects by the HIV-1 polymerase chain reaction assay and by the isolation of HIV-1 from the PBMCs according to the U.S. Centers for Disease Control and Prevention (CDC) guidelines.

Biologic Characteristics of the Virus

To assess the biologic properties of the type of virus that was favored for transmission, cellular tropism and growth characteristics of, at, and near birth isolates from 10 non-transmitting mothers and their infants were evaluated. The results indicate that all viral isolates were able to infect primary human macrophage cultures to varying degrees as indicated by the level of the reverse transcriptase (RT) activity in the culture fluids. In some cases, silent infection was observed and RT activity was detected in the culture fluids only after co-cultivation of the inoculated macrophages with phytohemagglutinin (PHA)-stimulated PBMCs. With respect to infectivity in T cell lines, none was found in the non-transmitting group. However, low levels of infection were detected in one or two of the three T cell lines with isolates from four transmitting mothers and three infants. Subsequent co-cultivation of these inoculated cells with human PBMCs did not lead to increased viral detection.

The ability to form cell syncytia has been associated with viral virulence as it is usually found during the symptomatic stage of the disease.6 The viral isolates were tested for their ability to induce syncytium with the MT-2 cell line. No syncytium formation was observed with any of the viral isolates.

The kinetics of virus replication in PBMCs were evaluated. The isolates from the transmitting mothers either replicated more rapidly and with a higher peak level of virus production or exhibited a higher peak level of replication than did those isolates from the non-transmitting mothers (Figure 1). The replication of each infant isolate was similar to that of its respective mother (Figure 2). The combined properties of the wide cellular host range and the efficient replication of the virus in PBMCs provide a basis for high viral load as a factor for the HIV-1 vertical transmission.

Immunologic Analysis of Maternal Sera

The birth or near-birth sera from both transmitting and non-transmitting mothers had antibodies to the HIV-1-specific proteins as detected by an immunoblot assay. The immunoblot pattern showed reactivity to HIV-1-specific proteins with all sera, in particular against HIV-1 core proteins p55 and p25. Reactivity to the envelope gp160 was detected in all sera from both groups at varying degrees.

The third hypervariable (V3) domain of the HIV-1 envelope contains the principal neutralizing domain (PND) at the crown of its loop-structure that elicits type-restricted neutralizing antibodies. These antibodies appear early after HIV-1 infection and contribute significantly to the neutralizing capacity of the infected host's sera. Some studies s uggested that anti-V3 reactivity in the mother's serum was associated with protection against HIV-1 transmission,7 while others did not.8 The maternal sera in this study were evaluated for their reactivity in both titer and binding affinity against the PND by peptide-ELISA methods. High titers against two (MN and the consensus sequences) or all three peptides were observed in all sera from the non-transmitting group but in only three of five sera from the transmitting group. However, no correlation between V3-reactivity titers or binding affinity and protection was indicated (p>0.5). The lack of association may be due to the variation between the sequences of the actual isolates and of those used in the assays. A small change in sequence in the V3 region could affect the binding affinity between the peptides.

The maternal sera were further tested for their neutralizing activity against their autologous HIV-1 isolates. These isolates were also tested for their susceptibility to neutralization by a reference-neutralizing serum from an infected individual with a high level of neutralizing activity. All viral isolates but one were neutralized by the refere nce HIV-1-positive serum at a titer of at least 50 (Table 1 [table not avilable through VINE]). The one isolate from a transmitting mother was resistant to neutralization even at the lowest dilution tested, 1:10, suggesting a drastic immunologic shift of the virus from that of a typical North American subtype.

The majority (8/10) of viral isolates from the non-transmitting mothers were neutralized by their autologous sera. In contrast, only two of five HIV-1 isolates from the transmitting group were neutralized (Table 1). Enhancement of infection9,10 was observed in one maternal isolate due to the woman's autologous serum. While the data suggest some as sociation between neutralization of autologous HIV-1 isolates by maternal sera and protection, the relationship was not statistically significant by Fisher's Exact test (p < 0.10)

Genetic Analysis

Most of a single HIV-1 isolate exists as a complex of closely related genetic variants called quasispecies.5 The degree of genetic heterogeneity in the quasispecies varies over the course of the disease. This provides a basis for how a variant can be selected from an apparent single phenotype. In particular, variations in the V3 domain can affect syncytium formation, tropism, growth kinetics, and neutralization.11,12 Several studies on genetic features of HIV-1 found in mothers and infants have shown a higher degree of genetic heterogeneity based on the V3 sequence in the mother isolates than in those of their infant counterparts.13,14 In most cases, similar results were found in this stud y

Conclusions

Our findings suggest that certain non-syncytium-inducing but efficiently replicating strains of HIV-1 are important in mother-to-child transmission. The virus may be selected further by the strains' resistance to maternal antiviral antibodies. Genetic variation in the V3 region of the viral envelope between these mother/infant pairs of isolates ma y reflect the immunologic or biologic selection. This study, although limited in size, provides directions for further evaluation of intervention strategies.

Acknowledgments

This research project was supported by grants from the National Institutes of Health (RO1 AI32446-01) and the Universitywide AIDS Research Program (R91SF264).

References

1. Report of a Consensus Workshop, Siena, Italy. Maternal factors involved in mother-to-child transmission of HIV-1. J AIDS 1992;5:1169-78.
2. European Collaborative Study. Risk factors for mother-to-child transmission of HIV-1. Lancet 1992; 339:1007-12.
3. Goedert JJ, Drummond JE, Minkoff HL, et al. Mother-to-infant transmission of human immunodeficiency virus type 1: association with prematurity or low anti-gp120. Lancet 1989;2:1351-54.
4. Rossi P, Moschese V, Broliden PA, et al. Presence of maternal antibodies to human immunodeficiency virus type 1 envelope glycoprotein gp120 epitopes correlates with the uninfected status of children born to seropositive mothers. Proc Natl Acad Sci USA 1989; 86:8055-58.
5. Holland JJ, De La Torre JC, Steinhauser DA. RNA virus populations as quasispecies. Current Topics in Microbiology and Immunology 1992;176:1-20.
6. Koot M, Vos AHV, Keet RPM, et al. HIV-1 biological phenotype in long-term infected individuals evaluated with an MT-2 cocultivation assay. AIDS 1992;6:49-54.
7. Devash Y, Calvelli TA, Wood DG, Regan KJ, Rubinstein A. Vertical transmission of human immunodeficiency virus is correlated with the absence of high-affinity/avidity maternal antibodies to the gp120 principal neutralizing domain. Proc Natl Acad Sci USA 1990;87:3445-49.
8. Parekh BS, Shaffer N, Pau C-P, et al. Lack of correlation between maternal antibodies to V3 loop peptides of gp120 and perinatal HIV-1 transmission. AIDS 1991;5:1179-84.
9. Homsy J, Tateno M, and Levy JA. Antibody-dependent enhancement of HIV infection. Lancet 1988;i:1285-86.
10. Shioda T, Levy JA, Cheng-Mayer C. Small amino acid changes in the V3 hypervariable region of gp120 can affect the T-cell-line and macrophage tropism of human immunodeficiency virus type 1. Proc Natl Acad Sci USA 1992;89:9434-9438.
11. Kliks SC, Shioda T, Haigwood NL, Levy JA. V3 variability can influence the ability of an antibody to neutralize or enhance infection by diverse strains of human immunodeficiency virus type 1. Proc Natl Acad Sci USA 1994;90:11518-22.
12. Grimaila RJ, Fuller BA, Rennert PD, et al. Mutations in the principal neutralization determinant of human immunodeficiency virus type 1 affect syncytium formation, virus infectivity, growth kinetics, and neutralization. J Virol 1992;66:1875-83.
13. Wolinsky SM, Wike CM, Korber BTM, et al. Selective transmission of human immunodeficiency virus type-1 variants from mothers to infants. Science 1992;255:1134-37.
14. Scarlatti G, Leitner T, Halapi E, et al. Comparisons of variable region 3 sequences of human immunodeficiency virus type 1 from infected children with the RNA and DNA sequences of the virus populations of their mothers. Proc Natl Acad Sci USA 1993;90:1721-5.