Perinatal transmission of major, minor, and multiple maternal human immunodeficiency virus type 1 variants in utero and intrapartum.

Previous studies have provided conflicting data on the presence of selective pressures in the transmission of a homogeneous maternal viral subpopulation to the infant. Therefore, the purpose of this study was to definitively characterize the human immunodeficiency virus type 1 (HIV-1) quasispecies transmitted in utero and intrapartum. HIV-1 envelope gene diversity from peripheral blood mononuclear cells and plasma was measured during gestation and at delivery in mothers who did and did not transmit HIV perinatally by using a DNA heteroduplex mobility assay. Children were defined as infected in utero or intrapartum based on the timing of the first detection of HIV. Untreated transmitting mothers (n = 19) had significantly lower HIV-1 quasispecies diversity at delivery than untreated nontransmittting mothers (n = 18) (median Shannon entropy, 0.711 [0.642 to 0.816] versus 0.853 [0.762 to 0.925], P = 0.005). Eight mothers transmitted a single major env variant to their infants in utero, and one mother transmitted a single major env variant intrapartum. Four mothers transmitted multiple HIV-1 env variants to their infants in utero, and two mothers transmitted multiple env variants intrapartum. The remaining six intrapartum- and two in utero-infected infants had a homogeneous HIV-1 env quasispecies which did not comigrate with their mothers' bands at their first positive time point. In conclusion, in utero transmitters were more likely to transmit single or multiple major maternal viral variants. In contrast, intrapartum transmitters were more likely to transmit minor HIV-1 variants. These data indicate that different selective pressures, depending on the timing of transmission, may be involved in determining the pattern of maternal HIV-1 variant transmission.

Primary HIV infection of infants: the effects of somatic growth on lymphocyte and virus dynamics.

Acute HIV infection is characterized by the appearance of high concentrations of virus in the peripheral blood. In adults, this high-level viremia spontaneously abates after several weeks. In contrast, after perinatal infection of infants, blood virus levels remain high for many months, during which the concentration of circulating CD4+ lymphocytes remains well above normal values for adults. Here we suggest an explanation for these differences, based on developmental factors including somatic growth and immunological ontogeny. Flow cytometric analysis revealed that at birth the thymus contains elevated levels of mature T lymphocytes, compared to the thymus after 3 months of age. A mathematical model is proposed incorporating immunological and virological data from longitudinally evaluated infants who acquired infection at the time of birth. This model explains the pattern of high-level viremia in infants as resulting from the replication of HIV within the progressively expanding lymphoid cell mass.

Decreased CD8 cell-mediated viral suppression and other immunologic characteristics of women who transmit human immunodeficiency virus to their infants.

CD8 T cell function, lymphocyte surface phenotype, serum markers of immunologic activation, and viral burden were assessed in 75 human immunodeficiency virus (HIV)-infected pregnant women, including 9 who transmitted infection to their infants. Serial studies during and after pregnancy showed no significant differences in levels of cell-surface or serum activation molecules in transmitting compared to nontransmitting mothers, with the exception of a postpartum increase in tumor necrosis factor alpha in transmitting women. The transmitting women had a median plasma viral load of 65,516 RNA copies/mL at delivery versus 5139 in nontransmitting women. During the third trimester, the CD8 cells of 81% of the nontransmitting and 44% of the transmitting mothers suppressed HIV production in vitro by >50%. Women with <50% suppression had a 3.4 times greater risk of transmitting HIV to their infants. CD8 suppression and viral load were interrelated, but when either CD4 percent or AZT use was controlled for, suppression was still significant.

Early prognostic indicators in primary perinatal human immunodeficiency virus type 1 infection: importance of viral RNA and the timing of transmission on long-term outcome.

The time of perinatal human immunodeficiency virus type 1 (HIV-1) transmission and the pattern of early plasma viremia as predictors of disease progression were evaluated in infected infants followed from birth. Cox proportional hazards modeling demonstrated that a 1-log higher HIV-1 RNA copy number at birth was associated with a 40% increase in the relative hazard (RH) of developing CDC class A or B symptoms (P = .004), a 60% increase in developing AIDS (P = .01), and an 80% increase in the of risk death (P = .023) over the follow-up period of up to 8 years. The peak HIV-1 RNA copy number for infants during primary viremia was also predictive of progression to AIDS (RH, 9.9; 95% confidence interval [95% CI], 1.8-54.1; P = .008) and death (RH, 6.9; 95% CI, 1.1-43.8; P = .04). The results indicate that high levels of HIV-1 RNA at birth and during primary viremia are associated with early onset of symptoms and rapid disease progression to AIDS and death in perinatally infected children.

Optimization of specimen-handling procedures for accurate quantitation of levels of human immunodeficiency virus RNA in plasma by reverse transcriptase PCR.

Human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma are currently widely used clinically for prognostication and in monitoring antiretroviral therapy. Accurate and reproducible results are critical for patient management. To determine the effects of specimen collection and handling procedures on quantitative measurement of HIV-1 RNA, we compared anticoagulants and sample processing times. Whole blood was collected from 20 HIV-1-infected patients in EDTA, acid citrate dextrose (ACD), and heparin tubes, aliquoted, and stored at room temperature. Plasma was separated from whole-blood aliquots prepared at < or =1, 3, 6, 24, and 48 h postcollection and then stored at -70 degrees C until use. HIV-1 RNA levels were determined by the AMPLICOR HIV-1 MONITOR assay. Heparinized plasma samples, which were pretreated with heparinase prior to analysis, had the lowest baseline HIV-1 RNA levels. In the first 6 h, HIV-1 RNA levels decreased by 10, 20, and 31% in EDTA, ACD, and heparin tubes, respectively. From 6 to 48 h postcollection, HIV-1 RNA levels decreased in all anticoagulants, albeit at a slower, more consistent rate. Our results indicate that EDTA should be the anticoagulant of choice for plasma HIV-1 RNA measurement by reverse transcriptase PCR, but ACD tubes are acceptable if the plasma is separated within 6 h of blood collection. Caution must be applied in the interpretation of absolute HIV-1 RNA copy number values obtained with suboptimal specimen collection and processing procedures.

Human immunodeficiency virus type 1 genetic evolution in children with different rates of development of disease.

The rate of development of disease varies considerably among human immunodeficiency virus type 1 (HIV-1)-infected children. The reasons for these observed differences are not clearly understood but most probably depend on the dynamic interplay between the HIV-1 quasispecies virus population and the immune constraints imposed by the host. To study the relationship between disease progression and genetic diversity, we analyzed the evolution of viral sequences within six perinatally infected children by examining proviral sequences spanning the C2 through V5 regions of the viral envelope gene by PCR of blood samples obtained at sequential visits. PCR product DNAs from four sample time points per child were cloned, and 10 to 13 clones from each sample were sequenced. Greater genetic distances relative to the time of infection were found for children with low virion-associated RNA burdens and slow progression to disease relative to those found for children with high virion-associated RNA burdens and rapid progression to disease. The greater branch lengths observed in the phylogenetic reconstructions correlated with a higher accumulation rate of nonsynonymous base substitutions per potential nonsynonymous site, consistent with positive selection for change rather than a difference in replication kinetics. Viral sequences from children with slow progression to disease also showed a tendency to form clusters that associated with different sampling times. These progressive shifts in the viral population were not found in viral sequences from children with rapid progression to disease. Therefore, despite the HIV-1 quasispecies being a diverse, rapidly evolving, and competing population of genetic variants, different rates of genetic evolution could be found under different selective constraints. These data suggest that the evolutionary dynamics exhibited by the HIV-1 quasispecies virus populations are compatible with a Darwinian system evolving under the constraints of natural selection.

Timing of lymphocyte activation in neonates infected with human immunodeficiency virus.

Human immunodeficiency virus (HIV) infection in children is associated with qualitative and quantitative changes in the peripheral lymphocyte surface phenotype beyond the normal maturational changes. Neonates, however, have been reported to have a delayed immune response to HIV compared to HIV-infected adults. We prospectively performed immunophenotyping of T lymphocytes by three-color immunofluorescent labeling and laser flow cytometry to determine the timing of phenotypic alterations in 112 neonates born to HIV-infected mothers. Serial testing was performed at birth (cord blood) and at 2, 6, and 12 weeks of age. Data were divided retrospectively for analysis into those for HIV-infected (n = 14) infants and those for exposed, uninfected infants. Our results show that both infected and uninfected infants had a decline in the percentages and numbers of CD4 cells beginning at 2 weeks of age but that the decline was greater in the HIV-infected group. The activation and differentiation of CD8 T cells in HIV+ infants were shown by a significant increase in CD45RA- CD45RO+ CD8+ cells by 6 weeks of age and by increases in CD8+ S6F1+ CD3+ cells and HLA-DR+ CD38+ CD8+ cells by 2 weeks of age. These results indicate that HIV-infected neonates show alterations in T-cell phenotype reflecting those reported for older HIV-infected children. Most importantly, neonatal T cells are able to respond to HIV within the first weeks of life.

Presence of human immunodeficiency virus (HIV) type 1 and HIV-1-specific antibodies in cervicovaginal secretions of infected mothers and in the gastric aspirates of their infants.

The presence of human immunodeficiency virus (HIV) in cervicovaginal secretions (CVS) may be a risk factor for perinatal transmission. CVS of 25 women were evaluated for HIV and HIV mucosal antibodies; 16 infants had gastric aspirates cultured. Maternal plasma HIV was measured by quantitative RNA polymerase chain reaction. Seven women (28%), 4 of 19 pregnant and 3 of 7 nonpregnant, had HIV in CVS. Two of 4 HIV-infected neonates had positive gastric aspirate cultures. The 4 pregnant women with HIV in CVS did not transmit infection. HIV-specific secretory IgA was present in CVS of 10 (42%) of 24 women (in 3 cases concurrent with virus). Plasma HIV RNA levels at delivery were higher among transmitters (mean, 68,921 copies/mL) than nontransmitters (mean, 9457 copies/mL). Intermittent HIV shedding in CVS occurred despite mucosal antibodies and did not necessarily correlate with maternal plasma HIV RNA copy number. The presence of HIV in newborn gastric aspirates may be a risk factor for perinatal infection.

Identification of levels of maternal HIV-1 RNA associated with risk of perinatal transmission. Effect of maternal zidovudine treatment on viral load.

OBJECTIVE: To determine if there are levels of human immunodeficiency virus type 1 (HIV-1) associated with a high or low risk of perinatal transmission and to ascertain the mechanism by which zidovudine treatment reduces perinatal transmission. DESIGN: A nonrandomized prospective cohort study. SETTING: University medical center and two general hospital affiliates from May 1989 to September 1994. PATIENTS: Ninety-two HIV-1-seropositive women (95 pregnancies) and their 97 infants. INTERVENTION: Forty-two mothers (43 pregnancies) received zidovudine therapy during pregnancy and/or during labor and delivery. Eleven infants received prophylactic zidovudine for the first 6 weeks after delivery. MAIN OUTCOME MEASURE: HIV-1 infection status of the infant. RESULTS: Twenty of the 97 infants were perinatally infected with HIV-1. Transmitting mothers were more likely to have plasma HIV-1 RNA levels higher than 50000 copies per milliliter at delivery than nontransmitting mothers (15 [75.0%] of 20 transmitters vs four [5.3%] of 75 nontransmitters; P < .001). None of the 63 women with less than 20000 HIV-1 RNA copies per milliliter transmitted. Twenty-two women treated with open-label oral zidovudine during gestation showed an eightfold median decrease in plasma RNA levels (median [25th and 75th percentile], 43043 [5699 and 63053] copies per milliliter before zidovudine vs 4238 [603 and 5116] HIV-1 RNA copies per milliliter at delivery; P < .001) and none transmitted. Four zidovudine-treated women with high HIV-1 levels transmitted despite the presence of zidovudine-sensitive virus in vitro in both the mothers and their infants. CONCLUSIONS: Maternal HIV-1 RNA levels were highly predictive of perinatal transmission risk and suggest that certain levels of virus late in gestation and/or during labor and delivery are associated with both a high risk and a low risk of transmission. Our results also suggest that zidovudine exerts a major protective effect by reducing maternal HIV-1 RNA levels prior to delivery and that further strategies are needed to prevent perinatal transmission in women with high or increasing virus levels and/or zidovudine-resistant virus.

Clearance of HIV infection in a perinatally infected infant.

BACKGROUND: We describe a child who was identified shortly after birth as infected with the human immunodeficiency virus type 1 (HIV-1), but whose infection appears to have completely cleared. Asymptomatic HIV-1 infection was diagnosed in the mother during the fourth month of pregnancy. The infant was delivered vaginally at 36 weeks, received no blood products, and was not breast-fed. METHODS AND RESULTS: HIV-1 was detected by culture of the infant's peripheral-blood mononuclear cells at 19 and 51 days of age. Plasma from the infant was also culture-positive for HIV-1 at 51 days of age by DNA polymerase chain reaction (PCR). Nucleotide-sequence analysis of HIV-1 DNA showed extremely close homology of the cultures obtained 32 days apart, and forensic markers of genetic identity for the two cultures were identical. Hence, inadvertent viral contamination or error in the collection of specimens was highly unlikely. At 12 months of age the infant was seronegative for HIV-1, and numerous subsequent cultures and tests by PCR have also been negative for HIV-1. The child is five years of age at this writing, is HIV-seronegative, and remains well, with normal growth and development and no laboratory or clinical evidence of HIV-1 infection. CONCLUSIONS: The infant we describe was infected perinatally with HIV-1, but the infection subsequently cleared and the infant remained without detectable HIV-1 infection five years later.

Rapid increases in load of human immunodeficiency virus correlate with early disease progression and loss of CD4 cells in vertically infected infants.

The relationship between viral burden, timing of transmission, and clinical progression was investigated in 110 children at risk for vertical human immunodeficiency virus (HIV) infection using quantitative polymerase chain reaction, coculture, and immune complex-dissociated p24 antigen assay. In a cross-sectional study, the mean HIV DNA copy number in 19 symptomatic children was significantly higher than in 31 infected, asymptomatic children (420 +/- 125 vs. 87 +/- 78; P < .0001). In a second group of 8 vertically infected infants followed prospectively from birth, 4 defined as infected in utero showed a more rapid increase in virus load, an accelerated loss of CD4 cells, and early progression to symptomatic disease (3-12 weeks) compared with 4 children with late in utero or intrapartum transmission (10-31 months). These data suggest that a direct relationship exists between HIV replication, the timing of transmission and onset and progression of HIV disease in vertically infected children.

Decreases in unintegrated HIV DNA are associated with antiretroviral therapy in AIDS patients.

Better markers are needed to monitor the efficacy of antiretroviral drugs in persons infected with human immunodeficiency virus (HIV). We investigated the effects of zidovudine (ZDV) and dideoxycytidine (ddC) on the presence of unintegrated HIV-1 DNA in peripheral blood mononuclear cells (PBMCs) from AIDS patients. DNA was extracted from PBMCs and separated into low molecular weight (unintegrated) and high molecular weight (integrated) chromosomal fractions. These DNA fractions were then amplified by a quantitative polymerase chain reaction (PCR) and the amount and percentage of unintegrated HIV DNA were determined. Very high levels of unintegrated HIV DNA were found in AIDS patients not receiving treatment with ZDV or ddC (median = 95% unintegrated HIV DNA). In contrast, most patients who had received 4 or more weeks of antiretroviral therapy had lower levels of unintegrated HIV DNA (median = 30% unintegrated HIV DNA for patients receiving ZDV). Paired samples taken from five patients before and after therapy showed a striking reduction in the percentage of unintegrated HIV DNA. The decrease in the proportion of unintegrated HIV DNA in AIDS patients was due to both a reduction in the copy number of unintegrated HIV DNA and an increase in the copy number of integrated HIV DNA. Thus, measurements of unintegrated and integrated HIV DNA may be useful in providing objective assessments of the effectiveness of antiretroviral therapies.

HIV-1 proviral copy number in blood mononuclear cells from AIDS patients on zidovudine therapy.

The effect of treatment with 400-1,200 mg/day of zidovudine (ZDV) on HIV DNA concentrations in patient peripheral blood mononuclear cells (PBMCs) was studied in six patients during a 5- to 14-month period of therapy. HIV DNA was measured in PBMCs at intervals using a recently developed quantitative polymerase chain reaction assay. The amount of HIV DNA ranged from 2,000 to 40,000 copies of provirus per microgram of cellular DNA. The HIV provirus copy number showed little change with time in five patients, and increased and then remained constant in one patient. Thus, prolonged treatment with ZDV does not decrease the levels of HIV DNA in PBMCs.

Vaccine protection of rhesus macaques against simian immunodeficiency virus infection.

Rhesus macaques (Macaca mulatta) immunized with an inactivated whole SIVmac vaccine and muramyl dipeptide (MDP), incomplete Freund's adjuvant (IFA), or aqueous suspension were challenged intravenously with 0.1 TCID50 of cell-free SIVmac. Whereas virus was readily recovered from the peripheral blood lymphocytes of 10 of 10 nonvaccinated controls following this challenge dose, virus was not recovered from the three animals that received the vaccine with MDP nor from one of two animals that received the vaccine with IFA and one of three animals that received the aqueous vaccine. The animals that were protected against challenge were those that had detectable SIV antibody response to the envelop, both the outer glycoprotein (gp120) and the truncated transmembrane glycoprotein (gp31). Protected monkeys tended to have higher titers of syncytial inhibition antibody prior to challenge. An anamnestic response after challenge was observed only in the vaccinated monkeys that became infected. Vaccinated animals that became challenge-infected tended to live longer than infected controls. These results confirm those at two other primate centers and indicate that killed whole SIV vaccines can protect against low challenge doses of SIV and prevent early death in those monkeys that do become infected. The mechanism of this protection remains undetermined. This finding adds optimism to the possibility of an eventual AIDS vaccine.

Laboratory tests used in diagnosis and treatment of AIDS.

Detailed knowledge of the structure of the human immunodeficiency virus (HIV) triggered a rapid development of methods for diagnosing the infection. The enzyme-linked immunoadsorbent assay (ELISA) determining the presence of antibodies to the protein components of the virus in toto is highly sensitive and provides thus the basic screening approach. It is however somewhat less specific and therefore all positive results are verified by the Western blot method which detects individual HIV proteins and possesses a 99.9% specificity. In sporadic cases the latent period between HIV infection and the possibility to establish antibody response may extend to six months and even longer. The polymerase chain reaction (PCR) revealing the presence of DNA HIV in infected cells is suitable for detecting these seronegative patients. It is also the method of choice in diagnosing HIV infection in children of infected mothers since HIV antibodies are in newborns mostly of maternal origin. HIV antibodies, particularly the antigen p 24, can be quantified in serum by using ELISA. As their amounts correlate with the severity of the disease determination of antigen p 24 concentration is an indicator of the therapeutic efficacy of preparations such as AZT. The recently developed quantitative PCR is a further potentially valuable method for assessing the therapeutic effect since treatment should reduce the amount of cellular HIV DNA. With the exception of testing the sensitivity of HIV strains to antiretrovirus preparations, HIV cultures are rarely set up since they are technically demanding and involve a considerable risk.

Quantitation of human immunodeficiency virus DNA by using the polymerase chain reaction.

The polymerase chain reaction was used to measure the DNA copy number of human immunodeficiency virus (HIV). Differences in polymerase chain reaction amplification efficiency were controlled by amplifying known amounts of HIV DNA in parallel with samples. This technique is a sensitive, accurate, and reproducible method for the quantitation of HIV DNA.