Replication and tropism of human immunodeficiency virus type 1 as predictors of disease outcome in infants with vertically acquired infection

Replication and tropism of human immunodeficiency virus type Jl as predictors of disease outcome in infants with vertically acquired infection A n i t a De Rossi, PhD, Carlo G i a q u i n t o , MD, Lucia O m e t t o , DrBSc, Fabrizio M a m m a n o , DrBSc, C a r l o Z a n o t t o , DrBSc, David Dunn, MSc, a n d Luigi C h i e c o - B i a n c h i , MD From the Institute of Oncotogy, JnterUniversity Center for Cancer Research, and the Department of Pediatrics, University of Padua; Padua, Italy; and the Department of Pediatric Epidemiology, Institute of Child Health, London, England In a series of 97 infants born to mothers who were seropositive for human immun o d e f i c i e n c y virus type I (HIV-I), 18 were identified as infected within the first 60 days of life on the basis of viral culture and polymerase chain reaction findings. We studied viral burden in vivo by quantitative polymerase chain reaction and the in vitro replication pattern of the HIV-I infecting strain by culturing patient cells with normal phytohemagglutinin-stimulated peripheral b l o o d mononuclear cells. A c c o r d i n g to the lag phase before p24 antigen d e t e c t i o n and the level of p24 production on peripheral b l o o d m o n o n u c l e a r cells, HIV-I isolates from these patients were classified as r a p i d / h i g h (R/H), slow/high (S/H), and s l o w / l o w (S/L). The pattern of HIV-I replication in vitro was significantly associated with the viral burden in vivo; the range of HIV-I copies per 105 peripheral b l o o d m o n o n u c l e a r cells was 10 to 38, 44 to 314, and 360 to 947 in children with isolates of the S/L, S/H, and R/H types, respectively. Viral tropism was assessed by culturing patient cells under end-point dilution conditions with either CD4 + T-lymphocytes or m o n o c y t e - d e r i v e d m a c r o p h a g e s . We found that children with S/L isolates harbored mainly m o n o c y t o t r o p i c variants; all infants with S/H or R/H isolates had T-lymphotropic variants and, in 7 of 11 cases, m o n o c y t o t r o p i c or amphitropic variants. All children with R/H isolates had HIV-related symptoms by the a g e of 4 months, and five had a c q u i r e d i m m u n o d e f i c i e n c y syndrome by the a g e of 1 year. At I year of age, four and no infants with S/H or S/L isolates, respectively, had HIV-1-related symptoms (p <0.001), and none had a c q u i r e d imm u n o d e f i c i e n c y syndrome (p = 0.006). (J PEDIATR1993;123:929-36)

In about one third of children with vertically acquired infection with human immunodeficiency virus type 1, acquired immunodeficiency syndrome develops within the

Supported by Istituto Superiore di Sanit~t, Progetto AIDS 1991, 1992, 1993. Submitted for publication April 21, 1993; accepted July 27, 1993. Reprint requests: Anita De Rossi, PhD, Institute of Oncology, University of Padua, Via Gattamelata 64, 35128 Padua, Italy. Copyright | 1993 by Mosby-Year Book, Inc. 0022-3476/93/$1.00 + .10 9/20/50313

first months of life; the disease progresses more slowly in other infants, most of whom remain clinically stable during the ensuing years. 1, 2 All HIV-1-infected infants are free of symptoms in the first weeks of life, and immunologic markers are normal, so it is not possible to predict which children will have early AIDS. Both viral load 35 and the biologic properties of infecting strains68 have been reported to play a role in disease outcome in HIV-1-infected adults. Primary HIV-1 isolates are highly heterogeneous in vitro with respect to replication rate, syncytium-inducing capacity, and cell tropism 9, 10;

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AIDS HIV- 1 MDM PBMC PCR R/H RPMI S/H S/L TCID

Acquired immunodeficiencysyndrome Human immunodeficiencyvirus type 1 Monocyte-derived macrophage Peripheral blood mononuclear cell Polymerase chain reaction Rapid/high Roswell Park Memorial Institute [medium] Slow/high Slow/low Tissue-culture-infective dose

strains isolated from symptom-free subjects usually replicate slowly in culture and are monocytotropic, but strains from patients with AIDS show rapid replication, the capacity to induce syncytia, and broad cytotropism.6, 11, 12 Moreover, studies of sequential HIV-1 isolates have shown that the emergence of fast-replicating and highly cytopathic T-lymphotropic variants precedes the onset of AIDS.8, 13, 14 We have found that primary HIV-1 isolates from children also show wide variation in biologic properties in vitro15; moreover, we have observed that the viral replication pattern on peripheral blood mononuclear cells in vitro is related to the viral load in vivo.~5 To evaluate the possible prognostic value of these viral characteristics in infants in terms of disease progression, we studied HIV-l-infected children prospectively from birth. METHODS Patients. Ninety-seven infants born to HIV-l-seropositive mothers were enrolled in this study. All the children were followed clinically and immunologically every month during the first 3 months of life and then every 2 to 3 months. According to the criteria of the European Collaborative Study, 1 children were classified as having HIV-related symptoms when oral candidiasis or any two of the following developed and persisted concurrently for more than 2 months: lymphadenopathy, hepatomegaly, splenomegaly, and failure to thrive. AIDS was diagnosed according to the criteria of the Centers for Disease Control and Prevention.16 Blood samples for viral culture and polymerase chain reaction were available in 52 cases within the first 15 days of life and in all 97 cases from 4 to 8 weeks of life. HIV-1 p24 antigen assay. Plasma samples were acid hydrolyzed, as detailed elsewhere, 17 to dissociate p24 antigen and anti-p24 antibody immunocomplexes, and then were assayed for p24 by means of an enzyme immunoassay (Coulter Corp., Hialeah, Fla.). Viral culture. The PBMCs were separated from blood on a Ficoll-Hypaque gradient (Pharmacia AB, Uppsala, Sweden). Both viral culture and PCR assay were performed on the same cell sample; when possible, aliquots of cells were

The Journal o f Pediatrics December 1993

frozen for further experiments. For 1 month 5 • 106 PBMCs from patients were cultured with an equal number of PBMCs from healthy donors after the cells had been pretreated for 48 hours with phytohemagglutinin protein (Difco Laboratories, Inc., Detroit, Mich.) in Roswell Park Memorial Institute medium supplemented with 10% fetal calf serum, 10% T-cell growth factor (Cellular Products, Buffalo, N.Y.), 1000 U anti-interferon alfa per milliliter (ICN Immunobiologicals, Costa Mesa, Calif.), and 1 #g polybrene per milliliter (Sigma Chemical Co., St Louis, Mo.). Cocultures were scored twice a week for the presence of syncytium formation; at least five fields (X200) per culture were examined. Supernatants were collected twice a week and assayed for the presence of p24 HIV- 1 core protein with the use of a commercially available kit (HIV-1 p24 core profile enzyme-linked immunosorbent assay; Du Pont Co., Wilmington, Del.). Polymerase chain reaction. The number of H IV-1 proviruses in patient PBMCs was determined by PCR as previously reported. 15 Briefly, PBMCs were washed in phosphate-buffered saline solution, and amplification by PCR was carried out directly in 1 X 105 lysed cells, using the primer pair specific for HIV-1 long-terminal-repeat 5 ' - 3 ' 501-518, 589-605 sequences. 18 For each PCR experiment, a standard reference curve was prepared with 8E51 cells, which contain one provirus per cell. 19 Hybridization of the amplified products was achieved with a 5" end oligonucleotide-specific probe labeled with phosphorus 32, as previously reported, 2~and the amount of the amplified products was determined by densitometer analysis. Optical-density values of reference samples were linearly related up to 1000 HIV-1 copies (r = 0.98); optical-density values of patient samples compared with a standard reference curve gave the number of proviruses per 105 PBMCs. Tropism of HIV-1 isolates for CD4 + T cells and MDM. Monocyte and CD4 + T lymphocyte-enriched cell populations were obtained from PBMCs of HIV-1 seronegative donors. Briefly, to obtain CD4 + lymphocytes, we separated PBMCs from blood as described above, suspended at 5 • 106 cells per milliliter in RPMI medium supplemented with 10% fetal calf serum, and stimulated with phytohemagglutinin (1 ~g/ml) for 48 hours; only nonadherent cells were harvested and then were depleted of CD8 + lymphocytes by the use of magnetic beads (Dynabeads; Dynal, Oslo, Norway). The CD4 + cells were cultured at a concentration of 1 X 106 cells per milliliter in RPMI medium supplemented with 10% fetal calf serum and 10% T-cell growth factor. To obtain monocytes, we separated PBMCs as described and seeded them into T25 flasks at a concentration of 5 x 106 ceils per milliliter in endotoxin-free lscove-modified Dulbecco medium supplemented with 10% human AB serum (Irvine Scientific, Santa Ana, Calif.). After 2 days

The Journal o f Pediatrics Volume 123, Number 6

nonadherent cells were removed and monocyte-derived macrophages were cultured at a concentration of 1 • 106 cells per milliliter in Iscove-modified Dulbecco medium supplemented with 10% AB human serum. The CD4 + T-lymphocytes and M D M ceils cultured at 1 • 106 cells per milliliter in 24-well plastic tissue culture plates (Costar Corp., Cambridge, Mass.) were cocultured for 28 days with equal amounts of serially diluted patient PBMCs. The PBMC-HIV titers on CD4 + lymphocytes and MDMs were calculated on the basis of the end-point dilution that gave a positive p24 result in CD4 + cells and MDMs, respectively, and were expressed as tissue-culture-infective doses per 106 PBMCs. 21 Statistical analysis. The cumulative proportion of children who progressed to HIV-related symptoms and to AIDS was derived by the Kaplan-Meier method, with censoring at the last clinical examination. Differences in rates of progression with respect to viral phenotypes were tested for statistical significance by an extension of the log-rank test to the comparison of three ordered groups. RESULTS

HIV-1 replication on primary PBMC cocultures. According to positive findings on viral culture and PCR, 18 (18.5%) of the 97 infants from 4 to 8 weeks of age were identified as infected with HIV-1. At this time, these children had no symptoms and had CD4 cell counts ranging from 1650 to 5400 cells per microliter. After coculture of cells from these patients with normal phytohemagglutininstimulated PBMCs, different patterns of viral replication emerged in reference to the lag phase of first positive detection of the p24 antigen and to the level of p24 production in the culture supernatant. Seven isolates, operatively defined as the rapid/high type, were detected after the first determination of p24 antigen at 3 days of culture and featured persistently high p24 levels (Fig. 1). Eleven isolates had a lag phase ranging from 7 to 21 days before releasing p24 antigen into the supernatant; after the initial delay (7 to 14 days), the slopes and plateau phases of p24 production curves in seven of these isolates superimposed those of R / H isolates and were defined as the slow/high type of isolate (Fig. 1). Four isolates, designated as slow/low, had a lag phase of 7 to 21 days and thereafter showed consistently low p24 levels (Fig. 1). In the PBMC cocultures, no S / L isolates showed syncytium-inducing capacity, which was instead observed in two of seven S / H and in six of seven R / H isolates. HIV-1 burden in vivo. The viral burden in vivo, as assessed by PCR, ranged from 10 to 947 HIV-1 copies per 105 PBMCs and was significantly associated with the in vitro viral growth pattern on PBMCs; HIV-1 proviral copy numbers per 105 PBMCs ranged from 360 to 947 (mean, 603 _+ 184)

De Rossi et al.

931

in children with R / H isolates, from 44 to 314 (mean, 158 + 114; p = 0.005, Mann-Whitney-Wilcoxon test) in children with S / H isolates, and from 10 to 38 (mean, 19 _+ 13; p = 0.008) in children with the S / L type of virus. At the time of PCR analysis, all children with R / H isolates had high levels of p24 antigen in plasma (>200 pg/ml); three infants with S / H and three with S / L isolates also had p24 antigen in plasma (range, 30 to 200 pg/ml). Tropism of primary HIV-1 isolates. To ascertain the tropism of the HIV- 1 variants in our patients, and to avoid any in vitro selection that might occur during primary coculture, we directly cocultured serial dilutions of patient PBMCs with CD4 + lymphocytes or MDMs obtained from the same donors. Stored frozen cell samples for this assay were available for five, six, and all four infants having isolates of the R / H , S / H , and S / L types, respectively. Even though viral yield in both indicator cell lines was low, all children with S / L isolates had a higher PBMC-HIV titer on MDMs (range, 1 to 5 TCID/106 cells) than on CD4 + T cells (range, <1 to 1 TCID/106 cells). All children with S / H or R / H isolates had an increase in PBMC-HIV titers on CD4 + T cells (ranges, 10 to 200 and 500 to 5000 TCID/106 cells, respectively); MDM cocultures were found to be positive for HIV-1 in only seven of these patients, with an PBMC-HIV titer ranging from 5 to 500 and from 10 to 200 TCID/106 cells, respectively (Fig. 2). The overall increase in PBMC-HIV titers in children with S / L , S / H , and R / H isolates paralleled that observed by PCR analysis. Although the two assays measured two different aspects of viral burden in vivo (i.e., the number of infected cells and the number of proviruses), the results in the same individual did not differ significantly. If the different sensitivities of the two assays are taken into account, this finding may indicate that most of the infected cells in vivo carry only a few copies of fully competent virus; this is in agreement with other reports. 4, 5 Although the real amount of T-lymphotropic and monocytotropic virus cannot be quantified because of the possible presence of amphitropic virus, which infects T cells and monocytesmacrophages equally well, 14,22 it is reasonable to assume that the relatively higher efficiency of HIV-1 recovery on CD4 + T cells or MDMs is attributable to a higher burden of variants with preferential T-lymphotropism or monocytotropism, respectively. Therefore the aforementioned findings indicate that children with S / L isolates may harbor low levels of mainly monocytotropic variants. In two children with the S / H type of virus, the PBMC-HIV titers on MDMs and on CD4 + T cells were the same (Fig. 2); this finding might reflect an equal number of monocytotropic and T-lymphotropic variants or an amphitropic virus. All children with an R / H type of virus had higher PBMC-HIV titers (>100-fold) on CD4 + T cells than on MDMs, thus

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The Journal of Pediatrics December 1993

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The Journal of Pediatrics Volume 123, Number 6

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indicating an expansion of T-lymphotropic variants devoid of monocytotropism in these infants. Clinical follow,up. All the children were followed clinically and immunologicallyfor a mean period of 22.9 months (range, 12 to 47 months). Progression to HIV-related symptoms was more rapid in children infected with the R / H type of isolate than in children with an S / H or S / L type of isolate (p <0.001) (Fig. 3). No child with an S / H or S / L type of isolate had AIDS by the age of 1 year, compared with 71% of the children with the R / H type of isolate (p = 0.006).

No significant relationship emerged between early PCRpositive results and disease outcome; 0nly 5 of 12 infected children tested within the first 15 days of life were found to have PCR findings that were positive for HIV-1 sequences,23 and only two of them had early manifestations of AIDS. DISCUSSION Extensive evidence that high-replicating and cytopathic T-lymphotropic viral variants play a role in disease progression has emerged from a large number of transectional

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The Journal of Pediatrics December 1993

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and sequential studies in adults infected with HIV-1. 614 A significant association also has been found between the number of HIV-l-infected cells in vivo and clinical status. 3-5 The present data confirm our previous findingsL5and indicate that the pattern of HIV-1 replication on primary PBMC coculture is influenced by the initial inoculum of viral particles in the culture systems. Nonetheless, the restricted efficiency of S / L isolates in spreading to PBMCs, where the T cells constitute more than 95% of the cell population, may indicate that intrinsic variation in viral tropism also plays a role in the viral growth pattern in vitro. We and others have demonstrated that S/L isolates are likely to contain more monocytotropic variants, and they exert transactivation activity better on monocytoid than on T-lymphoid cells. 12, 15A primary HIV-1 isolate can be constituted of viral variants, which differ in their tropism for T-lymphoid and monocytoid cells.14' 22 To avoid any in vitro selection that may occur during primary HIV-1 isolation, we directly cocultured patient PBMCs at end-point dilution with CD4 + lymphocytes or MDMs. By using this assay, we found that children with S / L isolates had low levels of mainly monocytotropic variants, as indicated by the relatively higher efficiency of HIV-1 recovery on MDMs than on lymphocytes. This finding may have important implications. Although PBMCs are the best cell target for HIV-1

detection and isolation, in some cases macrophages may be more appropriate target cells; thus, particularly when discordant results between PCR analysis and viral culture are obtained, a search for virus in MDM cells may be useful. In addition, the prevalence of monocytotropic variants in some infants suggests efficient transmission of them from mother to child, in agreement with a recent report of vertical transmission from a mother with undetectabte viremia. 24 Because mainly T-lymphotropic variants are transmitted in adults, z4 this finding suggests that the macrophages in the fetus or newborn may be more susceptible to HIV-1 infection than macrophages in adults. Further studies are needed to evaluate the role of the macrophages in vertically transmitted HIV-1 infections. In agreement with the increase in viral burden demonstrated by PCR analysis, an increase in T-lymphotropic variants and, in 7 of 11 cases, monocytotropic or amphitropic variants was observed in children with S / H and R / H isolates. In contrast to the children with an S / L type of isolate, those with R / H isolates had PBMC-HIV titers that were consistently higher (> 100-fold) in CD4 + T cells than in MDMs, indicating a predominance of T-lymphotropic variants that either were unable to infect macrophages or had a strong preferential tropism for T lymphocytes. We cannot determine whether these variants are transmitted directly from the mother or are the result of a selec-

The Journal o f Pediatrics Volume 123, Number 6

tive expansion d u r i n g primary infection, but their detection in infants is significantly correlated with early onset of disease. A t t h e time of testing, none of the infants h a d a C D 4 + c o u n t below the 5th percentile25; this finding f u r t h e r stresses the poor prognostic value of C D 4 + cell n u m b e r in infants.15, 26 All children with R / H isolates h a d persistently high levels of p24 antigen in plasma (not shown), so it is likely t h a t a high viral burden of replicating T-lymphotropic variants m a y cause an i m p a i r m e n t in C D 4 + cell function t h a t precedes the fall in cell count. T h e P C R and viral culture assays performed soon a f t e r b i r t h have a poor sensitivity for diagnosing HIV-1 infection.23, 27, 28 It has been suggested t h a t HIV-1 detection at b i r t h m a y depend on the different time at which infection occurs (in utero or perinatally) and m a y have some prognostic value. 29 In our infant series, detection of virus at b i r t h and severe progression of disease were not closely related. A l t h o u g h the poor sensitivity of P C R and viral culture in detecting HIV-1 in the first days of life m a y also limit their prognostic value a t t h a t time, b o t h assays are able to identify almost all H I V - 1 infected children when performed at 4 to 8 weeks of age. 23, 27 Despite the relatively low n u m b e r of children in this study, our findings indicate t h a t detection of a high viral b u r d e n of T-lymphotropie variants at this t i m e is predictive of early onset of A I D S . We thank Gabriella Miazzo and Enrico Schiavon for help in technical work.

REFERENCES 1. European Collaborative Study. Children born to women with HIV-1 infection: natural history and risk of transmission. Lancet 1991;337:253-60. 2. Tovo PA, De Martino M, Gabiano C, et al. Prognostic factors and survival in children with perinatal HIV-1 infection. Lancet 1992;339:1249-53. 3. Schnittman SM, Psallidopolous MC, Lane HC, et al. The reservoir for HIV-1 in human peripheral blood is the T cell that maintains expression of CD4. Science 1989;245:305-8. 4. Simmonds P, Balfe P, Peutherer JF, Ludlam CA, Bishop JO, Leigh Brown AJ. Human immunodeficieney virus-infected individuals contain provirus in small numbers of peripheral mononuclear ceils and at low copy number. J Virol 1990;64:86472. 5. Michael NL, Vahey M, Burke DS, Redfield RR. Viral DNA and mRNA expression correlate with the stage of human immunodeficiency virus (HIV) type 1 infection in humans: evidence for viral replication in all stages of HIV disease. J Virol 1992;66:310-6. 6. Asjo B, Morfeld-Manson L, Albert J, et al. Replicative capacity of human immunodeficiency virus from patients with varying severity of HIV infection. Lancet 1986;2:660-2. 7. Cheng-Mayer C, Seto D, Tateno M, Levy JA. Biological features of HIV-1 that correlate with virulence in the host. Science 1988;240:80-2.

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8. Tersmette M, Gruters RA, de Wolf F, et al. Evidence for a role of virulent human immunodeficiency virus (HIV) variants in the pathogenesis of acquired immunodeficiency syndrome: studies on sequential HIV isolates. J Virol 1989;63:211825. 9. Fenyo EM, Morfeldt-Manson L, Chiodi F, et al. Distinct replicative and cytopathic characteristics of human immunodeficiency virus isolates. J Virol 1988;62:4414-9. 10. Cloyd MW, Moore BE. Spectrum of biological properties of human immunodeficiency virus (HIV-1) isolates. Virology 1990;174:103-6. 11. Tersmette M, De Goede R, AI BJM, et al. Differential syncytium-inducing capacity of human immunodeficiency isolates: frequent detection of syncytium-inducing isolates in patients with acquired immunodeficiency syndrome (AIDS) and AIDSrelated complex. J Virol 1988;62:2026-32. 12. Schwartz S, Felber BK, Fenyo EM, Pavlakis GN. Rapidly and slowly replicating human immunodeficiency virus type 1 isolates can be distinguished according to target-cell tropism in T-cell and monocyte cell lines. Proc Natl Acad Sci USA 1989;86:7200-3. 13. Fiore JR, Calabr6 ML, Angarano G, et al. HIV-1 variability and progression to AIDS: a longitudinal study. J Med Virol 1990;32:252-6. 14. Schuitemaker H, Koot M, Kootstra NA, et al. Biological phenotype of human immunodeficiency virus type 1 clones at different stages of infection: progression of disease is associated with a shift from monocytotropic to T-cell tropic virus populations. J Virol 1992;66:1354-60. 15. De Rossi A, Pasti M, Mammano F, Ometto L, Giaquinto C, Chieco-Bianchi L. Perinatal infection by human immunodeficiency virus type 1 (HIV-1): relationship between proviral copy number in vivo, viral properties in vitro, and clinical outcome. J Med Virol 1991;35:283-9. 16. Centers for Diseases Control. Classification system for HIV infection in children under 13 years of age. M M W R 1987; 15:225-36. 17. De Rossi A, Ometto L, Mammano F, et al. Time course of antigenaemia and seroconversion in infants with vertical acquired HIV-1 infection. AIDS (in press). 18. Ou CY, Kowk S, Mitchell SW, et al. DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells. Science 1988;238:295-7. 19. Folks TM, Powell D, Lightfoote M, etal. Biological and biochemical characterization of a cloned leu-3 cell surviving infection with the acquired immunodeficiency syndrome retrovirus. J Exp Med 1986;164:280-90. 20. De Rossi A, Ades A, Mammano F, et al. Antigen detection, virus culture, polymerase chain reaction, and in vitro antibody production in the diagnosis of vertically transmitted HIV-1 infection. AIDS 1991;5:15-20. 21. Ho DD, Moudgil T, Alam M. Quantitation of human immunodeficiency virus type 1 in the blood of infected persons. N Engl J Med 1989;321:1621-5. 22. Gendelman HE, Baca LM, Huisayni H, et al. MacrophageHIV interaction: viral isolation and target cell tropism. AIDS 1990;4:221-8. 23. De Rossi A, Ometto L, Mammano F, Zanotto C, Giaquinto C, Chieco-Bianchi L. Vertical transmission of human immunodeficiency virus type 1 (HIV-1): lack of detectable virus in peripheral blood cells of infected children at birth. AIDS 1992;6:1117-20.

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24. Ariyoshi K, Weber J, Walters S. Contribution of maternal viral load to HIV-I transmission [Letter]. Lancet 1992;340: 435. 25. European Collaborative Study. Age-related standards for T-lymphocyte subsets based on uninfected children born to HIV-1 infected women. Pediatr Infect Dis J 1992;11:101826. 26. Leibovitz E, Rigaud M, Pollack H, et al. Pneumocystis carinii pneumonia in infants infected with the HIV with more than 450 CD4 T lymphocytes per cubic millimeter. N Engl J Med 1990;323:531-3. 27. Krivine A, Firtion G, Cao L, Francoual C, Henrion R, Lebon

P. HIV replication during the first weeks of life. Lancet 1992;339:1187-9. 28. Burgard M, Mayaux M J, Blanche S, et al. The use of viral culture and p24 antigen testing to diagnose human immunodeficiency virus infection in neonates. N Engl J Med 1992; 327:1192-7. 29. Rogers MF, Ou C-Y, Rayfield M, et al. Use of the polymerase chain reaction for early detection of the proviral sequences of human immunodeficiency virus in infants born to seropositive mothers. N Engl J Med 1989;320:1649-54.

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