HIV-1 Specific Cytotoxic T Lymphocyte (CTL) Responses in Pediatric Infection
Katherine Luzuriaga1 and John L. Sullivan2
Pediatrics and Molecular Medicine, University of Massachusetts Medical School, Biotech 2, Suite 318, 373 Plantation Street, Worcester, MA 01605
Background
Over the past decade, the number of HIV-1 infected infants and children has rapidly increased through the vertical (mother-infant) transmission of the virus. It is currently estimated that one thousand four hundred infants are newly infected each day throughout the world [UNAIDS/WHO Working Group on Global HIV/AIDS and STD Surveillance in collaboration with National AIDS Programmes]. Great interest has developed, therefore, in the development of effective strategies to preventvertical HIV-1 infection.
Proposed strategies for the reduction of the vertical transmission of HIV-1 have primarily focused on perinatal antiretroviral prophylaxis. Treatment of pregnant women and their infants with the reverse transcriptase inhibitor, zidovudine, can profoundly reduce the risk of vertical HIV-1 transmission [Connor 19946]. However, the cost and logistic intensity of this antiretroviral regimen renders it impractical for use in developing nations, where most pediatric infections occur. These strategies would also not prevent the vertical transmission of HIV-1 beyond the neonatal period (e.g., through breastfeeding). A safe and effective infant vaccine regimen, begun at birth, would therefore be more desirable and might also provide the basis for lifetime protection against HIV-1 infection. Improved understanding of the early infection process and the capability of the young human infant to generate HIV-1 specific immune responses are important for the development of such a vaccine.
The natural history of vertical HIV-1 infection and correlates of disease
In general, vertically-infected children experience more rapid disease progression than children infected at an older age or adults. Approximately 23% of vertically-infected children develop an AIDS-defining condition in their first 1-2 years of life (``rapid progressors'') and an additional 17% will develop an AIDS-defining condition by 4 years of age [Newell 199426]. The tempo of disease progression in the remainder (``nonrapid progressors''; 60%) is variable, although at most 10% of vertically-infected children survive to 8 years with intact CD4 counts and without symptoms of disease [Martin 199622].
Potential viral and host determinants of infection outcome have been studied. Within weeks of birth, rapid increases in the plasma HIV-1 RNA copy number to 105 to 107 copies per milliliter of plasma have been documented [Palumbo 199529; Shearer 199737]. Plasma HIV-1 RNA levels only gradually decrease over the first 1 to 2 years of life [Palumbo 199529; Shearer 199737] and mean plasma HIV-1 RNA levels remain greater than 105 copies per milliliter of plasma through at least the third year of life. A continued reduction in plasma HIV-1 RNA (mean -0.2 to -0.3 log decline per year) independent of clinical or immunologic status and antiretroviral therapy has been observed in vertically-infected children through 6 years of age [McIntosh 199624; Mofenson 199725]. The reason for prolonged elevation of plasma HIV-1 RNA
levels during the early years of vertical HIV-1 infection is unclear. It has been suggested that a larger pool of permissive host cells (activated CD4 T cells) in infancy and early childhood might explain this observation. While the observed decrease in plasma HIV-1 RNA levels (10 to 100-fold) exceeds the age-related 3-fold reduction in CD4 T cells observed over the same time period [McIntosh 199624], this could be due, in part, to the production of many virions by an infected cell. Infection at a time of reduced ability to generate effective virus-specific immune responses might also account for the apparent diminished control of viral replication in infancy and early childhood. Support for this
hypothesis is provided by studies that suggest that host immune responses may be potent selective forces for diversification [Ganeshan 199710; Wolinsky 199643], along with studies that have documented limited viral diversification over the six months of life [Salvatori 199735].
HIV-1 specific CTL and the pathogenesis of HIV-1 infection
Virus-specific CTL are important for the clearance of acute viral infection and suppression of viral replication in chronic infections though they likely act in synergy with other virus-specific or non-specific immune responses (Reviewed in [Oldstone 199528]). In adult primary infection, HIV-1 specific CTL are among the earliest documented host immune responses [Koup, 1994]. In established infection, HIV-1 specific CTL have been demonstrated in the absence of virus-specific in vitro stimulation [Walker 198741]. Additionally, an
individual may generate HIV-1 specific CTL directed against multiple structural (Envelope, Gag; reviewed in [Johnson, 1994]) or non-structural (Reverse Transcriptase; [Walker 198842]), Nef [Riviere 199433]) epitopes. Depletion or blocking studies indicate that HIV-1 Gag-specific CTL are CD8 T cell-mediated and HLA Class I restricted [Koup 198916]. Studies of HIV-1 Envelope-specific cytolysis have demonstrated classic CD8 T cell-mediated, HLA Class I restricted responses as well as NK cell or neutrophil-mediated cytolysis through ADCC [Riviere 198932].
The role of HIV-1 specific CTL in HIV-1 infection is unclear. The temporal association of the appearance of HIV-1 specific CTL with a reduction of blood viral load in adult primary viremia [Koup 1994; Borrow 19941; Borrow 19972] and the association of
high HIV-1 specific CTL precursor frequencies with preservation of CD4 counts [Greenough 199711] suggest a protective role. However, chronic, high-level viral replication may continue despite a broad and vigorous HIV-1 specific CTL response [Luzuriaga 199521].
Several factors may contribute to continued viral replication in the presence of an apparently vigorous HIV-1 specific CTL response. In vitro assay methods used
to detect HIV-1 specific CTL may not detect biologically relevant CTL activity, i.e., high avidity CTL [Speiser 199238]. Rapid and widespread dissemination of HIV-1 infection prior to the generation of CTL responses may ``outstrip'' the CTL response, particularly since effective neutralizing antibodies develop slowly in primary infection. Viral avoidance of CTL recognition may occur
through replication in immune-privileged sites, induction of immunosuppression, the down regulation of MHC Class I or adhesion molecules on infected cell surfaces, or amino acid sequence variation (reviewed in [Koup, 1994]). Finally, viral-specific CTL, including HIV-1 specific CTL have been implicated in pathological processes [Jassoy 199213], [Jassoy 199314] and CTL responses may contribute to HIV-1 associated CD4 depletion or disease.
HIV-1 specific CTL in Vertical Infection
Virus-specific CTL responses have not been well-defined in children. Historically, murine models suggested that young mice were tolerized following fetal or neonatal antigen exposure [Oldstone 198927]. More recent data indicate that young mice can generate CTL responses if antigen presentation is
accompanied by appropriate co-stimulatory signals and if appropriate antigen doses are used [Ridge 199631; Sarzotti 199636; Forsthuber 19968]. Cellular cytotoxic responses have been studied in human infants with acute respiratory syncytial virus (RSV) infection. After virus-specific in vitro
stimulation, Isaacs and colleagues [Isaacs 198712] detected RSV-specific, cell-mediated cytotoxic responses in 4 of 22 infants studied. Chiba and
colleagues [Chiba 19895] described the detection of age-dependent RSV-specific cellular cytotoxic responses, again after virus-specific in vitro stimulation. In these studies, however, effector cells and HLA restriction of these responses were not definitively characterized.
The large numbers of infants born to HIV-1 infected women each year has provided a unique opportunity to study the ability of infants children to generate virus-specific CTL, to characterize the CTL responses, and to evaluate their potential role in disease pathogenesis. HIV-1 specific CTL responses were first studied in older children with established disease. In an early study
[Luzuriaga 199120], activated Gag-specific HIV-1 specific CTL (i.e., those detected using freshly-isolated peripheral blood lymphocytes as effectors) were less commonly detected in vertically-infected children than in hemophilic children infected after the age of 2 years or in HIV-1 infected adults studied by the same laboratory [Koup 198916]. A subsequent study [Buseyne 19933] confirmed the less frequent detection of activated CD8 T cell-mediated, HIV-1 Gag and Pol-specific CTL in children compared with adults; direct Envelope-specific
cytotoxicity was detected in the peripheral blood of 12 (51%) children of 21 studied but the responsible effector cell(s) were not fully characterized. In a third study [McFarland 199423], circulating, activated HIV-1 Gag and Pol-specific CTL were detected in only 1 of the 11 children studied; activated HIV-1 Envelope-specific cytotoxic responses were detected in 5 of the 11 children but characterization of effector cells suggested that these responses were NK cell-mediated.
While activated HIV-1 specific CTL have been uncommonly detected in the circulation of HIV-1 infected children, HIV-1 specific CTL precursors (CTLp) recognizing at least 1 HIV-1 gene product have been detected in the majority (> 60%) of vertically-infected children using non-specific in vitro stimulation of PBMC [Buseyne 19933; Froebel 19949]. It must be noted that most of
the children studied thus far have been older than 2-4 years of age at first evaluation and thus primarily represent nonrapid progressors. In fact, at least 2 cross-sectional studies have documented CTLp in higher proportions of nonprogressor children than in children with rapid disease progression [Van De Perre 199240; Luzuriaga, Manuscript in Preparation]. Prospective studies of several cohorts are now in progress to examine the potential relationship between HIV-1 specific CTL and disease progression.
Quantitation of HIV-1 specific CTLp after non-specific in vitro stimulation has revealed HIV-1 Gag (50-630 CTLp per million PBMC) and Envelope (66-330 CTLp per million PBMC) specific CTLp frequencies similar to those measured in adults with established disease [Koup, 1991, 1994]. All CTL activity detected after in vitro stimulation in these studies has been CD8 T cell-mediated.
Little information is available on the fine specificity of HIV-1 specific CTL responses in children. Responses to 2 or more epitopes (within the same or different gene products) have been defined in most HIV-1 infected children studied thus far [Buseyne 19933; Luzuriaga et al unpublished data]. Again, it must be emphasized that most of these studies have been carried out in older children with less rapidly progressing disease and detailed, prospective studies of the CTL repertoire, beginning in infancy, are necessary.
Several groups have prospectively evaluated HIV-1 specific CTL responses in early vertical infection. Circulating, activated HIV-1 specific CTL are rarely detected in the first year of life [Luzuriaga 199521]. Of interest, HIV-1 specific CTLp have also been uncommonly detected in early infancy (i.e., < 4-6 mo), but
have been detected in the circulation of most HIV-1 infected infants by the end of the first year of life [Luzuriaga 199521]. This contrasts with the frequent detection of HIV-1 specific CTLp in the peripheral blood of adults within weeks of primary infection [Koup 198916; Borrow 19941; Borrow 19972].
Most vaccinia constructs used in assays to detect HIV-1 specific CTL express laboratory strain gene products. In a small cohort of young HIV-1 infected infants, the use of vaccinia vectors expressing Envelope sequences derived from first infant isolates allowed earlier detection of HIV-1 specific CTL than the use of vaccinia vectors expressing HIV-1 IIIB Envelope sequences [Pikora 199730].
Again, these data contrast with the frequent detection of HIV-1 specific CTLp in the peripheral blood of adults within weeks of primary infection using vaccinia vectors expressing HIV-1 IIIB Envelope sequences [Koup 198916; Borrow 19941; Borrow 19972], suggesting that type-specific responses might predominate in early infancy.
HIV-1 specific CTL have been detected in infants who acquired HIV-1 infection in utero, as well as in infants who acquired infection during birth [Luzuriaga 199521]. The detection of HIV-1 specific CTLp in the cord blood of an HIV-1 infected infant suggest that the capacity to generate virus-specific CTL
develops in fetal life although the exact timing is unclear. Recent murine studies suggest that the antigen presentation and co-stimulatory signals required for generation of a CTL response by an immature host are more stringent than those required in a mature host; given appropriate antigen presentation and
co-stimulatory signals, however, neonatal mice appear capable of generating antigen-specific CTL responses [Ridge 199631]. Data from studies of HIV-1 specific CTL in infants support this model, rather than the classic ``self versus nonself'' model, that would predict tolerance following fetal or neonatal exposure to antigen.
HIV-1 specific CTL in exposed, uninfected infants
In an effort to define immune responses which protect against the acquisition of HIV-1 infection, HIV-1 specific immune responses have been studied in cohorts of individuals who have not acquired infection despite high risk exposures. Several groups have reported the detection of cellular immune responses, including HIV-1 specific CTL, in uninfected children born to HIV-1 infected women. In the first group of studies, activated Envelope, Gag, and Nef-specific cytotoxic activities were repeatedly detected in the peripheral blood of 2 infants through 17-35 months of age [Cheynier, 1992]. In the second group of studies, activated HIV-1 specific cytotoxic T cell responses were detected in the peripheral blood of 6 (25%) of 23 uninfected infants born to HIV-1 infected women; age at detection ranged from 15 to 50 months [De Maria 19947]. Since no other evidence of HIV-1 infection was detected in the peripheral blood of the infants/children, these studies raise the question of how such vigorous CTL responses could be maintained in the absence of antigenic stimulation. Using virus-specific stimulation, Rowland-Jones [Rowland-Jones 199334] and colleagues have reported the detection of HIV-1 specific CTL of unspecified phenotype in an uninfected infant. At least 3 other groups, including our own, have been unable to detect these responses, even after virus-specific in vitro stimulation [Luzuriaga 199120; Buseyne 19933; McFarland 199423; Luzuriaga 1997]. Further analyses of exposed but uninfected infants and children are necessary in order to determine whether clearance of infection occurs and to examine virus-specific immune responses that are potentially associated with viral clearance. If HIV-1 specific CTL responses are detected, characterization of the responses (including the documentation of virus-specificity) would have important implications for neonatal vaccine development.
References
1. Borrow, P., H. Lewicki, B. H. Hahn, G. M. Shaw, and M. B. Oldstone. 1994. Virus specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection. Journal of Virology 68 6103-6110.
2. Borrow, P., H. Lewicki, X. Wei, M. S. Horowitz, N. Peffer, H. Meyers, J. A. Nelson, J. E. Gairin, B. H. Hahn, M.B.Oldstone, G.M. Shaw. 1997. Antiviral pressure exerted by HIV-1-specific cytotoxic T lymphocytes (CTLs) during primary infection demonstrated by rapid selection of CTL escape virus. Nature Medicine 3 205-211.
3. Buseyne, F., S. Blanche, D. Schmitt, C. Griscelli, and Y. Riviere. 1993. Detection of HIV-specific cell-mediated cytotoxicity in the peripheral blood from infected children. Journal of Immunology 150 3569.
4. Cheynier, R., P. Langlade-Demoyen, M. R. Marescot, S. Blanche, G. Blondin, S. Wain-Hobson, C. Griscelli, E. Vilmer, and F. Plata. 1992. Cytotoxic T lymphocyte responses in the peripheral blood of children born to human immunodeficiency virus-1 infected mothers. European Journal of Immunology 22 2211-2217.
5. Chiba, Y., Y. Higashidake, K. Suga, K. Honjo, H. Tsutsumi, and P. Ogra. 1989. Development of cell-mediated cytotoxic immunity to respiratory syncytial virus in human infants following naturally acquired infection. Journal of Medical Virology 28 133-139.
6. Connor, E. M., R. S. Sperling, R. Gelber, and the ACTG 076 Study Group. 1994. Reduction of maternal-infant transmission of human immunodeficiency virus type 1 with zidovudine treatment. New Engl J Med 331 1173-1180.
7. De Maria, A., C. Cirillo, and L. Moretta. 1994. Occurrence of human immunodeficiency virus type 1 (HIV-1)-specific cytolytic T cell activity in apparently uninfected children born to HIV-1-infected mothers. Journal of Infectious Diseases 170 1296-1299.
8. Forsthuber, T., H. C. Yip, and P. V. Lehmann. 1996. Induction of Th1 and Th2 immunity in neonatal mice. Science 271 1728-1730.
9. Froebel, K. S., M. C. Aldhous, J. Y. Q. Mok, J. Hayley, M. Arnott, and J. F. Peutherer. 1994. Cytotoxic T lymphocyte activity in children infected with HIV. AIDS Res Hum Retroviruses 10: S83-S90.
10. Ganeshan, S., R. E. Dickover, B. T. Korber, Y. J. Bryson, and S. M. Wolinsky. 1997. Human immunodeficiency virus type 1 genetic evolution in children with different rates of development of disease. J Virol 71 663-677.
11. Greenough, T. C., D. B. Brettler, M. Somasundaran, D. L. Panicali, and J. L. Sullivan. 1997. Human Immunodeficiency Virus type 1- specific cytotoxic T lymphocytes (CTL), virus load, and CD4 T cell loss: evidence supporting a protective role for CTL in vivo. Journal of Infectious Diseases 176 118-125.
12. Isaacs, D., C. R. M. Bangham, and A. J. McMichael. 1987. Cell-mediated cytotoxic response to respiratory syncytial virus in infants with bronchiolitis. Lancet 2(8562) 769-771.
13. Jassoy, C., R. P. Johnson, B. A. Navia, J. Worth, and B. D. Walker. 1992. Detection of a vigorous HIV-1-specific cytotoxic T lymphocyte response in cerebrospinal fluid from infected persons with AIDS dementia complex. Journal of Immunology 149 3113-3119.
14. Jassoy, C. J., T. Harrer, T. Rosenthal, B. A. Navia, J. Worth, R. P. Johnson, and B. D. Walker. 1993. HIV-1 specific cytotoxic T cells release interferon gamma, tumor necrosis factor (TNF)-alpha, and TNF-beta when they encounter their target antigens. J Virol 67 2844-2852.
15. Johnson, R. P., and B. D. Walker. 1994. Cytotoxic T lymphocytes in human immunodeficiency virus infection: responses to structural proteins. Current Topics in Microbiology and Immunology 189 35-63.
16. Koup, R. A., J. L. Sullivan, P. H. Levine, D. Brettler, A. Mahr, G. Mazzara, S. McKenzie, and D. Panicali. 1989. Detection of major histocompatibility complex class I restricted HIV-specific cytotoxic T lymphocytes in the blood of infected hemophiliacs. Blood 73 1909-1914.
17. Koup, R. A. 1994. Virus escape from CTL recognition. Journal of Experimental Medicine 180 779-782.
18. Koup, R. A., J. T. Safrit, Y. Cao, C. A. Andrews, G. McLeod, W. Borkowsky, C. Farthing, and D. D. Ho. 1994. Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome. J Virol 68 4650-4655.
19. Koup, R. A., R. M. Hesselton, J. T. Safrit, M. Somasundaran, and J. L. Sullivan. 1994. Quantitative assessment of human immunodeficiency virus type 1 replication in human xenografts of acutely infected Hu-PBL-SCID mice. AIDS Res Hum Retroviruses 10 279-84.
20. Luzuriaga, K., R. A. Koup, C. A. Pikora, D. B. Brettler, and J. L. Sullivan. 1991. Deficient human immunodeficiency virus type 1-specific cytotoxic T cell responses in vertically infected children. Journal of Pediatrics 119 230-6.
21. Luzuriaga, K., D. Holmes, A. Hereema, J. Wong, D. L. Panicali, and J. L. Sullivan. 1995. HIV-1-Specific cytotoxic T lymphocyte responses in the first year of life. Journal of Immunology 154 433-443.
22. Martin, N. L., R. Koup, R. Kaslow, J. Coffin, and A. Ammann. 1996. Workshop on perinatally-acquired human immunodeficiency infection in long-term surviving children: a collaborative study of factors contributing to slow disease progression. AIDS Res Hum Retroviruses 12 1565-1570.
23. McFarland, E. J., P. A. Harding, D. Luckey, B. Conway, R. K. Young, and D. R. Kuritzkes. 1994. High frequency of gag- and env-specific cytotoxic T lymphocyte precursors in children with vertically-acquired human immunodeficiency virus type 1 infection. Journal of Infectious Diseases 170 766-774.
24. McIntosh, K., A. Shevitz, D. Zaknun, J. Kornegay, P. Chatis, N. Karthas, and S. K. Burchett. 1996. Age- and time-related changes in extracellular viral load in children vertically infected by human immunodeficiency virus. Pediatric Infectious Disease Journal 15 1087-1091.
25. Mofenson, L. M., J. Korelitz, W. A. Meyer, J. Bethel, K. Rich, S. Pahwa, J. Moye, R. Nugent, and J. Read. 1997. The relationship between serum human immunodeficiency virus type 1 (HIV-1) RNA level, CD4 lymphocyte percent, and long-term mortality risk in HIV-1-infected children. Journal of Infectious Diseases 175 1029-1038.
26. Newell, M.-L., C. Peckham, D. Dunn, T. Ades, and C. Giaquinto. 1994. Natural history of vertically-acquired human immunodeficiency virus-1 infection. The European Collaborative Study. Pediatrics 94 815-819.
27. Oldstone, M. B. A. 1989. Viral persistence. Cell 56 517-520.
28. Oldstone, M. B. A. 1995. The role of cytotoxic T lymphocytes in infectious disease: history, criteria, and state of the art. Current Topics in Microbiology and Immunology 189 1-8.
29. Palumbo, P. E., S. Kwok, S. Waters, Y. Wesley, D. Lewis, N. McKinney, A. Bardeguez, E. M. Connor, and J. Oleske. 1995. Viral measurement by polymerase chain reaction-based assays in human immunodeficiency virus-infected infants. Journal of Pediatrics 126 592-595.
30. Pikora, C. A., J. L. Sullivan, D. Panicali, and K. Luzuriaga. 1997. Early HIV-1 envelope-specific cytotoxic T lymphocyte responses in vertically infected infants. Journal of Experimental Medicine 185 1153-1161.
31. Ridge, J. P., E. J. Fuchs, and P. Matzinger. 1996. Neonatal tolerance revisited: turning on newborn T cells with dendritic cells. Science 271 1723 -1726.
32. Riviere, Y., F. Tanneau-Salvadori, A. Regnault, O. Lopez, P. Sansonetti, B. Guy, M. P. Kieny, J. J. Fournel, and L. Montagnier. 1989. Human immunodeficiency virus-specific cytotoxic responses of seropositive individuals:distinct types of effector cells mediate killing of targets expressing gag and env. J Virol 63 2270-2277.
33. Riviere, Y., M. N. Robertson, and F. Buseyne. 1994. Cytotoxic T lymphocytes in human immunodeficiency virus infection: regulatory genes. Current Topics in Microbiology \& Immunology 189 65-74.
34. Rowland-Jones, S., D. F. Nixon, M. C. Aldhous, F. Gotch, K. Ariyoshi, N. Hallam, J. S. Kroll, K. Froebel, and A. McMichael. 1993. HIV-1 specific cytotoxic T-cell activity in an HIV-exposed but uninfected infant. Lancet 341 860-861.
35. Salvatori, F., S. Masiero, C. Giaquinto, C. M. Wade, A. J. Leigh-Brown, L. Checo-Bianchi, and A. De Rossi. 1997. Evolution of human immunodeficiency virus type 1 in perinatally infected infants with rapid and slow progression to disease. J Virol 71 4694-4706.
36. Sarzotti, M., D. S. Robbins, and P. M. Hoffman. 1996. Induction of protective CTL responses in newborn mice by a murine retrovirus. Science 271 1726-1728.
37. Shearer, W. T., T. C. Quinn, P. LaRussa, J. F. Lew, L. Mofenson, S. Almy, K. Rich, E. Handelsman, C. Diaz, M. Pagano, V. Smeriglio, and L. Kalish. 1997. Viral load and disease progression in infants infected with human immunodeficiency virus type 1. New England Journal of Medicine 336 1137-1349.
38. Speiser, D. E., D. Kyburz, U. Stubi, H. Hengartner, and R. M. Zinkernagel. 1992. Discrepancy between in vitromeasurable and in vivo virus neutralizing cytotxic T cell reactivities. Journal of Immunology 149 972-980.
39. Stoneburner, R. L., P. Sato, A. Burton, and T. Mertens. 1994. The global HIV pandemic. Acta Paediatrica Supplement 400 1-4.
40. Van De Perre, P., P. LePage, A. Simonon, and e. al. 1992. Biological markers associated with prolonged survival in African children maternally infected by the human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 8 435-442.
41. Walker, B. D., S. Chakrabarti, B. Moss, T. J. Paradis, T. Flynn, A. G. Durno, R. S. Blumberg, J. C. Kaplan, M. S. Hirsch, and R. T. Schooley. 1987. HIV-specific cytotoxic T lymphocytes in seropositive individuals. Nature 328 345-348.
42. Walker, B. D., C. Flexner, T. J. Paradis, T. C. Fuller, M. S. Hirsch, R. T. Schooley, and B. Moss. 1988. HIV-1 reverse transcriptase is a target for cytotoxic T lymphocytes in infected individuals. Science 240 64-66.
43. Wolinsky, S. M., B. T. Korber, A. U. Neumann, M. Daniels, K. J. Kunstman, A. J. Whetsell, M. R. Furtado, Y. Cao, D. D. Ho, J. T. Safrit, and R. A. Koup. 1996. Adaptive evolution of human immunodeficiency virus-type 1 during the natural course of infection. Science 272 537-542.