Human Immunodeficiency Virus Type 1 Subtypes Differ in Disease Progression
Phyllis J. Kanki, Donald J. Hamel, Jean-Louis Sankale´,
Departments of Immunology and Infectious Diseases and Harvard Chung-cheng Hsieh, Ibou Thior, Francis Barin,
AIDS Institute, Harvard School of Public Health, Boston, and Stephen A. Woodcock, Aı¨ssatou Gue`ye-Ndiaye,
University of Massachusetts Cancer Center, Worcester, Massachusetts; Laboratory of Virology, University of Tours, Tours, France; Er Zhang, Monty Montano, Tidiane Siby,
Laboratory of Bacteriology and Virology, Cheikh Anta Diop Richard Marlink, Ibrahima NDoye, Myron E. Essex,
University, and Institut Hygiene Sociale, Dakar, Senegal and Souleymane MBoup
At least 10 different genetic human immunodeficiency virus type 1 (HIV-1) subtypes (A–J)
are responsible for the AIDS pandemic. Much of the understanding of HIV-1 disease pro-
gression derives from studies in the developed world where HIV infection is almost exclusively
subtype B. This has led many to question whether the properties and consequences of HIV-
1 infection can be generalized across subtypes that afflict the majority of infected persons in
the developing world. From 1985 to 1997, a prospective study of registered female sex workers
in Senegal tracked the introduction and spread of HIV-1 subtypes A, C, D, and G. In clinical
follow-up, the AIDS-free survival curves differed by HIV-1 subtype. Women infected with a
non-A subtype were 8 times more likely to develop AIDS than were those infected with subtype
A (hazard ratio
ϭ 8.23; P ϭ .009), the predominant subtype in the study. These data suggest
that HIV-1 subtypes may differ in rates of progression to AIDS.

The human immunodeficiency virus (HIV) type 1 epidemic transmission potentials. This prospective study was undertaken in many parts of sub-Saharan Africa is at least several decades to determine the dynamics of various HIV-1 subtype infections old, considerably older than in other parts of the world, such in a high-risk population over a decade of observation.
as Asia. HIV burden for the African continent is estimated at The recognized differences in transmission and virulence of 15–20 million, with heterosexual and perinatal transmission the HIV-2, compared with HIV-1, indicate that HIV viruses can major routes of infection [1]. Most of the 10 known HIV-1 have different pathogenic potentials. Prospective studies that subtypes have been reported in sub-Saharan Africa, although identified and followed seroincident infection were critical to surveillance data on subtypes G, H, I, and J remain preliminary the observation that HIV-2 was less transmissible and slower [2–5]. Current information on the prevalence of HIV-1 subtypes in disease progression than HIV-1 [6–8]. This lends support to is cross-sectional, frequently based on small numbers of samples the hypothesis that genetically distinct subtypes of HIV-1 might that were readily available, and often derived from patients at also differ in their biologic properties. Subtype differences in different times during their HIV infection [2]. Thus, although transmission and disease potential would drastically alter our studies in certain African countries have described multiple predictions of the future of the HIV epidemic and its impact.
HIV-1 subtypes, it is not known whether subtypes entered these Estimates of the time to development of AIDS after HIV populations at different times or whether the distribution of infection are long, variable, and based primarily on data from subtypes reflects the dynamics of different subtype-specific US and European studies with subtype B–infected persons.
Although few data are available from the developing world,where non-B subtypes predominate, cross-sectional studies sug- Received 4 May 1998; revised 25 August 1998.
gest that HIV non-B subtypes are common in AIDS patients Presented in part: 12th World AIDS Conference, Geneva, Switzerland, [9, 10]. However, differences in pathogenicity between HIV sub- 28 June–3 July 1998 (abstract 277).
Informed consent was obtained from all study participants. The protocol types might be best discerned from prospective studies, where was approved by ethical review committees in Dakar, Senegal, and at Har- the time to AIDS development can be estimated and compared.
vard School of Public Health. Human experimentation guidelines of the US Given the known differences in the geographic distribution and Department of Health and Human Services were followed.
Financial support: US Army Medical Research and Materiel Command associated epidemics of different HIV subtypes, we asked (grants 17-87-7072, 17-90-C-0138, 17-95-C-5005) and NIH (grants AI- whether HIV-1 subtypes might differ in their rates of disease 30795, AI-35173). The study was conducted under the Inter-University development. This study is the first to evaluate disease pro- Convention for the Prevention of AIDS and Other Sexually TransmittedDiseases. I.T., A.G.-N. and J.-L.S. are Fogarty International fellows.
gression of different HIV-1 subtypes in a cohort of prospectively Reprints or correspondence: Dr. P. J. Kanki, Dept. of Immunology and followed persons with known time of infection.
Infectious Diseases, Harvard School of Public Health, 651 Huntington Ave.,Boston, MA 02115 (pkanki@hsph.harvard.edu).
The Journal of Infectious Diseases
1999; 179:68–73
Since 1985, all women registered as prostitutes at the Institut 1999 by the Infectious Diseases Society of America. All rights reserved.
d’Hygie`ne Sociale clinic in Dakar, Senegal, have been serologically screened for exposure to HIV-1 and HIV-2. The clinic provides Multiple alignments were done with the Clustal package (W 1.6) clinical examinations and treatments of sexually transmitted dis- with minor manual adjustment when necessary [14]. Phylogenetic eases (STDs) during biannual visits, which are required for the analysis was performed by the neighbor-joining method, and re- legal registration of prostitutes. All women who gave informed liability was estimated from 1000 bootstrap resamplings. Repre- consent were enrolled in the study, and a baseline questionnaire sentative sequences of various HIV subtypes A–I were included in and health evaluation was administered by study physicians. The the analysis [2]. The nucleotide sequences were submitted to dynamic nature of this clinic resulted in an open cohort design in GenBank (accession numbers: AF085284–085327 and AF020819– which subjects were continuously identified and enrolled over the study period. The study design, recruitment procedures, and var- In samples where envelope sequences could not be amplified, ious protocols have been described [6–8, 11].
nested PCR in the long-terminal-repeat (LTR) region was done for At the time of enrollment and at subsequent biannual visits, a subtype assignment. Oligonucleotide primers were designed on the complete physical examination was done that emphasized HIV- basis of an analysis of conserved regions within the LTR across related signs and symptoms. All biannual blood samples were tested the clades represented in the Los Alamos (NM) National Labo- for antibodies to each virus by immunoblot for HIV-1 and HIV-2 ratory HIV database (A–G, O). Upstream primers for the LTR antibodies in the Dakar and Boston laboratories. T cell subsets were PH5KPN. Downstream primers for the LTR were PH3 LTR were determined annually, and DNA was extracted from the pe- ripheral blood mononuclear cells that remained after T cell subset PH3 LTR: 5 -CTGAGGGATCTCTAGTTACCAGAG-3 ; PH56: analysis by proteinase K–phenol-chloroform extraction.
5 -ATTGAGGCTTAAGCAGTGGG-3 ). Samples were subjected Seroincident HIV-1 cases were defined as women who were pre- to 30–39 amplification cycles consisting of a denaturing step (94ЊC, viously HIV-negative and subsequently seroconverted to HIV-1.
30 s), a primer annealing step (50ЊC, 30 s), and a primer extension The time of infection, or seroconversion date, was estimated as the midpoint between the last seronegative and first seropositive sam-ples. HIV-1–positive person-years of observation was calculatedfrom the seroconversion date until the AIDS disease outcome.
AIDS was diagnosed by the CDC revised surveillance case defi- From February 1985 to December 1995, 1683 seronegative nition, which includes !200 CD4 cells [12]. Cases were censored at women were enrolled in the study and followed; 81 women their last clinic examination date or date of death (not caused byAIDS); the observation period ended on 1 January 1997. Kaplan- subsequently seroconverted to HIV-1. DNA samples were avail- Meier AIDS-free survival analysis and Cox proportional hazards able from 54 of the 81 HIV-1 seroconverters, and HIV-1 subtype models for AIDS outcome were done as described (Stata, College was determined. HIV-1 subtype was assigned based on the C2- V3 sequence in 53 of 54 samples; LTR sequences were used for HIV-1 subtype was determined by polymerase chain reaction the remaining sample to determine subtype after the envelope (PCR) amplification of ∼350 bp in the HIV-1 C2–V3 envelope sequences failed to provide subtype information. Subtypes were region. A nested PCR was done using two sets of primers: WT1, determined by phylogenetic tree analysis; based on 1000 boot- 5 -GCTGGTTTTGCGATTCTAAAGTGTA (6884–6908, posi- strap resamplings, high bootstrap values demonstrated the tions relative to HXB2) and WT2, 5 -CAATAGAAAAATTCC- branching order of sequences with known HIV-1 subtypes A CCTCCACAAT (7353–7377), for the first round; and published (93.4%), C (96.4%), D (92.1%), and G (99.0%) (figure 1).
primers, KK30–KK40, for the second round [13].
Subtype A was found in 37 (68.5%) of 54 seroconverters, The PCR reactions were in a 100-mL volume reaction mixture subtype C in 5 (9.3%), subtype D in 5 (9.3%), and subtype G containing 1 mg of DNA, 10 mL of 10ϫ PCR buffer II and 2.5 Uof Taq polymerase (Perkin-Elmer PCR Reagents, Roche Molecular in 6 (11.1%). One (1.8%) of the V3 envelope sequences had Systems, Branchburg, NJ), 0.2 mM of each dideoxy-nucleotide, 2 significant variation from other subtype A sequences. Exami- mM MgCl , and 20 pmol of each primer. Each reaction was sub- nation by the Recombinant Identification Program [15] re- jected to 30–35 cycles of denaturation (94ЊC, 45 s), annealing (45 vealed a mosaic virus composed of subtypes A and G, which s at 57ЊC and 55ЊC in the first and second rounds, respectively), was further verified by sequencing of the complete gp120 por- and extension (72ЊC, 45 s), followed by a final extension (72ЊC, 3 tion of the envelope. Most of the gp120 aligned with subtype min) in an automatic thermal cycler. Negative controls included A, but ∼300 bp in the V2–C2 region aligned with subtype G.
deionized water and negative peripheral blood lymphocyte DNA This woman was infected in 1994 and is currently healthy; she was not included in further analyses because of the mosaic The PCR product was purified and directly sequenced using the virus. While HIV-1 subtype assignment utilizing the V3 enve- second-round primers (KK30 and KK40). When necessary, the lope sequence has high specificity and sensitivity, it is inade- purified product was cloned in pCR2.1 vector (T/A cloning; In- quate to address the potential existence of recombinant or mo- vitrogen, San Diego). Sequences were determined by dye termi-nator cycle sequencing using Taq polymerase and an automatic saic viruses [16, 17]. Thus, it is possible that other women in sequencer ABI 373A (both Perkin-Elmer, Applied Biosystem Di- our study harbor viruses that are monotypic within the V3 vision, Foster City, CA). In all cases, sequences were determined region but mosaic or recombinant in other regions of the for both strands of DNA, and in many instances multiple clones The distribution of seroconverters over the 10-year period is Figure 1.
Phylogenetic tree analysis by neighbor-joining method of V3 envelope sequences from registered female sex workers who seroconverted between 1985 and mid-1995. Representative V3 envelope sequences of various HIV-1 subtypes A–I are indicated in underlined bold text [2].
Alignment was subjected to 1000 bootstrap resamplings; no. of resamplings is indicated at major branch points. Horizontal distance correspondsto genetic relatedness.
illustrated by HIV subtype designated at the estimated sero-conversion date (figure 2). In this high-risk group of women,the first HIV-1 seroconversions occurred in late 1987. Through-out the study period, subtype A was the most common HIV-1 subtype. The first seroconversion to subtype C occurred latein 1987; the last C seroconversion was observed in late 1993.
Similarly, subtype D was observed early in 1987 and then spo-radically until early 1994. Subtype G infections first appearedin mid-1992 and in subsequent years represented 20% of theincident HIV-1 subtypes.
We are cautious in our interpretation of the subtype dynamics in this cohort, since DNA samples were unavailable for 27 ofthe 81 HIV-1 seroconverters observed in the study. A logisticregression analysis, including age at registration, years of reg-istered prostitution, nationality, and year of seroconversion,showed that women of Ghanaian nationality were less likelyto be subtyped (P ϭ .02). Ghanaian women represented a smallfraction of all HIV-1 seroconverters in the study (18.8%), yetour results may be less generalizable to this subpopulation.
The prospective nature of the study allowed the clinical ob- servation of women after seroconversion. Overall, we observed11 cases of AIDS in the 54 women with subtype diagnosis.
These women were followed for 199 person-years. The HIV-1subtype-specific AIDS incidence rates per 100 person-years var-ied widely, from 3.45 (95% confidence interval [CI], 1.44–8.29)for subtype A to 15.95 for subtype C (95% CI, 5.14–49.45) and14.45 for subtype D (95% CI, 4.66–44.81). No cases of AIDSwere seen in the 6 women who were infected with subtype Grelatively late in the study period.
Kaplan-Meier disease-free survival analysis was performed on seroincident women grouped by HIV subtype (figure 3A).
The AIDS-free survival curves differed by HIV subtype (logrank test, P ϭ .06; Wilcoxon-Gehan, P ϭ .03). At 5 years ofinfection, 87.0% of women infected with subtype A were AIDS-free compared with 30.0% of subtype C– and 60.0% of subtypeD–infected women. To adjust for other potentially confoundingvariables, we constructed a Cox proportional hazards modelwith AIDS as the outcome, controlling for nationality, age atregistration, years of registered prostitution, and HIV subtype.
Figure 2.
HIV-1 subtypes in 54 registered female sex workers ob- The adjusted ratio for development of AIDS was 8.11 for sub- served from 1985 to 1997 with first seronegative visit (⅜), seroconver-sion date, subtype (A, C, D, G, and A/G), and AIDS status (᭢) in- type C (95% CI, 1.44–45.61; P ϭ .02) and 8.43 for subtype D dicated. Horizontal line to left of seroconversion date/subtype indicates negative person-years of observation. Horizontal line to right of sero- Since relatively few women were infected with subtypes C, conversion date indicates positive person-years of observation. Cal- D, and G, we analyzed AIDS-free survival probabilities in women with subtype A compared with those with non-subtypeA HIV (figure 3B). The AIDS-free survival curves were distinct: AIDS than those infected with subtype A (95% CI, 1.70–39.8; Women infected with subtype A had a longer AIDS-free sur- vival period than those infected with non-A subtypes; the 5- In the prospective cohort study design, the evaluation of loss year AIDS-free survival probability was 87% in subtype A– to follow-up is critical to the interpretation of the AIDS pro- infected women versus 52% for non-subtype A–infected women gression data. At the beginning of each calendar year, study (log rank test, P ϭ .02; Wilcoxon-Gehan, P ϭ .03). The Cox physicians and social workers actively investigated enrolled proportional hazards model estimated that women infected women who had not attended the clinic for 112 months. In the with a non-A subtype were 8.23 times more likely to develop 54 HIV-incident women with known HIV subtype, 4 (7.4%) Figure 3.
A, Kaplan-Meier AIDS-free survival in seroincident women by HIV-1 subtype (P ϭ .06, log rank; P ϭ .03, Wilcoxon-Gehan). B, Kaplan-Meier AIDS-free survival analysis in seroincident women with HIV subtype A vs. all non-A subtypes (P ϭ .02, log rank; P ϭ .03, Wilcoxon-Gehan).
were considered lost to follow-up after the 1997 active follow- global HIV pandemic. Differences in sexual practices, rates of up evaluation. We performed a sensitivity analysis in which STDs, and other determinants of HIV infection and transmis- these women were considered to have developed AIDS. Again, sion also likely contribute to the different HIV epidemic curves subtype A demonstrated a significantly longer AIDS-free sur- observed worldwide. However, to date, studies of different sub- vival and reduced rate ratio for AIDS development compared types have been limited to cross-sectional surveys. This is the with non-A subtypes (95% CI, 0.07–0.74; P ϭ .014). Thus, we first study that documents the introduction and spread of mul- do not believe that differential follow-up in our study popu- tiple HIV subtypes in a high-risk population studied over time.
lation was responsible for the subtype differences in progression Previous studies have estimated the rate of progression to AIDS in US and European cohorts. Although study designs Misclassification bias must also be considered in the evalu- and methods of analysis have varied, 65%–80% AIDS-free sur- ation of our study results. Multiple methods have been used vival at 5 years has been reported in cohorts before the extensive to assess viral subtype infection, including heteroduplex mo- use of antiviral therapy [20–23]. The rate of disease progression bility shift assays, complete sequencing of the viral genome, in populations in the developing world has been poorly studied and PCR sequencing of partial regions of the HIV genome [2, to date; our study cohort in Dakar, Senegal, provides the only 3, 18]. Sequence analysis of the V3 envelope region is one of data on AIDS progression in an incident cohort studied 15 the more common methods for molecular epidemiology studies years [8]. Studies concerning the natural history of chronic in- and has relatively high reliability in determining HIV-1 subtypes fections, such as HIV, are difficult to achieve. Nevertheless, our [2]. However, this does not rule out the possibility of mosaic study has been successful in minimizing loss to follow-up genomes, which might only be detected through full genomesequencing [15–17]. Current data suggest that the number and through regular active follow-up and the efforts of a dedicated complexity of HIV recombinants is ever expanding. The ma- jority of sequences from subtype G viruses originate in West We believe our study is the first to evaluate disease progres- and Central Africa; our data suggest this may be a more recent sion in non-B subtype infections with known time of infection.
HIV-1 subtype [2]. In addition, sequence analysis of subtype G In evaluating AIDS-free survival curves of women with incident viruses suggest that this subtype may frequently be a mosaic A, C, D, and G infection, we found distinct differences in AIDS- with subtype A [19]. In consideration of this, we grouped sub- free survival. The grouped comparison of non-A versus A sub- types A and G together and compared disease progression rates types demonstrated a significantly longer AIDS-free survival to those of subtypes C and D. Subtypes A and G demonstrated for women infected with subtype A. Due to the small sample a significantly longer AIDS-free survival and adjusted AIDS size per subtype, with few AIDS cases despite a lengthy study rate ratio (95% CI, 1.70–39.82; P ϭ .009). Therefore, despite period, our estimate of AIDS incidence should not be consid- the possibility of mosaic viruses, our classification of subtypes ered precise, and further study is clearly warranted. Cross-sec- based on potential mosaicism still suggests distinct differences tional studies indicate a significant proportion of AIDS cases with subtype A infection in West and East Africa (unpublisheddata) [24]. However, this is not inconsistent with our results Discussion
should the proportion of subtype A in the high-risk population In recent years, there has been a growing appreciation of the be quite elevated. Further study of HIV-1 subtype natural his- role of HIV-1 subtypes in the complex epidemiology of the tory and progression from different geographic regions is clearly needed to better evaluate the role of viral subtype differences 7. Donnelly C, Leisening W, Sandberg S, Kanki P, Awerbach T. Comparison of transmission rates of HIV-1 and HIV-2 in a cohort of prostitutes in Senegal. Bull Math Biol 1993; 55:731–41.
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John McNeil, and Boris Renjifo for critical review of this manuscript, 15. Siepel AC, Halpern AL, Macken C, Korber BTM. A computer program Rajiv Rawat for editorial assistance, and the Senegalese Ministry of designed to rapidly screen for HIV-1 intersubtype recombinant sequences.
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