Viral load differences in early infection with two HIV-1 subtypes
Hu, Dale J.a; Vanichseni, Suphakb; Mastro, Timothy D.a,c; Raktham, Suwaneeb; Young, Nancy L.a,c; Mock, Philip A.c; Subbarao, Shambavid; Parekh, Bharat S.d; Srisuwanvilai, La-ongb; Sutthent, Ruengpunge; Wasi, Chantaponge; Heneine, Walidd; Choopanya, Kachitb
From the aDivision of HIV/AIDS Prevention - Surveillance and Epidemiology, National Center for HIV, STD, and TB Prevention, Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, US, the bBangkok Metropolitan Administration, Bangkok, Thailand, the cHIV/AIDS Collaboration, Nonthaburi, Thailand, the dDivision of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA and eMahidol University, Bangkok, Thailand.
Received: 27 September 2000;
revised: 24 November 2000; accepted: 18 January 2001.
Sponsorship: This study was carried out with the financial support of the US Centers for Disease Control and Prevention as a part of a research collaboration with the Bangkok Metropolitan Administration and the Ministry of Public Health of Thailand
Correspondence to Dale J. Hu, MD, Division of HIV/AIDS Prevention, Mailstop E-45, National Center for HIV, STD, and TB Prevention, Centers for Disease Control and Prevention, 1600 Clifton Road, NE, Atlanta, GA 30333, USA. Tel: +1 404 639 6104; fax: +1 404 639 6127; e-mail: email@example.com
Note: Reprint requests to The HIV/AIDS Collaboration, DMS 6 Building, Ministry of Public Health, Tivanon Road, Nonthaburi 11000, Thailand.
Objectives: Information on early HIV-1 infection has come primarily from studies of persons infected with subtype B in North America and Europe; much less is known about other subtypes. The purpose of the present study was to compare the virologic and immunologic parameters following seroconversion among recently-infected persons infected with either of two different HIV-1 subtypes.
Method: A prospective cohort study was carried out at methadone treatment clinics administered by the Bangkok Metropolitan Administration, Thailand. A total of 130 HIV-1-infected seroconverters (103 with HIV-1 subtype E and 27 with subtype B) were included in the study. The main outcome measures were serial HIV-1 RNA viral load, natural killer cell percentage, CD4 and CD8 lymphocyte counts since seroconversion.
Results: The demographic and behavioral characteristics of persons with either subtype were similar. Median RNA viral levels at the earliest time within 3 months of seroconversion were more than three times higher for persons infected with subtype E than subtype B (63 100 versus 18 050 copies/ml, P = 0.001). However, this difference decreased over time such that viral loads were similar at 12, 18, and 24 months following seroconversion. The CD4 and CD8 lymphocyte counts were similar in infections with either subtype during the entire period up to 24 months post-seroconversion.
Conclusions: Higher viral loads associated with subtype E may result from inter-subtype biological differences; however, the epidemiological dynamics of transmission in Bangkok may have also contributed to this phenomenon.
Human immunodeficiency virus, type 1, exhibits a high degree of genetic variation and has been classified into a number of clades or subtypes . Although a variety of viral and host factors have been shown to influence pathogenesis [2,3], the relative role of HIV-1 genetic variation in influencing transmissibility and pathogenesis has been a topic of debate and a focus for research in recent years [1,4].
The variable course of early HIV-1 infection has been described primarily from studies of persons in North America and Europe who have recently become infected with HIV-1 subtype B [2,5–7]. Of the many factors associated with pathogenesis [2,3], plasma viral load has been shown to be one of the strongest predictors of disease progression and clinical outcome [5,8–12]. The viral RNA level established by 6 months after infection has been shown to be highly predictive of subsequent disease progression and appears to be influenced by both viral and host factors [2,5]. In addition, higher viral load has been closely associated with increased sexual  and perinatal [14,15] HIV-1 transmission.
To date, relatively few studies have examined the early events following seroconversion and the clinical progression of HIV-1 infection in developing countries, where most infections are occurring worldwide and are caused by subtypes other than B [4,11,16]. It is extremely important to understand, within the context of prevention and treatment whether HIV-1 disease progression differs in population groups around the world and by infecting subtype [4,16,17].
The HIV-1 epidemic in Thailand has been well-described with a very rapid initial epidemic among injection drug users (IDU) in Bangkok in the late 1980s caused primarily by HIV-1 subtype B followed by a much larger epidemic of HIV-1 subtype E among people at heterosexual risk of infection [18–20]. However, in recent years, subtype E has been documented to account for an increasing proportion of infections in IDUs [20–22]. Although a number of potential factors have been hypothesized to contribute to the increase in subtype E infections, it remains unknown whether HIV-1 subtype E differs significantly from subtype B in transmissibility and disease progression [23–25]. The objectives of this study were to describe and compare the virologic and immunologic events following seroconversion among IDUs who had recently become infected with either of two HIV-1 subtypes, B or E.
Materials and methods
The Bangkok Metropolitan Administration (BMA) manages a large municipal drug treatment program in Bangkok, Thailand where approximately 8000 drug users are seen annually. As described elsewhere, 1209 HIV-negative injecting heroin users were enrolled and followed in a prospective study at 15 BMA drug treatment clinics from May 1995 to December 1996 [21,26]. They were treated with methadone and received HIV prevention, education, and counseling. Participants in the original study were seen at 4-month intervals where they were interviewed and serum specimens were tested for antibodies to HIV-1 by enzyme immunoassay (EIA) at the BMA laboratory as described previously . Specimens that were found to be HIV-1-seropositive on EIA were tested with another EIA (Genetic Systems, Redmond, Washington, USA) and Western blot (Novapath HIV-1 Immunoblot; BioRad, Hercules, California, USA) at the HIV/AIDS Collaboration laboratory; specimens that were positive on EIA and Western blot were considered to be HIV-seropositive . Following positive serologic testing for HIV-1, subjects were offered, with voluntary informed consent, enrollment in a subsequent prospective study of HIV-infected persons where blood was collected as soon as possible, 1 month later, and then at 4-month intervals. The study protocols were approved by the Ethical Review of Research Committee, Ministry of Public Health, Nonthaburi, Thailand and an Institutional Review Board, Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA. As of December 1998, after 2308 person–years of observation, 133 IDUs seroconverted to HIV-1 yielding an incidence of 5.8 per 100 person–years . Of the seroconverters from the original cohort, 126 agreed to participate in the study. In addition, four IDU seroconverters were identified during 1998 from a sixteenth BMA clinic site yielding a total of 130 persons.
Data and specimen collection
At each study visit, participants were interviewed about risk behavior and were clinically evaluated and referred for treatment of any medical conditions as per BMA treatment guidelines . Serum specimens were collected at all visits for serological testing . Following the first seropositive visit, additional venous blood samples were collected into EDTA-anticoagulated tubes and Cell Preparation Tubes (Becton Dickinson, Franklin Lake, New Jersey, USA) for lymphocyte phenotyping, cell pellets, and separation into plasma and peripheral blood mononuclear cells (PBMC). The PBMC were frozen in liquid nitrogen and all cell pellets and plasma samples were frozen at −70°C within 8 h of collection. Genetic characterization of all viral isolates has been described previously . Specimens from the last seronegative visit, first seropositive visit, and all subsequent visits following seroconversion were tested for viral load determination by Amplicor HIV-1 Monitor Test version 1.5 (Roche Diagnostics, Branchburg, New Jersey, USA). The lower limit of quantitation was 400 RNA copies/ml. Lymphocyte immunophenotyping was carried out on fresh EDTA-anticoagulated venous samples following the first seropositive visit with the FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, California, USA) using a standard six-tube, two color monoclonal antibody panel (Becton Dickinson). Natural killer (NK) cells were defined as cells that were CD3−, CD16+, and CD56+. Characterization of viral strains was confirmed by genetic sequencing and phylogenetic analysis . Finally, to assess the possibility of differential sensitivity of the Amplicor Monitor viral load assay to either subtype B or E, we measured virion-associated reverse transcriptase (RT) activity at the first seropositive visit, which was the earliest time point following seroconversion, using the Amp-RT assay .
Demographic, behavioral and follow-up characteristics of seroconverters were compared by infecting HIV-1 subtype. Serial HIV-1 viral load and three immunological parameters (CD4 and CD8 lymphocyte counts and NK cell percentages) were analyzed over time in persons infected with either HIV-1 subtype B or E. Estimated date of seroconversion (EDS) was assumed to have occurred at the midpoint between the dates of the last EIA-negative test and the first EIA-positive test. Although no antiretroviral therapy was reported for any of the seroconverters during the first 12 months following seroconversion, antiretroviral therapy for HIV-infected persons increased substantially in Thailand during the latter period of this study. Therefore, all data observations from 2 months before and subsequent to the first reported antiretroviral therapy were right-censored so that analyses and comparisons reported in this study came from data contributed only by seroconverters when they were treatment naive.
The non-parametric Wilcoxon rank sum test was used to compare viral load and the immunological parameters at key time periods following EDS. These periods were the first determination within 3 months of EDS, and sequential determinations closest in time (within 3 months) to 6 months, 12 months, 18 months, and 24 months following EDS. We also compared the highest measured viral loads within 3 months of EDS. To evaluate temporal trends in viral load, we used a robust, locally weighted, non-parametric, smoothed regression (lowess) .
An alternative analysis for repeated measurements of HIV-1 RNA viral load, NK cell percentage, and CD4 and CD8 lymphocyte counts was carried out using a general linear mixed-effects regression model . We used a random intercept and slope (time) model such that each subject had their own parameters for baseline measurement (intercept) and rate of change (slope). The dependence structure was assumed to be exchangeable such that a common correlation between repeated observations and statistical inferences was based on robust standard errors. The infecting HIV-1 subtype variable was included as a fixed effect in the model. For NK cell percentage and CD4 and CD8 lymphocyte count, regression models were calculated from EDS to 12 months and to 24 months. For plasma viral load, the regression models were analyzed from 3 months after EDS to 12 months and to 24 months so as to avoid the effects of the non-linear early primary viremic phase. All statistical analyses were carried out using SAS version 6.12 (SAS Institute, Cary, North Carolina, USA) and Stata version 6 (StataCorp, College Station, Texas, USA).
Of the 130 participants who seroconverted between September 1995 and December 1998, 103 (79%) were infected with subtype E and 27 (21%) were infected with subtype B. All persons were of Thai nationality and the demographic and behavioral characteristics were similar, irrespective of infecting subtype (Table 1). Epidemiological data suggested that transmission through needle sharing accounted for the large majority of infections with both subtype B and E and that sexual behavior was not associated with increased risk for HIV-1 seroconversion .
Overall, the time from the last EIA-negative specimen to the first EIA-positive specimen was similar for participants infected with either subtype B [median 119 days; interquartile (Q1–Q3) range, 116–130 days] or subtype E (median 124 days; Q1–Q3 range, 119–139) (P = 0.12). As of December 1999, the number of days of total follow-up from EDS ranged from 95 to 1556 (median 855) days for subtype B and 84 to 1511 (median 943) days for subtype E. Follow-up times did not differ significantly by subtype (P = 0.89). Viral load testing of the last seronegative specimen yielded similar proportions that were HIV-1 RNA positive; five of 27 (18.5%) subtype B and 12 of 100 (12%) subtype E specimens (P = 0.4).
Figure 1 illustrates the levels of viral RNA over time since EDS for persons infected with either subtype B or E.. Mean and median viral loads were significantly higher within 6 months of seroconversion for persons infected with subtype E than those infected with subtype B (Table 2). Even when specimens from participants who were RNA-positive on the last seronegative visit were excluded, the viral loads remained higher for subtype E within 3 months of seroconversion (P = 0.006). However, at 12, 18, and 24 months following seroconversion, the differences were not statistically significant.
To assess the possibility of differential subtype sensitivity by the Amplicor Monitor, we compared the ratios of subtype-independent virion-associated RT activity as measured by the Amp-RT assay with the Amplicor Monitor viral load at the first determination following EDS. Similar ratios of Amp-RT to Amplicor Monitor viral load [medians of 0.10 (Q1–Q3 range, 0.028–0.31) for 21 subtype B specimens and 0.095 (Q1–Q3 range, 0.024–0.38) for 85 subtype E specimens tested (Wilcoxon rank sum test, P = 0.99)] suggested no significant assay bias by subtype.
Mean and median CD4 counts were similar for infected persons regardless of subtype during the entire 24 month period following EDS (Table 3). The CD8 counts were slightly higher for persons infected with subtype E, especially during the first 3 months after EDS, but the differences were not statistically different (Table 3). The NK cell percentages were significantly lower for persons infected with subtype E than B during the first 12 months but later became less significant (Table 3).
From one of the largest and best characterized cohorts of HIV-1 seroconverters in Asia and one of the few cohorts worldwide with substantial proportions of more than one infecting HIV-1 subtype, the most notable finding was the significantly higher viral load observed during the early period following seroconversion among persons infected with subtype E compared with those infected with subtype B. Although viral RNA levels remained higher in persons infected with subtype E, the magnitude of the intersubtype difference was not statistically significant during the period from 12 to 24 months.
Observed differences in inter-subtype viral loads can be the result of a number of factors. Therefore, it is important to evaluate the likelihood of any systematic biases prior to considering causative factors. One potential bias is the possibility of variable viral quantitation assay sensitivity for different HIV-1 subtypes. However, we believe that differential assay sensitivity is unlikely to account for the inter-subtype difference for several reasons. Although earlier versions of viral load assays were primarily based on subtype B sequences from North America and Europe and were less sensitive in quantifying viral load for certain non-B subtypes such as subtype E, any differential assay sensitivity would more likely result in underestimation rather than overestimation of viral load levels in subtype E-infected persons [30,31]. Furthermore, newer versions of viral load assays such as the Amplicor Monitor version 1.5 have improved sensitivities for non-B subtypes, such that sensitivities now seem comparable for the major HIV-1 subtypes [30–32]. In addition, as the magnitude of inter-subtype viral load differences decreased over time, a time-dependent bias in assay sensitivity seems unlikely. Finally, similar ratios and distributions of Amp-RT (which measures reverse transcriptase activity independent of which subtype is present) to Amplicor Monitor for both subtypes B and E also support the absence of any significant assay bias by subtype and thus support the validity of higher early viral loads in subtype E infections.
Another potential bias that may artefactually influence measured intersubtype viral load differences would be the systematic sampling of persons infected with one subtype closer to their actual time of seroconversion than those with the other subtype. The present results show that the times between the last seronegative and first seropositive test results were very similar. Although the proportion of subtype B specimens that were RNA-positive at their last seronegative visit was slightly higher, this difference was not significant, and when those seronegative polymerase chain reaction-positive specimens were excluded, the early viral load difference between subtypes B and E persisted. In the absence of any strong systematic biases, our observed findings more likely reflect the true higher early viral load levels for persons recently infected with subtype E than with B.
In general, our viral load measurements over time were similar in magnitude or slightly higher (0.5 to 1.0 log10) than those reported from other well-controlled prospective cohort studies of seroconverters from North America or Europe [6,33–36]. However, comparisons of early viral load between studies are difficult due to differences in the seroconversion intervals. On the other hand, CD4 lymphocyte counts among seroconverters in the present study were similar or lower than those reported from earlier cohort studies [33,37]. However, evidence of lower baseline CD4 counts and higher NK cell numbers in Asian populations compared with non-Asians has been reported [38,39].
The observation that intersubtype viral load differences decrease over time in the context of similar CD4 counts in persons infected with either subtype suggest that the host immune system can respond in a similar general manner to infections with distinct HIV-1 subtypes. Nevertheless, a trend in immunologic parameters, such as the higher early CD8 counts, although not statistically significant, and the significantly lower NK cell percentages in persons infected with subtype E as compared with B, may be consistent with slight differences in the early host immune response to higher viral loads associated with subtype E [40,41].
Very few prospective studies have been conducted to specifically address the issue of differences in clinical and immunologic progression by HIV-1 subtype. Although significant differences in viral load and disease progression between HIV-1 and HIV-2 have been well documented, analogous evidence of significant inter-subtype differences is far from clear [42,43]. Not only is there relatively little information on the clinical progression of HIV-1 infection in most developing countries, where the predominant subtypes are not subtype B [42,44,45], studies of otherwise similar persons infected with different subtypes living in similar settings are limited. Although one study from West Africa reported that women infected with subtype A appeared less likely to develop AIDS at 5 years post-seroconversion than women infected with subtypes C, D, and G, this study did not report viral load comparisons . A prospective study of female sex workers in northern Thailand infected primarily with HIV-1 subtype E showed similar or slightly faster progression compared with populations infected with subtype B in Western countries before highly active antiretroviral therapy was available . Continued follow-up of our cohort will be useful to compare our data with other studies in Thailand [11,45] to assess potential clinical progression differences by subtype or transmission mode as well as to confirm whether higher initial viral loads in persons infected with subtype E are correlated with subsequent intersubtype differences in disease progression.
It is unclear what factors contribute to the higher observed viral loads associated with subtype E infection. Although research has shown that viral genetic variation can influence phenotypic properties such as cell-tropism, co-receptor usage, and the ability to form syncytia [46–50], these properties have generally not been associated with genetic subtype [4,49]. For example, although initial reports had suggested that isolates of HIV-1 env subtypes E infected Langerhans cells (found in the genital tract) more readily than isolates of subtype B , two subsequent studies failed to show any subtype-specific differences [51,52]. On the other hand, recent characterization of subtype B and E viruses from our cohort of seroconverters showed major inter-subtype differences in the proportions of different envelope V3 motifs as well as predicted co-receptor usage and phenotype from genetic sequence data . In addition, reported inter-subtype differences in the function of long terminal repeat (LTR) transcriptional promoters suggest that subtype E may replicate more efficiently than subtype B in certain circumstances .
Although subtype-specific biologic characteristics may account for our observed viral load differences, it is possible that higher viral loads associated with subtype E may also be influenced by the dynamics of the HIV-1 epidemic among IDU in Bangkok. As subtype E was introduced more recently with a higher incidence in this population of IDU, one might predict a higher proportion of persons with recent infection and hence higher mean viremia among the reservoir of source partners infected with subtype E than with subtype B [54,55]. Indeed, our own cohort data indicate that the proportion with higher viral loads among persons infected with subtype E is greater than those with subtype B. Thus, if these recently infected persons in our study are similar to those serving as source partners, then regardless of the reasons for it, the subtype E reservoir of infection sources is richer in higher viral loads than for the subtype B reservoir. As higher viral load has been associated with not only higher rates of transmission through a variety of modes [15,56] but higher subsequent viral loads in the recipient in animal models [57,58], we hypothesize that a person infected by someone with subtype E would be more likely to have encountered someone with a higher viral load or inoculum, which in turn would result in higher initial viral loads in the seroconverter, which is consistent with our findings. Further research will be helpful in understanding the dynamics of transmission and the public health significance of inter-subtype viral load differences. In addition to providing very important information on the clinical progression among persons infected with two different HIV-1 subtypes, this study will provide useful background data within the context of an ongoing phase III HIV vaccine efficacy trial in Thailand .
The authors gratefully thank the participants of the study and all of the staff affiliated with this study from the Bangkok Metropolitan Administration, the HIV/AIDS Collaboration (HAC), Mahidol University, the World Health Organization, the Joint United Nations Programme on HIV/AIDS, and the Centers for Disease Control and Prevention for administrative, clinical, laboratory, and data management support. Specifically, we would like to acknowledge the study coordinators from HAC: Pongsri Virapat, Wanitchaya Kittikraisak, and Natapakwa Skunodom, Nartlada Chantharojwong (HAC) and Robert Nelson (CDC) for data management, Vedapuri Shanmugam for performing the Amp-RT assays, Marie Morgan (CDC) for editorial assistance and Timothy Dondero (CDC) for critical review of the manuscript and overall support.
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