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Long-Term Intrapatient Viral Evolution during HIV-2 Infection

  1. Adam MacNeil1,
  2. Jean-Louis Sankalé1,
  3. Seema Thakore Meloni1,
  4. Abdoulaye Dieng Sarr1,
  5. Souleymane Mboup2 and
  6. Phyllis Kanki1
  1. 1Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts
  2. 2Laboratoire de Bacteriologie et Virologie, Universite Cheikh Anta Diop, Dakar, Senegal
  1. Reprints or correspondence: Dr. Phyllis Kanki, Dept. of Immunology and Infectious Diseases, Harvard School of Public Health, 651 Huntington Ave., Boston, MA 02115 (pkanki{at}hsph.harvard.edu).
 
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Abstract

Background. Disease progression and transmission of human immunodeficiency virus (HIV) type 2 are attenuated, compared with HIV-1, which is consistent with the lower plasma viral loads observed in HIV-2 infection. Although numerous studies have characterized the intrapatient evolution of viral sequences during HIV-1 infection, prospective studies examining intrapatient evolution during HIV-2 infection have been limited.

Methods. We examined viral sequence evolution in the C2V3C3 region of the viral env gene in 8 HIV-2- infected individuals from Dakar, Senegal, over the course of ∼10 years. To compare results with HIV-1 infection, we reanalyzed data from our previous study that prospectively examined intrapatient viral evolution in HIV-1- infected individuals from the same population.

Results. HIV-2 sequences from early and late time points were phylogenetically intermixed for all subjects. No distinct trends were observed in terms of increases or decreases in fragment size or the number of N-linked glycosylation sites, and ratios of synonymous substitutions per synonymous site to nonsynonymous substitutions per nonsynonymous site suggested selection to be neutral or negative. In homologous env C2V3 sequences, rates of viral divergence and diversification were slower in individuals infected with HIV-2 than in those infected with HIV-1.

Conclusions. Viral evolution occurs slowly in HIV-2 infection, which is consistent with the slow disease progression of HIV-2 and supports the notion that viral evolution may be a relevant correlate for disease progression.

HIV-1 and HIV-2 are distinct human lentiviruses that cause AIDS. Previous studies have shown that HIV-2 is less pathogenic than HIV-1, with a significantly slower CD4+ T cell count decrease and a longer time to the development of AIDS [1–4]. Consistent with this observation, people infected with HIV-1 have higher plasma viral loads than those infected with HIV-2 [5–9]. By contrast, the proviral loads in peripheral-blood mononuclear cells (PBMCs) are similar between people infected with HIV-1 and those infected with HIV-2 [8, 10, 11]. On the basis of these observations, it has been suggested that HIV-1 replicates at a higher rate than HIV-2 in vivo [8].

Because of the high degree of replication in vivo and the error-prone process of replication, lentiviruses undergo rapid evolution in an infected host. Studies of HIV-1 have shown that, during initial infection, viral sequences are relatively homogenous. After infection, viral sequences diverge from earlier sequences in a constant manner through the asymptomatic phase of infection. Similarly, the diversity of the HIV-1 population in an infected person increases at a relatively constant rate during this stage of infection [12, 13].

Plasma viral loads have been shown to be a strong predictor of HIV-1 disease progression [14]. Studies have also found that rates of intrapatient HIV-1 sequence divergence correlate with disease progression [15–17]. By contrast, other researchers have reported high rates of sequence divergence to be associated with slow disease progression [18–20]. Similarly, multiple studies have reported that HIV-1 populations diversify faster in people progressing slowly to AIDS than in those who develop disease more rapidly [16, 19, 21–24] and that genetic changes appear to be associated with stronger positive selection in people who progress slowly to AIDS [18–21, 25–27]. However, it has also been reported that rates of viral diversification are higher in HIV-1 progressors than in nonprogressors [17]. These differing conclusions in terms of divergence, diversification, and disease progression may be partially accounted for by differences in disease stage, definitions of progression, and populations examined in various studies. In our laboratory, Mani et al. [28] compared the evolution of env C2V3 between groups of asymptomatic HIV-1-infected individuals who had low and high viral set points and found higher rates of divergence and diversification in those with high viral set points

Prospective [29–31] and cross-sectional [32] studies examining intrapatient evolution of HIV-2 have been limited. For the present study, we examined the intrapatient HIV-2 evolution of env C2V3C3 in 8 individuals from Dakar, Senegal, over the course of ∼10 years per subject. Because it is believed that HIV-2 maintains a lower rate of replication in vivo than HIV-1, we hypothesized that intrapatient viral evolution occurs more slowly in HIV-2 infection than in HIV-1 infection. To test this hypothesis, we compared rates of intrapatient HIV-2 divergence and diversification between groups of HIV-1- and HIV-2-infected individuals from Dakar, Senegal.

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Subjects, Materials, and Methods

Sample acquisition. HIV-2-infected study subjects were selected from a cohort of female sex workers in Dakar, Senegal, that has been monitored since 1985, as described elsewhere [33]. Informed consent was obtained from all study subjects, in accordance with the human-experimentation guidelines of the US Department of Health and Human Services for the conduct of clinical research. All subjects enrolled in the study were naive for antiretroviral therapy and remained free of dual infection with HIV-1 throughout the course of the study. For each subject, PBMC samples were acquired at 2 time points separated by 8.1–11.4 years. HIV-2 plasma loads were determined as described elsewhere [6].

DNA amplification and sequencing. Proviral DNA was isolated from PBMC samples (QIAmp DNA Blood Midi Kit or QIAmp DNA Blood Mini Kit; Qiagen), and DNA concentrations were determined by the optical density at 260 nm. Approximately 1 µg of DNA was used for polymerase chain reaction (PCR) amplification, and a fragment of the C2V3C3 region of the viral env gene was amplified in a nested PCR using first-round primers AM2E1f (5′-GAGACATCAATAAAACCATGTGTC-3′) and AM2E1r (5′-ACCCAATTGAGGAACCAAGTCA-3′) and second-round primers AM2E2f (5′-GATACTGTGCACCACCGGG-3′) and AM2E2r (5′-TCTCCTCTGCAGTTAGTCCAC-3′). To avoid cross-contamination, first- and second-round PCRs were performed in separate rooms, and PCRs from early and late time points were performed on separate days. Three independent PCRs were performed on each sample, to minimize resampling bias. Amplified products were cloned (pCR2.1; Invitrogen) and transformed into competent cells (TOP10; Invitrogen). Individual colonies were selected; cloned plasmids were purified (SNAP MiniPrep kit; Invitrogen) and sequenced using M13 primers (Invitrogen). A total of 8–13 sequences were acquired for each study subject at each time point.

HIV-2 sequence analysis and calculations. Sequences were aligned using CLUSTAL X software (version 1.81) [34], and manual adjustments were made using MacClade software (version 4; Sinauer) [35]. Of 160 sequences, 4 contained stop codons and were excluded for distance calculations and for calculations of the ratio of synonymous substitutions per synonymous site to nonsynonymous substitutions per nonsynonymous site (DS:DN). Similar conclusions were reached even with the inclusion of these sequences in the distance and DS: DN calculations. For phylogenetic analysis and distance calculations, aligned sequences were trimmed to remove gaps. Findmodel [36] was used to evaluate evolutionary models. A maximum-likelihood tree was generated by TREE-PUZZLE (version 5.2) [37] using a general time-reversible evolutionary model, incorporating γ to account for nucleotide rate variation across sites [38]. Bootstrap support was generated with 1000 resamplings in a neighbor-joining tree in PAUP software (version 4.0b10; Sinauer), using a general time-reversible evolutionary model incorporating γ. Distance calculations were performed in MEGA software (3.1) [39], using the Tamura-Nei model of evolution [40] incorporating g. Intrapatient diversification rates were calculated by dividing the difference in the mean intrasample pairwise nucleotide distance at the later time point and the mean intrasample pairwise nucleotide distance at the early time point for a study subject by the follow-up time between samples for that respective individual. Intrapatient divergence rates were calculated by dividing the mean pairwise nucleotide distance between early and late samples for a study subject by the follow-up time between samples for that respective individual. N-linked glycosylation sites were identified using N-GLYCOSITE [36, 41], and the DS:DN was calculated using the program SNAP [36] in accordance with the method of Nei and Gojobori [42] and incorporating a statistic developed by Ota and Nei [43].

Comparison of divergence and diversification rates among individuals with a high HIV-1 set point, a low HIV-1 set point, and HIV-2 infection. Previously, Mani et al. [28] prospectively examined HIV-1 evolution in the env C2V3 in groups of HIV-1-infected individuals from the cohort described above who had high (n=7) and low (n=5) viral set points. HIV-1 sequences were acquired from proviral DNA samples, using 3 independent PCRs per time point. Median observation times for HIV-1-infected subjects with high and low viral set points were 2.4 and 3.2 years, respectively. Because HIV-1-infected subjects were selected on the basis of viral set point by Mani et al., we elected to place these subjects in high viral set point and low viral set point groups, as described elsewhere [28], for comparison with HIV-2 infection. For comparison, distance calculations were performed on the homologous env C2V3 sequence of all HIV-1- and HIV-2-infected subjects, using MEGA software (version 3.1) [39] and the Tamura-Nei model of evolution [40] incorporating γ.

Nucleotide sequence accession numbers. HIV-2 env C2V3C3 sequences were deposited in GenBank (accession numbers DQ825803-DQ825962).

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Results

Characteristics of study subjects. We prospectively examined viral evolution in 8 individuals from Dakar, Senegal, who were infected with HIV-2. All subjects were infected with HIV-2 subtype A viruses, remained free of HIV-1 infection, were naive for antiretroviral therapy, and remained free of AIDS throughout the study period. Observation times were 8.1–11.4 years (median, 10.3 years) (table 1). Four of 8 individuals had known dates of seroconversion; the time from seroconversion to the first sample date was 4 months-6.2 years. The other 4 individuals were seropositive at the time of enrollment in our cohort, with a range of 2.3–10.0 years between enrollment and acquisition of the first sample for the study. CD4+ T cell counts were moderately stable for all subjects. Although a slight decrease occurred for some individuals (5/8), CD4+ T cell counts remained >350 cells/µL for all individuals throughout the course of the study. Plasma HIV-2 loads were acquired for study subjects at the approximate midpoint of the study period. For all subjects, plasma viral loads were low; 4 of 8 subjects had undetectable plasma viral loads (<100 copies of RNA/mL of plasma), and the remaining 4 had 200–1200 copies of RNA/mL of plasma (data not shown).

Table 1
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Table 1

Clinical characteristics and summary data of HIV-2-infected subjects.

Phylogenetic analysis. To examine the intrapatient phylogenetic relationship of env C2V3C3 sequences between time points for each individual, we constructed a maximum-likelihood tree (figure 1). For each individual, distinct monophyletic branches were observed, supported by high bootstrap values, which suggested that each individual was infected by a single genetically distinct virus (i.e., no superinfection) and ensured that there was no cross-contamination between subjects in the amplification process. After ∼10 years of follow-up for each subject, intermixing between viral populations from early and later time points was observed for all study subjects. Among half of the study subjects, a subset of samples from the later time point formed subclusters on distinct branches within the respective subject's clade.

Figure 1
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Figure 1

Maximum-likelihood tree of HIV-2 sequences. Individual sequences from the early time point (white circles) and the late time point (black squares) are shown for each study subject. The observation time between the early and late time points is shown in parentheses. Bootstrap support was generated on the basis of 1000 resamples in a neighbor-joining tree; values >75 are shown for clusters in agreement with maximum-likelihood topology.

Diversification and divergence. To examine the evolution of viral sequences, we calculated the diversification rate and divergence rate for each subject (table 1). Rates of viral diversification were low for all subjects; however, 6 of 8 individuals had higher diversity at the later time point. Interestingly, viral diversification rates were higher among subjects who had a decrease in CD4+ T cell counts over the course of the study period, with rates of diversification positively associated with rates of CD4+ T cell count decrease (Spearman rank correlation, rs=0.8095; P=.0149). Similar to diversification, rates of sequence divergence were also low but consistent among subjects (mean, 0.23% per year [95% confidence interval,±0.09%]). There was no association between rates of divergence and CD4+ T cell decrease (Spearman rank correlation, rs=0.5238; P=.1827).

Amino acid evolution in the env C2V3C3. Among study subjects, no individuals had a change in consensus fragment size between the early and late time points. However, 2 sequences from subject p1704 had an insertion of 3 aa at the later time point—a single sequence from subject p517 had an amino acid deletion at the later time point—and 2 sequences at the early time point from subject p2082 had a nucleotide insertion. All observed insertions and deletions occurred within the C3 region. Interestingly, the size variant sequences from subjects p1704 and p517 formed distinctly outlying phylogenetic branches within their respective individual's clade in phylogenetic analysis, even though phylogenetic trees were constructed using gap-stripped sequences. Additionally, both sequences from subject p1704 had evidence of hypermutation based on a high ratio of G→A to A→G mutations and contained stop codons within the coding sequence.

Among the env C2V3C3 sequences, the numbers of N-linked glycosylation sites was 8–11. Intrapatient variance did exist in the number of N-linked glycosylation sites; 7 of 8 individuals had a variable number of N-linked glycosylation sites between sequences for at least one of the time points. However, there was no difference in the median numbers of glycosylation sites between time points and, therefore, no evidence of an intrapatient increase or decrease in N-linked glycosylation over time.

Evolutionary selection within the env C2V3C3. The DS: DN is a measure of evolutionary selection, with values >1 indicative of purifying selection, values near 1 indicative of neutral selection, and values <1 indicative of positive selection. To examine intrapatient selective pressures within the env C2V3C3, we calculated DS:DN values, as described elsewhere [18]. All observed DS:DN values were >1 (median, 3.37; P=.0156, sign test), which suggests that positive selection is not a dominant factor in evolution of the env C2V3C3.

Viral diversification and divergence rates, compared with those for HIV-1. A previous study examined intrapatient evolution in 7 HIV-1-infected women with high viral set points (mean, 87,782 RNA copies/mL of plasma) and 5 HIV-1-infected women with low viral set points (mean, 1822 RNA copies/ mL of plasma) from our cohort of female sex workers in Dakar, Senegal, and we found higher rates of diversification and divergence in the env C2V3 in individuals with high viral set points [28]. Similar to the HIV-2-infected subjects in the present study, all HIV-1-infected individuals were free of AIDS and naive for antiretroviral therapy during the observation time between samples. To compare rates of intrapatient evolution between HIV-1 and HIV-2, we calculated viral diversification and divergence rates for the HIV-1 sequences generated by Mani et al. and the HIV-2 sequences in the present study for homologous env C2V3 sequences.

The rate of viral diversification was significantly higher in the group with high HIV-1 set points than in the HIV-2-infected group (P=.0128, 2-sided Wilcoxon rank sum test) (figure 2A). By contrast, there was no difference in viral diversification rates between the group with low HIV-1 set points and the HIV-2-infected group (P=.4208, 2-sided Wilcoxon rank sum test). Rates of sequence divergence were higher in the group with high HIV-1 set points than in the HIV-2-infected group (P=.0015, 2-sided Wilcoxon rank sum test) (figure 2B), as well as in the group with low HIV-1 set points than in the HIV-2-infected group (P=.0923, 2-sided Wilcoxon rank sum test). Among these 3 groups, a clear trend was observed, with the highest divergence rates in the group with high HIV-1 set points, followed by the group with low HIV-1 set points, and finally the HIV-2 group (Spearman rank correlation, rs=-0.7872; P<.0001).

Figure 2
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Figure 2

Comparison of rates of intrapatient diversification (A) and divergence (B) in overlapping env C2V3 sequence between HIV-2-infected individuals and those infected with HIV-1 with high low set points, as described by Mani et al. [28]. Corresponding 2-sided Wilcoxon rank sum P values are shown.

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Discussion

For the present study, we examined viral evolution over the course of ∼10 years in 8 people infected with HIV-2. Because HIV-2 is believed to maintain a low rate of replication in vivo, we expected that viral evolution would occur slowly during HIV-2 infection. The long observation period of the study was highly advantageous, because it allowed us to observe evolutionary changes that may have been otherwise too subtle to characterize. Additionally, the individuals examined in the study were relatively homogeneous in terms of disease stage; all study subjects remained free of AIDS and had relatively stable CD4+ T cell counts throughout the observation period. Studies of HIV-1 have indicated that viral evolution occurs at a relatively constant rate during this phase of infection [13].

Mani et al. previously reported [28] the presence of distinct phylogenetic separation in viral sequences between early and late observation time points in numerous HIV-1-infected individuals from our cohort, even after a relatively short observation time, demonstrating a rapid turnover of viral populations. Despite the long observation time of the present study, we observed intermixing of HIV-2 env C2V3C3 sequences between early and late time points for all subjects. Our data indicate that turnover of viral populations occurs extremely slowly during asymptomatic HIV-2 infection. Data from Brandin et al. [44] implied high turnover of HIV-2 during AIDS, which suggests that HIV-2 turnover rates may differ depending on the stage of disease.

HIV-1 is believed to develop latent infection within resting memory CD4+ T cells, a stable reservoir in which the integrated proviral genome can persist for decades (reviewed in [45, 46]). The lack of phylogenetic separation that we observed in HIV-2 sequences between time points is likely indicative of latent infection within a long-lived cellular compartment, and the fact that a high proportion of sequences from the later time point clustered with early time point sequences suggests that a high proportion of HIV-2 proviral genomes are present in a latent form in vivo. Similarly, the phylogenetically distinct subcluster of sequences from the later time point observed in some individuals likely corresponds to actively replicating viral populations in those subjects.

In the HIV-1 env gene, intrapatient changes in N-linked glycosylation and amino acid length are thought to alter sensitivity to neutralizing antibodies [47–49]. Although we observed some variance in the number of N-linked glycosylation sites, there was no clear trend toward an increase or decrease in N-linked glycosylation sites over time. Similarly, we did not observe a trend with regard to amino acid insertions or deletions. These data may suggest the absence of effective neutralizing antibodies against the HIV-2 env C2V3C3 in vivo and, thus, a limited selective pressure for resulting changes during the asymptomatic phase of HIV-2 infection. Additionally, it is possible that there are more constraints in the HIV-2 envelope, which limits the number of selectively advantageous changes that can evolve within the env C2V3C3. Consistent with these notions, the DS:DN values observed in the present study are indicative of neutral and purifying selection in HIV-2 env C2V3C3. Similar findings based on intrapatient data from a single time point were reported by Barroso and Taveira [32].

We observed 2 subjects (p517 and p1704) who had insertions or deletions among a minority of sequences that corresponded with phylogenetic outlying branches. Interestingly, the remaining sequences for these subjects have extremely little diversity, even for HIV-2. It is possible that the evolution of the viral populations within these subjects is highly constrained because of a large fitness cost associated with divergence from the founding viral population or a broad immune response limiting evolution of the virus. Additionally, the lack of diversity for these subjects may be representative of extremely limited viral replication.

Previously, in HIV-1-infected subjects fromthe same cohort, Mani et al. [28] found rates of divergence and diversification to be higher in individuals with high viral set points than in those with low viral set points, which suggests that viral evolution is a function of replication. For the HIV-2-infected subjects we examined, viral evolution, in terms of sequence divergence between time points, was consistently low for all subjects. Furthermore, the rates of intrapatient HIV-2 divergence in env C2V3 were significantly lower than the rates in the group with high HIV-1 set points and was modestly lower than those in the group with low HIV-1 set points. Given the low viral loads observed in HIV-2 infection, the low rates of HIV-2 divergence are consistent with the notion that replication is restricted during in vivo HIV-2 infection.

Across HIV-2 env C2V3C3, the majority of study subjects (6/8) had higher sequence diversity at the later time point, which suggests that viral populations increase in diversity through the asymptomatic phase of HIV-2 infection. Additionally, we observed higher rates of HIV-2 sequence diversification to be associated with decreases in CD4+ T cell count. This observation indicates that viral sequence diversification may be a marker of disease progression in HIV-2 infection.

In comparison to the group with high HIV-1 loads, rates of sequence diversification were significantly lower in HIV-2-infected subjects. A number of studies of HIV-1 have suggested that higher rates of viral diversification are a marker of slow disease progression [16, 19, 21–24]. Given the slow disease progression in HIV-2 infection, it may be expected that HIV-2 would have a high rate of diversification. However, diversification is dependent on viral replication, and low rates of replication for HIV-2 could possibly explain the low rate of diversification observed in the present study. Bailey et al. [50] recently reported extremely low diversity in the env gene in HIV-1-infected long-termnonprogressors, who maintain stable CD4+ T cell counts and viral loads <50 copies/mL in the absence of therapy—a group that closely models HIV-2-infected individuals in terms of viral load and CD4+ T cell maintenance.

We acknowledge distinct limitations of the present study. First, because of the long observation period, the potential exists for a bias in the selection of subjects favoring those who do not progress to disease. However, disease progression in HIV-2 infection is known to occur extremely slowly; we have previously estimated the rate of long-term nonprogression (>8 years symptom free and with stable CD4+ T cell counts >500 copies/mL) in HIV-2-infected women in our cohort to be 95% [3]. Therefore, we believe that the subjects in the study are representative of the typical course of HIV-2 infection. Second, for our analysis, it was assumed that HIV-2 evolution occurred at a constant rate over the course of the observation period. This assumption is consistent with observed data for HIV-1 in which viral divergence and diversification occur at a constant rate during the asymptomatic phase of infection [13]. Finally, because of low plasma viral loads in subjects infected with HIV-2, viral sequences for the present study were acquired from proviral DNA, not plasma RNA. Although the possibility exists that some proviral sequences are defective and are not completely representative of the circulating viral population, it is not technically feasible to acquire sequences from plasma RNA for most people infected with HIV-2, because of the low to absent plasma viremia. A study conducted in this manner would be heavily biased toward the inclusion of only HIV-2-infected individuals with high plasma viral loads.

To our knowledge, this is the most extensive study to examine intrapatient viral evolution in people infected with HIV-2. Rates of viral evolution were very low and were consistent with a virus that maintains a low rate of replication in vivo. Although the reasons for the attenuated virulence of HIV-2 remain unknown, these findings suggest that viral evolution is a marker of differences in the pathogenesis of HIV disease.

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Acknowledgments

We thank Shaun Rodriguez for helpful suggestions and Beth Chaplin and Christopher Mullins for technical assistance.

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: National Institute of Allergy and Infectious Disease, National Institutes of Health (grant AI46274-04).

  • Received June 29, 2006.
  • Accepted October 19, 2006.
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  1. J Infect Dis. (2007) 195 (5): 726-733. doi: 10.1086/511308
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