By definition, HIV-1 infection with more than one strain is classified as a coinfection or as a superinfection when the acquisition of a new variant occurs prior/simultaneously to or after seroconversion, respectively. Multiple HIV-1 infections are typically associated with increased viral load levels and faster decline in CD4+ T cells [1–13]. There is some debate, however, on the impact of superinfection on HIV controllers (HIC), a rare group of individuals who can efficiently suppress systemic HIV-1 replication and maintain RNA viral loads in the undetectable (<50–80 copies/ml; elite controllers) or detectable (50–2000 copies/ml; viremic controllers) range in the absence of antiretroviral therapy (ART) . Some cross-sectional studies detected a relatively high frequency of dually subtype B infected HIC [15–17], suggesting the ability of these individuals to control replication of different viruses of the same subtype. A few longitudinal analyses, however, described a sharp increase in plasma viremia and concomitant loss of CD4+ T cells after both intrasubtype and intersubtype superinfection in HIC [18–20], resembling the pattern observed in HIV noncontrollers [1–13].
Longitudinal studies in HIV noncontrollers individuals revealed that superinfection may have a high impact on HIV DNA reservoir reseeding and viral evolution, although the outcome of the original and superinfecting viruses seems to greatly vary across individuals. In some individuals, the secondary infecting strain is only transiently detected and declines rapidly, becoming undetectable few months after superinfection [13,21]. In other individuals, both original and superinfecting viruses cocirculate for several months after superinfection . Finally, in other individuals, there is a complete turnover of the viral quasispecies within a few months after superinfection, and the initial HIV-1 strain is virtually replaced by the new infecting virus and/or by recombinants between initial and secondary strains [13,21–26]. To date, the impact of superinfection on cell-associated HIV DNA diversity in HIC has not yet been characterized.
HIV-1 Brazilian epidemic has been characterized by the circulation of different subtypes and recombinant forms, a scenario that favours the occurrence of intersubtype superinfection events . In a recent cross-sectional study, we identified two Brazilian HIC dually infected with subtypes B and F1 viruses . Here, we performed an extensive longitudinal analysis of these two cases of dual HIV-1 infection to distinguish between coinfection and superinfection. In addition, we evaluated the short and long-term impact of intersubtype HIV-1 dual infections on viremia, reservoir reseeding, viral evolution and disease progression. We showed that HIC analysed in this study were susceptible to intersubtype HIV-1 superinfection and that the event was associated to an extensive turnover of viral population in the plasma, whereas the impact of superinfection on cell-associated HIV DNA diversity varied highly across individuals. Furthermore, both HIC were able to maintain control of viremia and suppress evolution of both initial infecting (subtype B) and superinfecting (subtype F1) HIV-1 strains for several years after superinfection, without evidence of disease progression.
Materials and methods
Individuals EEC09 and VC32 were followed-up at the Instituto Nacional de Infectologia Evandro Chagas (INI) from Rio de Janeiro (Brazil) and selected from a previous study , as they were dually infected with subtype B and F1 viruses. Both individuals were adults and provided written informed consent documents approved by the INI Institutional Review Board (Addendum 049/2010) and the Brazilian National Human Research Ethics Committee (CONEP 14430/2011). Individual EEC09 was a 48-year-old homosexual male who was diagnosed with HIV-1 infection in 2001 and remained treatment-naive until June 2015, when he initiated combined ART (cART) after the proposal of the clinicians following the recent recommendation of the Brazilian Ministry of Health. This determination follows the global strategy to control HIV/AIDS pandemic, in which all HIV-infected individuals should be treated, independent of the CD4+ T-cell counts or viral load measurements. At enrolment in our cohort in February 2009, individual EEC09 was classified as an elite controller, as most (≥70%) of his plasma viral load determinations were below the limit of detection for the available assays (<50–80 copies/ml). VC32 was a 39-year-old homosexual male who received the diagnosis of HIV-1 infection in 2004 and had remained antiretroviral-naive until the present study (personal decision). At study entry in April 2012, individual VC32 was classified as a viremic controller, as most (≥70%) of his plasma RNA viral load determinations were between 80 and 2000 copies/ml. At each follow-up visit, samples of peripheral blood mononuclear cells (PBMC) were obtained using Histopaque-1077 (Sigma, St. Louis, Missouri, USA) by density gradient centrifugation and stored in liquid nitrogen until further use, and plasma samples were separated and stored at −80°C. PBMC and plasma samples of individuals EEC09 and VC32 collected over 7 (2009–2015) and 6 (2012–2016) years, respectively, were analysed. To avoid cross-sample contamination, nucleic acid extraction and amplification from each PBMC and/or plasma sample were performed in individual experiments as described in the following sections.
Quantification of total cell associated HIV DNA
Total DNA was extracted from PBMC samples (1 × 107 cells) using the QIAamp DNA Mini Kit (Qiagen, Hilden, North Rhine-Westphalia, Germany) on each visit. Cell-associated HIV-1 DNA was quantified using the Generic HIV DNA cell Kit (Biocentric, Bandol, Var, France), following the manufacturer's recommendations. Results were reported either as actual numbers of HIV DNA copies/106 cells or as the threshold value of detection.
Single genome amplification and sequencing of HIV DNA sequences
Single genome amplification (SGA) of PBMC-associated HIV DNA env sequences was performed by limiting dilution nested PCR using previously described conditions . The PCR products were sequenced using the ABI BigDye Terminator v.3.1 reaction Kit (Applied Biosystems, Foster City, California, USA) in an ABI PRISM 3100 automated sequencer (Applied Biosystems). Chromatograms were assembled into contigs using the SeqMan Pro 11 software (DNASTAR Inc., Madison, Wisconsin, USA). Sequences resulting from chromatograms with double peaks or showing APOBEC3G/F-mediated hypermutation as determined using Hypermut software  were discarded.
HIV RNA haplotype prediction using next-generation sequencing data
Total RNA from plasma samples was extracted using the QIAamp Viral RNA Mini Kit (Qiagen) if viral load was at least 200 copies/ml or the QIAamp UltraSens Virus Kit (Qiagen) if viral load was less than 200 copies/ml, according to the manufacturer's instructions. cDNA was obtained by RT-PCR using SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, California, USA) and subjected to nested PCR for env amplification as described above. cDNA library preparation was performed using the Nextera XT DNA Library Prep Kit (Illumina, San Diego, California, USA) and each sample was subjected to barcoding with a unique index from the Nextera XT Index Kit (Illumina). High-throughput sequencing was performed with MiSeq Reagent Nano Kit, v2 (250 nt paired-end reads). Demultiplexed reads were trimmed to remove adaptors, low-quality regions (Q < 25) and short reads (<100 bp). The remaining reads were mapped against SGA consensus sequences from each patient using Geneious software v.9.1.8 , ensuring high mapping quality (MAQ ≥30) and a minimum overlapping identity of 90%. Alignment regions with at least 500× coverage (Supplemental Fig. 1, http://links.lww.com/QAD/B405) were exported in BAM format and used for haplotype reconstruction using QuasiRecomb 1.2  employing the flag ‘-conservative’ to increase specificity while considering the recombination process. Only haplotypes with frequency of at least 1% were used for further analyses.
Analyses of env sequences and coreceptor tropism prediction
All SGA and NGS sequences were screened for potential cross-contamination using BLAST against both local and public repositories of HIV-1 sequences as previously described . Viral env sequences obtained from SGA and NGS approaches for each patient's visit were aligned with HIV-1 subtype reference sequences using ClustalW and then manually edited, yielding a final alignment covering positions 7008–7650 relative to the HXB2 reference genome. Maximum-likelihood phylogenetic trees were reconstructed with the PhyML 3.0 program  as previously described . The genetic complexity of cell-associated HIV DNA quasispecies was characterized by calculating the mean nucleotide diversity (π) using MEGA7  as described previously , and the mean nucleotide divergence was determined by performing linear regression analysis of the root-to-tip distances against sampling time using the program TempEst . ‘Viral dating’ was estimated from the nucleotide divergence of each individual virus to the most recent common ancestor of the HIV-1 subtype B pandemic as described previously [16,36,37]. The V3 region of env nucleotide sequences was translated using MEGA7 , and viral tropism was predicted using Geno2pheno (http://coreceptor.bioinf.mpisb.mpg.de/cgi-bin/coreceptor.pl) with a false positive rate (FPR) cutoff of 5% .
Linear regression analyses were performed using GraphPad v6 (Prism Software, La Jolla, California, USA) to verify evidence of clinical (decline in CD4+ T-cell counts) progression after superinfection events. P value of 0.05 or less was considered significant.
Availability of data and material
HIV-1 sequences generated during the current study were deposited in GenBank under the accession numbers MH244562–MH244833. The data generated with NGS were made available in the SRA database under the accession number SRP139352.
Longitudinal clinical monitoring of the patients
In a previous cross-sectional study, we described two HIC that were dually infected with subtype B and F1 variants . Longitudinal analysis of plasma viremia revealed episodes of viral load increase during follow-up of both individuals. Individual EEC09 displayed undetectable plasma RNA viral load (<50–80 copies/ml) in most (21/24; 83%) measurements between HIV-1 diagnosis in January 2001–December 2012 (Fig. 1a), confirming the classification of this individual as an elite controller. After 10 years of follow-up, individual EEC09 presented six consecutive time points of detectable viral load (114–581 copies/ml), reaching a peak in July 2015. At this time, individual initiated cART following the recent recommendation of the Brazilian Ministry of Health to treat all HIV-infected individuals irrespective of viral load or CD4+ T-cell counts. Despite the loss of elite control, patient EEC09 maintained the status of viremic controller and displayed stable CD4+ T-cell counts within the normal range (595–2469 cells/μl) during follow-up (Fig. 1a). Individual VC32 displayed relatively stable plasma RNA viral load in the low range (>50–170 copies/ml) from HIV-1 diagnosis in July 2004 to April 2013 (Fig. 1b). After April 2013, plasma RNA viral load increased progressively up to 722 copies/ml in June 2015 and then gradually decreased, reaching undetectable levels in November 2017. Individual VC32 refuses to initiate cART and continuously fulfilled the viremic controller profile criteria and displayed stable CD4+ T cell counts within the normal range (469–1005 cells/μl) at all time points (Fig. 1b). No clinical manifestation was reported in the medical records of both individuals that could be associated with viremia increase after several years of stable control. To better understand whether the increase in viremia was related to a superinfection event in the dually infected individuals EEC09 and VC32, we longitudinally assessed viral quasispecies diversity in the PBMC and plasma.
Evidence of intrasubtype and intersubtype HIV-1 superinfection in individual EEC09
We obtained 170 env sequences derived from samples collected at seven visits over a period of 6.5 years (February 2009–July 2015) before initiation of cART. ML phylogenetic analysis revealed that env sequences from individual EEC09 branched in three highly supported [approximate likelihood ratio test (aLRT) > 0.95] independent monophyletic clades, with two within-subtype B clade (hereafter called as B1 and B2 variants) and one within-subtype F1 clade (Fig. 2a). This finding together with the high mean env genetic distance between variants B1 and B2 (17.3%) indicated that this individual was actually triple infected. Variants B2 and F1 were detected in both PBMC and plasma, whereas variant B1 was only detected in PBMC (Fig. 2a). Prediction of viral tropism showed that variants B1 and F1 were exclusively R5-tropic, whereas variant B2 was entirely X4-tropic (Fig. 2a and Supplemental Table 1, http://links.lww.com/QAD/B405). The mean quasispecies genetic diversity (π) of each HIV-1 variant was very low in both PBMC (0.2–1.4%) and plasma (0.1–0.3%) (Supplemental Table 2, http://links.lww.com/QAD/B405), consistent with the low number of B2 and F1 haplotypes reconstructed per visit in the plasma (n = 2–4) (Supplemental Fig. 2a, http://links.lww.com/QAD/B405). We did not detect a measurable increase in genetic divergence over time for any of the viral variants (Fig. 2b). Both subtype B variants were detected at similar frequencies (47% B1 and 53% B2) in PBMC in February 2009 (Visit 1; Fig. 3a). The frequency of variant B2 in PBMC reservoir reduced progressively in subsequent visits but was the dominant variant in the plasma in December 2012 (Visit 9) and September 2013 (Visit 10) (Fig. 3b). Because this individual was already infected with both HIV-1 subtype B variants at enrolment in the cohort study, we estimated the probable timing of infections by performing viral dating of the HIV DNA sequences sampled between 2009 and 2015. Viral dating of B1 sequences (1983–1991) resulted in much older dates than those of B2 sequences (2006–2007) (Fig. 3c), thus, indicating that individual EEC09 was initially infected by the B1 variant (probably in the 1980s) and superinfected by the B2 variant (probably in the 2000s). We first detected the subtype F1 variant in PBMC in September 2013 at a high frequency (42%) (Fig. 3a), indicating that this individual was superinfected with the subtype F1 variant between December 2012 and September 2013. In subsequent visits, the new infecting subtype F1 virus virtually replaced the subtype B strains in both PBMC (Fig. 3a) and plasma (Fig. 3b). The estimated superinfection with the B2 virus roughly coincided with detection of a plasma RNA viral load blip of 276 copies/ml in March 2008 (Fig. 1a), whereas superinfection with the subtype F1 variant coincided with the virologic breakthrough after 10 years of elite control of viremia (Fig. 1a) and with a transient increase in the total HIV DNA levels (Fig. 3a). By contrast, we did not observe a significant decline in CD4+ T-cell counts after the estimated superinfection events and before the initiation of cART (Fig. 3d).
Evidence of intersubtype HIV-1 superinfection in individual VC32
We obtained 179 env sequences derived from samples collected at six visits over a period of 4 years (April 2012–April 2016). ML phylogenetic analysis revealed that env sequences from both PBMC and plasma branched in two highly supported (aLRT > 0.95) independent monophyletic clades within subtypes B and F1 (Fig. 4a), which confirm that individual VC32 was dually infected. Prediction of coreceptor usage showed that all subtype B and F1 env sequences derived from PBMC and plasma were R5-tropic (Fig. 4a and Supplemental Table 1, http://links.lww.com/QAD/B405). The mean π estimated from cell-associated HIV DNA quasispecies of HIV-1 subtype B (2.7–3.6%) was higher than the mean π of subtype F1 (≤0.1%), which was consistent with an older infection by the subtype B variant (Table SII, http://links.lww.com/QAD/B405). The mean π of viral quasispecies in the plasma was very low (<0.5%; Supplemental Table 2, http://links.lww.com/QAD/B405), which was consistent with a low number of haplotypes per visit reconstructed for both subtype B (n = 1–2) and F1 (n = 3–6) populations (Supplemental Fig. 1b, http://links.lww.com/QAD/B405). No evidence of divergence over time was detected for subtype B or subtype F1 populations (Fig. 4b). Subtype B was the only variant detected between April 2012 and January 2014 in PBMC (Fig. 5a) or plasma (Fig. 5b). Subtype F1 was detected for the first time in both PBMC and plasma in June 2015 (Visit 6) and was the dominant variant (≥90%) in plasma in June 2015 and January 2016 (Visit 7). Subtype B, however, remained as the most prevalent clade (≥89%) in PBMC across all time points. These results clearly support that individual VC32 was infected with a subtype B variant before enrolment in the cohort study in April 2012 and superinfected with a subtype F1 variant between January 2014 and June 2015. The emergence of the subtype F1 variant coincided with a transient peak in plasma viremia (722 HIV-1 copies/ml; Fig. 1b), but it was not associated with a significant increase in total HIV DNA levels (Fig. 5a), nor with a significant decline in CD4+ T-cell counts (Fig. 5c).
In the present study, we report two cases of HIC that preserved virologic and immunologic control despite intrasubtype and/or intersubtype HIV-1 SI. Individual EEC09 was an elite controller who was probably sequentially infected with two subtype B strains (B1 and B2 variants) prior to his entry in our cohort in 2009 and then superinfected with a subtype F1 variant around 2012–2013. Individual VC32 was a viremic controller initially infected with a subtype B strain before 2004 (date of HIV diagnosis) and superinfected with a subtype F1 variant around 2014–2015. Previous studies have already identified some cases of dual infections in HIC [15–20,39,40] and of triple infection in HIV noncontrollers [41–46], but this is the first report of triple infection in an HIC.
HIV-1 superinfection typically has detrimental consequences for clinical outcome in both HIC  and noncontrollers [1–13]. Although individual EEC09 had lost his elite controller status after the second superinfection event and individual VC32 displayed a transient peak of viremia, both individuals maintained viremic control (<2000 copies/ml) of infecting viruses and presented no evidence of immunologic progression for at least 2 years after the intersubtype superinfection event. Individual EEC09 initiated cART around two years after the intersubtype superinfection event despite no signs of clinical progression, whereas plasma viremia in individual VC32 (who remained cART-free) gradually decreased after the peak and reached undetectable levels 2–3 years after superinfection. Furthermore, we found no measurable increase in viral divergence over time, which reinforces the extraordinary ability of these individuals to control the evolution of both initial and superinfecting viral strains. Although previous studies have already documented the ability of some HIC to maintain persistent virological and immunological control after dual infection [16,17,40] and superinfection [18,39] with HIV-1 subtype B viruses, this is the first report, to the best of our knowledge, of HIC who maintain sustained control of viral replication and evolution after intersubtype superinfection.
These results indicate that clinical consequences of superinfection in HIC could be different among individuals and may depend on the underlying mechanisms responsible for the natural control of viremia in each individual. Superinfection with a defective virus has been suggested as a factor associated with the low clinical impact of HIV-1 superinfection in one elite controller . The increase in viremia and rapid turnover of viruses in the plasma following superinfection in individuals EEC09 and VC32, however, argue against superinfection with a defective subtype F1 virus. It is also remarkable that infection  or superinfection  with X4-tropic and dual-tropic subtype B variants has been associated with rapid disease progression. Our analyses, however, indicates that individual EEC09 was probably initially infected with the R5-tropic B1 variant and maintained elite control after superinfected with the X4-tropic B2 variant. Overall, these evidences support the relevance of host mechanisms in the natural control of viremia in individuals EEC09 and VC32, similar to that previously shown for other HIC [15,48,49].
Studies on HIV noncontrollers found that superinfection may have a variable impact on proviral reservoir composition [13,21–26], and our findings in HIC confirm these observations. In individual EEC09, the superinfecting B2 strain co-circulated with the original B1 strain between 2009 and 2012 but declined continuously in PBMC and was undetectable in 2013, whereas it was the dominant variant in the RNA plasma until 2013. Superinfection with the subtype F1 strain around 2013 was associated with a subsequent transient increase in total HIV DNA load and a complete turnover of the viral quasispecies in both PBMC and plasma. In contrast to individual EEC09, we did not detect significant changes in the size and composition of PBMC-associated HIV DNA reservoir in individual VC32. Although the superinfecting subtype F1 virus was the dominant variant in the plasma after superinfection, it failed to replenish the PBMC reservoir that was continuously dominated by the original subtype B virus. The divergent dynamics of PBMC-associated DNA and plasma RNA viral quasispecies corroborate that other reservoirs (probably lymph nodes and gut-associated lymphoid tissue), apart from PBMC, are a source of plasma viremia in HIC .
In summary, this study reports that some HICs are unable to prevent HIV-1 superinfection but seem to be able to repeatedly control viral replication and evolution of different infecting viral subtypes and further prevent disease progression for several years after HIV-1 superinfection. Identifying the host mechanisms associated with the natural control of HIV-1 replication and evolution following primary infection and whether these mechanisms are the same that lead to a sustained control after superinfection could offer important clues for the development of innovative therapeutic vaccines towards HIV remission.
The authors thank the patients, who participated in the study, as well as all the technical staff involved in the clinical follow-up of these patients. We thank the Plataforma Genômica de Sequenciamento de DNA - RPT01A-PDTIS/FIOCRUZ, Plataforma de Sequenciamento de Ácidos Nucléicos de Nova Geração - RPT01J-PDTIS/FIOCRUZ and the Programa de Pós-Graduação em Genética/Departamento de Genética, IB/UFRJ by the nucleotide sequencing, NGS Library preparation and sequencing run with Miseq, respectively. We also thank Ms Marilia Alves Figueira de Melo for the excellent technical support in NGS Library preparation. We thank Dr Caroline Passaes for the critical reading of the manuscript.
G.B. conceived and designed the study and supervised the experiments. S.S.D.A. conducted experiments and analysed the data. E.D. participated in the quantification of total cell associated HIV-1 DNA, N.G.S. experiments and its analysis. F.H.C. participated in sample processing and determination of CD4+ T-cell counts. B.H., B.G. and V.G.V. conducted the patient recruitment and follow-up. T.M.L.S. and M.G.M. contributed to the study design and provided intellectual input. S.S.D.A. and G.B. wrote the first draft and all authors assisted with the writing and approved the final manuscript.
This work was supported by the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro – FAPERJ (grant number E-26/110.123/2014) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (Grant Number 401220/2016-8). S.S.D.A. was supported by funding from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro – FAPERJ. E.D. was supported by a Postdoctoral fellowship from the ‘Programa Nacional de Pós-Doutorado (PNPD)’/CAPES-Brazil.
Conflicts of interest
There are no conflicts of interest.
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