Evolution of human immunodeficiency virus type 2 coreceptor usage, autologous neutralization, envelope sequence and glycosylation

Yu Shi1, Eleonor Brandin1, Elzbieta Vincic2, Marianne Jansson2, Anders Blaxhult3, Katarina Gyllensten3, Lars Moberg4, Christina Broström3, Eva Maria Fenyö2 and Jan Albert1

1 Department of Virology, Swedish Institute for Infectious Disease Control and Microbiology and Tumorbiology Center, Karolinska Institutet, SE-171 82 Solna, Sweden
2 Unit of Virology, Division of Medical Microbiology, Department of Laboratory Medicine, Lund University, SE-223 62 Lund, Sweden
3 Department of Infectious Diseases/Solna, Karolinska University Hospital, Karolinska Institutet, SE-171 76 Stockholm, Sweden
4 Department of Infectious Diseases/Huddinge, Karolinska University Hospital, Karolinska Institutet, SE-141 86 Stockholm, Sweden

Correspondence
Jan Albert
Jan.Albert{at}smi.ki.se


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To investigate why human immunodeficiency virus type 2 (HIV-2) is less virulent than HIV-1, the evolution of coreceptor usage, autologous neutralization, envelope sequence and glycosylation was studied in sequentially obtained virus isolates and sera from four HIV-2-infected individuals. Neutralization of primary HIV-2 isolates was tested by a cell line-based assay and IgG purified from patients' sera. Significant autologous neutralization was observed for the majority (39 of 54) of the HIV-2 serum–virus combinations tested, indicating that neutralization escape is rare in HIV-2 infection. Furthermore, sera from 18 HIV-2 patients displayed extensive heterologous cross-neutralization when tested against a panel of six primary HIV-2 isolates. This indicates that HIV-2 is intrinsically more sensitive to antibody neutralization than HIV-1. In line with earlier reports, HIV-2 isolates could use several alternative receptors in addition to the major coreceptors CCR5 and CXCR4. Intrapatient evolution from CCR5 use to CXCR4 use was documented for the first time. Furthermore, CXCR4 use was linked to the immunological status of the patients. Thus, all CXCR4-using isolates, except one, were obtained from patients with CD4 counts below 200 cells µl–1. Sequence analysis revealed an association between coreceptor usage and charge of the V3 loop of the HIV-2 envelope, as well as an association between the rate of disease progression and the glycosylation pattern of the envelope protein. Furthermore, HIV-2 isolates had fewer glycosylation sites in the V3 domain than HIV-1 (two to three versus four to five). It is proposed here that HIV-2 has a more open and accessible V3 domain than HIV-1, due to differences in glycan packing, and that this may explain its broader coreceptor usage and greater sensitivity to neutralizing antibodies.

An amino acid sequence alignment of the V1, V2 and V3 domains of the HIV-2 envelope protein is available as supplementary material in JGV Online.

The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this study are DQ213026–DQ213040.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The global human immunodeficiency virus (HIV) epidemic is dominated by HIV type 1 (HIV-1), but HIV-2 infections are also common in certain countries, such as Guinea-Bissau and Portugal (Reeves & Doms, 2002). There are many similarities, but also important differences, between HIV-1 and HIV-2. Thus, HIV-2 is less virulent than HIV-1 and is associated with slower rates of disease progression, mortality and transmission (Andreasson et al., 1993; Kanki et al., 1994; Marlink et al., 1994; Pepin et al., 1991; Reeves & Doms, 2002). Furthermore, HIV-2 infection is associated with lower plasma-virus levels than HIV-1 infection (Andersson, 2001; Andersson et al., 2000; Berry et al., 1998; Reeves & Doms, 2002).

The reasons for the differences between HIV-1 and HIV-2 infection remain unclear, but several factors have been suggested to contribute. Better immune control of HIV-2 than HIV-1 is an obvious possibility. Indeed, cross-sectional studies have indicated that autologous neutralizing-antibody responses are more common in HIV-2 infection than in HIV-1 infection (Björling et al., 1993; Fenyö & Putkonen, 1996; Tamalet et al., 1995). Differences have also been reported in cytotoxic T-lymphocyte (CTL) responses, levels of apoptosis, production of {beta}-chemokines and capability of CD4+ and CD8+ lymphocytes to produce interleukins 2 and 4, as well as gamma interferon (Andersson, 2001; Reeves & Doms, 2002). However, a recent study reported no differences in cellular immune responses between HIV-1- and HIV-2-infected Gambian patients (Jaye et al., 2004).

Prior to this study, there have been no studies on the evolution of autologous neutralizing-antibody responses in HIV-2 infection. However, for HIV-1, it has been shown that escape from neutralization by antibodies is frequent and rapid in early infection (Albert et al., 1990; Richman et al., 2003; von Gegerfelt et al., 1991; Wei et al., 2003) and that autologous neutralizing-antibody responses are frequently low or absent later in infection (Ariyoshi et al., 1992; Fenyö & Putkonen, 1996; Homsy et al., 1990; Scarlatti et al., 1993; von Gegerfelt et al., 1991). Similarly, there is very limited information on heterologous neutralization of primary HIV-2 isolates, whereas it is known that heterologous neutralization of primary HIV-1 isolates is often absent or of low titre, except in some long-term non-progressors (Carotenuto et al., 1998; Dreyer et al., 1999; Scarlatti et al., 1993; Weber et al., 1996).

For effective infection of target cells, most HIV-1 and HIV-2 isolates require binding to a chemokine coreceptor, in addition to the CD4 receptor. The CCR5 receptor is used by the majority of primary HIV-1 isolates (R5 viruses), but some isolates from patients with more advanced immunodeficiency use CXCR4 instead of (X4 viruses) or in addition to (X4R5 viruses) CCR5 (Åsjö et al., 1986; Björndal et al., 1997; Tersmette et al., 1988; Zhang et al., 1996). The presence of X4 virus, as well as a switch from CCR5 to CXCR4 usage, is associated with accelerated rate of disease progression (Connor et al., 1997; Koot et al., 1993). CCR5 and CXCR4 are the major coreceptors for HIV-1, but a minor proportion of primary HIV-1 isolates can also utilize other alternative coreceptors (CCR1, CCR2, CCR3, CXCR6 or BOB) in vitro (Björndal et al., 1997; Rucker et al., 1997). In contrast to HIV-1, HIV-2 isolates can frequently use alternative coreceptors in vitro (Blaak et al., 2005; McKnight et al., 1998; Mörner et al., 1999b). However, CCR5 and CXCR4 also appear to be the major coreceptors for HIV-2 (Blaak et al., 2005; Mörner et al., 2002). Some primary HIV-2 isolates can infect coreceptor-positive cells in the absence of CD4 (Clapham et al., 1992; Reeves et al., 1999) and such isolates are highly sensitive to neutralizing antibodies (Thomas et al., 2003).

In this study, we provide the first data on evolution of autologous neutralizing-antibody responses and coreceptor usage of viruses isolated sequentially from four HIV-2-infected individuals. Moreover, we examined the env gene sequences of 15 HIV-2 isolates. We propose that the HIV-2 V3 domain has a more open and accessible configuration than that of HIV-1. This may explain the higher sensitivity to neutralizing antibodies and the broader use of alternative coreceptors.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Study subjects and virus isolates.
Four HIV-2-infected Swedish individuals were selected from a cohort of approximately 20 Swedish HIV-2 patients (Brandin et al., 2003) based on availability of sequential virus isolates and serum samples. All four patients were female immigrants from different West African countries. The treatment history and HIV-2 pol gene evolution in three of the four patients has been described in detail elsewhere (Brandin et al., 2003). Basic clinical, immunological and virological characteristics are shown in Table 1. Plasma HIV-2 RNA levels were determined by using an experimental assay that was kindly provided by Karen Young at Roche Molecular Systems, Alameda, CA, USA. HIV-2 was isolated by cocultivation of peripheral blood mononuclear cells (PBMCs) from the patients and phytohaemagglutinin-activated blood-donor PBMCs as described previously (Albert et al., 1990, 1996).


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Table 1. Patient characteristics and coreceptor usage of HIV-2 isolates

Abbreviations: AZT, zidovudine; 3TC, lamivudine; d4T, stavudine; NFV, nelfinavir; ddI, didanosine; ABC, abacavir; LPV/r, ritonavir-boosted lopinavir; RTV, ritonavir; ND, not done.

 
Thirteen additional sera from 11 other Swedish HIV-2-infected patients were used for heterologous-neutralization experiments (Brandin et al., 2003). Four of these patients were infected with HIV-2 of genetic subtype A, whereas the genetic subtype was unknown for the other patients. The heterologous-neutralization experiments included one isolate from each of the four principal study subjects, as well as the primary HIV-2 isolates 6669 (subtype A) and 1653 (subtype B) (Albert et al., 1987, 1996).

The study was approved by the medical ethics committee of the Karolinska Institute (nos 96-189 and 99-462).

Virus stocks and coreceptor usage.
Virus stocks were prepared as described previously (Shi et al., 2002). The infectivity of the HIV-2 isolates was determined by triplicate titration on U87.CD4 cells expressing CXCR4 or CCR5 (Shi et al., 2002). The coreceptor usage of the HIV-2 isolates was determined by using U87.CD4 cells expressing CCR1, CCR2, CCR3, CCR5 or CXCR4 and GHOST(3) cells expressing CCR3, CCR5, CXCR4, CXCR6 or BOB (Karlsson et al., 2003). Parental U87.CD4 and GHOST(3) cells without coreceptors were also included. The GHOST(3) cells were tested with and without the CXCR4 antagonist AMD3100. Presence of many syncytia and high HIV-2 p27 antigen levels were scored as strong usage of a specific coreceptor. Presence of low HIV-2 p27 levels in the absence of syncytia was scored as weak coreceptor usage. Absence of both syncytia and HIV-2 p27 antigen was scored as no usage of a specific coreceptor. All tests were repeated at least twice. HIV-2 p27 antigen levels were tested by using Murex HIVag mAb (Murex Biotech).

Neutralization assay.
The levels of autologous neutralizing antibodies were determined by using a recently developed assay based on plaque formation in U87.CD4 cells (Shi et al., 2002). Briefly, the virus stocks were diluted to contain a final concentration of 20–50 p.f.u. per well. In all neutralization experiments, we used IgG that had been purified from patients' sera to avoid false-positive neutralization due to the presence of antiretroviral drugs. IgG was purified by using protein G–sepharose 4 Fast Flow (Pharmacia Biotech) according to the instructions of the manufacturers and quantified by using an in-house ELISA. The purified IgG was used at a concentration that corresponded to a 1 : 30 dilution of the original serum. Virus–IgG mixtures were kept at 37 °C for 1 h and then further diluted in two to three fivefold-dilution steps. Each virus–IgG dilution (200 µl) was distributed into triplicate wells (or duplicate wells when the volume of serum was limited) containing the U87.CD4-CXCR4 or U87.CD4-CCR5 cells. Results are presented as means of parallel determinations. Positive virus controls consisted of wells with cells and virus, but no serum; negative virus controls consisted of wells with virus only; cell controls consisted of wells with cells only. The experiment was terminated on day 3 or day 4 by fixation with methanol : acetone (1 : 1). The number of p.f.u. was determined following haematoxylin staining. The neutralizing capacity of the serum was calculated by the formula [1–(p.f.u. with serum/p.f.u. without serum) x100] and thus expresses the degree of reduction in p.f.u. in the presence of serum relative to wells without serum. The cut-off for neutralization for this assay has been derived statistically and was determined to be a 30 % reduction in the number of plaques. The assay qualitatively compares well with traditional PBMC-based HIV-neutralization assays and has good reproducibility (Shi et al., 2002). However, the percentage plaque reduction in our assay does not translate directly into traditional neutralizing titres and therefore we focused on qualitative, rather than quantitative, aspects of the neutralization results.

HIV-2 env gene sequencing.
HIV-2 RNA was extracted from 200 µl virus-infected supernatant from PBMC cultures by using a NucliSense Isolation kit (Organon Teknika) according to the manufacturer's recommendations. A 1588 bp fragment encompassing the entire gp125 coding sequence was amplified by nested RT-PCR. The PCR was carried out in a 50 µl reaction mixture containing 1x PCR buffer (Applied Biosystems), 2·5 mM MgCl2 (Applied Biosystems), 50 µM each dNTP (Amersham Pharmacia), 0·1 µM each primer (Table 2), 1 U AmpliTaq DNA polymerase (Applied Biosystems), 5 U reverse transcriptase (M-MuLV; Roche Diagnostics) and 28 U rRNasin RNase inhibitor (Promega). The RT-PCR profile consisted of reverse transcription at 37 °C for 60 min, denaturation at 92 °C for 5 min, 30 amplification cycles of 20 s at 92 °C, 20 s at 55 °C and 1 min at 72 °C, and final elongation at 72 °C for 5 min. A portion of the PCR product (2·5 µl) was used as the template for nested PCR; the mixture for the nested PCR contained the same reagents as the mixture for the first PCR, except for reverse transcriptase and RNasin. The amplification profile of the nested PCR was identical to that the first PCR, except that the initial reverse-transcription step at 37 °C was omitted.


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Table 2. PCR and sequencing primers used for amplification and sequencing of the HIV-2 gp125 region

Y in primer JA255 denotes a position that was synthesized with a C/T wobble.

 
The PCR amplicons were purified with a QIAquick PCR purification kit (Qiagen). Sequencing reactions were carried out with an ABI Prism BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems) with the sequencing primers displayed in Table 2. The cycle-sequencing profile was denaturation for 5 min at 96 °C and 30 cycles of 10 s at 96 °C, 5 s at 50 °C and 4 min at 60 °C. Sequencing was performed on an ABI 3100 genetic analyser (Applied Biosystems). For some virus isolates, it was difficult to sequence through the V1 and V2 regions because of the simultaneous presence of virus variants with different V1 or V2 lengths within single isolates. However, all sequences could be resolved by the use of additional sequencing primers (see Table 2). The sequences have been submitted to GenBank under the accession numbers DQ213026–DQ213040.

The Sequencher software (Gene Codes Corporation) was used to edit the sequences and construct sequence contigs. The BioEdit software (Hall, 1999) was used to construct alignments of HIV-2 env gene sequences. Phylogenetic trees were constructed from a gap-stripped alignment by using the MEGA v2.1 software (Kumar et al., 2001) with the Kimura nucleotide-substitution model (Kimura, 1980) and the neighbour-joining method (Saitou & Nei, 1987). Reference sequences for these phylogenetic trees were obtained from the Los Alamos National Laboratory HIV sequence database (www.hiv.lanl.gov).

Potential N-linked glycosylation sites were identified by using the N-GLYCOSITE software available at the website of the Los Alamos National Laboratory HIV sequence database. The isolates were not clonal in sequence and some of the intrasample sequence polymorphisms involved potential N-linked glycosylation sites. In our calculations of the number of potential N-linked glycosylation sites, we have not distinguished between glycosylation sites that were present in part or all of the virus population. Changes in glycosylation sites over time were calculated by comparing each sequence to the closest previous sequence from the same patient. Glycosylation sites were considered to have moved (counted as one event) if a glycosylation site was lost and gained within three amino acid positions, whereas losses and gains of glycosylation sites at longer distances were counted as two events.

Statistical analyses.
Statistical analyses were complicated by the fact that observations on our 15 HIV-2 isolates could not be considered to be independent because the isolates were obtained sequentially from four individuals. For this reason, we deliberately avoided extensive statistical testing. When statistical analyses were performed, we used the Statistica v6.1 software (Statsoft Inc.).


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Characteristics of the study subjects
The study involved 15 HIV-2 isolates that had been obtained sequentially from four HIV-2-infected Swedish individuals over periods that ranged from 4 to 11 years (Table 1). Patients 1 and 2 had moderately progressive HIV-2 disease, moderately suppressed CD4+ T-lymphocyte (CD4) counts and no clinical symptoms. In contrast, patient 4 and, in particular, patient 8 had advanced immunodeficiency, severe clinical symptoms and high HIV-2 RNA levels in plasma, despite combination antiretroviral therapy. Patient 1 had not received antiretroviral treatment, whereas patients 2, 4 and 8 underwent therapy with some interruptions.

HIV-2 coreceptor usage associated with the degree of immunodeficiency
The coreceptor usage of the HIV-2 isolates was tested on U87.CD4 and GHOST(3) cells expressing different chemokine coreceptors (Table 1). In accordance with earlier studies of HIV-2 coreceptor usage, most isolates could use several alternative coreceptors (CCR1, CCR2, CCR3, CXCR6 and BOB) in addition to one or both of the two major coreceptors (CCR5 and CXCR4). However, none of the isolates replicated in the parental U87.CD4 or GHOST(3) cells that lacked coreceptors.

In two patients (patients 1 and 4), we observed an acquisition of the ability to use the CXCR4 coreceptor. Interestingly, the ability of the isolates to infect through CXCR4 appeared to be associated with the immunological status of the patients. Thus, all isolates, except one, that could infect through the CXCR4 receptor were obtained at time points when the CD4 counts of the respective patients were below 200 cells µl–1. Conversely, R5 isolates were obtained at time points when the patients had higher CD4 counts.

Autologous-neutralization escape is rare in HIV-2 infection
The evolution of autologous neutralization was tested in a chequerboard fashion with purified IgG from sequentially collected sera by using a recently described method based on plaque formation in U87.CD4 cells (Shi et al., 2002). Significant autologous neutralization (>30 % plaque reduction) was observed for the majority (39 of 54) of the tested serum–virus combinations (Fig. 1). Importantly, we observed no consistent pattern of neutralization escape in our four study subjects. Thus, many sera could neutralize autologous viruses that had been isolated many years later.



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Fig. 1. Autologous HIV-2 neutralization. Virus isolates (V) and sera (S) are named by time of sampling. The assay was based on reduction of plaque formation in U87.CD4-CCR5 or U87.CD4-CXCR4 cells; R5 viruses (see Table 1) were tested on U87.CD4-CCR5 cells and X4 and X4R5 viruses (see Table 1) were tested on U87.CD4-CXCR4 cells. The cut-off for neutralization was plaque reduction by 30 % and is indicated by a dashed line.

 
The clinical and immunological status of the patients was associated with the ability of their sera to neutralize autologous virus. Thus, patient 8, who had the most advanced disease, showed the weakest neutralization. However, exceptions to this pattern also existed, e.g. the 2001 isolate from patient 4, which was sensitive to neutralization even though the CD4 counts were low at the time of virus isolation. We observed no clear association between neutralization and coreceptor usage. Thus, some X4-using viruses were difficult to neutralize, e.g. the isolates from patient 8, whereas others were sensitive to neutralization, e.g. the 2000 and 2001 isolates from patient 4. However, most R5 isolates were sensitive to autologous neutralization.

Broad neutralization of heterologous primary HIV-2 isolates
To investigate whether HIV-2 sera also can neutralize heterologous primary HIV-2 isolates, we tested the ability of IgG purified from 18 HIV-2 sera from 15 Swedish patients to neutralize six heterologous HIV-2 isolates (Table 3). The isolates consisted of one isolate from each of our four study subjects, the reference HIV-2 subtype A isolate 6669 (Albert et al., 1987, 1996) and the HIV-2 subtype B isolate 1653 (Albert et al., 1996). The main observation was that significant neutralization was observed for the majority of the virus–IgG combinations, i.e. 84 of 97 tests (87 %). Furthermore, there was no obvious association between the neutralization results and the CD4 count of the patient from whom the serum was drawn. Thus, serum P4-2001, which was obtained when the patient had a CD4 count of 30 cells µl–1, could neutralize all five heterologous HIV-2 primary isolates. Similarly, there was no obvious association between the neutralization results and the coreceptor usage of the virus. A few interesting details from the heterologous-neutralization experiments should be noted. The X4 isolate P4-V2000 was comparably resistant to heterologous neutralization, as significant neutralization was observed with only six of 15 sera, even though this isolate was neutralized by all four autologous sera (Fig. 1). Furthermore, virus P8-V1998, which was neutralized poorly by autologous sera (Fig. 1), was neutralized efficiently by all heterologous HIV-2 sera. This indicates that the poor autologous neutralization that we observed in patient 8 was due to the immunological status of this patient, rather than the characteristics of the virus. Finally, isolate 1653, which is of HIV-2 subtype B, was neutralized by 13 of 15 sera, even though they, when known, were obtained from patients infected with subtype A virus (Brandin et al., 2003). Thus, the ability of HIV-2 sera to cross-neutralize heterologous HIV-2 isolates appears to extend to other HIV-2 subtypes.


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Table 3. Heterologous neutralization of six primary HIV-2 isolates

ND, Not done; results shown in bold represent neutralization below the cut-off of 30 % plaque reduction.

 
V1/V2 length is associated with clinical progression
The intrapatient evolution of the HIV-2 envelope gene was investigated by direct sequencing of the virus isolates (see Supplementary Figure, available in JGV Online). These sequences represent bulk population sequences of the virus isolates, rather than sequences of individual viral clones. Phylogenetic-tree analysis showed that all of our newly generated HIV-2 env sequences belonged to subtype A of HIV-2 (data not shown). Furthermore, the sequences showed a patient-specific clustering, indicating that the sequences were authentic and that no PCR contamination or sample confusion had occurred. The HIV-2 env gene sequences displayed no stop codons or other clearly inactivating rearrangements. The lengths of the translated HIV-2 SU proteins varied between 492 and 522 aa (Table 4). Length variations were concentrated in the V1 and V2 regions (Fig. 2).


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Table 4. Characteristics of the translated gp125 (SU) proteins of the HIV-2 isolates

 


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Fig. 2. Amino acid sequence of the V1, V2 and V3 domains of the HIV-2 envelope protein of sequential HIV-2 isolates from the four study subjects (P1, P2, P4 and P8). All sequences were obtained from PBMC-passaged stocks of primary HIV-2 isolates. Dashes indicate identity with the P1-1986 isolate, letters represent differences relative to the P1-1986 isolate and dots indicate gaps introduced to align the sequences. X, Polymorphic amino acid. Potential N-linked glycosylation sites are shaded in grey. The boxed area in the V3 panel denotes position 19, at which the presence of positively charged amino acids (K, lysine; R, arginine) correlated with X4 coreceptor usage. The charge of the V3 loop (which spans positions 1–35) was calculated according to the formula {[(no. arginine and lysine residues) x1]+[(no. glutamic acid and aspartic acid acid residues) x–1]}.

 
The length of the V1/V2 region of the SU protein was associated with the rate of disease progression and immunological status of the patients. Thus, the V1/V2 region increased in length over time in patients 1 and 2, who displayed slower disease progression and less advanced immunodeficiency. The most striking example was seen in patient 1, where the virus acquired 16 new amino acids in the V1 region between 1986 and 1991, mainly as a result of introduction of threonine residues. In contrast, the length of the V1/V2 region was stable over time in patients 4 and 8, who had more advanced HIV-2 disease. There was no clear association between V1/V2 length and chemokine coreceptor usage or neutralization sensitivity.

V3 sequence is associated with coreceptor usage
We found an association between the overall charge of the HIV-2 V3 loop and coreceptor usage (Fig. 2). All isolates that could use CCR5 efficiently had V3 loops with a net charge of +5 or +6, whereas all isolates that were unable to use this receptor efficiently had a charge of +7. A reverse dependence was observed between V3 charge and CXCR4 usage. Thus, all isolates that could use CXCR4 efficiently had V3 loop charges of +7, except for the dual-tropic isolate P4-2001, which had a charge of +6. Isolates that were unable to use CXCR4 efficiently had V3 loop charges of +5 or +6.

We also found that all six isolates that lacked the ability to infect efficiently through the CCR5 receptor displayed positively charged amino acids (arginine or lysine) at position 323 in the V3 loop (corresponding to position 19 in Fig. 2), whereas all nine isolates that could use CCR5 displayed neutral amino acids (valine or isoleucine) at this position. Furthermore, positively charged amino acids at position 323 characterized six of seven isolates that could infect efficiently through the CXCR4 receptor, but none of the CXCR4-negative isolates.

Changes in overall glycosylation pattern associated with disease progression
Recent data indicate that neutralization escape in HIV-1 infection may involve changes in glycosylation pattern (Wei et al., 2003). N-linked glycosylation may also influence coreceptor usage in HIV-1 (Nabatov et al., 2004; Ogert et al., 2001; Pollakis et al., 2001; Polzer et al., 2002). For this reason, we examined changes in potential N-linked glycosylation sites in our HIV-2 isolates in relation to neutralization, coreceptor usage and clinical progression.

The number of potential N-linked glycosylation sites varied between 24 and 29 (Table 4) and there was a tendency for the glycosylation sites to increase in number over time. Furthermore, viruses with longer V1/V2 regions tended to have a higher number of potential N-linked glycosylation sites.

We also examined changes in the positioning and presence of glycosylation sites over time (Table 4; Supplementary Figure). There were substantial differences in the number of changes of N-linked glycosylation sites between isolates and patients. The highest number of changes was observed in the 1991 isolate of patient 1 (compared with the preceding 1986 isolate). The lowest number, i.e. no change at all, was observed for the March 2001 sample and the 2002 sample from patient 8.

The number of changes in N-linked glycosylation sites was linked to the clinical and immunological status of the patients. Thus, the highest number of changes was observed in patients 1 and 2, who had moderate immunodeficiency, an intermediate number was observed in patient 4 and the lowest number was observed in patient 8, who had the most advanced immunodeficiency.

Glycosylation of the V3 region differed between HIV-1 and HIV-2
The glycosylation pattern of our HIV-2 isolates was also inspected for possible correlation with coreceptor usage and neutralization. One glycosylation site just downstream of the HIV-2 V3 loop, i.e. at position 40 in Fig. 2, appeared to be associated with the coreceptor usage of our HIV-2 isolates. However, this association was probably coincidental, because it does not hold true for previously published HIV-2 isolates with known env sequence and coreceptor usage (data not shown). Similarly, the neutralization sensitivity of our isolates did not correlate with the overall number of N-linked glycosylation sites or the absence or presence of specific glycosylation sites. The most important finding was that the HIV-2 isolates had fewer potential glycosylation sites in the V3 domain than have been reported for HIV-1, i.e. two to three for HIV-2 versus four to five for HIV-1 (Figs 2 and 3). This is interesting because the glycosylation pattern in the HIV-1 V3 domain has been reported to influence the neutralization sensitivity and coreceptor usage of HIV-1 isolates (McCaffrey et al., 2004; Nabatov et al., 2004; Polzer et al., 2002; Schønning et al., 1996).



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Fig. 3. Schematic figure showing the reduced glycan shielding and greater accessibility of the HIV-2 V3 domain compared with the HIV-1 V3 domain. The HIV-1 figure is based on the consensus sequence of HIV-1 subtype B and the HIV-2 figure is based on the consensus sequence of HIV-2 subtype A (www.hiv.lanl.gov). Numbering is according to the HIV-1HxB2 and HIV-2ROD sequences (GenBank accession nos K03455 and M15390, respectively).

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we have provided the first data on the evolution of autologous neutralizing-antibody responses, coreceptor usage and env gene sequence in HIV-2 infection. We show that neutralization escape is rare in HIV-2 infection and that HIV-2 sera are capable of broadly neutralizing heterologous primary HIV-2 isolates. Furthermore, we have documented for the first time that HIV-2 can switch within the same infected individual from CCR5 to CXCR4 coreceptor usage and that the coreceptor usage of HIV-2 appears to be linked intimately to the immunological status of the patients. Finally, we have found that the charge of the HIV-2 V3 loop appears to determine the coreceptor usage of HIV-2.

Our study is the first investigation of the evolution of autologous neutralizing-antibody responses in HIV-2 infection. The main finding is that neutralization escape appears rare in HIV-2 infection, even though some primary HIV-2 isolates are more difficult to neutralize than others. This is in contrast to HIV-1 infection, where neutralization escape is common and patients' sera are rarely capable of neutralizing contemporaneous autologous virus isolates (Albert et al., 1990; Ariyoshi et al., 1992; Fenyö & Putkonen, 1996; Homsy et al., 1990; Richman et al., 2003; Scarlatti et al., 1993; von Gegerfelt et al., 1991; Wahlberg et al., 1991; Wei et al., 2003). However, it should be pointed out that our patients were not followed from the time of infection, thus it is possible that neutralization escape early after infection may have been missed. Furthermore, sera from HIV-2-infected patients with varying severity of disease were capable of cross-neutralizing a panel of six heterologous primary HIV-2 isolates. This also differs from the findings in HIV-1 infection (Carotenuto et al., 1998; Dreyer et al., 1999; Scarlatti et al., 1993; Weber et al., 1996). Dreyer et al. (1999) found that antiretroviral drugs contributed to neutralization in some sera. Such an influence from antiretroviral therapy can be ruled out in our study, because we used IgG that had been purified from the sera of the HIV-2-infected individuals. In our study, all HIV-2 sera neutralized the majority of the six heterologous primary HIV-2 isolates, including the HIV-2 subtype B isolate 1653. Some HIV-2 sera were even able to cross-neutralize HIV-1 (Weiss et al., 1988). Taken together, our and previous studies show that there are fundamental differences between HIV-1 and HIV-2 in the induction of and sensitivity to neutralizing antibodies. Natural HIV-2 infection, but not HIV-1 infection, appears to induce broadly neutralizing antibody responses. Furthermore, HIV-2 appears intrinsically less able than HIV-1 to evade these neutralizing-antibody responses. Whether these important differences are a cause or a consequence of the lower virulence of HIV-2 remains to be elucidated.

In this study, we have shown for the first time that HIV-2 can evolve from CCR5 use to CXCR4 use in infected patients. The acquisition of CXCR4 usage was linked closely to the immunological status of the patients. Thus, all CXCR4-using isolates, except one, were isolated when the CD4 counts of the patients were lower than 200 cells µl–1. This is in agreement with a large number of studies on HIV-1, which have shown that CXCR4-using viruses are rare in early HIV-1 infection, but can be isolated from approximately 50 % of patients with AIDS (Åsjö et al., 1986; Björndal et al., 1997; Tersmette et al., 1988; Zhang et al., 1996). In HIV-1 infection, the emergence of CXCR4-using virus variants is associated directly with an accelerated rate of decline of CD4 counts (Connor et al., 1997; Koot et al., 1993). It is possible that the same is true for HIV-2 infection, but larger studies are needed to explore this.

Our study shows that many primary HIV-2 isolates can use one or several alternative coreceptors (CCR1, CCR2, CCR3, CXCR6 or BOB) in addition to CCR5 and/or CXCR4. This confirms the findings of earlier studies (Blaak et al., 2005; McKnight et al., 1998; Mörner et al., 1999b). We have chosen to refer to these receptors as ‘alternative coreceptors’, because it is unclear whether they are utilized in vivo (Blaak et al., 2005; Mörner et al., 2002). Some HIV-2 isolates displayed an R5X4 phenotype, but we did not investigate whether individual viral clones were dually tropic or whether the isolates contained a mixture of R5 and X4 clones.

We found that HIV-2, like HIV-1 (Fouchier et al., 1992; Nabatov et al., 2004; Pollakis et al., 2001), displays an association between the charge of the V3 loop and coreceptor use. Thus, all of our HIV-2 isolates with low V3 charge (+5 or +6) used CCR5, whereas isolates with higher V3 charge (+7) preferred CXCR4. These results are in agreement with our earlier finding that rapid/high HIV-2 isolates had a higher V3 charge than slow/low HIV-2 isolates (Albert et al., 1996), as well as with the results of Isaka et al. (1999), who also showed that the HIV-2 V3 domain contains determinants for coreceptor use, as exchange of the C-terminal half of the V3 loop between the laboratory HIV-2 strains ROD and GH-1 altered the coreceptor use reciprocally. At present, it is unclear whether the exact positioning of positively charged amino acids in the HIV-2 V3 loop also influences the coreceptor usage. However, we saw a preference for positively charged amino acids (lysine or arginine) at position 19 in the V3 loop in X4 HIV-2 isolates, similar to the preference for positively charged amino acids at positions 11 and 25 in X4 HIV-1 isolates (De Jong et al., 1992; Fouchier et al., 1992).

We believe that the high sensitivity to neutralizing antibodies, the inability to escape neutralization and broad coreceptor use of HIV-2 may be due to differences in the structure of the V3 domain between HIV-1 and HIV-2. As described above, the V3 domain is a determinant for coreceptor use of both HIV-1 and HIV-2. Furthermore, the V3 domain of both HIV-1 and HIV-2 contains neutralizing epitopes (Goudsmit et al., 1988; Javaherian et al., 1989; McKnight et al., 1996; Mörner et al., 1999a; Rusche et al., 1988). We observed that HIV-2 isolates only have two or, in a few cases, three potential N-linked glycosylation sites in and around the V3 loop, whereas HIV-1 isolates have four or five (Fig. 3). In HIV-1, these glycosylation sites appear to be utilized (Ogert et al., 2001; Polzer et al., 2002), but it is unknown whether the same is true for HIV-2. We propose that these differences in glycan packing confer a more open and accessible V3 domain on HIV-2 compared with HIV-1. The more open envelope configuration may explain the broader coreceptor usage and greater sensitivity to neutralizing antibodies of HIV-2. In support of this hypothesis, it has been shown for HIV-1 that deglycosylation of the V3 loop may lead to a broadening of the coreceptor repertoire to include CCR3 (Pollakis et al., 2001). Furthermore, several studies have shown that removal of glycans in and around the HIV-1 V3 loop may increase the sensitivity to neutralizing antibodies (Benjouad et al., 1992; McCaffrey et al., 2004; Nabatov et al., 2004; Polzer et al., 2002; Schønning et al., 1996). An additional interesting observation is that HIV-2 isolates that can infect coreceptor-positive cells in the absence of CD4 are highly sensitive to neutralizing antibodies (Clapham et al., 1992; Thomas et al., 2003).

We studied four HIV-2-infected patients who had moderate to advanced HIV-2 disease. Clearly, it would have been interesting to also study patients with fully asymptomatic HIV-2 infection, but such patients from our cohort could not be included in this study because attempts to isolate virus were generally unsuccessful. This difficulty in isolating virus from asymptomatic HIV-2 carriers is in agreement with results from other researchers (Simon et al., 1993). However, it has been reported that HIV-2 may be isolated more frequently if CD8 cells are depleted from the cultures. Recent studies on neutralization of HIV-1 have utilized plasma-derived SU proteins in recombinant virus-neutralization assays (Richman et al., 2003; Wei et al., 2003). We are developing such an assay for HIV-2, but we will still be unable to study asymptomatic HIV-2 carriers because they typically have undetectable plasma HIV-2 levels (Andersson et al., 2000; Berry et al., 1998; Brandin et al., 2003; Popper et al., 1999).

We noted that the immunological status of the patients was associated with the length of the V1/V2 domain and changes in glycosylation pattern. Thus, the virus carried by patients 1 and 2, who had relatively high CD4 counts, displayed a marked elongation of the V1/V2 domain and a high number of glycosylation changes. In contrast, patient 8, who had advanced immunodeficiency, displayed a stable V1/V2 length and few changes in glycosylation pattern. Interestingly, the elongation of the V1/V2 domain was primarily due to insertions of threonine residues and thereby increased the number of potential O-linked glycosylation sites. Similar changes in the V1/V2 domain correlate with neutralizing-antibody responses and neutralization escape in experimental simian immunodeficiency virus (SIVsm and SIVmne) infection (Chackerian et al., 1997; Rybarczyk et al., 2004). Both of these viruses are members of the HIV-2/SIVsm family and thus are related closely to HIV-2. It is known that the V1/V2 domain of HIV-2 also contains neutralizing epitopes (McKnight et al., 1996). Many studies show that neutralization escape in HIV-1 and SIV frequently involves changes in glycosylation pattern (Benjouad et al., 1992; Chackerian et al., 1997; Derdeyn et al., 2004; McCaffrey et al., 2004; Nabatov et al., 2004; Polzer et al., 2002; Schønning et al., 1996; Wei et al., 2003). Thus, it is possible that the changes that we observed in the V1/V2 region of HIV-2 could be immunologically driven, but this possibility appears to be contradicted by the fact that we did not observe neutralization escape. However, it is possible that the putative neutralizing epitopes in the HIV-2 V1/V2 region resemble subdominant CTL epitopes (Goulder et al., 2001), so that mutations in these epitopes do not lead to complete neutralization escape.

The hypothesis that we present concerning the mechanisms for evolution and interdependence between HIV-2 neutralization escape, coreceptor usage and glycosylation pattern is testable. Thus, we aim to produce infectious clones of HIV-2 with different biological characteristics. Through mutagenesis and construction of HIV-2 chimeras, we will try to verify formally whether the HIV-2 V3 domain has a more open configuration than that of HIV-1, and also to identify the molecular determinants for the biological properties listed above.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Swedish Physicians against AIDS Research Foundation and the Swedish Medical Research Council. Grants were also received from the Swedish International Development Cooperation Agency/Department for Research Cooperation (SIDA/SAREC). We thank Kajsa Aperia, Maj Westman and Zhong He for excellent technical assistance.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 13 June 2005; accepted 12 August 2005.



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