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Resistance to antibody neutralization in HIV-2 infection occurs in late stage disease and is associated with X4 tropism

Marcelino, José M.a,c; Borrego, Pedrob,c; Nilsson, Charlottad; Família, Carlosb; Barroso, Helenab,c; Maltez, Fernandoe; Doroana, Manuelaf; Antunes, Franciscof; Quintas, Alexandreb; Taveira, Nunob,c

doi: 10.1097/QAD.0b013e328359a89d
BASIC SCIENCE
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Objectives: To characterize the nature and dynamics of the neutralizing antibody (NAb) response and escape in chronically HIV-2 infected patients.

Methods: Twenty-eight chronically infected adults were studied over a period of 1–4 years. The neutralizing activity of plasma immunoglobulin G (IgG) antibodies against autologous and heterologous primary isolates was analyzed using a standard assay in TZM-bl cells. Coreceptor usage was determined in ghost cells. The sequence and predicted three-dimensional structure of the C2V3C3 Env region were determined for all isolates.

Results: Only 50% of the patients consistently produced IgG NAbs to autologous and contemporaneous virus isolates. In contrast, 96% of the patients produced IgG antibodies that neutralized at least two isolates of a panel of six heterologous R5 isolates. Breadth and potency of the neutralizing antibodies were positively associated with the number of CD4+ T cells and with the titer and avidity of C2V3C3-specific binding IgG antibodies. X4 isolates were obtained only from late stage disease patients and were fully resistant to neutralization. The V3 loop of X4 viruses was longer, had a higher net charge, and differed markedly in secondary structure compared to R5 viruses.

Conclusion: Most HIV-2 patients infected with R5 isolates produce C2V3C3-specific neutralizing antibodies whose potency and breadth decreases as the disease progresses. Resistance to antibody neutralization occurs in late stage disease and is usually associated with X4 viral tropism and major changes in V3 sequence and conformation. Our studies support a model of HIV-2 pathogenesis in which the neutralizing antibodies play a central role and have clear implications for the vaccine field.

Supplemental Digital Content is available in the text

aUnidade de Microbiologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon

bCentro de Investigação Interdisciplinar Egas Moniz (CiiEM), Instituto Superior de Ciências da Saúde Egas Moniz, Monte de Caparica

cUnidade de Retrovírus e Infecções Associadas, Centro de Patogénese Molecular, Faculdade de Farmácia de Lisboa, Lisbon, Portugal

dDepartment of Microbiology, Tumor and Cell Biology, Karolinska Institutet and Swedish Institute for Communicable Disease Control, Solna, Sweden

eServiço de Doenças Infecciosas, Hospital de Curry Cabral

fServiço de Doenças Infecciosas, Hospital de Santa Maria, Lisbon, Portugal.

Correspondence to Nuno Taveira, PharmD, PhD, Unidade dos Retrovírus e Infecções Associadas, Centro de Patogénese Molecular, Faculdade de Farmácia de Lisboa, Avenida das Forças Armadas, 1649-019 Lisbon, Portugal. Tel: +351 217946400; fax: +351 217934212; e-mail: ntaveira@ff.ul.pt

Received 23 January, 2012

Revised 9 August, 2012

Accepted 23 August, 2012

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (http://www.AIDSonline.com).

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Introduction

In contrast to HIV-1-infected patients, the majority of HIV-2-infected individuals have low or absent viremia, reduced general immune activation, and generally exhibit a much slower rate of CD4 decline and disease progression [1–7]. Hence, HIV-2 infection is viewed as an example of how the human immune system can control HIV replication and disease progression. The control of virus replication in HIV-2 patients might be related to a more effective and preserved antiviral innate, cellular, and humoral immune responses [8–10]. Unlike HIV-1, most chronically infected HIV-2 patients elicit the production of potent and broadly neutralizing antibodies suggesting that these antibodies might directly contribute to suppress virus replication and control disease progression [11–16]. However, the neutralizing antibody (NAb) response has still not been documented in disease progressing patients with low CD4+ T-cell counts and X4 isolates. Data are also lacking on the longitudinal dynamics of the NAb response within a single individual, on the nature and sequence of the neutralizing epitopes, and on HIV-2 resistance to antibody neutralization. This information is crucial for vaccine production and to understand the impact of neutralizing antibodies in viral evolution and pathogenesis.

The antibody specificities that mediate HIV-2 neutralization in vivo are still elusive. Using different methods, the V3 region in the envelope gp125 has been identified as a potent neutralizing domain by some investigators [12,13,17–21]. Other weakly neutralizing epitopes were identified in V1, V2, V4, and C5 regions as well as in the CD4-binding site in gp125 and in the COOH-terminal region of the gp41 ectodomain [12,13,18,19,22]. Recently, we found that mice immunized with a C2V3C3 recombinant polypeptide from a HIV-2 CCR5-using isolate produce potent neutralizing antibodies against R5 viruses but not against X4 viruses [23]. This suggested that the C2V3C3 region comprises a potent neutralizing domain that is differently presented in the envelope complex of X4 and R5 viruses and established a new association between HIV-2 tropism and susceptibility to antibody neutralization. Whether this neutralizing domain is formed in natural HIV-2 infection and whether susceptibility to antibody neutralization in HIV-2 patients is related with virus tropism is still not known.

The aims of this study were to characterize the evolution and dynamics of the NAb response (autologous and heterologous) and escape in chronically HIV-2-infected patient and identify neutralizing determinants in the envelope glycoproteins.

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Participants and methods

Study population

Twenty-eight HIV-2 patients attending different hospitals in Lisbon were analyzed in this study. The characteristics of the patients are shown in Table 1. Samples were collected one to four times per year over a 4-year period. HIV-2 viremia in the plasma was quantified with a quantitative-competitive reverse transcriptase-PCR assay as described elsewhere [24]. Ethical approval was obtained from Hospital Curry Cabral Ethics Committee and each participant gave written informed consent before entry into the study.

Table 1

Table 1

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Purification and quantification of immunoglobulin G

The serum samples were diluted (1 : 1 ratio) in 500 mmol/l NaCl (binding buffer) and mixed with equal volume (200 μl) of protein G Sepharose four Fast Flow (GE Healthcare, Lisbon, Portugal). The beads were washed three times with binding buffer and one time with phosphate-buffered saline (PBS) and the antibodies were eluted with 200 μl of 100 mmol/l glycine-HCl elution buffer (pH 2.7) for 30 s. The beads were then centrifuged for 30 s and the acid-eluted solution containing immunoglobulin G (IgG) was quickly removed and placed into a separate tube containing 1 mol/l Tris (pH 9.0) buffer to reach pH 7.0–7.4. IgG nephelometry Turbox kits (Turbox plus; Orion Diagnostica, Espoo, Finland) were used to evaluate the concentrations of IgG fractions, following the manufacturer's instructions.

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Virus isolation and coreceptor usage

Viruses were isolated by coculture of patient peripheral blood mononuclear cells (PBMCs) with PBMCs from uninfected individuals as described elsewhere [25]. Coreceptor usage of the HIV-2 isolates was determined in ghost cells expressing CD4 and CCR1, CCR2b, CCR3, CCR4, CCR5, CXCR4, Bonzo/STRL33, or BOB/GPR15. Virus replication in these cells was determined with an in-house ELISA for detection of HIV-2/simian immunodeficiency virus (SIV) antigen [26]. CCR5 and CXCR4 usage were confirmed by entry inhibition assays using the CCR5 antagonists maraviroc and TK779 and the CXCR4 antagonist AMD3100 as described elsewhere [27].

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Neutralization assay

The neutralizing activity of IgG antibodies against HIV-2 primary isolates was analyzed in quadruplicate using a luciferase reporter gene assay in TZM-bl cells as reported previously [23,28,29]. Briefly, the eluted IgG samples (concentrations tested ranged from 0.05 to 100 μg/ml) were mixed with 2–15 ng of HIV-2 isolates (autologous and heterologous). After 1 h of incubation at 37°C, the IgG-virus mixture was added to the cells. Forty-eight hours later, cells were lysed directly in the culture plate and 100 μl of ONE-Glo luciferase assay substrate reagent (Promega, Madison, Wisconsin, USA) was added. Plates were immediately analyzed for luciferase activity on a luminometer. Neutralizing activity was displayed as the percentage inhibition of viral infection (luciferase activity) at each antibody concentration compared to an antibody-negative control: percentage inhibition = [1 − (luciferase with antibody/luciferase without antibody)] × 100. The fifty percent inhibitory concentrations (IC50) were calculated through a dose–response curve fit with nonlinear function (four-parameter logistic equations) using GraphPad Prism version 5.1 software (GraphPad Software Incorporated, San Diego, California, USA).

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Homology modeling

Protein structure coordinates of C2V3C3 patient sequences were produced with SWISS-MODEL homology modeling server [30–32] in automated mode using as template the structure of an unliganded and fully glycosylated SIV gp120 envelope glycoprotein (PDB file 2BF1) [33]. Secondary structure was assigned with DSSP software (http://swift.cmbi.ru.nl/gv/dssp/) [34,35] and imported to the correspondent loaded structure within Pymol (The PyMOL Molecular Graphics System, Version 1.2r3pre; Schrödinger, LLC, http://www.pymol.org/pymol.) using in-house built scripts, after which three-dimensional images of the superimposed closely related models were produced.

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Statistical analyses

Statistical analyses were performed using GraphPad Prism version 5.01 with a level of significance of 5%. The Mann–Whitney U-test was used to compare means between variables. Contingency tables were analyzed with Fisher's exact test. To study how two variables varied together, linear regression was performed and Spearman correlation coefficients were computed.

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Results

Plasma viremia is associated with lower CD4+ T-cell counts and X4 tropism

Twenty eight chronically HIV-2-infected patients were studied between 2003 and 2006. Nineteen patients were on antiretroviral therapy with protease and/or reverse transcriptase inhibitors (Table 1). Viral load was undetectable (<200 HIV-2 RNA copies/μl) throughout the study period in 24 patients; median viral load in the remaining four patients who had a positive viral load on at least one occasion was 5525 HIV-2 RNA copies/μl (range 484–160 559; Table 1). Median number of CD4+ T cells at study entry was 363 cells/μl (range 15–1523). The median number of CD4+ T cells was significantly higher in aviremic patients compared with viremic patients (440 cells/μl, range 66–1523 vs. 63 cells/μl, range 15–342; P = 0.0127).

Twenty-five new primary HIV-2 isolates were obtained from 12 patients. Eight patients harbored viruses that preferentially or exclusively used CCR5, whereas isolates from the remaining four patients preferentially or exclusively used CXCR4 (Table 1). Out of 10 X4 isolates, three isolates from three different patients were able to use at least five coreceptors, whereas R5 isolates could use only one or two additional coreceptors (Table 1). Patients infected with X4 isolates tended to have lower median number of CD4+ T-cell counts than patients infected with R5 isolates, but this did not reach statistical significance (median number of CD4+ T cells in patients harboring X4 isolates, 78 cells/μl; range 15–342 vs. 270 cells/μl; range 43–615; P = 0.0868). Strikingly, however, all patients infected with X4 isolates had detectable viral load on at least one occasion, whereas patients infected with R5 isolates were aviremic. These results revealed a significant association between plasma viremia and X4 tropism (P = 0.002).

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Antibody neutralization is related with disease stage and viral tropism

Autologous neutralization, employing IgG samples, was investigated in 12 HIV-2 patients. Autologous NAb activity was found in six (50%) patients infected with R5 isolates (Fig. 1a). The autologous NAb activity was sustained over time (up to 4 years in patient 20) in three patients who were followed longitudinally suggesting that their R5 isolates were unable to escape neutralization. Most patients produced NAbs with IC50 in the micromolar range (median IC50 = 3.91 μg/ml; range = 0.05–38.0 μg/ml). Patient 18 was the exception with IC50 in the nanomolar range (50 ng/ml; Fig. 1b). Of note, we found no autologous neutralizing activity in the four patients infected with X4 isolates (patients 10, 19, 27, and 28) as well as in two patients infected with R5 isolates (patients 1 and 7; Fig. 1a).

Fig. 1

Fig. 1

The neutralizing activity of plasma IgGs from 28 patient samples (one time point per patient, year 2003) was tested against 11 heterologous primary isolates, three X4 and eight R5. All X4 isolates (PT19–03, PT27–03, and PT28–03) and two R5 isolates (PT01–03 and PT07–03) resisted neutralization with up to 100 μg/ml of purified IgG. These isolates were named X4-resistant (X4-R) and R5-resistant (R5-R). With the exception of patient 28 who did not neutralize any of the isolates, all patients produced IgG antibodies that neutralized at least two out of six R5-sensitive isolates (R5-S; Table 2). In total, of the 164 IgG/R5-S virus combinations, only 26 (16%) were negative for neutralization. Remarkably, the majority of the patients (18/27; 68%) could neutralize all R5-S isolates. NAb IC50 range was 0.06–49.08 μg/ml indicating wide variability in the neutralizing response between patients and that some patients elicit very potent heterologous NAb responses (Table 2). A strong negative correlation was found between the breadth of response and median IC50 (Spearman rank, r = −0.7209; P < 0.0001; Fig. 2a), indicating that the most potent neutralizing antibodies are also those with higher breadth. Importantly, the number of CD4+ T cells was negatively associated with median IC50 (Spearman rank, r = −0.155; P = 0.0279) and positively associated with breadth of neutralizing response (proportion of viruses neutralized; Spearman rank, r = 0.5249; P = 0.0041; Fig. 2b and c). Thus, a sustained and potent autologous and heterologous NAb response targeting exclusively R5 isolates is found in most chronically infected HIV-2 patients. The breadth and potency of neutralizing antibodies decrease as HIV-2 disease progresses and X4 viruses emerging in late stage disease are fully resistant to antibody neutralization.

Table 2

Table 2

Fig. 2

Fig. 2

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The neutralizing antibody response in HIV-2 patients is directed to the C2V3C3 envelope region

Previously, we have shown that most of our patients produce high titers of IgG antibodies that bind to polypeptides comprising the C2V3C3 region in envelope gp125 (rpC2-C3 polypeptide) and the gp36 ectodomain (rgp36 polypeptide) [36]. To search for the envelope determinants of neutralization, we have analyzed the association between the neutralizing and binding antibodies. Remarkably, a significant inverse association was found between the median IC50 of heterologous IgG NAbs and the titer (Spearman r = −0.4729, P = 0.0262) and avidity (Spearman r = −0.6136, P = 0.0024) of IgG antibodies binding to the rpC2-C3 polypeptide (Fig. 3a). This association was not found for rgp36 binding antibodies (NAb IC50 vs. IgG binding titer, Spearman r = −0.3968, P = 0.0675; NAb IC50 vs. IgG avidity, Spearman r = −0.3199, P = 0.1466; Fig. 3b). The results suggest that the IgG NAbs in HIV-2 patients target the C2V3C3 envelope region.

Fig. 3

Fig. 3

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V3 correlates of X4 tropism and neutralization resistance

To investigate the interplay between the NAb response and the evolution of virus isolates in each individual and to try to gain some insight into the molecular basis of neutralization sensitivity and escape in HIV-2, the consensus C3V3C3 amino acid sequences of the patient isolates were aligned with the reference HIV-2ALI sequence (an R5 isolate) (Supplemental Digital Content 1, http://links.lww.com/QAD/A264). Clonal nucleotide sequences of these isolates were previously published in Borrego et al.[37]. The most interesting amino acid changes possibly associated with neutralizing susceptibility were found in the V3 loop. NAb-sensitive R5 isolates (R5-S) had a median V3 loop net charge of 7 (range 6–7), which was lower than the V3 loop charge of NAb-resistant isolates (R5-R and X4-R; median 9, range 8–11; Supplemental Digital Content 2, http://links.lww.com/QAD/A264). In addition, all X4-R isolates had a 1–3 amino acid insertion at the tip of the V3 loop.

To explore further the determinants of tropism and neutralization susceptibility in HIV-2, the three-dimensional structure of C2V3C3 amino acid sequences from R5 and X4 isolates was determined by homology modeling using the structure of an unliganded SIV gp120 envelope glycoprotein as template [33]. The V3 loops of X4-R isolates fit in two major structural motifs, a β-α-β motif with higher β-sheet content present in patients 10, 27, and 28 and a helix-loop-helix motif with higher α-helix content present in patient 19 (Supplemental Digital Contents 3 and 4, http://links.lww.com/QAD/A264). The V3 loops of R5-S isolates fit into two main motifs that were markedly different from those of X4-R isolates. One structural pattern present in patients 2, 18, and 20 was characterized by absence or low amounts of regular secondary structural elements; the other was characterized by a high percentage of extended β-strands (patients 6, 11, and 16; Supplemental Digital Contents 4 and 5, http://links.lww.com/QAD/A264). However, V3 loop structures from neutralization-resistant R5 isolates (patients 1 and 7) were similar to one of the structures found in R5-S isolates (Supplemental Digital Contents 4 and 6, http://links.lww.com/QAD/A264).

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Discussion

In this study, the autologous and heterologous neutralizing IgG antibody responses were investigated in 28 HIV-2-infected patients from Portugal followed for a period of 1–4 years. Patients covered the full spectrum of disease, a significant proportion being severely immune compromised [19 (68%) patients had lower than 500 CD4+ T cells/μl; eight (29%) had lower than 200 CD4+ T cells/μl]. Although, as expected, most patients (eight of 12, 67%) harbored viruses that preferentially used CCR5, four (33%) patients harbored X4 isolates and plasma viral load was detectable only in these patients. Viremic patients with X4 viruses had a significantly lower number of CD4+ T cells compared with aviremic patients, providing further support for the association between viral X4 tropism and HIV-2 disease progression [16,38].

Similar to other studies, most (68%) patients elicited potent neutralizing antibodies against the majority of R5 isolates (neutralization-sensitive R5 isolates termed R5-S) [11–14]. This is a significantly higher proportion compared to chronic HIV-1 patients in whom only 10–31% make such broad NAbs [39–42]. As shown here, the potent and broad neutralizing activity in the plasma of HIV-2 patients is essentially due to the IgG antibody fraction. Patients with the most potent neutralizing IgGs were also those with higher neutralizing breadth as found previously in HIV-1 subtype B-infected individuals [43] and HIV-2 patients from Guinea Bissau [11]. Importantly, breadth and potency of the neutralizing antibodies were positively associated with the number of CD4+ T cells. Hence, CD4+ T-cell depletion and disease progression lead to a significant decrease in neutralizing activity in HIV-2-infected patients. This may be due to a selective decrease in NAb production or maturation associated with the expansion of regulatory CD4+ T cells [44], which negatively regulate B-cell function [45–47].

Autologous neutralizing IgG antibody responses were found in half of the patients who were tested and this response was sustained over the years in some patients. Of note, one patient produced very potent autologous NAbs that inhibited viral replication at nanogram concentration (patient 18, IC50, 50 ng/ml). The results confirm that some chronic HIV-2-infected patients can elicit autologous neutralizing antibodies [13,15,16] and demonstrate that this neutralizing activity is due to the IgG antibody fraction and can be very potent and sustained over the course of infection. This is in contrast to HIV-1 infection in which autologous NAbs are undetectable in most chronically infected patients because the virus easily escapes neutralization by these antibodies during acute infection [40,48–50]. Remarkably, however, a significant proportion (50%) of our patients could not produce neutralizing antibodies targeting their autologous and contemporaneous virus isolates. This was neither due to an intrinsic immune defect nor to low levels of antibody production, because for one exception (patient 28), all patients elicited neutralizing antibodies against heterologous virus isolates. Rather, the results are consistent with effective virus escape from autologous antibody neutralization, which has not been detected before in HIV-2-infected patients. Importantly, most (four of six, 67%) of the patients with evidence of resistance to autologous antibody neutralization were infected with CXCR4-using isolates. These isolates were also fully resistant to neutralization by heterologous IgGs. Thus, neutralization-resistance in HIV-2 isolates is strongly associated with X4 virus tropism. Assuming that these four patients were originally infected with R5 isolates, our results suggest that the selective pressure exerted by the neutralizing antibodies in HIV-2 patients favors the selection of neutralization-resistant X4 isolates. This is in contrast to HIV-1, in which X4 viruses are usually easier to neutralize than R5 viruses [51–53] and escape from antibody neutralization has only rarely been associated with changes in viral tropism [53–56]. The association between X4 tropism and resistance to antibody neutralization has not been detected previously because late stage disease HIV-2 patients with low CD4+ T-cell counts and X4 viruses have not been enrolled in previous studies. It has, however, been observed in mice immunized with envelope antigens derived from an R5 HIV-2 isolate [23]. These mice were unable to elicit antibodies against X4 isolates. Our studies support a model of HIV-2 pathogenesis in which a significant decrease in the number of CD4+ T cells and selective expansion of regulatory T cells occurring after many years of infection lead to the production of less potent neutralizing antibodies, which favors the emergence of neutralization escape mutants able to use CXCR4 and other alternative coreceptors and infect other cell types. These X4 viruses replicate faster and are more pathogenic and promote faster disease progression [16,57].

To try to identify the determinants of antibody neutralization in the HIV-2 envelope, we have first analyzed the nature of the association between the patient's neutralizing antibodies and antibodies binding to recombinant polypeptides comprising the C2, V3, and C3 regions (rpC2-C3) or the gp36 ectodomain (rgp36). Remarkably, a significant direct association was found between the potency of heterologous IgG NAbs and the titer and avidity of C2V3C3-specific binding IgG antibodies. These results suggest that the neutralizing IgG antibodies in HIV-2 patients are mostly directed to the C2V3C3 region in gp125. It remains unclear whether C2V3C3-specific antibodies actually neutralize HIV-2 or simply represent a marker for other Env-specific neutralizing antibodies such as those that target V1, V2, V4, C5, and CD4-binding site in gp125 [12,13,18,19,22]. However, the observation that mice immunized with the C2V3C3 polypeptide elicit a potent and broadly NAb response supports the former hypothesis that the C2V3C3 region is a potent neutralizing domain in HIV-2 [23].

To further investigate the molecular and structural determinants of neutralization sensitivity and resistance in the C2, V3, and C3 regions of HIV-2, we analyzed an extensive number of clonal sequences of the C2V3C3 region obtained from our patients. Neutralization-resistant isolates (X4 isolates and two R5 isolates) had a higher charged V3 loop compared to neutralization-sensitive R5 isolates. In addition, X4 isolates had a larger V3 loop due to a 1–3 amino acid insertion. The results confirm that the charge and size of the V3 loop are crucial determinants of CCR5 and CXCR4 use by HIV-2 [58]. More importantly, they suggest that resistance to antibody neutralization in X4 isolates is also determined by the charge and size of the V3 loop. Early studies have shown that amino acids FHSQ (V3 loop positions 315–318) and WCR (positions 329–331) in the HIV-2 V3 stem interact with one another to form a conformational neutralizing epitope [18,19]. The 1–3 amino acid insertions found in the X4-R isolates were placed immediately after the FHSQ motif and may prevent the proper assembly of this epitope, thereby conferring resistance to the V3-directed neutralizing antibodies.

Alternative V3 conformations that may be responsible for the selective interactions with CCR5 or CXCR4 have been identified in HIV-1 [59]. HIV-1 escape from antibody neutralization is usually not related with cell tropism because the neutralizing antibodies that target the V3 region are able to bind to both V3 β hairpin conformations [59,60]. We showed that the V3 loop structures of X4-R and R5-S HIV-2 primary isolates are markedly different, which supports a direct role of V3 loop conformation in the different susceptibility of these viruses to antibody neutralization. Hence, in contrast to HIV-1, R5 to X4 transition in HIV-2 primary isolates seems to involve a significant change in V3 loop charge, size, and conformation that might prevent efficient binding of the neutralizing antibodies that target this region.

In conclusion, most HIV-2 patients infected with R5 isolates produce C2V3C3-specific neutralizing antibodies whose potency and breadth decrease as the disease progresses. Resistance to antibody neutralization occurs in late stage disease and is associated with X4 viral tropism and major changes in V3 loop sequence and conformation. Our studies support a new model of HIV-2 pathogenesis in which the neutralizing antibodies play a central role and have clear implications for the vaccine field.

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Acknowledgements

J.M.M., C.N., A.Q., and N.T. conceived and designed the experiments. J.M.M., P.B., C.F., H.B., F.M., M.D., F.A., and A.Q. performed the experiments. J.M.M., C.N., A.Q., and N.T. analyzed the data and wrote the article. J.M.M. and P.B. were supported by PhD grants from Fundação para a Ciência e Tecnologia, Portugal. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: rVV/ROD from Dr Mark J. Mulligan; TZM-bl from Dr John C. Kappes, Dr Xiaoyun Wu and Tranzyme Inc.

This work was supported by grants PTDC/SAU-FCF/67673/2006 and PTDC/SAU-FAR/115290/2009 from Fundação para a Ciência e Tecnologia (FCT) (http://http://www.fct.pt), Portugal, and by Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN), from the European Union.

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Conflicts of interest

The authors have no commercial or other association that might pose a conflict of interest.

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Keywords:

C2V3C3 neutralizing domains; HIV-2 infection; neutralizing antibodies and disease progression; X4 tropism and resistance to neutralizing antibodies

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