Cheila Rochaa,b, Pedro Borregoa,b, Rita Caladoa,b, Marta Caladoa, Fernando Maltezc, José M. Azevedo-Pereiraa and Nuno Taveiraa,b
a
Unidade dos Retrovírus e Infecções Associadas, Centro de Patogénese Molecular, Faculdade de Farmácia de Lisboa, Portugal; bCentro de Investigação Interdisciplinar Egas Moniz (CiiEM), Instituto Superior de Ciências da Saúde Egas Moniz, Monte de Caparica, Portugal; cServiço de Doenças Infecciosas, Hospital Curry Cabral, Centro Hospitalar Lisboa Central - EPE, Lisboa, Portugal
Research article
Abstract
Objective: CXCR4-tropic HIV-2 viruses isolated from patients with advanced disease were
found to be highly resistant to IgG antibody neutralization but whether this is an intrinsic characteristic of these viruses remains to be determined. In this study we aimed to characterize the neutralization phenotype of an extended set of primary X4 isolates obtained from HIV-2 patients in diverse disease stages.
Design and methods: Seven X4 variants were obtained from five chronically infected
patients in late disease stages and two vertically infected children (early infection). Neutralization susceptibility of X4 isolates was determined using a luciferase reporter gene assay in TZM-bl cells with 16 plasmas from unrelated HIV-2 infected patients and compared to that of primary R5 isolates.
Results: Early and late infection X4 viruses were significantly more resistant to Nabs than R5
viruses (median reciprocal log10 IC50 neutralization titers: 1.60 vs 3.95, P<0.0001). Late infection X4 isolates were significantly more resistant to Nabs than early infection X4 isolates. Compared to R5 isolates, X4 viruses had all of the following characteristics in V3: a mutation at position 18, the mutation V19K/R, an insertion at position 24 and a higher global net charge. Nab resistance was also associated to a significant gain in secondary structure in V3.
Conclusions: Nab resistance is an intrinsic characteristic of X4 HIV-2 viruses that is likely
determined by major sequence and/or conformational changes in the V3 region in the envelope glycoprotein.
Introduction
In contrast to HIV-1, most HIV-2 infected patients have normal and stable CD4+ T cell counts, low or undetectable viral loads and absence of clinical disease [134, 139, 148]. These features might be related to a more effective cellular and humoral immune response generated against HIV-2 [138, 140, 141, 151, 152, 268, 313, 314, 368]. With disease progression, CD4+ T cell depletion occurs at a similar level relative to HIV-1 and the mortality rate is also equivalent to HIV-1 [145, 146].
HIV-2 cell entry usually requires the interaction of the envelope glycoproteins with the CD4 receptor and with co-receptors that belong to the family of chemokine receptors. Among those, CCR5 and CXCR4 are the most important HIV-2 co-receptors [83, 84, 95, 96, 98, 418] but some primary isolates from asymptomatic patients may infect peripheral blood mononuclear cells (PBMCs) independently of these co-receptors [419]. Other isolates may even infect CD4-negative cells using CCR5 or CXCR4 as main receptors [83]. Most chronically infected HIV-2 patients (usually asymptomatic and aviremic) harbour CCR5-using (R5) strains [89, 420]. CXCR4-tropic (X4) HIV-2 isolates have only been found in patients with advanced disease and low CD4+ T cell counts and are strongly associated with faster disease progression compared to R5 isolates [84, 89, 97, 420]. The V3 loop in the gp125 envelope glycoprotein is the main determinant of HIV-2 co-receptor usage and tropism and its charge, size and structural conformation seem to directly influence interaction with CCR5 or CXCR4 [84, 97, 102, 368, 421].
In contrast to HIV-1, most HIV-2 patients in chronic stage of infection have a potent and broad Nab response against R5 isolates [84, 97, 109, 180, 268, 269, 313]. Nonetheless, the potency of the Nab response against R5 isolates decreases with CD4+ T cell depletion and a minority of R5 isolates and all X4 viruses isolated from patients with advanced disease are highly resistant to plasma autologous and heterologous IgG neutralization [97]. Interestingly, mice immunized with envelope antigens from the reference R5 isolate (HIV-2ALI) produced antibodies that potently neutralized heterologous R5 primary isolates but not X4 isolates [368]. These results suggest that the susceptibility of HIV-2 to antibody neutralization is associated with co-receptor usage and that resistance to antibody neutralization is an intrinsic property of HIV-2 isolates that use the CXCR4 co-receptor.
In the present study we aimed to characterize the neutralization phenotype of an extended set of primary X4 isolates obtained from HIV-2 patients in diverse disease stages. We provide definitive evidence that Nab resistance is an intrinsic characteristic of X4 HIV-2 viruses that is likely determined by major sequence and/or conformational changes in the V3 region in the envelope glycoprotein.
Methods
Viruses, study subjects and ethics
Five X4 variants were obtained from five adult chronically infected patients in late disease stage (median CD4+ T cells/µl=78; interquartile range=31.5-221) and two additional X4 variants were obtained from two vertically infected children at age 5 (early infection) (median CD4+ T cells/µl=319.5; interquartile range=44-595). Three primary R5 isolates were obtained from chronically infected patients with median CD4+ T cells/µl=275; interquartile range=66-615). Sixteen plasmas from unrelated HIV-2 infected patients (median CD4+ T cells/µl=333; interquartile range=194.5-480) were used to neutralize the X4 and R5 viruses. Informed consent for blood collection and participation in the study was obtained from all participants or their mothers (in the case of the two vertically infected children). The study was approved by the Ethical Board of the Hospital de Curry Cabral, Lisbon.
Neutralization assay
Neutralization assays were performed using a luciferase reporter gene assay in TZM-bl cells as described previously [290, 368, 409]. Briefly, the cells [10,000 cells in 100 µl of complete growth medium (GM) that consists of DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS)], were added to each well of 96-well flat-bottom culture plates (Nunc) and allowed to adhere overnight. 100 µl of each virus (corresponding to 200 TCID50) were incubated for 1 h at 37°C with 2-fold serial dilutions of heat-inactivated patients sera in a total volume of 200 µl of GM containing DEAE-Dextran (20 µg/ml). The lowest serum dilution used in the assays was 1:40. Forty-eight hours later, plates were analysed for luciferase activity on a luminometer (TECAN) using the One-Glow Luciferase Assay System (Promega,
Madison, WI). Medium-only control wells were measured as background, and virus-only control wells were included as 100% infection. Nonspecific inhibition was assessed by testing all viruses against HIV-negative plasmas and all plasma samples from HIV-2 patients against HIV-1 pseudotyped with the vesicular stomatitis virus (VSV) envelope (using pSG∆env plasmid as a backbone). HIV-negative plasmas failed to neutralize HIV-2 strains and HIV-2 plasmas failed to neutralize VSV envelope pseudotyped HIV-1, indicating the absence of nonspecific inhibitory activities in these samples. Neutralization titers were expressed as the reciprocal of the plasma dilution that inhibited virus infection by 50% (IC50). IC50 was estimated by the sigmoidal dose–response (variable slope) equation in Prism version 5.0 [392].
Statistical analyses
Statistical analysis was performed with GraphPad Prism 5.0 [392] with a level of significance of 5%. Non parametric Mann Whitney U test was used to compare medians of reciprocal log10 IC50 neutralization titers between X4 viruses, X4 viruses from late infection, X4 viruses from early infection and R5 viruses.
Results
X4 viruses from late and early infection are more resistant to antibody neutralization than R5 viruses
Nab sensitivity of a panel of seven X4 viruses, including five isolates from patients in late disease stage and two variants from early infant infection, was analysed in parallel with three R5 variants. CD4+ T cell counts were not significantly different between patients providing the X4 and R5 strains (median, 78 vs 275 CD4+ T cells/μl, P=0.2667). All patients infected with R5 variants and four out of five infected with X4 strains had undetectable viral loads (< 200 RNA copies/ml). One individual chronically infected with an X4 virus and the two children also infected with X4 variants had viral loads of 4792, 20968 and 1250 RNA copies/ml, respectively.
(a)
Figure 5.1 – Antibody neutralization of the X4 isolates. Panel A – A heat map of the median reciprocal log10
IC50 neutralizing titer of each heterologous plasma sample (right) against the seven X4 isolates and the three R5 isolates (bottom) is shown. The reciprocal log10 IC50 value is colour-coded. The darkest colour represents
the highest neutralizing titer; the lightest colour indicates that there was no detectable neutralization above 50% with the lowest plasma dilution tested (1/40). Panel B – Dot-plot graphic showing the median reciprocal log10 IC50 neutralizing titers and interquartile range of 16 heterologous plasma samples against X4 isolates
from early and late stage disease and R5 isolates as controls. Mann-Whitney U test was used to compare the median log10 reciprocal IC50 values.