Introduction
HIV-2 causes localized infections in Western African countries, mostly in Guinea-Bissau, Gambia and Senegal, and in a few other countries with former colonial ties with these countries, including Portugal and France. There is limited knowledge on the natural history of HIV-2 infection because no study has investigated the full course of infection from the time of seroconversion. The available data indicate that the majority of HIV-2-infected individuals behave like HIV-1 controllers (long-term nonprogressors and elite suppressors) having normal CD4+ T-cell counts, low or absent plasma viremia and absence of clinical disease [1,2] HIV-2 infection has a minor effect on survival in most adults [3,4] [5–9] [10–12] HIV-2 infection worldwide [13,14]
The mechanisms underlying immune control of HIV-2 replication and disease progression are still not fully understood. Robust polyfunctional T-cell responses, mainly directed to the Gag protein, have been recently associated with viremia control in HIV-2 infection [15,16] [17] [5] [18] in vivo [9] [19–21]
Chronic immune activation is a characteristic of HIV disease progression and an important driving force of HIV-1 and HIV-2 pathogenesis [22–27] [28–31] [1] [32]
Very few studies have been published on the nature and dynamics of the antibody response against HIV-2. In general, the antibody response against the viral proteins in HIV-2 chronically infected patients does not seem to be different from that of HIV-1 infection. Indeed, the majority of HIV-2 patients produce IgG antibodies against the structural proteins of the virus and this forms the basis for their serologic diagnosis [33–35] HIV-2 infection compared with HIV-1 infection. This has been suggested in one study [36] HIV-2 infection .
Previously, we have used the new enzyme-linked immunosorbent assay (ELISA)–HIV2 assay to show that the majority of HIV-2 patients produce IgG antibodies against rpC2–C3 and rgp36, two recombinant polypeptides representing the gp125 and gp36 envelope glycoproteins [33] HIV-2 infection .
Patients and methods
Patients and samples
We analyzed 30 HIV-2-infected patients from three hospitals in Lisbon and 50 seronegative individuals (blood donors). Two patients were children with perinatal HIV-2 infection . The characteristics of the patients enrolled in the study are described in Table 1 . Patients were born in Portugal (16), Guinea Bissau (12), Cape Vert Islands (1) and Mozambique (1). All patients are living in Lisbon for over a decade.
Table 1: Characteristics of the HIV-2-infected adults and children included in this study.
HIV seropositivity was determined with VIDAS HIV DUO (Bio-Mérieux, Lyon, France). HIV-1 and HIV-2 differentiation was done by Western Blot 2.2 (Genelabs Diagnostics, Science Park, Singapore, Singapore), New LAV Blot II and Peptilav 1-2 (Bio-Rad, Hercules, California, USA). HIV-2 infection in children was determined by virus isolation, as described previously [37]
Quantification of CD4+ T cells, plasma viremia and IgA and IgG
CD4+ T-cell counts were determined in total blood samples by flow cytometric analysis using FACSCalibur (Becton Dickinson, Franklin Lakes, New Jersey, USA). HIV-2 viremia in the plasma was quantified with a quantitative-competitive reverse transcriptase-PCR assay as described elsewhere [38]
IgA and IgG antibody reactivity against the HIV-2 antigens rgp36 and rpC2–C3
The location of the recombinant rpC2–C3 (165 amino acids in gp125) and rgp36 (128 amino acids in the gp36 ectodomain) polypeptides used in this work was described previously [33] [33]
IgG subclass reactivity against rgp36 and rpC2–C3
Reactivity of the different IgG subclasses against rgp36 and rpC2–C3 was determined with ELISA-HIV-2 test with the following modifications. After incubation of plasma samples, HRP-conjugated sheep antihuman IgG1, IgG2, IgG3 or IgG4 antibody (The Binding Site Ltd., Birmingham, UK) was added and incubated at room temperature. The anti-IgG1 antibody was diluted at 1: 6000 and the others at 1: 3000.
Envelope glycoprotein-specific antibody avidity
The avidity index values of rgp36 and rpC2–C3-specific IgG1 antibodies were determined by measuring the resistance of IgG1–rgp36 or IgG1–rpC2–C3 complexes to dissociation with 6 mol/l urea as previously described [39] [40]
DNA extraction, PCR amplification, cloning and sequencing
Proviral DNA was extracted from peripheral blood mononuclear cells (PBMCs) with the Wizard Genomic DNA Purification kit (Promega, Madison, Wisconsin, USA). A fragment of the C2V3C3 region (394 bp) of the HIV-2 env gene was amplified by PCR, cloned and sequenced as described previously [41]
Statistical analysis
Statistical analyses were performed with GraphPad Prism 4.02. Nonparametric tests were used to compare means between variables: Mann–Whitney U -test was used to compare IgA and IgG antibody response against both polypeptides and the correlation between antibody concentration, titer and avidity to rgp36 and rpC2–C3 was determined using the Spearman rank test. Deming linear regression was used to study the overall variation (slopes) of dynamics of CD4 cells and IgG response against rpC2–C3 of each patient as a function of time (longitudinal analysis). All P values are two tailed and P values below 0.05 were considered significant.
Results
Total concentrations of plasma IgA and IgG in HIV-2 infection
The total IgG and IgA plasma concentrations were determined in 50 healthy controls (blood donors) and 28 HIV-2-infected adult patients residing in Portugal showing different clinical, virologic and epidemiologic features (Table 1 ). Total IgG plasma concentrations were significantly higher in HIV-2 patients compared with uninfected controls (mean 19.2 ± 6.3 vs. 10.1 ± 2.3 g/l, P < 0.0001). Total IgA concentrations were similar in HIV-2 patients and uninfected controls (mean 2.4 ± 1.1 vs. 2.1 ± 0.8 g/l, P = 0.1626). In HIV-2-infected patients, total IgG concentration was negatively correlated to the number of CD4+ T cells (r = −0.5829, P < 0.0001) (Fig. 1 a). No association was found between the total IgA concentration and the number of CD4+ T cells (r = 0.1392, P = 0.2540). Total IgA and IgG concentrations were positively correlated in healthy individuals (r = 0.4132, P = 0.0039). In HIV-2 patients, there was no correlation between the total IgA and IgG concentrations (r = −0.1927; P = 0.1).
Fig. 1: Total and envelope-specific antibody response in HIV-2 infection . (a) Correlation between CD4+ T-cell counts in peripheral blood and total concentration of IgA and IgG in plasma of HIV-2-infected patients; (b) level of IgA and IgG antibodies produced against the polypeptides rgp36 and rpC2–C3; (c) reactivity of the four human IgG subclasses (IgG1, IgG2, IgG3 and IgG4) against rgp36 and rpC2–C3; (d) avidity index of rgp36 and rpC2–C3–IgG1-specific antibodies.
IgG and IgA antibody response against the HIV-2 envelope glycoproteins
The presence of envelope-specific IgG and IgA antibodies was investigated in 28 HIV-2-infected patients by using the ELISA-HIV-2 test [33] Fig. 1 b). The magnitude of the IgG response was significantly higher against rgp36 than against rpC2–C3 (median optical density/cutoff 16.09 for rpC2–C3 vs. 34.22 for rgp36, P < 0.0001). The IgA response against both polypeptides was significantly lower when compared with the IgG response (P < 0.0001) (Fig. 1 b). There was, however, no significant difference in IgA reactivity against both polypeptides (P = 0.3988). IgA and IgG antibody responses against both polypeptides were not directly correlated (data not shown).
The level of reactivity of the different IgG subclasses produced against the two polypeptides was also investigated (Fig. 1 c). All patients had IgG1 antibodies binding to rgp36; 26 (92.8%) patients also produced IgG1 to rpC2–C3. The response against rgp36 was significantly stronger as compared with rpC2–C3 (mean optical density/cutoff 29.24 for rgp36 vs. 16.14 for rpC2–C3, P < 0.0001). Ten patients produced IgG2 against rgp36; six patients produced also IgG2 to rpC2–C3. However, the antibody reactivity against both polypeptides was very weak (mean optical density/cutoff 1.43 for rgp36 and 1.48 for rpC2–C3). Twenty patients produced IgG3 antibodies against rgp36; only three patients produced IgG3 against rpC2–C3. Similar to IgG1, IgG3 reactivity was stronger against rgp36 as compared with rpC2–C3 (mean optical density/cutoff 8.23 for rgp36 vs. 1.51 for rpC2–C3, P < 0.0001). IgG4 antibodies against rgp36 were detected in two patients only (patient numbers 4 and 11).
Correlation between antibody concentration, titer and avidity to rgp36 and rpC2–C3
The titer (log10 ) of the IgG antibodies produced against rpC2–C3 ranged from 3.08 to 4.32 and ranged from 3.65 to 5.45 to rgp36. Significantly higher IgG antibody titers were obtained against rgp36 than against rpC2–C3 (median titer 4.57 for rgp36 vs. 3.27 for rpC2–C3, P < 0.0001). There was a positive correlation between the antibody titers against both polypeptides (r = 0.5641, P = 0.0027).
The avidity index of the IgG1 antibodies binding to rgp36 was significantly higher as compared with rpC2–C3 (mean avidity index 0.96 for rgp36 vs. 0.89 for rpC2–C3; P = 0.0032) (Fig. 1 d). The relationship between IgG antibody titer, concentration and avidity was investigated. There was a strong positive correlation between antibody titer and concentration for rpC2–C3 (r = 0.8779, P < 0.0001) and rgp36 (r = 0.4875, P = 0.0099). Antibody titer and avidity were marginally associated only for rpC2–C3 (r = 0.3703, P = 0.0626).
The antibody response against the HIV-2 Env C2–C3 region is a sensitive marker of disease progression
The antibody response against rpC2–C3 and rgp36 and the number of CD4+ T cells was investigated prospectively for a period of 4 years in 16 adult HIV-2 patients (Table 1 ; Fig. 2 a and b). There was a significant inverse correlation between the dynamics of CD4 cell counts and IgG response against rpC2–C3 over time (Deming regression analysis, F = 5.817; P = 0.0345) (Fig. 2 c). No such correlation was found for the IgG response against rgp36 (data not shown).
Fig. 2: Longitudinal analysis of antirpC2–C3 IgG response and CD4+ T-cell counts in HIV-2-infected patients. Anti-C2–C3 antibody response was plotted as a function of the number of CD4+ T cells in patients with at least 500 CD4+ T cells/μl (a), patients with less than 500 CD4+ T cells/μl (b); Deming regression analysis of the dynamics of the C2–C3-specific IgG response and CD4+ T-cell counts along the course of HIV-2 infection (c); comparison of the annual variation (slopes) in the number of CD4+ T cells/μl in antiretroviral-treated and untreated patients (Mann–Whitney test) (d). Dotted lines indicate C2–C3-specific IgG level.
Antiretroviral therapy (ART) has a modest impact on CD4 cell recovery in HIV-2 patients [42,43] + T cells (ART patients, median variation = −21.24 CD4+ cells per year; range −187.0–50.26 vs. drug-naive patients, median = −11.58; range −49.61–83.50; Mann–Whitney test, P = 0.154) (Fig. 2 d). ART also did not impact the within-patient evolution of the C2–C3-IgG response (data not shown).
The IgG response against the C2–C3 and rgp36 antigens was measured retrospectively in two children with perinatal infection for a mean period of 7.5 years (Fig. 3 ). Both children exhibited a rapid decline in the number of CD4+ T cells and progressed rapidly to AIDS, children C1 dying at the age of 8 years (Table 1 ) [44] + T cells (Fig. 3 a). Antibody response against rpC2–C3 was always weak in patient C1 being almost undetectable at the time of death. Patient C2 produced a strong antibody response against gp36 and C2–C3 in a setting of significant decline in the number of CD4+ T cells (Fig. 3 b).
Fig. 3: Kinetics of IgG antibody response against rgp36 and rpC2–C3 and CD4+ T-cell counts in two children infected by mother-to-child transmission.
Sequencing of the env C2V3C3 regions
Clonal sequences were obtained from the env C2–C3 region of most patients. To try to find an explanation for the absence of IgG antibodies binding to the rpC2–C3 polypeptide in patient numbers 27 and 28, we compared the C2–C3 amino acid sequence of these patients with those of the other patients and of the HIV-2ALI isolate that was used to produce the rpC2–C3 polypeptide [33] Fig. 4 ). Nine unique amino acid substitutions and one insertion were found in the C2–C3 regions of patient numbers 27 and 28. These polymorphisms occurred mostly (six out of 10) at the core of the V3 region.
Fig. 4: Alignment of the consensus C2V3C3 amino acid sequences of patient numbers 27 and 28 with the reference HIV-2ALI sequence and with a consensus sequence of all the remaining patients included in this study. The polymorphisms found exclusively in patient numbers 27 and 28 sequences are shown in bold letters.
Discussion
In this study, we examined, for the first time, the unspecific and Env-specific IgA and IgG responses in acute and chronic HIV-2 infection . As previously found in Senegalese patients [36] HIV-2 infection . Total IgG concentration correlated inversely with CD4+ T-cell counts establishing, for the first time, a link between CD4 cell loss and B-cell activation in HIV-2 infection . However, this is not a polyclonal activation as contrary to HIV-1 infection in which the levels of all immunoglobulin isotypes are increased in association with immunological and/or virological failure [45,46] [47] HIV-2 infection suggests that the gastrointestinal immune system is not as severely affected in HIV-2 as it is in HIV-1 infection [23,48]
The human antibody response against rpC2–C3 and rgp36, two polypeptides representing the most antigenic regions of the HIV-2 gp125 and gp36 envelope glycoproteins, was analyzed in two infants infected with HIV-2 by their mothers and in 28 chronically infected adults. In both infants, seroconversion to gp36 occurred during the first year of age but at different levels. For the infant who is still controlling the infection (patient C2) antibody response to gp36 and C2V3C3 rose to levels similar to those found in chronic patients, even in a context of progressive CD4 cell count decline. In contrast, de-novo production of anti-C2V3C3 antibodies did not occur in infant C1 who progressed quickly to AIDS and death. Hence, these results indicate that an early, strong and sustained antibody response to C2V3C3 in gp125 is important to prevent progression to AIDS and death. It is likely that patient C1 was infected in early pregnancy and that this prevented the adequate development of the immune system. The weakened antibody response may have contributed significantly for the rapid evolution of the infecting virus to a highly aggressive phenotype, which caused rapid immune deficiency and death [44,49,50]
In two patients (number 27 and 28), we could not detect IgG antibodies reacting with the rpC2–C3 polypeptide. A high number of amino acid changes were detected in the C2–C3 region of these patients compared with the other patients and with the rpC2–C3 antigen used in the ELISA-HIV2 assay. These occurred mostly at the core of the V3 region, which is the principal antigenic determinant in the HIV-2 surface glycoprotein [51]
All but one patient that exclusively produced antigp36 IgA, produced IgA antibodies reacting with both the C2–C3 and gp36 polypeptides. These findings are consistent with a healthy gastrointestinal immune system in HIV-2 infection (see above) and signal an important difference for HIV-1 infection as only 32–91% of HIV-1 patients produce serum IgA antibodies against the gp41 and/or gp120 envelope glycoproteins, this being inversely related to the stage of disease [45,52–54] HIV-2 infection [20]
IgG titers against rgp36 and rpC2–C3 were strongly correlated, which indicates that in the native envelope glycoprotein complexes the corresponding epitopes are presented in a similar way to the B lymphocytes. Nonetheless, the IgG response was predominantly directed to gp36 both qualitatively (avidity) and quantitatively (titer and concentration). Gp36 also induces higher levels of all IgG subclasses when compared with gp125. Together, these results extend our previous observations providing definitive evidence for the immunodominant role of the gp36 ectodomain in HIV-2 infection [20,33]
We found that IgG1 is the predominant antibody subclass produced against both HIV-2 envelope glycoproteins. However, unlike in HIV-1 infection, IgG3 and not IgG2 was the second most reactive subclass to gp36 [55–59] [55,56,59–61] [62] + T cells. Instead, we found a significant inverse association between C2 and C3-specific IgG antibody production and the number of CD4+ T cells. Importantly, this association was also found in one pediatric patient with progressive infection. How can the loss of CD4+ T cells be associated with the IgG response against the C2V3C3 region? Cavaleiro et al . [32,63] HIV-2 infection [32]
In agreement with most HIV-2 reports, we have shown here that chronic HIV-2 infection usually courses without evidence for viral replication in the plasma [9] + T cells is the only available marker to monitor disease progression in these patients. However, as shown here and elsewhere [9] + T-cell counts without sign of disease progression. Our results show that the anti-C2–C3 IgG response adequately reflects the immunological and clinical progression in the HIV-2 patients. We suggest, therefore, that antibody concentration against the C2–C3 envelope region should be a useful marker to monitor disease progression in HIV-2 infection .
Acknowledgements
The present work was supported by Fundação para a Ciência e Tecnologia (project POCTI/ESP/48045). José Marcelino is the recipient of a PhD scholarship from Fundação para a Ciência e Tecnologia (FCT), Portugal. The Instituto Português do Sangue (IPS), Portugal, is gratefully acknowledged for the provision of seronegative plasma samples. We thank Luisa Papoila for statistical support.
J.M.M., C.N. and N.T. designed the study. J.M.M. performed all antibody assays. H.B. and P.B. provided analytical reagents and nucleotide sequences. F.M., L.R., F.A. and M.D. contributed clinical data from the patients. P.G. performed the viral load assays. H.B., P.B., F.M., L.R., F.A., M.D. and P.G. assisted in data analysis. J.M.M., C.N. and N.T. analyzed the data and wrote the paper.
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