The specificity and variability of antibodies (Abs) is achieved by affinity maturation through somatic hypermutation and clonal selection. This process proceeds for weeks after acute infections or vaccination. There is much debate about the mutation levels and chronic antigen exposure needed to generate broadly neutralizing Abs (bNAbs) against HIV-1 . In adults, bNAbs usually take 2–4 years to develop and are associated with high viremia and low CD4+ T-cell counts [2–7]. In contrast to HIV-1, most HIV-2-infected patients develop potent bNAb responses [8–12]. In addition, HIV-2 infection usually courses with undetectable viral load and very slow rate of CD4+ T-cell decline even in the absence of antiviral treatment [13,14]. Therefore, understanding the generation of broad and potent neutralizing Abs in this unique model of naturally occurring control of viremia is relevant for the vaccine field.
We aim to investigate the relationship between memory B cells and neutralizing Ab (NAb) response in untreated HIV-2-infected adults. Previously, we have shown that these patients featured contraction of the memory B-cell compartment in direct association with the degree of CD4+ T-cell depletion and markers of immune activation, suggesting the occurrence of lymphoid tissue disruption . Here, we asked how this finding relates with HIV-2-specific Abs.
We analysed the NAb response in 27 untreated HIV-2-infected patients, included in a previous report . Twenty-one patients (78%) had undetectable viral load (detectable in six patients, ranging from 1189 to 13 627 RNA copies/ml), and 20 (74%) had CD4+ T-cell counts above 350 cells/μl (median 568, range 52–1511 cells/μl). Informed consent for blood collection and participation in the study was obtained from all participants, under the approval of the Ethical Board of the Faculty of Medicine, University of Lisbon. HIV-2 viremia was quantified by reverse transcriptase-PCR (detection threshold: 40 RNA copies/ml), as described previously [16,17]. The neutralizing activity was analysed in plasma against a panel of four heterologous primary CCR5-tropic isolates (03PTHCC6, 03PTHCC12, 03PTHCC19, 03PTHSM2). Origin and characterization of these isolates, and their sensitivity to IgG NAbs, have been described elsewhere [10,18,19]. A luciferase reporter gene assay in TZM-bl cells was used, as described previously [20,21]. Additionally, binding Abs against HIV-2 envelope glycoproteins gp125 (C2V3C3 region) and gp36 were quantified in samples from a subset of the patients using an ELISA HIV-2 assay as described previously [15,22]. Memory B-cell populations were assessed based on the expression of CD27 and surface IgD by flow cytometry, with class-switched memory B cells defined as CD19+CD27+IgDneg and unswitched memory B cells as CD19+CD27+IgDpos, as described previously . Serum B-cell activating factor (BAFF) levels were quantified by ELISA as described previously . Statistical analysis was performed with Prism, version 5.0 (GraphPad Software Inc., San Diego, California, USA). P values were two-tailed and significance was defined as P value less than 0.05. Linear regression was performed and Spearman correlation coefficients were computed.
All patients produced NAbs against the panel of four primary R5 isolates, with a median reciprocal log10 IC50 neutralization titer of 2.89 (interquartile range 2.76–3.18). NAb potency and breadth were directly associated (N = 27, Spearman rank, r = 0.4916, P = 0.0092; Supplemental Digital Content 1, http://links.lww.com/QAD/B170). Moreover, NAb potency and breadth were not associated with CD4+ T-cell counts (N = 27, Spearman rank, r = −0.1902, P = 0.3419 and r = −0.0325, P = 0.8723, respectively). NAb potency has generally not been associated with the number of CD4+ T cells in previous HIV-2 studies [9,12], except when patients representing the full spectrum of disease were analysed .
NAb potency increased in direct association with the increase in C2V3C3-specific binding Abs (N = 12, Spearman rank, r = 0.7250, P = 0.0098; Supplemental Digital Content 2 – blue, http://links.lww.com/QAD/B170). However, no association was found between NAb potency and gp36-specific binding Abs (N = 12, Spearman rank, r = 0.4483, P = 0.1446; Supplemental Digital Content 2 – black, http://links.lww.com/QAD/B170). Our findings confirm the presence of broad and potent NAbs targeting the C2V3C3-envelope region during chronic HIV-2 infection, as described previously .
NAb potency was found to increase significantly with the loss of switched (N = 27, Spearman rank, r = −0.6436, P = 0.0003) and unswitched (N = 27, Spearman rank, r = −4162, P = 0.031) memory B-cell populations (Fig. 1, top and bottom, respectively). In accordance with the association between NAbs and binding Abs, the loss of both switched and unswitched memory B-cell populations was directly correlated with the increase in C2V3C3-specific binding Abs (N = 12, Spearman rank, r = −0.7622, P = 0.0055 and r = −0.6434, P = 0.0278, respectively; Supplemental Digital Content 3, http://links.lww.com/QAD/B170). No association was found between switched and unswitched memory B-cell populations and gp36-specific binding Abs (N = 12, Spearman rank, r = −0.5455, P = 0.0708 and r = −0.2517, P = 0.4303, respectively; Supplemental Digital Content 3, http://links.lww.com/QAD/B170). No association was found between NAb titers and BAFF levels (N = 11, Spearman rank, r = 0.1455, P = 0.6731; Supplemental Digital Content 4, http://links.lww.com/QAD/B170), despite the previous report of BAFF increase in parallel with B-cell disturbances in HIV-2 infected individuals .
To our knowledge, this is the first study addressing the relationship between NAb responses in HIV-2-infected patients and memory B-cell disturbances. In spite of the limitations imposed by the number of patients and sample availability that only allowed to test four viral isolates, we were able to reveal a strong direct association between memory B-cell exhaustion and the generation of NAbs as well as C2V3C3 binding Abs. These findings suggest that multiple rounds of HIV-2 infection are important for the generation of highly specific Abs, and that this happens at the expense of the memory B-cell pool. The lack of hypogammaglobulinemia  is likely because of the persistence of long-lived plasma cells, as shown in other clinical settings . Although the levels of plasma viral load were reduced in HIV-2 infection even in the absence of antiretroviral therapy, we have shown that the extent of viral reservoirs is similar in HIV-2 and HIV-1 individuals [17,24]. Moreover, HIV-2 has been shown to infect and induce cytopathicity in lymphoid tissues , suggesting that there is contained local viral replication . The prolonged time of infection that characterizes HIV-2 pathogenesis favors the continuous exposure to envelope antigens (either present in plasma viruses or bound to the surface of infected cells) and persistent immune activation . This setting is ideal for the generation of highly specific Abs through affinity maturation, but ultimately leads to immune exhaustion.
N.T. and A.E.S. designed research. C.R., J.M.M., R.C., and R.T. performed experiments. C.R. and J.D. analyzed and interpreted the data. C.R. and P.B. performed the statistical analysis. E.V. contributed with patients’ data. C.R., J.D., A.E.S., and N.T. wrote the article. All authors revised and approved the final manuscript.
The work was supported by grants PTDC/SAU-FAR/115290/2009, PTDC/SAU-EPI/122400/2010, and PIC/IC/82712/2007 from Fundação para a Ciência e Tecnologia (FCT) (http://www.fct.pt), Portugal and by Programa Operacional Ciência e Inovação 2010 (POCI2010). C.R., P.B., R.C., and R.T. were supported by PhD grants from FCT. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: TZM-bl from Dr John C. Kappes, Dr Xiaoyun Wu, and Tranzyme Inc.
Conflicts of interest
There are no conflicts of interest.
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