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Viral control in chronic HIV-1 subtype C infection is associated with enrichment of p24 IgG1 with Fc effector activity

Chung, Amy W.a,b,*; Mabuka, Jenniffer M.c,d,*; Ndlovu, Bongiwed; Licht, Annaa; Robinson, Hannaha; Ramlakhan, Yathishad; Ghebremichael, Musiea; Reddy, Taryleee; Goulder, Philip J.R.f; Walker, Bruce D.a,d; Ndung’u, Thumbia,c,d,g,*; Alter, Galita,*

Author Information
doi: 10.1097/QAD.0000000000001812

Abstract

Background

In 2016 alone, there were an estimated 1.8 million new HIV-1 infections globally; a figure that highlights the relentless spread of this virus and the urgent need for an efficacious vaccine (http://www.unaids.org/en/resources/fact-sheet). In 2009, the RV144 trial, a phase III HIV-1 human vaccine trial, was the first to show modest efficacy [1]. Immune correlate analysis identified binding antibodies to the viral envelope variable loops 1 and 2 as the primary correlates of reduced risk from infection [2]. However, in secondary analyses, Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) preferentially mediated by IgG3 was also associated with reduced risk of infection in the absence of IgA [3,4], suggesting a potentially critical role for nonneutralizing antibody Fc effector activities in protection from HIV-1 infection.

In addition to ADCC, antibodies mediate additional extra-neutralizing functions relevant in HIV-1 infection, such as antibody-mediated cellular phagocytosis (ADCP), through their capacity to interact with Fc receptors expressed on innate immune cells [5,6]. Importantly, both the magnitude and breadth of the ADCC response has been linked to slow disease progression [7–11], and higher ADCC activity has been observed in rare individuals capable of controlling viral replication in the absence of antiretroviral therapy (ART) [12]. Furthermore, HIV-1 transmission from mothers to their breastfeeding infants is significantly reduced in women with high ADCC activity in breast milk [13]. In animal models, vaccine-induced ADCC and antibody-dependent cell-mediated viral inhibition responses have been associated with protection and reduced viral set point [14–19]. Moreover, prevention of simian–human immunodeficiency virus infection via vaccination has also been linked to enhanced ADCP activity [18,19], which was also enriched in RV144 vaccinees [4,20]. Together, these observations have renewed interest in dissecting the importance of Fc effector functions in natural infection, particularly, against clades that contribute to the greatest HIV-1 burden globally.

The antibody response to HIV-1 proteins is largely dominated by IgG1 antibodies, with variable levels of other antibody subclasses (IgG2, IgG3 and IgG4) [21]. In natural infection, however, levels of gp120-specific antibodies do not correlate with markers of disease progression despite their potential antiviral activity [22–30]. In contrast, anti-p24 antibody levels have been shown to correlate with viral control in several studies, largely focused on subtype B cohorts (summarized by French et al.[31]). However, as p24 is not expressed on the cell surface, the direct contribution of p24-specific antibodies to antiviral control is unclear, but has also been proposed as a surrogate marker of an intact CD4+ T-cell response [32].

As the humoral immune response may differ between clade B-infected and clade C-infected individuals [33], here we aimed to define the Fc receptor-mediated functional profiles of gp120-specific and p24-specific antibody responses in the context of HIV-1 subtype C infection. Similar to previous studies [31,32], we found that levels of p24-specific but not gp120-specific IgG1 were associated with lower viral loads and higher CD4+ cell counts. Furthermore, individuals with high-p24 IgG1 levels had higher Fc-mediated responses that showed a trend towards viral control and higher CD4+ cell counts. The association between levels of p24 IgG1 and viral loads and CD4+ counts was maintained even after adjusting for the impact of Gag-specific CD4+ and CD8+ T-cell responses and protective human leukocyte antigen (HLA) class I alleles (HLA-I). These data indicate that p24-specific IgG1 may independently contribute to viral control and further studies are warranted to ascertain their direct or indirect roles in antiviral immunity.

Materials and methods

Study participants

Three hundred and sixty-one archived plasma samples from the Sinikithemba cohort [34], a study of HIV-1 subtype C-infected, ART-naive individuals, established in Durban, South Africa were included in this study. All study participants provided written informed consent, and the study protocol was approved by the Biomedical Research Ethics Committee of the University of KwaZulu-Natal.

Customized IgG subclass-binding assay

To determine the relative levels of each HIV-1-specific isotype/subclass in plasma, we adopted a customized subclass-binding assay described previously [35]. We used carboxylated microspheres (luminex) coupled to HIV-1 recombinant proteins of interest: p24 subtype B/C and gp120 consensus C (Immune Technology Corp., New York, New York, USA).

Antibody-dependent cellular phagocytosis

The ability of plasma antibodies to mediate ADCP was determined as previously described using gp120 consensus C and p24 subtype B/C protein (Immune Technology Corp.) [36]. Antigen-bound fluorescent neutravidin beads (Invitrogen) were incubated with 1 : 100 dilution of plasma antibodies in the presence of THP-1 cells. HIV-negative plasma and media alone were used as negative controls and pooled HIV-positive plasma was used as a positive control. The cells were analyzed for bead uptake by flow cytometry and the phagocytic score representing iMFI values (integrated mean fluorescence intensity = frequency × MFI) [37] was calculated. Flow cytometry-gating strategy is shown in Supplementary Figure 1a, http://links.lww.com/QAD/B255.

Although previous studies [36] have confirmed that gp120-specific ADCP activity is mediated by FcγR mechanisms, this has not been confirmed whenever using p24 protein as an antigen. Therefore, as an additional control, six randomly selected HIV-positive plasma samples were purified for IgG using Melon Gel (Thermo Fisher Scientific, Waltham, Massachusetts. USA). THP-1 cells were preincubated with FcγRII (Abcam, Cambridge, Massachusetts, USA) or FcγRIII (Sigma, St Louis, Missouri, USA)-blocking antibodies for 30 min prior to assaying for p24-specific ADCP responses as described above (Supplementary Figure 1B, http://links.lww.com/QAD/B255).

Natural killer cell degranulation as a surrogate for antibody-dependent cellular cytotoxicity

We used a modified ELISA-based protocol for detection of CD107a as a surrogate marker of natural killer cell-mediated cytolysis [38]. This assay has previously been shown to be associated with FcγRIIIa-binding capacity of antigen-specific antibodies [39,40]. A 96-well plate was coated overnight at 4 °C with 150 ng of recombinant protein per well 2% BSA-blocked plates were used as antigen controls. The next day, the plates were washed six times with PBS, 50 μl of plasma (diluted 1 : 100) was added to each well, and incubated at 37 °C for 2 h. HIV-negative plasma samples or media alone were used as negative controls, whereas HIVIG (pooled HIV Immunoglobulin G, NIH AIDS Reagents Program) was used as a positive control. The plates were washed and 5 × 104 natural killer cells enriched via negative selection from healthy blood donors (RosetteSep, Stemcell Technologies, Cambridge, Massachusetts, USA) were added to each well in the presence of Brefeldin A (Biolegend, San Diego, CA, USA), Golgi stop, and anti-CD107a-PE-Cy5 (BD Biosciences, San Jose, California, USA). The plate was incubated for 5 h at 37 °C and 5% CO2. Following incubation, cells were stained with anti-CD3-AF700, anti-CD56-PE-Cy7, anti-CD16-APC (BD Biosciences), fixed with Perm A, permeabilized using Perm B (Invitrogen, Carlsbad, California, USA), and stained with anti-IFNγ-APC and anti-MIP1β-PE (BD Biosciences). The cells were then fixed with 2% paraformaldehyde and analyzed by flow cytometry (gating strategy shown in Supplementary Figure 2, http://links.lww.com/QAD/B255).

Gag-specific CD4+ and CD8+ T-cell responses

Gag-specific IFN-γ CD4+ T-cell responses were detected by intracellular cytokine staining (ICS) using freshly isolated peripheral blood mononuclear cells (PBMCs) as previously described [41] staining for antihuman CD4+ APC and antihuman IFNγ FITC. Gag-specific IFNγ CD8+ T cells were detected by IFNγ enzyme-linked immunosorbent spot (ELISpot) using overlapping peptides spanning Gag subtype C as previously described [42].

Statistical analysis

Nonparametric Spearman's rank tests were used to test for correlations, and a two-tailed Mann–Whitney test was used to evaluate unmatched groups. P values less than 0.05 were considered significant. To assess the effect of p24 IgG1 on viral load and CD4+ cell count, multiple linear regression was used. IgG1p24 values were z score-normalized (standardized) to have a mean of zero and a standard deviation of one. We considered a variable to be a potential confounder of the effect of p24 IgG1 if it was shown to have a significant relationship with the outcome in univariate analysis or if its inclusion in the model resulted in a 10% or greater change in the estimated coefficients. A square root transformation was applied to CD4+ cell count and a log transformation was applied to viral loads, which resulted in approximately normally distributed values. Analysis was performed using Graphpad Prism version 6 (Graphpad Software, La Jolla, California, USA) and Stata Statistical Software: Release 13 (StataCorp LP, College Station, Texas, USA).

Results

p24 IgG1 levels predict viral control in chronic HIV-1 subtype C infection

Previous studies, largely in clade B-infected cohorts, have shown that p24-specific but not gp120-specific antibody levels are a prognostic marker of disease progression [24,27–31]. Thus, to first define whether similar relationships were observable in clade C infection, a cross-sectional study including a cohort of 361 HIV-infected patients (median viral load: 4.8 log10 HIV-RNA copies/ml, interquartile range (IQR) 3.9–5.3; median CD4+ cell count: 339 cells/μl, IQR 226–492) were profiled for gp120-specific or p24-specific antibodies. HIV-specific IgG to both proteins were detected in all individuals, however, neither gp120-specific nor p24-specific total IgG antibody levels correlated with either viral loads or CD4+ cell counts (Fig. 1b and d, left).

Fig. 1
Fig. 1:
p24 IgG1 levels predict viral control in chronic HIV-1 subtype C infection.Using recombinant protein coupled to fluorescent beads and diluted plasma, a customized luminex assay was performed to determine the levels of HIV-1 gp120- and p24 specific antibodies. (a) Description of percentage of positive responders. A positive threshold was determined as 3× mean +2SD of HIV negatives. The relationship between the mean fluorescence intensity (MFI) of gp120 total IgG and isotype-specific-binding antibodies and viral loads or CD4+ cell counts is depicted in panels (b) and (c) whereas p24 is shown in panels (d) and (e). Plots with red and blue background represent significant positive and negative correlations, respectively, wheraes a white background denotes nonsignificant relationships. P values were calculated by Spearman's correlation.

Next we determined whether relative differences in gp120-specific or p24-specific Ig subclass-specific responses correlated with measures of viral control. Similar to reports from other subtypes [21], the antibody responses directed at both gp120 and p24 were dominated by IgG1 responses. IgG3 directed to p24 was also observed in all individuals, and gp120-IgG3 responses were detected in most individuals (99%). Conversely, levels of other subclasses were lower and less frequently detected: p24-specific and gp120-specific IgA were detected in 94 and 99% of the cohort, respectively Conversely, levels of other subclasses were lower and less frequently detected: gp120-specific and p24-specific IgA were detected in 94 and 99% of the cohort, respectively, whereas gp120-specific and p24-specific IgG2 were detected in 80 and 94% of patients, respectively (P = 0.0032), and gp120-specific and p24-specific IgG4 were detected in 99 and 90%, respectively (P = 0.0052) (Fig. 1).

To further probe the relationship between antigen-subclass-specific responses and viral control, the relationships between individual antigen-specific subclasses and markers of disease progression were assessed. Higher gp120-specific IgG2 and IgG4 responses were associated with higher viral loads (P = 0.0002, r = 0.206 and P = 0.035, r = 0.115, respectively; Fig. 1b) and were negatively associated with CD4+ cell counts (P = 0.014, r = −0.133 and P = 0.034, r = −0.115, respectively) (Fig. 1c). Similarly, increasing levels of IgG3 responses targeting p24 were also associated with higher viral loads (P = 0.0002, r = 0.205; Fig. 1d) and lower CD4+ cell counts (P = 0.006, r = −0.151) (Fig. 1e). In contrast, Gag p24 IgG1 levels were negatively correlated with viral loads (P = 0.01, r = −0.139; Fig. 1d) and positively correlated with CD4+ cell counts (P = 0.025, r = 0.121; Fig. 1e). These data suggest that higher levels of p24-IgG3 subclass selection profiles in chronic subtype C infection are associated with poor viral control, whereas p24-specific IgG1 responses in chronic subtype C infection may be a marker of viral control.

Elevated p24 IgG1 levels track with enhanced Fc effector activity

Although antibodies directed against internal structural proteins of the virion have a limited capacity to mediate neutralization, these antibodies may contribute to viral control via nonneutralizing antiviral functions [43,44]. Thus, we next profiled the ADCP and ADCC activity of these antibodies and found that p24-specific phagocytic activity was detectable in all individuals whereas only low-level p24-specific ADCC activity was observed, in a minority of patients. Detectable p24-specific natural killer cell degranulation (CD107a) was observed in 34% of the tested patients, and IFNγ and MIP1β secretion was detected in 47 and 16% of the patients, respectively, with strong correlations observed between all three functions (P ≤ 0.0001) (Fig. 2a). We then determined the relationship between antibody levels and functional activity and found that p24 IgG1 was positively correlated with both ADCP phagoscore and CD107a expression (P < 0.0001 and P = 0.033, respectively, Fig. 2b). Interestingly, there was a clear distribution of high and low IgG1 levels whenever considering ADCP phagoscore. Using the median value as a cutoff for p24 IgG1 levels, we found that individuals with high p24 IgG1 levels were able to mediate significantly higher ADCP and ADCC (CD107a) responses (P < 0.0001 Fig. 2c, left and P = 0.027, respectively; Fig. 2c, right). These data suggest that p24 antibodies may be effective at mediating Fc effector functions.

Fig. 2
Fig. 2:
p24 IgG1 levels correlate with both antibody dependent cellular phagocytosis phagoscore and CD107a expression levels.(a) Antibody-mediated natural killer cell activation measured by expression of CD107a degranulation as a surrogate for ADCC, IFNγ cytokine secretion and MIP1β were assayed in the presence of p24 protein as a surrogate for ADCC activity. All measurements of natural killer activation were high correlated with each other. (b) Antibody-dependent cellular phagocytosis (ADCP) of fluorescently labelled bead conjugated to p24 protein by monocyte cell line THP-1 was assessed. The relationship between the mean fluorescence intensity (MFI) of p24-specific IgG1 with ADCP phagoscore (left) and CD107a levels (right) are depicted. The grey dotted line on each graph represents the median p24-specific IgG1 levels. (c) Using the median, individuals were dichotomized into two groups with high and low p24 IgG1. The differences in magnitude of p24 ADCP (left) and ADCC (right) responses in the groups are shown, respectively. Correlations between two variables were calculated by Spearman's test. Difference between two groups were calculated by two-tailed Mann–Whitney test. ADCC, antibody-dependent cellular cytotoxicity; ADCP, antibody-dependent cellular phagocytosis.

Higher p24-specific Fc effector activity trends with markers of disease progression

Given that higher p24 IgG1 levels were associated with higher Fc effector activity, we next investigated the relationship between p24-specific Fc effector activity and markers of disease progression. Although we did not observe a significant relationship between the expression of CD107a and viral load (P = 0.562, r = −0.055; Fig. 3b), a trend towards higher CD4+ cell counts (P = 0.065, r = 0.172) was observed (Fig. 3d). In addition, the magnitude of p24-specific ADCP responses trended towards a negative correlation with viral load (P = 0.086, r = −0.157; Fig. 3a) and a positive correlation with CD4+ cell count (P = 0.082, r = 0.158; Fig. 3c), suggesting that p24-specific antibody functions are associated, albeit weakly, to viral control.

Fig. 3
Fig. 3:
Higher p24-specific Fc effector activity trends with markers of disease progression.Diluted plasma was used to cross link and initiate phagocytosis of FITC fluorescent beads conjugated to p24 protein (ADCP) by THP-1 cells. Antibody mediated natural killer activation (detection of CD107a used as a surrogate for ADCC) was measured in the presence of p24 protein. The relationship between the magnitude of ADCP and ADCC responses and viral loads shown in panels (a) and (b) whereas panels (c) and (d) show the relationship with CD4+ cell counts. P values were calculated by Spearman's correlation. ADCC, antibody-dependent cellular cytotoxicity; ADCP, antibody-dependent cellular phagocytosis.

p24 IgG1 levels are not a simple surrogate of protective human leukocyte antigen class I antiviral activity

To next determine whether p24-specific antibodies may contribute to antiviral control, we examined the relationship between p24-specific IgG1 responses and traditional markers of HIV-1 control. Specifically, particular HLA class I alleles have been linked to slower disease progression, through the induction of superior CD8+ T-cell activity in this cohort [34,42,45]. Of the 361 individuals, those with protective HLA class I alleles (HLA-B*57:01, HLA-B*58:01, HLA-B*81:00 and HLA-B*81:01) had significantly lower viral loads (P = 0.002) and higher CD4+ cell counts (P = 0.003). Thus, to define whether p24 IgG1 was independently associated with antiviral control, individuals with protective HLA-I alleles were removed from the analysis (n = 24). Interestingly, individuals with high p24 IgG1 levels maintained higher ADCP (P < 0.0001) and ADCC (P = 0.035), (Fig. 4a and b) and continued to exhibit lower viral loads (P = 0.004) and higher CD4+ cell counts (P = 0.019; Fig. 4c and d), indicating that the relationship between p24-specific IgG1 responses and enhanced viral control was not solely influenced by protective HLA-I alleles, and therefore, may represent an independent predictor of enhanced viral control.

Fig. 4
Fig. 4:
Relationship between human leukocyte antigen alleles, CD4+ and CD8+ Gag responses and Gag p24 IgG1 levels.Patients with protective HLA alleles (HLA-B*57:01, HLA-B*58:01 and HLA-B* 81:00/01) were removed from this analysis and individuals with no protective alleles were dichotomized into groups of high and low p24 IgG1 levels. The panels show the difference in the magnitude of ADCP (a) and ADCC (b) responses, viral loads (c) and CD4+ cell counts (d). Gag CD4+ and CD8+ activity was measured by ICS and ELISpot, respectively, and reported as percentage of cells positive for IFNγ or as number of spot-forming units (SFUs/million PBMCs), respectively. Differences in CD4+ and CD8+ Gag responses in the dichotomized groups of individuals with high and low p24 IgG1, respectively, are shown in panels (a) and (b). P values were calculated by two-tailed Mann–Whitney test. ADCC, antibody-dependent cellular cytotoxicity; ADCP, antibody-dependent cellular phagocytosis; HLA, human leukocyte antigen.

p24 IgG1 levels independently mark protective HIV-1 control

Both CD4+ and CD8+ T-cell responses have also been linked to protective immunity in this cohort [34,41,46]. Moreover, because enhanced helper CD4+ T-cell responses may promote more effective antibody responses in addition to viral control, it is possible that elevated p24 antibodies may simply represent a marker of preserved T-cell immunity rather than a direct antiviral mechanism of control. Thus, to parse out the relationship between T-cell-mediated antiviral control and p24-specific antibody responses, we next examined the inter-relationship between p24 IgG1 responses and both arms of the T-cell response in these individuals. The frequency of Gag-specific CD4+ T cells (by ICS) and magnitude of CD8+ T cells (by ELISpot) was associated with lower viral loads (P < 0.0001 for both) and higher CD4+ cell counts (P = 0.0002 and 0.138), respectively, in these individuals, as previously observed [34]. Moreover, whenever individuals were split into those with high or low-p24 IgG1 levels, patients with high-p24 IgG1 levels had significantly higher Gag-specific CD4+ (P = 0.037) and CD8+ (P = 0.049) responses (Fig. 4e and f) suggesting that p24-specific humoral immune responses are induced in parallel to protective Gag T-cell immunity.

To gain insights, however, into whether p24-specific IgG1 immunity was simply a surrogate of a more effective T-cell immune response or if it represented an independent marker of enhanced viral control, factors that were associated with viral control and elevated CD4+ cell counts in univariate analyses including IgG1p24 levels, CD4+/CD8+ T-cell Gag responses (dichotomized as having any or no response) and the presence of protective HLA-I alleles, were considered in a multivariate analysis. The CD4+ T-cell Gag response remained a significant predictor of viral load and CD4+ T-cell counts (P = 0.002 and P < 0.001, respectively; Table 1). Yet, most interestingly, even after adjusting for CD4+ or CD8+ Gag responsiveness and carriage of protective HLA-I alleles, IgG1 p24 antibody responses remained significantly associated with lower viral loads (P = 0.030) and higher CD4+ cell counts (P = 0.005, Table 1). Hence, p24 IgG1 antibodies independently predict viral control and preservation of CD4+ cells and likely complement T-cell–mediated antiviral immunity in the control of viral replication.

Table 1
Table 1:
Univariate and multivariate analysis of relationships with viral loads or CD4+ counts.

Discussion

Despite the growing appreciation for the role of nonneutralizing antibody functions in both antiviral control [8–12] and protection from HIV-1 infection [3,4,13], little is known about the role of these functions in the context of HIV-1 subtype C viral control, which predominates the current global epidemic. Thus, here, we aimed to define the role of Fc-mediated antibody activities in chronic subtype C infection in a cross-sectional cohort of HIV-1-infected participants at the heart of the South African epidemic, in Durban. Similar to previous studies of other viral subtypes [22–30], the levels of gp120-specific total IgG and other IgG subclasses did not correlate with viral control. Instead, as previously reported [31] p24-specific IgG1 responses were associated with antiviral control and slower progression to disease. Moreover, these antibodies have the capacity to mediate both cytotoxicity and clear infected cells via phagocytosis, providing two potential antiviral mechanisms by which these antibodies may contribute to antiviral control. Finally, although elevated CD4+ and CD8+ T-cell responses were induced among individuals with the highest p24-specific IgG1 responses, multivariate modeling demonstrated that the p24-specific IgG1 levels independently predicted HIV-1 control, suggesting for the first time that p24-specific functional IgG1 antibodies may contribute directly to antiviral immunity in a manner complementary to known mechanisms of cellular immune control of HIV-1.

The selection of the poorly functional gp120 IgG2 and IgG4 was associated with poor viral control, likely by dampening antibody functionality because of their lower affinity for Fc-receptors, as has been previously reported [4]. However, interestingly, although IgG3, the most functional antibody subclass, has been linked to reduced risk of HIV-1 infection in RV144 vaccinees [3,4], here, elevated gp120-specific or p24-specific IgG3 levels were not observed in patients with markers of slower disease progression. Rather, elevated p24-specific IgG3 responses were associated with higher viral loads and lower CD4+ T-cell counts. As IgG3 is the first subclass in the immunoglobulin heavy locus, the selection of IgG3 antibodies is a signature of recently selected B cells, that have not rearranged to IgG1, IgG2 or IgG4 [47]. Thus, these data suggest that p24 IgG3 antibody responses in chronic infection may represent a marker of a less mature B-cell response, which lack provision of B-cell cross switch recombination to other subclasses, whereas, in contrast, enhanced p24 IgG1 responses may contribute most effectively to postinfection antiviral immunity in the setting of Clade C HIV-1 infection.

Although the association between p24-specific antibody levels and markers of disease control has been reported in several studies [31], the mechanism by which p24-specific IgG1 antibodies contribute to antiviral immunity remains unclear. One possibility includes a scenario where higher virus loads drive the formation of p24-containing immune complexes; although, this hypothesis has been refuted by a study done by Fenouillet et al.[48] who showed that acid dissociation of immune complexes did not increase p24 levels. Alternatively, p24-specific IgG1 antibodies may only be a marker of an intact immune response, where preserved p24-specific CD4+ helper T-cell responses sustain elevated levels of p24-specific B-cell responses p24-specific IgG1 but do not individually contribute to viral control [26,49]. Our data, however, challenges this last hypothesis. Although we show that although p24-specific IgG1 levels are associated with higher CD4+ T-cell numbers (Fig. 1d) as well as elevated CD4+ T-cell help (Fig. 4e), the ability to predict lower viral loads is sustained after controlling for CD4+ T-cell numbers and Gag-specific CD4+ responses (Table 1). Thus, our data indicates that p24-specific IgG1 responses, beyond being only a marker of a more effective overall immune response, may also contribute independently to viral control.

Whether Gag is exposed on the virion surface remains a subject of debate. Although rare studies [50–52] have detected cell surface p24 on infected cells, these studies have only been conducted on cell lines, and surface expression was only observed at high, nonphysiological rates of infection. However, it is possible that Gag may be exposed during delivery of HIV-1 Gag to the plasma membrane for virion assembly [53], particularly in the setting of early apoptosis when the inner-cell lipid layer flips out to expose phosphotidyl serine, potentially exposing cytosolic anchored membrane proteins as well [54]. It is also plausible that in addition to native protein expression on the cell surface, processed Gag peptides may be recognized by antibodies in the groove of major histocompatibility complex (MHC) molecules on the surface of infected cells to induce ADCP or ADCC, though this has yet to be demonstrated. Importantly, Fc effector responses to other nonenvelope protein derived-peptides have been reported [11,43,44,55–57]. Indeed, Tjiam et al.[44] recently showed p24 IgG1 and IgG2-enhanced dendritic cell-mediated opsonophagocytic responses in individuals who controlled viremia and these same opsonophagocytic responses were associated with early viral control, interestingly specifically mediated by p24 IgG1 [43]. In our study, only p24-specific IgG1 levels were associated with higher ADCP and ADCC responses that trended towards viral control. Unlike Tjiam's study, p24-specific IgG2 levels did not correlate with Fc function, potentially because of the different assays used, which measure different Fc functions and utilize different effector cells. Although in our study, these p24-specific Fc effector functions did not achieve significance with markers of disease progression (Fig. 4, P value range = 0.062–0.086), antibodies are capable of mediating multiple other Fc-driven effector functions beyond ADCP and ADCC, including complement fixation, neutrophil activation, dendritic cell activation as well as recruiting multiple other FcγR-expressing effector cells. Furthermore, all our assays were tested utilizing total plasma, which includes multiple different immunoglobulin isotypes including IgA, IgM, IgE along with other serum proteins including complement, which were not assessed. Future studies testing for alternative Fc effector functions using purified IgG and isolated p24-specific IgG1 could provide greater insights.

Additionally, antibodies have recently been shown to not only drive extra-cellular antiviral activity but also to target pathogens within cells. In fact, the intracellular host restriction factor TRIM21 is an intracellular cytoplasmic Fc receptor that can drive rapid and profound autophagy. In this context, interactions between TRIM21 and cytosolic antibody-coated antigens triggers an innate immune intracellular signaling cascade that ultimately results in the induction of autophagy [58]. Thus, it is plausible that p24-specific antibodies able to gain access to the cytoplasm may drive the rapid elimination of infected cells. Taken together, these observations suggest that p24-specific IgG1 antibodies may contribute to antiviral control via a spectrum of diverse mechanisms, thereby driving viral control by unconventional Fc-driven means.

This study used a large cohort that has been well characterized for cellular responses associated with disease outcome, enabling a holistic understanding of the relationship between HIV-specific antibody responses and function with markers of slower disease progression. Importantly, the study demonstrated the independent predictive value of p24-specific IgG1 responses on markers of disease progression, highlighting a potential role for Gag-specific antibodies in antiviral control. However, as this study used cross-sectional samples from a cohort of chronically infected individuals with unknown dates of infection, it remains unclear whether p24-specific antibodies actively contribute to reduced disease progression. Thus, future longitudinal studies on acutely infected patients may shed light on the impact of p24-specific IgG1 antibodies on rates of disease progression.

In conclusion, these data confirm the importance of antibody isotype selection in HIV-1 clade C infection and show that p24-specific IgG1 and not gp120-specific antibodies are associated with markers of slower HIV-1 disease progression. Moreover, p24-specific antibodies have the capacity to mediate Fc effector functions and predict viral control independent of Gag-specific CD4+ and CD8+ T-cell responses or the presence of protective HLA-I alleles. Determining the mechanisms of antiviral activity of Gag-specific antibodies will provide us with a greater understanding of its potential as a target for an antibody-based vaccine, particularly in light of its more conserved nature.

Acknowledgements

We thank the study participants, the clinical and laboratory staff of the HIV Pathogenesis Programme. This work was supported by the National Institute of Health (R01 AI080289 and R01 A102660-01), the Bill and Melinda Gates Foundation CAVD (OPP1032817: Leveraging Antibody Effector Function), the Ragon Institute of MGH, MIT and Harvard, the South African Research Chairs Initiative, the Victor Daitz Foundation and an International Early Career Scientist Award from the Howard Hughes Medical Institute to T.N., National Health and Medical Research Council and the American Foundation for AIDS Research Mathilde Krim Fellowship to A.W.C.

Author contributions: A.W.C., J.M.M., P.G., B.W., T.N. and G.A. conceived the study, A.W.C., J.M.M., B.N., A.L., H.R. and Y.R. conducted experiments. A.W.C., J.M.M., M.G. and T.R. conducted the analysis. A.W.C., J.M.M., T.N. and G.A. wrote the manuscript.

Conflicts of interest

There are no conflicts of interest.

This work was presented in part at the AIDS Conference, 7–10 October 2013 in Barcelona Spain. Title; ‘p24 specific Antibody Dependent Cellular Phagocytosis in Associated with Antiviral Control in Chronic HIV-1 Subtype C Infection.’

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* Amy W. Chung, Jenniffer M. Mabuka, Thumbi Ndung’u and Galit Alter contributed equally.

Keywords:

antibody; antibody-dependent cellular cytotoxicity; antibody-dependent cellular phagocytosis; chronic; Gag; HIV-1 subtype C; natural killer cell degranulation; p24

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