ABSTRACT

HIV-1 Gag p24 has long been identified as an informative biomarker of HIV replication, disease progression, and therapeutic efficacy, but the lower sensitivity of immunoassays in comparison to molecular tests and the interference with antibodies in chronic HIV infection limit its application for clinical monitoring. The development of ultrasensitive protein detection technologies may help in overcoming these limitations. Here, we evaluated whether immune complex dissociation combined with ultrasensitive digital enzyme-linked immunosorbent assay (ELISA) single-molecule array (Simoa) technology could be used to quantify p24 in plasma samples from people with HIV-1 infection. We found that, among different immune complex dissociation methods, only acid-mediated dissociation was compatible with ultrasensitive p24 quantification by digital ELISA, strongly enhancing p24 detection at different stages of HIV-1 infection. We show that ultrasensitive p24 levels correlated positively with plasma HIV RNA and HIV DNA and negatively with CD4-positive (CD4+) T cells in the samples from people with primary and chronic HIV-1 infection. In addition, p24 levels also correlated with plasma D-dimers and interferon alpha (IFN-α) levels. p24 levels sharply decreased to undetectable levels after initiation of combined antiretroviral treatment (cART). However, we identified a group of people who, 48 weeks after cART initiation, had detectable p24 levels despite most having undetectable viral loads. These people had different virological and immunological baseline characteristics compared with people who had undetectable p24 after cART. These results demonstrate that ultrasensitive p24 analysis provides an efficient and robust means to monitor p24 antigen in plasma samples from people with HIV-1 infection, including during antiretroviral treatment, and may provide complementary information to other commonly used biomarkers.
IMPORTANCE The introduction of combined antiretroviral treatment has transformed HIV-1 infection into a manageable condition. In this context, there is a need for additional biomarkers to monitor HIV-1 residual disease or the outcome of new interventions, such as in the case of HIV cure strategies. The p24 antigen has a long half-life outside viral particles, and it is, therefore, a very promising marker to monitor episodes of viral replication or transient activation of the viral reservoir. However, the formation of immune complexes with anti-p24 antibodies makes its quantification difficult beyond acute HIV-1 infection. We show here that, upon immune complex dissociation, new technologies allow the ultrasensitive p24 quantification in plasma samples throughout HIV-1 infection at levels close to those of viral RNA and DNA determinations. Our results further indicate that ultrasensitive p24 quantification may have added value when used in combination with other classic clinical biomarkers.

INTRODUCTION

Human immunodeficiency virus (HIV) capsid p24 is a 24- to 25-kDa protein encoded by the gag gene and corresponds to the most abundant viral antigen. Detection of p24 is considered essential for the diagnosis of HIV infection, especially in individuals under preexposure prophylaxis and newly infected individuals (1). Fourth-generation antibody-antigen assays detecting p24 in addition to anti-HIV antibodies are currently recommended for HIV testing (2), as they reduce the time between infection and HIV diagnosis, allowing people with HIV (PWH) to start antiretroviral treatment as early as possible (3). Prior to the development of nucleic acid tests (NAT), p24 was used as a surrogate marker of HIV replication, disease progression, and therapeutic efficacy in the clinical monitoring of PWH (410). At primary infection, p24 levels are increased and were shown to correlate with HIV RNA (4, 1116). At chronic HIV infection, PWH positive for p24 antigen showed a higher risk of developing AIDS than those who remain antigen negative (12, 17, 18). The quantification of plasma p24 has shown superiority to RNA viral determination in anticipating decline in CD4-positive (CD4+) T cell counts in some studies (5, 18) and has shown to better correlate with CD8+ T cell activation in another (6). Moreover, p24 is sometimes detected dissociated from viral particles (i.e., in the absence of viral RNA), which might provide a more extensive estimation of the infection burden in the organism (6, 19).
However, although HIV-1 p24 is recognized as an informative biomarker, its detection by classical enzyme-linked immunosorbent assays (ELISAs) is less sensitive than HIV nucleic acid tests, in part due to the small sample volume that can be tested. In addition, after seroconversion, the development of anti-HIV humoral responses makes the detection of p24 inaccurate by immunoassays due to the formation of antigen-antibody immune complexes (1). The development of single-molecule array (Simoa) technology represents an important recent advance in ultrasensitive protein detection, reaching detection at femtomolar concentrations (20, 21). With this technology, single-molecule HIV-1 Gag p24 detection is 1,000-fold more sensitive than current available ELISAs. We have previously tested the sensitivity of this assay in HIV-infected cells and culture supernatants. We showed that viral proteins produced by a single infected cell could be detected by an ultrasensitive p24 assay, which opens new opportunities for the study of HIV pathogenesis (22). To date, the applicability of an ultrasensitive HIV p24 assay in patients’ samples was demonstrated only in serum from HIV-infected individuals during acute infection positive for HIV RNA but nonreactive for anti-HIV antibodies (23). More recently, the single-molecule digital p24 assay showed enhanced sensitivity compared to the currently approved fourth-generation antigen/antibody combination and stand-alone p24 antigen assays in a panel of serially diluted RNA-positive/antibody-negative plasma-derived culture supernatants (24). The applicability and relevance of ultrasensitive HIV p24 detection in serum/plasma samples from chronically infected individuals remains unknown.
We show here that, upon immune complex dissociation (ICD) with an approach compatible with the single-molecule assay, p24 can be detected at ultrasensitive levels in plasma from most individuals during primary and chronic HIV-1 infection. The levels of p24 dropped sharply upon antiretroviral treatment initiation but remained detectable in some individuals despite undetectable RNA viral load. The increased sensitivity of this technique could bring new insights into the predictive value of p24 as an informative biomarker of viral replication, inflammation, and treatment efficacy in people with primary and chronic HIV-1 infection.

RESULTS

Acid dissociation to disrupt antigen-antibody immune complexes is compatible with ultrasensitive p24 quantification.

Immune complex dissociation is required to increase the sensitivity of p24 antigen assays during chronic HIV-1 infection. We therefore assessed whether common methods for ICD are compatible with the digital ELISA technology. We tested (i) heat, (ii) heat with sodium dodecyl sulfate (SDS) and diethylenetriaminepentaacetic acid (DTPA), and (iii) acid dissociation with glycine-HCl, pH 1.8 or 2.5. Heat treatment denatures antibodies, but high and nonspecific loss of antigen and protein coagulation due to this procedure was reported (25). The treatment with a solution containing SDS and DTPA for diluting samples prior to heat treatment aimed at reducing these problems. SDS reduces protein coagulation by giving them a negative charge, and DTPA chelates iron and other ions, preventing further interference in the p24 assay (26).
We first evaluated the impact of these different methods on the ultrasensitive p24 standard curve. Based on the average enzyme per bead (AEB) and the shape of the standard curves obtained after different treatments for ICD, we observed that heat alone or in combination with SDS plus DTPA importantly interfered with p24 determination, flattening the standard curves and increasing the background signal in the absence of p24 (Fig. 1A and B). Higher AEB values were also observed when testing only the SDS with DTPA solution in the absence of p24 protein. The other reagents used for sample dilutions and virus inactivation did not show any impact (Fig. 1C). We next evaluated the effect of heat mediated with SDS and DTPA and acid dissociation methods in plasma from HIV-1-negative donors. We confirmed that heat mediated with SDS plus DTPA treatment, but not acid dissociation, increases the background signal for p24 detection in plasma samples (Fig. 1D). These results suggest that heat-mediated dissociation, with or without SDS plus DTPA, is not compatible with the Simoa technology and may overestimate p24 levels in clinical samples. It is possible that heating itself alters the p24 conformation or that heating-mediated protein coagulation promotes nonspecific binding of assay antibodies. Treatment with SDS/DTPA did not prevent these problems; conversely, it contributed to an increase of nonspecific signal.
FIG 1
FIG 1 Acid dissociation to disrupt antigen-antibodies immune-complexes is compatible with ultrasensitive p24 quantification. (A) Bars denote the average enzyme per beads (AEB) for 0, 0.01, 0.02, 0.07, 0.24, 0.67, 2.39, and 13.17 pg/ml of p24 standards tested in different experimental conditions for immune complex dissociation as follows: nontreated, black; heat mediated, red; heat mediated with SDS and DTPA, orange; and acid dissociation with glycine-HCl, pH 1.8, blue, or pH 2.5, violet. (B) Four-parameter logistic regression curves obtained from different experimental conditions for immune complex dissociation as follows: nontreated, black; heat mediated, red; heat mediated with SDS and DTPA, orange; and acid dissociation with glycine-HCl, pH 1.8, blue, or pH 2.5, violet. (C) AEB values obtained for standard A (p24 = 0 pg/ml, negative control) (dark gray), standard A plus Triton X-100 2% (light gray), sample diluent manufactured by Quanterix (dark blue), PBS 1× (light blue), and the solution of 7 mM SDS plus 1.5 mM DTPA pH 7.2 (orange). Dashed line indicates the median value obtained for standard A condition. (D) AEB values obtained from plasma samples from HIV-1 negative donors nontreated (gray), after heat-mediated dissociation with SDS and DTPA (orange), and after acid dissociation with glycine-HCl pH 1.8 (green). Dashed line indicates the median value obtained for nontreated condition.
In contrast, the curves obtained after acid-mediated dissociation with glycine-HCl displayed the same profile as nontreated samples (Fig. 1A, B, and D). AEB values were comparable and standard curves obtained with nontreated samples, and samples undergoing acid-mediated ICD presented equal distributions (Fig. 1A and B). Therefore, the acid-mediated ICD methods described here can be used for p24 ultrasensitive quantification by digital ELISA, while other classical methods previously validated for ICD in classical ELISAs are not compatible with this new technology. No differences were observed between glycine at pH 1.8, and pH 2.5, and we kept using the glycine, pH 1.8, as protocol for further analyses.

Acid-mediated ICD combined with usp24 assay enhances p24 quantification in plasma samples of people with HIV.

After defining the best experimental conditions to quantify the levels of p24 in the plasma, we evaluated the sensitivity of Simoa p24 detection in plasma samples from PWH. We first determined a cutoff to best discriminate between positive and negative samples by analyzing plasma from HIV-negative donors (n = 15). Based on the results obtained, we determined 24 fg/ml (calculated as 2.5 standard deviations from the mean signal in plasma from HIV negative donors; see Materials and Methods for further detail) as the cutoff to consider an ICD-treated plasma sample as positive for p24 in our experimental conditions (Fig. 2A), and we used this cutoff in our analyses. This value is higher than the lower limit of quantification (10 fg/ml) and the lower limit of detection (LoD; 3 fg/ml) proposed for the assay, likely due to interference by some blood components in protein quantification.
FIG 2
FIG 2 Sensitivity of ultrasensitive p24 quantification in plasma samples. (A) p24 signal in plasma samples of HIV-1-negative donors. Samples received acid dissociation treatment with glycine-HCl, pH 1.8. Median is indicated by black line. Dashed line denotes the cutoff value of 0.024 pg/ml calculated as 2.5 standard deviations from the mean of p24 signal in the plasma from HIV-negative donors. (B) p24 levels detected in plasma samples from HIV-1-infected individuals nontreated for immune complex dissociation (NT, open symbols) and after acid dissociation with glycine-HCl, pH 1.8 (ICD, green dots). (C) Relationship between positive p24 and HIV RNA in plasma samples from HIV-1-infected individuals. Correlation was calculated using a nonparametric Spearman test.
We next evaluated the levels of p24 in plasma samples from 25 PWH displaying a wide range of detectable viremia, ranging from 1.7 to 7 HIV-1 RNA log copies/ml. The treatment of plasma samples with glycine, pH 1.8, for ICD increased the p24 levels detected compared to nontreated samples (P = 0.0026) (Fig. 2B). After ICD, 19/25 samples had p24 levels above our experimental cutoff. While p24 was often detected in samples with viral load (VL) >104 copies/ml (18/19 positive samples), the sensitivity dropped sharply for samples with lower VL (1/6 positive samples). Indeed, we found a strong correlation between p24 values and the corresponding plasma viral loads (Fig. 2C;  r = 0.86, P < 0.0001). The correlation was still statistically significant when we considered only the samples with p24 values above the cutoff (r = 0.76, P < 0.0001). This correlation with viral RNA was not observed when we used the p24 values obtained in samples not treated by ICD (r = 0.41, P = 0.12). These analyses showed that immune complex dissociation enhanced the ultrasensitive quantification of p24 in the plasma of PWH.

Ultrasensitive p24 detection in a cohort of PWH during primary HIV-1 infection.

To validate our approach, we analyzed the ultrasensitive p24 quantification in 92 plasma samples from a subcohort of people with primary HIV-1 infection from the ANRS CO6 PRIMO cohort (27). Samples from 92 individuals displaying highly variable plasma RNA viral loads were analyzed (Fig. 3A; median, 48,263 copies/ml; range, 24 to 211,360,000). The median time since estimated HIV acquisition was 52 days (range, 20 to 183 days). We detected ultrasensitive p24 (usp24) values above the experimental cutoff in 72 of the 92 samples (Fig. 3A). There was a wide range of p24 antigen concentrations (median, 1.013 pg/ml; interquartile range [IQR], 0.027 to 191.4). As before, a strong correlation was observed between viral RNA and usp24 in the plasma samples analyzed (Fig. 3B; r = 0.7791; P < 0.0001). We also found a strong positive correlation between usp24 levels and the cell-associated HIV DNA (Fig. 3C; r = 0.64, P < 0.0001). These correlations were still statistically significant when we considered only the samples with usp24 values above the cutoff (r = 0.69, P < 0.0001 and r = 0.61, P < 0.0001 for HIV RNA levels and HIV DNA levels, respectively). A weak negative correlation was observed between usp24 levels and the CD4+ T cell counts corresponding to these samples (Fig. 3D;  r = −0.22, P = 0.03), although only a trend was found with samples with above cutoff p24 levels (r = −0.22, P = 0.07).
FIG 3
FIG 3 Ultrasensitive p24 detection in a cohort of acutely HIV-infected individuals. (A, Left) p24 levels in plasma samples from 92 individuals acutely infected with HIV-1 (ANRS PRIMO cohort). Acid dissociation with glycine-HCl, pH 1.8, was used to disrupt immune complexes. Samples below the limit of quantification were given an arbitrary value of 0.024 pg/ml based on the established cutoff. (A, Right) HIV RNA levels in plasma samples from the same 92 individuals acutely infected with HIV-1. Median is indicated by black line. (B to D) Relationship between p24 and HIV RNA (B) p24 and HIV DNA (C), and p24 and CD4+ T cell counts (D) in plasma samples from individuals acutely infected with HIV-1. Correlations were calculated using a nonparametric Spearman test.
These data showed that usp24 can be quantified after ICD in plasma samples from PWH in different stages of primary infection. Moreover, the p24 levels were associated with other classic biomarkers of HIV-1 infection.

Ultrasensitive p24 quantification in PWH at chronic HIV-1 infection prior to and after cART initiation.

ICD should enable the quantification of p24 during chronic infection despite the presence of anti-p24 antibodies. We thus quantified usp24 in plasma samples from 137 volunteers with chronic HIV-1 infection (viral loads are depicted in Fig. 4A) who were enrolled in the ANRS Reflate tuberculosis (TB) trial and initiated cART treatment. Before cART (W0), p24 values above the cutoff were detected in 98 out of 137 samples (Fig. 4A). There was again a statistically significant correlation between usp24 and the RNA viral load in the plasma samples analyzed (Fig. 4B; r = 0.42, P < 0.0001). We also found a positive correlation between usp24 levels and CD4+ T cell-associated HIV DNA levels (Fig. 4C; r = 0.32, P = 0.0004) and a trend for a weak negative correlation with CD4+ T cell counts (Fig. 4D; r = −0.15, P = 0.07).
FIG 4
FIG 4 Ultrasensitive p24 detection in a cohort of chronically HIV-infected individuals. (A, Left) p24 levels in plasma samples from 137 individuals chronically infected with HIV-1 (ANRS 12180 Reflate TB trial). Acid dissociation with glycine-HCl, pH 1.8, was used to disrupt immune complexes. Samples below the limit of quantification were given an arbitrary value of 0.024 pg/ml based on the established cutoff. (A, Right) HIV RNA levels in plasma samples from the same 137 individuals chronically infected with HIV-1. Median is indicated by black line. (B to D) Relationship between p24 and HIV RNA (B), p24 and HIV DNA (C), and p24 and CD4+ T cell counts (D) in plasma samples from individuals chronically infected with HIV-1. Correlations were calculated using a nonparametric Spearman test.
Next, we aimed to analyze usp24 values after cART treatment. The participants at the ANRS Reflate trial were coinfected with tuberculosis. They initiated antiretroviral treatment (raltegravir [400 mg], raltegravir [800 mg], or efavirenz [600 mg], with tenofovir and lamivudine) 2 to 8 weeks after treatment for TB was initiated. We compared the usp24 values prior to cART initiation to those 24 and 48 weeks after cART initiation in the 108 participants with samples available throughout the follow-up (W0, W24, and W48). There was a dramatic decrease of usp24 levels at weeks 24 and 48 after cART initiation, in line with the decrease in RNA viral load levels observed at the same time points (Fig. 5A and reference 28). The usp24 became undetectable for 89/108 participants after 48 weeks on cART (Fig. 5A). Among the 19 individuals with usp24 above the cutoff at W48, 10 also had detectable usp24 at W24. Five had detectable viral RNA loads at W48. Overall, 14/19 samples with detectable usp24 levels at W48 were characterized by persistent levels of antigen and/or transient viral RNA relapse on cART. We observed several differences when we compared the characteristics before cART initiation of these 19 participants with the characteristics of the other participants whose p24 levels were detectable before cART and dropped to undetectable levels with treatment (n = 59). Although both subgroups of participants had similar RNA viral loads and usp24 levels before treatment, we found a trend for a lower HIV RNA/usp24 ratio in the participants who maintained detectable usp24 after 48 weeks of treatment (Fig. 5B). Moreover, these participants had, before treatment, lower cell-associated HIV DNA levels (Fig. 5C) and higher CD4+ T cell counts (Fig. 5D) than the participants with undetectable usp24 levels after cART. No differences were observed for the same parameters at W48. The baseline demographic and clinical characteristics of the participants with positive or negative usp24 at W48 after cART initiation are shown in Table 1.
FIG 5
FIG 5 p24 levels in patients chronically infected with HIV prior to and after cART initiation. (A) p24 levels in plasma samples from 108 individuals chronically infected with HIV-1 (ANRS 12180 Reflate TB trial) prior to antiretroviral treatment initiation (W0) and longitudinally monitored at weeks 24 and 48 after cART. (B to D) Baseline (W0) differences between patients who presented detectable or undetectable p24 levels 48 weeks after cART initiation. (B) HIV RNA levels (left), p24 (middle), and the ratio HIV RNA/p24 (right); C) HIV DNA; D) CD4+ T cell counts. Median is indicated by black lines. **, P < 0.01; ****, P < 0.0001; ns, not significant.
TABLE 1
TABLE 1 Baseline demographic and clinical characteristics of participantsa enrolled in the ANRS 12180 Reflate TB trial with usp24 positive or negative at W48 after cART initiation
CharacteristicParticipants with positive p24 at W48 (n = 19)Participants with negative p24 at W48 (n = 89)P value
Age (median [IQR])32 (27–42)38 (31–44)0.1607
Gender (no. [%])  0.8376
°Male13 (68.4)63 (70.8) 
°Female6 (31.6)26 (29.2) 
cART arm (no. [%])  0.5580
°Efavirenz8 (42.1)32 (36.0) 
°Raltegravir (400 mg)4 (21.1)30 (33.7) 
°Raltegravir (800 mg)7 (36.8)27 (30.3) 
Response to TB treatmentb (no. [%])  0.8921
°Cure3 (15.8)13 (14.6) 
°Treatment completed16 (84.2)75 (84.3) 
°Treatment failure0 (0)1 (1.1) 
IRISc (no. [%])  0.5080
°Yes1 (5.3)9 (10.1) 
°No18 (94.7)80 (89.9) 
Resistance to ARV treatment at follow-up (no. [%])  0.6862
°Yes/potential3 (15.8)11 (12.4) 
°No16 (84.2)78 (87.6) 
Resistance to ARV treatment at W48 (no. [%])  0.4677
°Yes/potential1 (5.3)2 (2.2) 
°No18 (94.7)87 (97.8) 
AUC viral load (median [IQR])255,302 (77,868−1,002,300)180,736 (73,990–745,161)0.6597
Frequency detectable viral load (>50 copies/ml) (no. [%])   
°W49 (47.4)50 (56.2)0.4837
°W124 (21.1)18 (20.2)0.9352
°W243 (16.7)12 (13.5)0.7227
°W486 (31.6)8 (9)0.0078
a
Participants for whom samples prior to cART initiation and at 24 and 48 weeks after cART initiation were available (108 participants).
b
Response to TB treatment was defined as follows: cure, participant with negative sputum smear in the last month of treatment and on at least one previous occasion; treatment completed, participant who has completed treatment but who does not meet the criteria to be classified as a cure or a failure; treatment failure, participant with positive sputum smear at 5 months or later during treatment and also a participant with negative sputum smear before starting treatment but with positive sputum smear after completing the initial phase of treatment.
c
IRIS, immune reconstitution inflammatory syndrome; AUC, area under the concentration-time curve.
We analyzed the association of usp24 levels with some of the main plasma markers associated with inflammation and coagulation in PWH measured in the same samples (28). At W0, a weak significant correlation was observed between usp24 levels with D-dimers (r = 0.24, P = 0.006) and IFN-α (r = 0.38, P < 0.001) (Fig. 6A), and these correlations were still statistically significant when we took into consideration only the usp24 values above the cutoff (r = 0.23, P = 0.03 and r = 0.25 P = 0.02 for D-dimers and IFN-α, respectively). We did not find correlations between usp24 and C-reactive protein (CRP), interleukin 6 (IL-6), or soluble CD14 (sCD14). No correlations were found between usp24 and soluble plasma markers at W48. We then studied the subgroup of participants who remained detectable for p24 at W48. They also differed for the levels of some inflammatory markers before treatment initiation. While the participants whose usp24 levels became undetectable with cART had higher IFN-α levels at W0 (Fig. 6B), the participants whose usp24 levels remained detectable were characterized by higher plasma levels of CRP (Fig. 6C) and tended to have higher levels of D-dimers (Fig. 6D) and IL-6 (Fig. 6E). These differences were lost after 48 weeks of cART (not shown). Altogether, these results show that usp24 can be monitored throughout HIV-1 infection and may have a complementary value to other biomarkers. Moreover, the detection of p24 in the plasma of PWH under cART may reveal the presence of a subset of people with particular immunovirological characteristics.
FIG 6
FIG 6 Association of p24 levels and inflammation markers in patients chronically infected with HIV prior to and after cART initiation. (A) Relationship between p24 and D-dimers (left) and IFN-α (right) in plasma samples from 137 individuals chronically infected with HIV-1 (ANRS 12180 Reflate TB trial) prior to cART initiation (W0). Correlations were calculated using a nonparametric Spearman test. (B to D) Baseline (W0) differences between 108 patients who presented detectable or undetectable p24 levels 48 weeks after cART initiation. (B) IFN-α; (C) CRP; (D) D-dimers; (E) IL-6. Median is indicated by black lines. *, P < 0.05; **, P < 0.01.

DISCUSSION

Plasma HIV RNA and CD4+ T cell counts are commonly used biomarkers to monitor HIV-1 infection. However, these markers do not always reflect the evolution of infection, in particular, the occurrence of comorbidities or viral reactivation events during antiretroviral treatment. Here, we developed a method that allowed quantification of usp24 despite the presence of anti-p24 antibodies. We show that ultrasensitive p24 quantification is a robust virologic marker associated with HIV-1 RNA and DNA during primary and chronic HIV-1 infection, which recapitulates viral suppression observed upon cART, but may also provide added value regarding events dissociated from viral replication.
In the present work, we report a higher frequency of p24 detection in samples with viral loads higher than 103 copies/ml, which correspond to an increment of at least 1 log compared to conventional ELISA methods (23, 24, 29). Overall, we estimate that current standard immunoassays would allow p24 detection in a very small fraction (15%) of the samples included in this study. Here, the quantification of p24 was possible in >70% of tested samples. Compared to nucleic acid quantification, the sensitivity of the usp24 assay is still limited, due in part to the amount of plasma that can be tested. The inclusion of a prior ultracentrifugation step represents an increase in the hands-on time, but it efficiently increases the sensitivity of immunoassays for quantification of p24 in plasma samples with RNA viral loads at the magnitude of 102 copies/ml (data not shown).
Numerous studies have shown that chronic HIV-related immune activation and inflammation are associated with increased risk of morbidity and mortality in PWH (3035). Moreover, some plasma markers better predict evolution of HIV-1 infection than viral load or CD4+ T cell counts (3638). We found that usp24 was correlated with plasma levels of the coagulation marker D-dimer during chronic infection. We also found a correlation between usp24 and IFN-α, which could also be quantified by the ultrasensitive digital assay in the same samples. Some recent studies have shown that markers of HIV-1 transcription may be better associated with systemic inflammation than viremia or the levels of integrated proviruses (3942). The relative association of available biomarkers of infection with the levels of inflammation may depend on the various factors that sustain chronic inflammation (33, 43).
Strong correlations were found between HIV RNA and usp24 in the samples obtained during primary and chronic infection, and the overall usp24 detection rate was similar in the samples from the chronically infected cohort than in those from the people with primary HIV infection (PHI) cohort (72% versus 78%). However, while in the PHI cohort, we could detect p24 in most samples with VL >104 RNA copies/ml (64/68 samples with detectable usp24), the usp24 detection rate was lower in the chronic samples with equivalent VL (94/121 samples with detectable usp24, P = 0.008). The relative half-lives of HIV-1 p24 and RNA diverge during the course of infection (44), and the difference of antibody abundance and avidity between PHI and chronic infection could also impact the ICD efficacy (26). It was indeed previously described that ICD can be incomplete in some individuals, depending on the concentration and avidity of specific anti-HIV antibodies in the samples (6). It is also important to notice that the samples corresponding to chronic infection came from participants coinfected with HIV-1 and TB, which were characterized by higher inflammation levels than PWH with no TB coinfection, even after 48 weeks of suppressive antiretroviral treatment (28). Additional studies, including PWH without coinfections, should be done in the future to elucidate whether usp24 may in some cases better reflect viral activity in blood and/or in lymphoid tissues than viral RNA. Further studies might also analyze its potential contribution to chronic inflammation. In any case, HIV-1 usp24 and RNA levels may provide complementary readouts to monitor infection. It is reasonable to imagine that p24 levels during chronic infection can indicate the presence of viral particles, in particular, without cART, but also antigen production by cells carrying nonproductive viruses, which frequency increases with duration of infection (4547). The latter might particularly be the case during antiretroviral treatment. The half-life of p24 outside viral particles was shown to be around 42 days and thus was longer than for viral RNA, which may allow the detection of p24 even after viral replication is suppressed (13). It was indeed intriguing to observe that, in some samples, p24 remained detectable under cART despite undetectable viral RNA levels. These samples came from participants with marked immunological and virological differences before treatment initiation. The observation that, in these individuals, there was a trend toward lower RNA/p24 ratio before antiretroviral therapy (ART) initiation and that, in most cases, usp24 could be detected both in the W24 and W48 samples invites to think that these individuals might carry infected cells with higher antigen production. It is tempting to speculate that this may have led to enhanced stimulation of immune responses before treatment initiation (and perhaps at a lesser extent also under cART), which may explain higher levels of inflammatory markers at a lower frequency of infected cells at that point. Unfortunately, no analyses of T or B cell activation or HIV-specific responses were available for this study. The use of usp24 as a biomarker in HIV infection and persistence will require validation in other cohorts of PWH.
In conclusion, this study shows that digital ELISA combined with acid-ICD allows the monitoring of usp24 in individuals with primary and chronic HIV-1 infection. This novel tool may provide helpful information to monitor the burden of infection during cART. Moreover, this marker could be helpful to investigate the presence of HIV antigens in samples from participants undergoing interventions aiming at HIV remission or eradication, such as HIV reservoir reactivation in “shock and kill” strategies (48).

MATERIALS AND METHODS

Cohorts and ethics statement.

Blood samples from HIV-negative controls were obtained from the French Blood Bank (Etablissement Français du Sang) in the context of a collaboration agreement with the Institut Pasteur (C CPSL UNT; number 15/EFS/023). Plasma samples from people with acute and chronic HIV-1 infection monitored for virologic markers at the Hospital Necker-Enfants malades (France) were used to determine the efficacy of ICD and the sensitivity of the Quanterix Simoa p24 assay in plasma samples. Plasma samples from the French National Agency for Research on AIDS and Viral Hepatitis (ANRS) CO6 PRIMO cohort (49) and 12180 Reflate TB trial (50) were used to validate the protocol for ultrasensitive p24 detection combined with ICD. People with primary HIV infection (PHI) were enrolled in the ANRS CO6 PRIMO cohort following the inclusion criteria as described in reference 49. The cohort was approved by the Ile-de-France-3 Ethics Committee, and all participants gave their written informed consent. The ANRS 12180 Reflate TB trial was carried out in accordance with the ANRS Ethical Chart for Research in Developing Countries, the Brazilian regulatory requirements for clinical trials, and the Declaration of Helsinki. The protocol was approved by national and local ethics committees in Brazil (Comissão Nacional de Ética em Pesquisa [CONEP] and Comitê de Ética em Pesquisa [CEP] at IPEC/FIOCRUZ) and France (Comité de Protection des Personnes de Paris Ile-de-France-I). The experiments were conducted with the understanding and the written informed consent of each participant.

Samples.

EDTA blood samples were collected from each individual analyzed. Plasma samples were collected after centrifugation at 1,800 rpm for 20 min and stored at −80°C.

Immune complex dissociation.

Heat-mediated dissociation (51) with SDS and DTPA (26) and acid dissociation (5254) methods were tested for compatibility with ultrasensitive HIV-1 Gag p24 detection.

(i) Heat-mediated immune complex dissociation.

Plasma samples (100 μl) were diluted 1/3 with distilled water and incubated for 5 min at 95°C in a water bath and then cooled to room temperature prior to p24 quantification.

(ii) Heat-mediated immune complex dissociation with SDS and DTPA.

Plasma samples (100 μl) were diluted 1:3 with a solution of 7 mM sodium dodecyl sulfate (SDS) and 1.5 mM diethylenetriaminepentaacetic acid (DTPA), pH 7.2. Samples were incubated for 5 min at 95°C in a water bath and then cooled to room temperature prior to p24 quantification.

(iii) Acid dissociation.

Plasma samples (100 μl) were diluted 1:1 with 1.5 M glycine-HCl, pH 1.8, incubated 60 min at 37°C, and then neutralized with 1.5 M Tris-HCl, pH 9.0. In parallel, we tested 1.5 M glycine-HCl, pH 2.5, and neutralization with 1.5 M Tris-HCl, pH 7.5.

usp24 digital immunoassay.

Plasma concentration of HIV-1 Gag p24 was determined on a Simoa HD-1 analyzer using the Simoa HIV p24 kit (Quanterix, USA) following the manufacturer’s instructions. Plasma samples were thawed at room temperature, centrifuged at 3,000 rpm for 5 min, and then inactivated with Triton X-100 (final concentration, 2%) prior to p24 quantification. Four-parameter logistic (4PL) regression fitting was used to estimate the concentration of p24. Samples below the limit of quantification were given an arbitrary value of 0.024 pg/ml based on the established cutoff (cutoff was determined based on the p24 AEB signal in plasma from HIV-negative donors. It was calculated as 2.5 standard deviations from the mean of p24 AEB signal. Figure 2A represents the range of background p24 signal in HIV-negative samples, all below the established cutoff of 0.024 pg/ml).

IFN-α ultrasensitive digital immunoassay.

Plasma samples from the ANRS CO6 PRIMO cohort and the ANRS Reflate TB trial were assayed for IFN-α. Plasma samples were thawed at room temperature, centrifuged at 3,000 rpm for 5 min, and then inactivated with Triton X-100 (final concentration, 2%) prior to IFN-α quantification. Plasma concentration of IFN-α was determined on an HD-1 analyzer using the Simoa human IFN-α kit (Quanterix, USA) following the manufacturer’s instructions. Four-parameter logistic regression fitting was used to estimate the concentration of IFN-α.

Clinical parameters and inflammation biomarkers.

ANRS PRIMO cohort viral RNA loads in plasma were determined with the Cobas AmpliPrep/Cobas TaqMan HIV-1 test, v2.0 (Roche Diagnostics, Germany). Total cell-associated HIV-1 DNA was quantified with the generic HIV-1 DNA cell kit (Biocentric, Bandol, France). CD4+ T cell counts were determined by flow cytometry as previously described (49, 55). The date of infection was estimated based on the date of symptom onset minus 15 days or, in asymptomatic participants, the date of the incomplete Western blot analysis finding minus 1 month or the midpoint between a negative and a positive ELISA result (56).
The clinical parameters and inflammation biomarkers for the participants to the ANRS Reflate TB trial were determined as described in references 50 and 28. Briefly, CD4+ T cell counts were determined by flow cytometry. Plasma HIV RNA was determined either with the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test, v2.0, or the Versant HIV-1 RNA 3.0 assay (bDNA; Bayer, USA). Total cell-associated HIV-1 DNA was quantified with the Generic HIV-1 DNA cell kit. High-sensitivity C-reactive protein (hsCRP) and IL-6 levels were measured with the high-sensitivity Tina-quant C-reactive protein Gen 3 and the Elecsys IL-6 immunoassay kits, respectively. D-dimers were quantified using the STA-Liatest D-Di Plus. Human sCD14 was measured with the hCD14 Quantikine ELISA kit.

Statistical analyses.

Statistical analyses were performed using GraphPad Prism, version 8.3.1. Comparisons between groups were based on the nonparametric Mann-Whitney test. Correlations were performed using the Spearman test. Differences were considered significant at a P value of <0.05.

ACKNOWLEDGMENTS

This study was funded by the ANRS, the French National Agency of research on AIDS and Viral Hepatitis, and by MSDAvenir to A.S.-C.
This study was developed in the context of the ANRS RHIVIERA program.
We declare no competing interests.

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Information & Contributors

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Published In

cover image Journal of Virology
Journal of Virology
Volume 95Number 1424 June 2021
eLocator: 10.1128/jvi.00016-21
Editor: Frank Kirchhoff, Ulm University Medical Center

History

Received: 6 January 2021
Accepted: 23 April 2021
Accepted manuscript posted online: 5 May 2021
Published online: 24 June 2021

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Keywords

  1. HIV/AIDS
  2. HIV p24 antigen
  3. Gag p24
  4. Simoa
  5. ELISA
  6. biomarker
  7. plasma
  8. plasma marker

Contributors

Authors

Caroline Passaes [email protected]
Institut Pasteur, HIV Inflammation et Persistance, Paris, France
Héloïse M. Delagreverie
INSERM U944, Université de Paris, Paris, France
Laboratoire de Virologie, Hôpital Saint Louis, Assistance Publique-Hôpitaux de Paris, Paris, France
Véronique Avettand-Fenoel https://orcid.org/0000-0002-7022-2990
Assistance Publique-Hôpitaux de Paris, Service de Microbiologie Clinique, Hôpital Necker-Enfants Malades, Paris, France
Université de Paris, Faculté de Médecine, Paris, France
INSERM, U1016, CNRS, UMR8104, Institut Cochin, Paris, France
Annie David
Institut Pasteur, HIV Inflammation et Persistance, Paris, France
Valérie Monceaux
Institut Pasteur, HIV Inflammation et Persistance, Paris, France
Asma Essat
Centre de Recherche en Epidémiologie et Santé des Populations (CESP), Université Paris-Sud, Université Paris-Saclay, INSERM, Le KremLin-Bicêtre, Paris, France
Assistance Publique-Hôpitaux de Paris, Hôpital de Bicétre, Service de Santé Publique, le Kremlin Bicetre, France
Michaela Müller-Trutwin
Institut Pasteur, HIV Inflammation et Persistance, Paris, France
Darragh Duffy
Institut Pasteur, Translational Immunology Lab, Paris, France
Nathalie De Castro
Département de Maladies infectieuses, Hôpital Saint Louis, Assistance Publique-Hôpitaux de Paris, Paris, France
Linda Wittkop
ISPED, Inserm, Bordeaux Population Health Research Center, Team MORPH3EUS, UMR 1219, CIC-EC 1401, Bordeaux University, Bordeaux, France
CHU de Bordeaux, Pole de Santé Publique, Service d'information médicale, Bordeaux, France
Christine Rouzioux
Assistance Publique-Hôpitaux de Paris, Service de Microbiologie Clinique, Hôpital Necker-Enfants Malades, Paris, France
Jean-Michel Molina
INSERM U941, Université de Paris, Paris, France
Département de Maladies infectieuses, Hôpital Saint Louis, Assistance Publique-Hôpitaux de Paris, Paris, France
Laurence Meyer
Centre de Recherche en Epidémiologie et Santé des Populations (CESP), Université Paris-Sud, Université Paris-Saclay, INSERM, Le KremLin-Bicêtre, Paris, France
Assistance Publique-Hôpitaux de Paris, Hôpital de Bicétre, Service de Santé Publique, le Kremlin Bicetre, France
Constance Delaugerre
INSERM U944, Université de Paris, Paris, France
Laboratoire de Virologie, Hôpital Saint Louis, Assistance Publique-Hôpitaux de Paris, Paris, France
Institut Pasteur, HIV Inflammation et Persistance, Paris, France

Editor

Frank Kirchhoff
Editor
Ulm University Medical Center

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