Altered Antibody Responses in Persons Infected with HIV-1 While Using Preexposure Prophylaxis

Abstract

Preexposure prophylaxis (PrEP) is an effective HIV prevention tool, although effectiveness is dependent upon adherence. It is important to characterize the impact of PrEP on HIV antibody responses in people who experience breakthrough infections to understand the potential impact on timely diagnosis and treatment. Longitudinal HIV-1-specific antibody responses were evaluated in 42 people who inject drugs (PWID) from the Bangkok Tenofovir Study (BTS) (placebo = 28; PrEP = 14) who acquired HIV while receiving PrEP. HIV-1 antibody levels and avidity to three envelope proteins (gp41, gp160, and gp120) were measured in the plasma using a customized Bio-Plex (Bio-Rad Laboratories, Hercules, CA) assay. A time-to-event analysis was performed for each biomarker to compare the distribution of times at which study subjects exceeded the recent/long-term assay threshold, comparing PrEP and placebo treatment groups. We fit mixed-effects models to identify longitudinal differences in antibody levels and avidity between groups. Overall, longitudinal antibody levels and avidity were notably lower in the PrEP breakthrough group compared to the placebo group. Time-to-event analyses demonstrated a difference in time to antibody reactivity between treatment groups for all Bio-Plex biomarkers. Longitudinal gp120 antibody levels within the PrEP breakthrough group were decreased compared to the placebo group. When accounting for PrEP adherence, both gp120 and gp160 antibody levels were lower in the PrEP breakthrough group compared to the placebo group. We demonstrate hindered envelope antibody maturation in PWID who became infected while receiving PrEP in the BTS, which has significant implications for HIV diagnosis. Delayed maturation of the antibody response to HIV may increase the time to detection for antibody-based tests.

Clinical Trial Registration Number, NCT00119106.

Introduction

Preexposure prophylaxis (PrEP) has been shown to be an effective prevention strategy for populations at high risk for HIV infection, including men who have sex with men, serodiscordant couples, and people who inject drugs (PWID).^1–4 Truvada^®, a Food and Drug Administration (FDA)-approved combination of tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC), is recommended as a daily oral prophylactic for people at high risk for HIV infection to prevent infection.^2,5,6 Studies have shown that consistent daily PrEP use significantly decreases the risk of HIV transmission and that concentrations of tenofovir (TFV) in the blood correlate with protection.^7–9

The Bangkok Tenofovir Study (BTS) investigated the efficacy of daily oral TDF in preventing HIV transmission in PWID.^3,9,10 A 49% reduction in transmission was observed in the PrEP treatment study group compared to the placebo group, which increased to an 83.5% reduction when adjusted for adherence.⁹ These data confirm that although effective at reducing HIV transmission, HIV infection in the presence of PrEP is still possible due to variable adherence or other biological factors.^11,12 As PrEP use expands, the impact of PrEP on the early immune response to HIV and timely diagnosis needs to be explored fully.

HIV infection is characterized by the sequential emergence of viral markers in the host, beginning with viral RNA, p24 antigen, and finally seroconversion to HIV antibody.¹³ The antibody response continues to mature throughout the course of infection as a result of ongoing exposure to viral antigens, leading to an increased breadth of HIV antibody specificity and avidity.^14–16 The distinct stages of HIV biomarker positivity have been used to diagnose and characterize the disease state during the course of HIV-1 infection.^13,17

People who initiate antiretroviral therapy early in the course of HIV infection may have reduced viral loads and delayed detection of infection using traditional antibody-based diagnostic platforms.^18–20 An altered immune response to the virus could prove to be problematic for the accurate diagnosis of HIV in individuals who exhibit breakthrough infections and become infected while receiving PrEP. In a Simian HIV rhesus macaque model of HIV transmission, oral or subcutaneous PrEP led to reduced viral loads and blunted antibody avidity maturation in animals that exhibited breakthrough infection.²¹ Furthermore, women who became HIV infected while using an intravaginal TFV gel in the CAPRISA 004 PrEP randomized trial demonstrated delayed antibody avidity maturation.²²

Traditionally, HIV antibodies have been measured using the western blot, enzyme-linked immunosorbent assay or enzyme immunoassay (EIA). The development of customizable bead-based assays such as the HIV-1 Multiplex assay, which is based on the Bio-Plex platform (Bio-Rad Laboratories, Inc., Hercules, CA), allows for the sensitive detection of multiple HIV antibody responses using minimal sample volume.^23,24 The objective of this study was to determine the potential impact of PrEP on the development or maturation of serological biomarkers, which may impact the detection of HIV-1 infection by serology-based diagnostic tests. We evaluated antibody levels and avidity in a longitudinal cohort of PWID from the BTS using our customized HIV-1 Multiplex assay.²³ Time to seroconversion and longitudinal HIV-1 antibody responses were compared between the PrEP and placebo treatment groups to determine the impact of PrEP during breakthrough infection. As PrEP use expands, such studies are of critical importance to inform diagnostic strategies needed for the accurate diagnosis of HIV infection in people taking PrEP.

Methods

Study population

Forty-two persons who seroconverted to HIV-1 during the BTS were evaluated in this study, based on remaining sample volume of archived specimens.^3,10 Study participants, enrolled between June 2005 and July 2010, received daily oral TDF (n = 14) or placebo (n = 28). The BTS preceded the U.S. FDA approval of Truvada for PrEP and World Health Organization's initial recommendations for PrEP use, both released in 2012 (Ref.²⁵). PrEP adherence and HIV infection were monitored throughout the trial participation.

Study participants were tested for HIV infection at enrollment and monthly visits using oral fluid OraQuick Rapid (OFOQ) HIV-1/2 Antibody test (OraSure Technologies, Inc., Bethlehem, PA). For participants with a nonreactive OFOQ test, whole blood was collected at enrollment, months 1, 2, and 3, and every 3 months thereafter. For participants who had a reactive OFOQ test result during the trial (both PrEP and placebo groups), whole blood was collected at the time of test positivity, months 1, 2, and 4, and every 4 months thereafter. HIV infection was confirmed by EIA (Genetic Systems HIV-1/HIV-2 Plus OEIA; Bio-Rad, Redmond, WA) and western blot (Bio-Rad) using whole blood. For persons confirmed HIV positive, stored whole blood was tested for HIV-1 RNA (COBAS TaqMan, Roche Molecular Systems) to determine viral load and the last HIV-negative and first HIV-positive specimens.

In this substudy, plasma samples collected at baseline (last HIV-negative test), first positive nucleic acid test (NAT), first positive OFOQ test, and postseroconversion (months 1, 2, 4, and every 4 months thereafter) were available for most participants in both PrEP and placebo groups. PrEP adherence was determined based on previously collected data on the detection of TFV in the plasma at the time of seroconversion³ or the first positive NAT.²⁶ Plasma TFV concentrations were measured, using ultrahigh-performance liquid chromatography tandem mass spectrometry with a lower limit of detection of 0.31 or 5 ng/mL, as part of the original study³ and upon receipt of the specimens at the U.S. Centers for Disease Control and Prevention (CDC) for a follow-up study, respectively.^3,26,27

A study participant was considered adherent if TFV was detected in the plasma at the time of the original study or upon testing of the specimens at the CDC. Plasma TFV levels measured at the CDC have been published elsewhere.²⁶

The Thailand Ministry of Public Health, Bangkok Metropolitan Administration Ethical Review Committees and CDC Institutional Review Board approved the study. All samples included in this substudy were unlinked from personal identifiers and, upon CDC review, it was determined that the CDC was not engaged in human subject research.

HIV-1 Bio-Plex assay

The HIV-1 Bio-Plex assay was performed on plasma samples, as described.^23,28 Magnetic carboxylated beads (Bio-Rad Laboratories) were coupled to the recombinant HIV-1 proteins p24, gp120, gp160, and gp41 (Immunodiagnostics, Inc., Woburn, MA). The recombinant proteins were derived from subtype B strains HIV-1 IIIB (p24, gp120, and gp160), and HIV-1 MN (gp41). All plasma samples were tested in duplicate, with and without diethylamine for measurement of avidity. Normalized mean fluorescent intensity (MFI) values (n-MFI or -n) and avidity index (AI or -a) were calculated as previously described.^23,28 High and low positive controls were included in each run to validate the assay performance and assess interrun variation. The control samples were each an individual patient specimen selected from a previously characterized proficiency testing panel composed of serum from HIV-1-positive individuals.²⁹

Statistical analysis

Differences in median plasma HIV-1 viral loads (RNA copies/mL) at first positive NAT between the PrEP and placebo groups were evaluated using the Wilcoxon rank sum test. A Wilcoxon rank sum test was also used to compare the median time from HIV-1 RNA detection to seroconversion for PrEP and placebo groups as measured by the OFOQ and Bio-Plex analytes. Study subjects were excluded from the analysis if the first RNA-positive specimen was not available for antibody testing or first RNA and antibody positive test dates were the same. The EIA was positive at the time of the first NAT positive date for 79% (22 out of 28) of the placebo and 86% (12/14) of the PrEP participants; therefore, time to seroconversion as measured by EIA was not included in the analyses. We conducted all analyses using SAS version 9.4.

A comparison of HIV-specific antibody responses between the PrEP and placebo groups, as measured by the HIV-1 Multiplex assay, was performed using two separate statistical methods. A time-to-event (survival) analysis was performed for each analyte to compare the distribution of times, at which study subjects reached a specific assay threshold between PrEP and placebo treatment groups (n-MFI value or AI) (Fig. 1). The assay threshold corresponds to a timepoint in the course of HIV-1 infection used to define “recent” HIV-1 infection based on the HIV-1 Multiplex assay parameters. The recency threshold or cutoff was chosen to evaluate HIV-1 antibody responses at a common timepoint or frame of reference between the study groups; the point where a person transitions from an assay determination of “recent” to “long-term” HIV-1 infection.

FIG. 1.

Survival analysis comparing the distribution of times, at which study subjects exceeded the recent/long-term assay threshold between PrEP and placebo treatment groups. Product limit survival estimates for gp120 and gp160 antibody levels (A), and for gp120, gp160, and gp41 antibody avidity (B) by time after viral RNA positivity. The PrEP and untreated control group are displayed in solid red and blue lines, respectively, with 95% confidence intervals shaded. -a, avidity; -n, normalized value; PrEP, preexposure prophylaxis.

The analyte cutoffs that were used for this analysis are as follows: gp120-n = 2.8, gp160-n = 2.6, gp120-a = 20%, gp160-a = 40%, and gp41-a = 65%. Gp41-n was not considered for the evaluation, as antibodies to gp41 peak early in the course of infection and do not distinguish recent versus long-term infection.²³

For visual comparison of longitudinal antibody responses between treatment groups, smooth curves were plotted using the scatterplot smoother locally estimated scatterplot smoothing (LOESS) for each analyte (Fig. 2). Longitudinal differences in antibody levels and avidity between groups were evaluated using two mixed-effects models for repeated measures (Table 2). Treatment group was included as an independent variable within the first model. The second model recategorized the treatment group into control group, PrEP adherent group, or PrEP nonadherent group, and included viral load at first NAT positive date categorized by group [<1,000 (reference group), 1,000–10,000, or >10,000/RNA copies/mL].

FIG. 2.

Longitudinal HIV-1 envelope antibody responses among BTS participants. Antibody levels (A) and avidity index values (B) for each individual subject are plotted for the PrEP treatment (red lines) and untreated control (blue lines) groups for gp120, gp160, and gp41. LOESS curves for the PrEP and untreated control group are displayed as bold red and blue lines, respectively. BTS, Bangkok Tenofovir Study; LOESS, locally estimated scatterplot smoothing.

Table 2.

Comparison of Longitudinal Antibody Response Between Preexposure Prophylaxis and Placebo Treatment Groups in the Bangkok Tenofovir Study (p values)

Analyte	Placebo vs. PrEP	Placebo vs. PrEP adherent	Placebo vs. PrEP nonadherent	Placebo vs. PrEP VL >10,000	Placebo vs. PrEP VL 1,000–10,000	Placebo vs. PrEP VL <1,000
gp120-n	0.012	0.002	0.962	0.186	0.296	0.758
gp160-n	0.156	0.005	0.312	0.153	0.368	0.722
gp41-n	0.801	0.205	0.145	0.389	0.271	0.900
gp120-a	0.305	0.780	0.977	0.762	0.778	0.234
gp160-a	0.631	0.109	0.064	0.213	0.279	0.276
gp41-a	0.600	0.334	0.234	0.908	0.706	0.183

Results

Detection of HIV biomarkers

The median time from HIV-1 RNA detection to seroconversion, as measured by OFOQ, was 56 days [interquartile range (IQR) = 29–128; N = 17] for the placebo group and 224 days (IQR = 56–448; N = 9) for the PrEP group (Table 1; p = .0187). For gp120, the median time from RNA detection to seroconversion was 42 days (IQR = 28–84; N = 7) for the placebo group and 56 days (IQR = 29–105; N = 6) for the PrEP group with a p = .5122 testing for a difference between the two groups. The majority of study participants in both the placebo group (82%, 23/28) and PrEP group (93%, 13/14) had detectable antibodies to gp160 and gp41, as measured by the HIV-1 Multiplex assay, at the same time as the first NAT positive date or the first NAT-positive specimen was not available for testing; therefore, time to seroconversion could not be accurately assessed. The median plasma RNA copies/mL (log10) for the placebo group at the first detectable NAT was 5.34 versus 4.02 for the PrEP group (p = .0023).

Table 1.

Characteristics of Bangkok Tenofovir Study Participants

	Placebo (N = 28)	PrEP (N = 14)	p
Female/male	6/22	2/12	ND
Median follow-up visits (minimum to maximum)	11 (6–21)	10.5 (5–21)	ND
Median follow-up time in days (minimum to maximum)	784.5 (113–2,210)	1,178 (504–2,294)	ND
Median time to seroconversion-OraQuicka	56 (IQR = 29–128)	224 (IQR = 56–448)	.0187
Median time to seroconversion-gp120b	42 (IQR = 28–84)	56 (IQR = 29–105)	.5122
Median VL at 1st RNA positive (log10 RNA copies/mL)	5.34	4.02	.0023
TDF detected in plasma/serum	NA	8	ND

^aMedian days between 1st HIV RNA positive and OraQuick test (N = 17 placebo, N = 9 PrEP).

^bMedian days between 1st HIV RNA positive and Bio-Plex gp120 analyte (N = 7 placebo, N = 6 PrEP).

IQR, interquartile range; NA, not applicable; ND, not done; PrEP, preexposure prophylaxis; TDF, tenofovir disproxil fumarate; VL, viral load.

HIV-1 antibody maturation

A summary of the time-to-event analyses, comparing the proportion of study participants with a HIV-1 Multiplex assay n-MFI or AI that reached a recent/long-term assay threshold, is shown in Figure 1. The PrEP group exhibited a delay in reaching the HIV-1 Bio-Plex assay cutoffs compared to the placebo group for gp120-n (log-rank p < .0001), gp160-n (log-rank p = .0002), gp120-a (log-rank p < .0001), and gp160-a (log-rank p = .0014) antibody responses. There was no observed difference between the placebo and PrEP groups for gp41-a (log-rank p = .596).

Longitudinal anti-HIV IgG antibody responses for each study participant in the placebo and PrEP groups are shown in Figure 2. The LOESS curves demonstrate the trends in antibody reactivity over time for each of the study groups. An overall increase in antibody levels (n-MFI) and avidity (AI) was observed over time from the first RNA-positive test result for gp41, gp120, and gp160. Gp120 n-MFI and AI showed a notable distinction between the PrEP and placebo groups throughout the course of infection, with the PrEP group exhibiting lower n-MFI and AI values compared to the placebo group. Antibody levels also appeared to be lower in the PrEP group for gp160-n, gp160-a, and gp41-a, when visually comparing LOESS curves.

Differences in the longitudinal antibody responses between treatment groups are summarized in Table 2. The PrEP group exhibited lower gp120 antibody levels compared to the placebo group (p = .012); however, no significant difference was observed for all other analytes. When accounting for PrEP adherence, PrEP users exhibited lower gp120 (p = .002) and gp160 antibody levels (p = .005) compared to the placebo group; no significant differences were observed among viral load (VL) groups.

Discussion

In this study, we demonstrated the impact of daily oral TDF on the development of the HIV antibody response in a longitudinal Thai cohort of PWID. Using two distinct statistical methods to evaluate antibody responses, our results suggest that PrEP delays the development or detection of antibodies to HIV envelope, particularly those directed to gp120. The statistical methods used in this study allowed for a comparison of antibody responses at an early point in the course of HIV infection and longitudinally, throughout the course of sample collection. We observed that, compared to the placebo group, PrEP was associated with lower levels of gp120 antibodies throughout the course of the BTS study, as well as gp160 antibody levels when accounting for adherence. These data support prior studies suggesting a delay in HIV envelope antibody maturation^21,22 and highlight the importance of evaluating testing strategies for individuals on PrEP.

Recently, for the 53 HIV-1 seroconverters enrolled in the BTS, there was a reported median delay of 126.5 days for OFOQ test seroconversion relative to the estimated time of infection.³⁰ The delay in test positivity was associated with participant assignment to the PrEP study arm. Similar findings were reported by Suntharasamai et al., although differences in the timing of OFOQ positivity were also noted between subtypes.³¹ Here, we confirmed a delay in the detection of seroconversion by the OFOQ in a subset of 42 seroconverters from the BTS, when comparing the placebo to PrEP treatment study groups.

In many cases, HIV antibody was detected at the time of first NAT-positive result indicating that the sample collection intervals were not frequent enough to assess whether time to detection was delayed with the EIA and most HIV-1 Bio-Plex assay analytes. This prevented measuring the interval between detection by NAT and seroconversion. It has been reported that the OFOQ demonstrates delayed detection of HIV relative to laboratory/rapid tests requiring plasma or whole blood, including the OraQuick used with whole blood; therefore, it was not surprising to observe the significant delay in detection of seroconversion in the BTS.^32–34 However, in the Partners study, a randomized clinical trial of oral PrEP in Kenya and Uganda, PrEP was associated with delayed detection of HIV by whole-blood rapid tests.³⁵

Given that it can be difficult to capture the relatively short period of time between HIV acquisition and detection by diagnostic tests, evaluation of longitudinal antibody responses is useful for the characterization of the impact of PrEP on the evolution of markers that are commonly used for HIV diagnosis.

An advantage of the customized HIV-1 Bio-Plex assay is the ability to detect multiple analytes and to concurrently measure both antibody levels and avidity. The assay components primarily included HIV envelope antigens, as gp120, gp160, and/or gp41 are represented in most antibody-based diagnostic tests. Although we observed decreased antibody responses to envelope markers, particularly gp120, it is not known whether the impact of PrEP on the immune response is unique to the envelope. HIV-1 p24 antibody was included in initial analyses; however, p24 antibody responses tended to be highly variable throughout the course of infection and among individuals (data not shown).

In two prior studies evaluating the impact of PrEP on the antibody response, delayed maturation of antibody avidity was observed in the PrEP breakthrough groups although antibody levels were not significantly different.^21,22 Here, we found delayed development in both gp120 and gp160 longitudinal antibody responses when accounting for adherence. Several factors may explain the differences in findings between these studies, including differences in modality (topical vs. oral), study model (monkey vs. human study), and adherence. In addition, variation in the immune response among individuals may contribute to the difficulty in discerning statistically significant differences between groups without a large number of subjects. A limitation of our study is the relatively small sample size included in the study analyses.

It is well documented that antiretroviral use during early HIV infection leads to reduced viral loads and antibody levels.^19,36–38 In the presence of PrEP, it is plausible that detection of seroconversion is delayed due to PrEP-induced virus suppression. We noted a significant decrease in VL in the PrEP breakthrough group at the time of first positive RNA test; however, an association between VL and delayed development of the HIV envelope antibody response was not found. In the BTS, PrEP was discontinued at the time of diagnosis; therefore, virus suppression due to PrEP was unlikely to be sustained. Ruone et al. reported that PrEP exposure in breakthrough infections from the CDC TDF2 PrEP trial with daily FTC/TDF limited HIV env diversity; however, a similar analysis in seroconverters from BTS study found no effect with single agent TDF.^26,39

Regardless of the mechanism, it is clear that PrEP has some impact on the development of the immune response. It is yet to be determined whether this impact will lead to significant challenges in diagnosing newly infected individuals who exhibit breakthrough infections on PrEP.

In summary, delayed maturation of the antibody response to HIV may prolong the time to detection for antibody-based tests; therefore, further research may be needed to evaluate diagnostic test performance in populations with high PrEP coverage.

Author Disclaimer

The findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the U.S. Centers for Disease Control and Prevention.

Author Disclosure Statement

J.G.G-L is named in U.S. Government patents on “Inhibition of HIV infection through chemoprophylaxis”, and in U.S. Government patent applications on “HIV postexposure prophylaxis” and “HIV pre-exposure prophylaxis”. The other authors declare no conflicts of interest.

Funding Information

This work was supported by CDC intramural funds.