Curtis, Kelly A PhD*; Kennedy, M Susan BS*; Luckay, Amara MS*; Cong, Mian-er MD*; Youngpairoj, Ae S BS*; Zheng, Qi MS*; Smith, James PhD*; Hanson, Debra MS†; Heneine, Walid PhD*; Owen, S Michele PhD*; García-Lerma, J Gerardo PhD*
The HIV/AIDS epidemic remains a great public health challenge. In the absence of an available HIV vaccine, much focus has been placed on the development of new biomedical interventions that can complement existing HIV prevention strategies.1 Emerging biomedical interventions include male circumcision, microbicides, and antiretroviral drugs used as either treatment to reduce infectiousness or as pre- or post-exposure prophylaxis. The use of antiretrovirals for HIV prevention has been well documented.2 Antiretroviral treatment during pregnancy or breastfeeding has proven efficacious in reducing mother-to-child transmission.3,4 Furthermore, an 80% reduction in heterosexual transmission has been observed with serodiscordant couples when the infected partner was receiving highly active antiretroviral therapy (HAART).5 An ongoing Phase III clinical trial, involving approximately 1750 couples, will evaluate the efficacy of antiretroviral therapy in preventing sexual transmission in serodiscordant couples to expand on such findings.6 Postexposure prophylaxis with antiretroviral drugs also prevented HIV transmission in healthcare workers accidentally exposed to HIV through percutaneous injury.7 One retrospective study demonstrated an 81% reduced risk of infection when the antiretroviral drug zidovudine was taken immediately after exposure.7
Pre-exposure prophylaxis (PrEP) is a novel intervention strategy to reduce the incidence of HIV in populations that are at high risk of infection. Globally, several clinical trials evaluating the safety and efficacy of oral PrEP are currently at various stages of completion.8,9 All trials are using the HIV reverse transcriptase inhibitors tenofovir disoproxil fumarate (TDF) alone or in combination with emtricitabine (FTC). The results from these trials are widely anticipated because if PrEP is found to be safe and effective, it may represent an important biomedical intervention to prevent HIV infection. Recently, the iPrEx study, a Phase III efficacy trial, demonstrated a 44% reduction in HIV-1 transmission among 2499 high-risk men who have sex with men or transgender women who received TDF in combination with FTC once daily.10 The results from this study led to the publication of a Morbidity and Mortality Weekly Report recommending interim guidelines for healthcare providers electing to provide PrEP to high-risk men who have sex with men.11
The SIV or simian/human immunodeficiency virus (SHIV)/nonhuman primate model of HIV infection has also provided valuable information regarding the efficacy of PrEP.12-14 Studies evaluating daily and intermittent PrEP regimens with TDF, FTC, or combination FTC/TDF have all demonstrated efficacy against SHIV transmission in rhesus macaques.8,13,15 Of note, however, the PrEP-treated macaques that exhibited breakthrough infections demonstrated reduced acute virus loads relative to untreated animals.13,15 These findings suggested that in the absence of complete protection, PrEP might have an additional benefit of reducing the risk of HIV transmission by suppressing acute virus loads in individuals who exhibit breakthrough infections and possibly decreasing the rate of disease progression. Little is known, however, about the impact of suppressed acute viremia on the development of early immune response to the virus or the impact this may have on disease progression.
The natural course of primary HIV infection has been characterized by stages of viral marker emergence, in which RNA and p24 antigen reach detectable levels consecutively followed by seroconversion.16 The antibody response continues to mature throughout the course of infection, leading to an increased breadth of antibody specificity and avidity as a result of ongoing exposure to viral antigens.17,18 Few studies have fully addressed the impact of early antiretroviral treatment on the maturation of the HIV-specific antibody response. Existing data suggest that the evolution of antibody avidity and/or levels may be affected.19-22
In this study, we explored the impact of PrEP on seroconversion and the maturation of the SHIV-specific antibody response. We evaluated SHIV-specific neutralizing antibody titer, binding antibody, and avidity index in rhesus macaques as a model of HIV breakthrough with PrEP.
Viral and immunologic dynamics during early infection were evaluated in a total of 23 Indian rhesus macaques infected during repeated, low-dose atraumatic rectal exposures to SHIVSF162p3. The SHIVSF162P3 chimeric virus contains the tat, rev, and env coding regions of HIV-1SF162 in a background of SIVmac239 (National Institutes of Health AIDS Research and Reference Reagent Program23). Of the 23 animals, six received daily PrEP with FTC (n = 4) or FTC/TDF (n = 2) and six received intermittent PrEP with one (n = 1) or two (n = 5) weekly doses of FTC/TDF.13,15 Drugs (10 mg/kg FTC or 22 mg/kg TDF) were administered at human equivalent doses subcutaneously or by oral gavage as previously described.13,15 Infected animals remained on PrEP for a median of 26 weeks (range, 17-34 weeks) after infection. Two animals receiving daily PrEP with FTC (AG46) or Truvada (AI54) developed the M184V/I mutation associated with FTC resistance 3 to 10 weeks after the first detectable virus RNA (data not shown)13. A control group of 11 macaques did not receive any drug treatment. Figure 1 shows the characteristics of the study population, including frequency of specimen collection and drug administration as well as time of first nucleic acid and serologic positivity. The Institutional Animal Care and Use Committee of the Centers for Disease Control and Prevention approved all animal procedures.
Simian HIV Real-Time Reverse Transcriptase-Polymerase Chain Reaction Assay
Quantification of SHIV RNA from rhesus macaque plasma was performed as previously described.12 As an internal control, 3 × 105 virus particles of the HIV-1 CM240 virus stock were added to each plasma sample before RNA extraction. Reverse transcription and amplification of SHIV and HIV-1 CM240 were carried out using primers specific for SIVmac239 gag and CM240 env, respectively.12 The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health: CM240 from Dr Nelson Michael.24-26 The limit of detection for the assay was 50 RNA copies/mL.
Neutralizing Antibody Assay
Autologous neutralizing antibody responses were measured using a cloned env-pseudotyped virus containing the SHIV162p3env gene, as previously described.27 The pseudotyped virus was produced by cotransfection of 293 T cells with an Env expression plasmid and an env-deficient plasmid (pSG3 Env) containing the luciferase reporter gene. The reduction of luciferase gene activity as compared with the virus control was measured after a single-round infection of TZM-bl cells with the plasma-treated pseudovirus. Titers of neutralizing antibodies were calculated as the reciprocal of the plasma dilution resulting in 50% reduction of the luciferase signal, as described.27 A plasma sample with a known neutralizing antibody titer was used as a positive control. Sample with values greater than 20 were considered positive for neutralizing antibody.
BIO-RAD HIV-1/HIV-2 PLUS O EIA
The presence of SHIV-specific antibody was measured using the Genetic Systems HIV-1/HIV-2 Plus O enzyme immunoassay (EIA) (Bio-Rad Laboratories, Redmond, WA) according to the manufacturer's instructions.
SHIV-SPECIFIC BIO-PLEX ASSAY
A SHIV-specific multiplex panel was created by coupling recombinant SIV p27 (mac251) and HIV-1 envelope proteins gp41 (MN), gp120 (IIIB), and gp160 (IIIB) (Immunodiagnostics, Inc, Woburn, MA) to Bio-Plex COOH microspheres (Bio-Rad Laboratories, Hercules, CA) using the Bio-Plex Amine coupling kit (Bio-Rad Laboratories). Briefly, 10 μg of recombinant protein was added to each coupling reaction containing 1.25 × 106 microspheres. For sample addition and nonspecific binding controls, coupling reactions were also performed using goat antihuman IgG (Invitrogen, Carlsbad, CA) and bovine serum albumin (Sigma-Aldrich, St Louis, MO), respectively.
Plasma samples were diluted 1:50 in phosphate-buffered saline with 1% bovine serum albumin, 0.5% polyvinyl alcohol (Sigma-Aldrich), and 0.8% polyvinylpyrrolidone (Sigma-Aldrich) and incubated for 15 minutes at room temperature on a shaker. The microspheres and prediluted plasma samples were added to a 1.2-μm filter membrane 96-well plate (Millipore, Danvers, MA) and incubated for 30 minutes at room temperature. The microspheres were washed twice with 200 μL of assay buffer (phosphate-buffered saline with 1% bovine serum albumin), resuspended in 100 μL of assay buffer containing 4 μg/mL phycoerythrin-labeled antihuman IgG (Sigma-Aldrich), and incubated for 30 minutes at room temperature on a shaker. The microspheres were washed, resuspended in assay buffer, and analyzed on a Bio-Plex 200 System (Bio-Rad Laboratories).
To control for run-to-run variation, a calibrator was included in every assay plate. The assay calibrator was created by pooling plasma samples from five different HIV-1-infected donors (ZeptoMetrix Corp, Buffalo, NY). The mean fluorescent intensity values obtained by the Bio-Plex System for the rhesus macaque plasma samples were divided by the mean fluorescent intensity of the calibrator to calculate a normalized value.
To calculate a cutoff value for each analyte of the SHIV-specific multiplex assay that distinguishes seropositive from seronegative, plasma samples from 33 SHIV-naïve rhesus macaques were tested. The cutoff values were determined using the following formula: average normalized value of SHIV seronegative samples + 2 standard deviations. Seroconversion for each individual analyte was defined as weeks at which a normalized value surpassed the cutoff value.
Calculation of Avidity Index
The Bio-Plex assay was modified by including an additional incubation step with diethylamine after incubation of the plasma samples with the microsphere mixture. Briefly, the microspheres were washed once with assay buffer and 100μL of 0.1 M diethylamine (in assay buffer) was added to one well for each sample. For comparison purposes, 100 μL of assay buffer was added to a duplicate well. The microspheres were incubated at room temperature for 15 minutes on a shaker with protection from light. All subsequent assay steps were performed as described previously.
Antibody avidity or avidity index was calculated for each sample using the following formula: (normalized value of diethylamine-treated well/normalized value of buffer-treated well) × 100.
Length of follow-up was constant across study groups. Differences in virus loads between study groups (control vs PrEP and daily vs intermittent PrEP) were evaluated by comparing the distribution of peak viremia levels (log10-transformed) using nonparametric Wilcoxon rank sum statistics.
The time interval between first RNA positive test and seroconversion was calculated for each antibody and avidity measure. Group differences in the distribution of the seroconversion interval were evaluated using log-normal regression.
Group differences in total HIV antibody (normalized values) and antibody avidity maturation were evaluated by comparing slopes estimated from implementation of a linear random coefficients model,28 Data transformations of natural logarithm of weeks since the week before the first electroimmunoassay positive test and natural logarithm of antibody and avidity values were used to linearize the data and homogenize variability.
Additionally, neutralizing antibody, peak total antibody and avidity levels, defined as the highest values achieved within 20 weeks, were compared between study groups using the Wilcoxon rank sum nonparametric test statistic.
Study animals included 23 rhesus macaques infected with SHIV162p3, 12 of which were infected during daily or intermittent PrEP treatment and 11 were untreated controls. This study population was selected from a larger group of animals based on availability of specimens with a sufficient follow-up period.13,15 The characteristics of each study subject, including first detectable RNA and EIA, duration of observation, and drug treatment, are described in Figure 1. Virus loads were monitored on a weekly basis, unless otherwise indicated, and peak virus loads were compared between study groups. As was recently reported in a study involving a larger number of animals, the median peak virus load was significantly lower in the PrEP treatment group compared with untreated controls (5.84 vs 7.54 log10 RNA copies/mL, P = 0.002; Fig 2).13 No significant difference in peak virus loads was found between the daily and intermittent PrEP treatment groups (data not shown; P > 0.05).
Time to Seroconversion
The time from first RNA positive test to seroconversion was compared between study groups. Seroconversion was defined as the first time point that a positive test result was identified by the Bio-Plex assay for each individual analyte or by the HIV-1/HIV-2 Plus O EIA. As measured by the Bio-Plex assay, median time to seroconversion for gp41, p27, gp160, and gp120 was not significantly different between the PrEP-treated animals and untreated controls (see Table, Supplemental Digital Content 1, http://links.lww.com/QAI/A176) or between daily and intermittent PrEP treatment (data not shown). Similarly, no difference was found between the study groups when seroconversion was determined by enzyme immunoassay.
Neutralizing Antibody Response
The effect of PrEP treatment on the development of the neutralizing antibody response was first evaluated by comparing the maximum neutralizing antibody titers achieved in PrEP treated and untreated controls (Fig 3). Despite the lower acute viremias seen in PrEP failures (Fig 2), maximum neutralizing antibody titers in these animals (median, 99; range, less than 20-5600) were similar to those seen in untreated controls (median, 266; range, less than 20-688; P = 0.4).
Total Binding Antibody
For semiquantitative, multiplexed detection of SIV p27 and HIV-1 gp120, gp160, and gp41 binding antibody, a modified Bio-Plex assay was used. The normalized values achieved over the course of the study for each study group are displayed in Figure 4A. Each analyte exhibited a distinctive range and magnitude of antibody response. No significant difference in the rate of change of normalized values for any of the four analytes was found between the PrEP-treated subjects and untreated controls or between the daily and intermittent PrEP treatment groups. Additionally, peak normalized values achieved during the 20 weeks since seroconversion were compared (see Figure, Supplemental Digital Content 2, http://links.lww.com/QAI/A177). No statistical significance in the distribution of peak values was found between study groups.
The effect of PrEP treatment on the maturation of the antibody response was also evaluated by comparing SHIV-specific antibody avidity between study groups. The Bio-Plex assay was used to calculate an avidity index for anti-p27, gp120, gp160, and gp41. The avidity index values for each individual subject are shown in Figure 4B. All four analytes exhibited significantly lower maturation of avidity index values in the PrEP groups as compared with the untreated controls (P < 0.05). The rate of avidity maturation for gp160 and gp41 was significantly lower in the daily as compared with the intermittent treatment group (P = 0.04 and 0.008, respectively). Peak avidity index values achieved during the 20 weeks postseroconversion were significantly lower for p27 and gp160 (see Figure, Supplemental Digital Content 2, http://links.lww.com/QAI/A177). No difference in peak avidity index values was observed between the daily and intermittent treatment groups (data not shown).
PrEP is a novel HIV intervention strategy that is currently being evaluated in several clinical trials.9,13 A recently completed trial evaluating the efficacy of daily Truvada (TDF + FTC) among men who have sex with men has provided the first indication that oral PrEP is protective in humans.10 In this trial, the incidence of HIV-1 was reduced by 44% among participants who took Truvada; efficacy was substantially higher (73%) among study participants who reported greater than 90% adherence.10 Ongoing clinical trials in different high-risk populations will demonstrate whether PrEP is protective against HIV acquisition by other routes of transmission.8,9 Because it is unlikely that PrEP will prove 100% efficacious, it is essential to understand the impact of PrEP on the development of the immune response to HIV for individuals who become infected despite treatment. In this study, we evaluated the effects of PrEP treatment on seroconversion and maturation of antibody responses against multiple structural and functional antigens in a nonhuman primate model. The use of the SHIV/nonhuman primate model has provided us with a unique opportunity to monitor virus loads and antibody responses longitudinally at defined intervals, allowing precise determination of viral and immunologic dynamics during acute infection.
As was recently reported with a larger number of animals, acute infections in the context of PrEP were associated with blunted acute viremia.13 The attenuated viremia seen in PrEP breakthroughs is noteworthy and likely reflects continued and strong antiretroviral activity by the PrEP regimen.13,15 Our findings showing that the frequency and magnitude of autologous neutralizing antibody responses in PrEP failures and controls were similar also suggests that the attenuated viremia resulting from PrEP may not be sufficient to impact the early development of neutralizing antibody responses. Interestingly, we recently noted in macaques that PrEP with FTC or Truvada significantly limited env sequence evolution and diversification during early infection, likely reflecting the antiviral activity of drugs in reducing virus replication. It will be important to evaluate if such limited virus evolution will reduce the ability of the virus to escape from neutralizing antibody responses.29
The appearance of virus-specific antibody was monitored using a conventional EIA and for a semiquantitative measure of binding antibody, a modified Bio-Plex assay was used that measures the response to multiple antigens in a single test. Regardless of assay format, PrEP treatment did not affect the timing of seroconversion as defined in this study, indicating sufficient antigenic stimulation for a B cell response to develop despite lower virus loads. This is an important finding, because the ability to detect HIV infection using standard serological assays is crucial for evaluation of PrEP efficacy in future or ongoing clinical trials. Additionally, the pattern of HIV-1 antibody appearance was similar to that observed in HIV-infected humans with gp41 among the first to develop and gp120 appearing approximately 1 week later.30 Some studies have suggested that low virus loads associated with HAART alter antibody production by decreasing titers or preventing antibody responses to certain viral antigens, possibly as a result of the lack of ongoing antigenic exposure.19,31 In contrast, we found no detectable difference in neutralizing and binding antibody levels between PrEP-treated and untreated subjects. It is important to note that HAART regimens usually decrease virus replication below the limit of detection of the more sensitive RNA assays. In contrast, the PrEP failures still exhibited significant levels of virus replication, which are likely to be sufficient to elicit a strong and sustained immune response.
Although antibody quantity was not affected by PrEP treatment, a significant impact on the maturation of antibody avidity was observed for all analytes evaluated. These results are interesting for a number of reasons. First, antibody avidity is used in some assays to measure HIV incidence and, thus, altered kinetics of avidity maturation may adversely affect seroepidemiologic studies on HIV incidence.32-34 Second, similar delays in antibody maturation have been noted in patients treated with HAART during acute HIV infection.21,22 Although the mechanism responsible for the observed delay is not known, it has been suggested that reduced virus replication might limit the production of cytokines that stimulate B cells such as interleukin-6 and interferon.22,35 Supporting this observation is the increase in avidity index noted in acute HIV-infected persons after discontinuation of HAART.21 The clinical implications of these findings are not known because nonprogressing elite controllers have been shown to be immunologically heterogeneous, some patients exhibiting immature antibody avidity profiles.36 Interestingly, a significantly lower avidity maturation rate was also observed for gp160 and gp41 in the daily versus intermittent PrEP group. Because a relatively small number of animals were included in each treatment group, further studies are needed to evaluate the relevance of these findings.
One limitation of this study is that significant differences in total antibody levels between study groups may be difficult to observe as a result of large individual variations in antibody responses and limited numbers of study animals. Additionally, the SHIV/nonhuman primate model provides only a limited ability to evaluate the impact of PrEP on disease progression, because a less pathogenic virus (SHIV162p3) was used in relation to HIV infection of humans. In addition to the altered kinetics of antibody avidity maturation seen here, macaques that failed PrEP demonstrated higher CD4+ T cell counts and more multifunctional, diverse SHIV-specific T cell responses.37 These findings suggest that PrEP regimens might also improve T cell immunity even when infection is not prevented, thus having the potential to impact virus control and transmissibility after PrEP discontinuation. All of these observations highlight the need to closely monitor immune parameters and their significance during current PrEP human clinical trials.
In summary, we used a macaque model of SHIV transmission to explore the potential impact that PrEP may have on early antibody responses. The finding that time to seroconversion, as defined by detection with EIA or Luminex, was not affected by ongoing drug exposure was reassuring and suggested that PrEP may not impact the ability of current serologic diagnostic assays to detect HIV infection. The delayed maturation of antibody avidity raises the concern that PrEP may affect the performance of assays that measure HIV incidence based on the kinetics of antibody avidity 32,33,38 and suggest that alternative testing strategies may be required for serosurveillance studies.
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