Sexual intercourse represents one of the major routes for HIV transmission in both men and women1 and especially among men who have sex with men (MSM).2 The colorectal mucosa is the primary portal of entry for HIV in MSM, and the risk of transmission during unprotected anal intercourse has been estimated to be at least 10 times greater than that for vaginal intercourse.3–5 Effective prevention strategies are urgently needed to block anal rectal transmission. In addition to mucosal vaccine development, topical preexposure prophylaxis with proper antiretrovirals (ARVs) has been shown as promising alternative.6–14
Similar to highly active antiretroviral therapy (HAART), ARVs that exert effects at different phases during HIV infection, including receptor (miniCD415) and coreceptor binding [maraviroc (MVC)9,10], viral fusion,11–14 reverse transcription [tenofovir (TFV)6–8], integration,16 protein clipping (indinavir),17 have been formulated into microbicides and explored to prevent mucosal transmission. Among which, TFV is one of the most extensively evaluated ARVs. Vaginally applied TFV gel was shown to be able to reduce HIV-1 acquisition by 54% in women with high adherence in the CAPRISA 004 clinical trial.18 Moreover, TFV also has been shown to prevent rectal transmission under single high-dose challenge circumstances in both monkey19 and BLT mouse6 models.
Of note, most previous studies focused on the efficacy for the prevention from HIV/simian immunodeficiency virus (SIV) infection and pharmaceutical characterization of candidate microbicides.20,21 No attention actually has been paid to their potential influences on the immune system. Indeed, the topical application of microbicides may exert influences on the local microbiota22,23 and thereby alter the microenvironment, including the microimmune environment. In addition, the coexistence of microbicides and pathogens (such as HIV and other viruses) at mucosal sites could occur during the repeatedly application of microbicides, and the microbicide may modify the immune properties of pathogens and thereby regulate pathogen-specific immune responses. In addition, any influence from microbicide on the mucosal pathogen-specific immune responses may serve as a priming and could be further amplified into significant differences in both mucosal and systemic immune responses by a subsequent systemic infection and thereby alter the disease progress and survival.
We tested this concept in a 2-phase study. In the first phase, we tested the prophylactic efficacy of microbicides formulated with TFV, sifuvirtide (SFT), and MVC in Chinese rhesus macaques in repeated challenge with low-dose Simian-Human Immunodeficiency Virus (SHIV-1157ipd3N4). This creates a setting with repeated coexistence of microbicide and SHIV, which amplifies the chances for microbicides to modify the immune properties. SFT is a novel HIV-1 fusion inhibitor,24 whose in vitro efficacy and safety have been evaluated in our previous work.25 In the second phase, all monkeys were rechallenged with a single high dose of SIVmac239 to determine whether and how the previous microbicide efficacy study could influence subsequent infection and its survival outcome. To simplify our model, we selected SIVmac239 as our challenge virus, which will allow us to minimize the effects from neutralizing antibodies and to mainly examine the potential T-cell effects from the first phase study.
The study protocol (No. ILAS-VL-2012-002) was approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (ILAS, CAMS). All animal experimental procedures were performed in an Animal Bio-Safety level 3 (ABSL-3) laboratory, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), International. This study was performed in strict compliance with the “Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Science (est. 2006)” and “The use of nonhuman primates in research of the Institute of Laboratory Animal Science (est. 2006)” to ensure personnel safety and animal welfare. All nonhuman primates used in this study were negative for SIV, Simian Retrovirus (SRV), Simian T-Lymphotropic Virus (STLV), B virus, tuberculosis, parasites (eg, Entameba), in accordance with national regulations (GB14922-2001).
Sixteen Chinese rhesus macaques were equally divided into 4 groups. Only one Mamu-A*01–positive (MHC class I typing was performed by allele-specific polymerase chain reaction as described26) monkey was involved in this study, and it was grouped into the SFT-treated group. Candidate microbicide active components (MVC, TFV, and SFT) were formulated with hydroxyethyl cellulose (HEC) gel, and the dose and inoculation route are shown in Table 1. Monkeys were pretreated with microbicides or placebo and 30 minutes later challenged with 10 TCID50 [titrated with rhesus peripheral blood mononuclear cells (PBMCs)] SHIV-1157ipd3N4 intrarectally. SHIV-1157ipd3N4, carrying the HIV-1 clade C env gene, was kindly provided by Dr R. Ruprecht (obtained through the NIAID, NIH). In week 39, the monkeys were rechallenged with 1 × 105 TCID50 SIVmac239 intravenously or intrarectally (see Table S1, Supplemental Digital Content, https://links.lww.com/QAI/A752) without microbicide treatment. Peripheral blood was routinely collected to monitor plasma viral load and immune responses (Fig. 1A). Monkeys were followed up for 104 weeks (2 years) to determine their disease progression.
Microbicide Candidates and Repeated Low Dose SHIV Challenge
The concentrations of TFV and MVC used in our microbicides were in accordance with the clinical dosage.18,27 SFT level in the microbicide was determined by the consideration of both efficacy and safety. Our cell experiments in vitro showed that IC90 is ∼1 μM at cell experiments, and we extrapolate the effective concentration in vivo at 1–10 mM (103–104 folds to cell experiments). In safety evaluations, we identified that the maximum nontoxic concentration of SFT used in local mucosal tissue should not exceed 1 mM; otherwise, it would induce abnormal inflammatory response.25 Therefore, 1 mM SFT was chosen as the concentration used in the microbicide.
Microbicides gels were prepared according to a procedure described previously.25 Briefly, SFT, TFV, and MVC were first dissolved in phosphate-buffered saline (PBS) (pH = 4.5) at the indicated concentration, to which HEC was slowly added for a final concentration of 1.5%. While adding HEC, the solution was rapidly stirred until a translucent gel was formed. Before microbicide application, monkeys were anaesthetized, and their recta were washed with 6 mL of sterile PBS. Then, 6 mL of gel (containing 1 mM SFT, 6 mM MVC, or 35 mM TFV) was administered rectally to each monkey. Half an hour later, 10 TCID50s of SHIV-1157ipd3N4 were applied to the rectum. The microbicide-challenge processes were repeated for 5 times as illustrated in Figure 1A.
Virus Load Assay
Plasma viral load was determined by real-time reverse transcription polymerase chain reaction. Viral RNA was prepared from EDTA-anticoagulated, cell-free plasma using the QIAamp viral RNA mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The viral loads were quantified with primers (SGAG21: 5′-GTCTGCGTCATCTGGTGCATTC-3′, SGAG22: 5′-CACTAGGTGTCTCTGCACTATCTGTTTTG-3′) and probes [pSGAG23: 5′-(FAM)CTTCCTCAGTGTGTTTCACTTTCTCTTCTGCG-(BHQ 1)-3′] that were annealed to SIVmac239-gag, and RNA standard kindly provided by Dr Lifson.28 All viral loads were repeatedly quantified in 2 independent experiments. Reactions were performed in duplicate for each sample. The threshold of detection for this assay was 200 copy equivalents of viral RNA per milliliter of plasma.
Intracellular Cytokine Staining
Intracellular cytokine staining (ICS) was performed as one of the tools to quantify the SIV-specific CD8+ T-cell responses. PBMCs of 1 × 106 were stimulated with Gag, Nef, or Vpx peptide pools derived from SIVmac239 (15-mers overlapping by 11 amino acids; Cat# 12364, 8762, and 6450, kindly provided by NIH AIDS reagent and reference program). The final concentration of each individual peptide was 5 μg/mL. During the stimulation, 1 μg/mL brefeldin A and 1 μM monensin (both Sigma, St Louis, MO) were added, and the plate was incubated at 37°C for 8 hours in total. After incubation, the cells were first stained with 2 cell surface markers: anti-CD3-Pacific Blue (clone SP34-2; BD pharmingen, San Jose, CA) and anti-CD8a-APC-eFluor780 (clone RPA-T8; eBioscience, San Diego, CA). After surface staining, the cells were washed twice with PBS (containing 2% fetal bovine serum), then fixed, and permeabilized with BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Cytofix/Cytoperm Plus). Next, the cells were washed twice with 1× perm/wash buffer, and intracellular cytokine interferon (IFN)-gamma was stained with anti-IFN-γ-PE-Cy7 (clone B27; BD pharmingen). Data analysis was performed using FlowJo X software (TreeStar Inc., San Carlos, CA).
IFN-γ ELISPOT Assay
IFN-γ enzyme-linked immunospot (ELISPOT) assays were performed in parallel with ICS assays to quantify the SIV-specific CD8+ T-cell responses. PBMCs were plated at 2 × 105 cells per well in multiscreen 96-well plates coated with an IFN-γ capture antibody (BD ELISPOT, human IFN-γ set). PBMCs were stimulated in duplicate with peptide pools derived from SIVmac239 Gag, Nef, and Vpx (the same with peptide pools as used in ICS). Plates were incubated overnight at 37°C and developed according to the manufacturer's instructions. Spots representing IFN-γ producing T cells were counted with an automated ELISPOT plate reader (Saizhi ChampSpot III Elispot Reader, Beijing, China).
Microarray-Based Peripheral Blood Transcriptome Profiling
On the day of SIVmac239 challenge, peripheral blood was collected from 2 monkeys of each group before virus inoculation, and preserved in PAXgene Blood RNA Tubes (QIAGEN) and stored at −80°C until use. Total RNA was extracted using PAXgene Blood RNA Kit (QIAGEN). Microarray-based transcriptome assay was performed by the Gene Tech (Shanghai, China) company using Affymetrix GeneChip Rhesus Macaque Genome Array (Cat# 900657). The average gene expression of HEC group was taken as control; genes that had a fold change ≥3.0 were selected from TFV, MVC, and SFT groups and defined as differentially expressed genes.
All statistical analyses in this study were performed using GraphPad software (Prism 5; San Diego, CA). The different groups were compared by unpaired t tests. For groups with P values of homogeneity of variance <0.05, Mann–Whitney tests were performed. Survival comparisons between different groups were tested with log-rank (Mantel-Cox) tests. A significant difference was defined as P < 0.05.
Monkeys in SFT-Formulated Microbicide-Treated Group Showed Better Survival Than Other Groups After a High-Dose SIVmac239 Challenge
The experimental design is illustrated in Figure 1A and detailed above. The overall survival for all groups is shown in Figures 1B, 2A. All monkeys survived throughout the 39-week microbicide efficacy study. The first death was recorded 10 weeks (week 49) after SIVmac239 challenge for a monkey in the control group that was treated with HEC gel alone. All monkeys in both TFV and MVC groups died during the 104-week observation, 4 monkeys in the TFV group died at weeks 50, 52, 53, and 95, respectively, and 4 monkeys in the MVC group at weeks 54, 56, 67, and 78. In contrast, the first death in the SFT group occurred later at week 95, and the remaining 3 monkeys (75%) survived during the 104-week observation. Interestingly, 2 monkeys in the control group died early at weeks 49 and 53, and the 2 remaining monkeys survived throughout the observation period. Overall, the SFT group showed significantly better survival than the TFV group (P = 0.0169) and the MVC group (P = 0.0067).
Because we used the identical dose of SIVmac239 for all challenges for all groups during the second phase of the study, the discrepancies observed above are likely resulting from the different treatments during the first phase of the study. These data suggest that the administration of different microbicides may significantly influence the survival of subsequent SIV infection.
Repeated Mucosal SHIV-1157ipd3N4 Challenges Failed to Result in Productive Infection in Monkeys
To determine what causes these differences, we first investigated whether different microbicides provided different protective efficacies during the first phase study. As shown in Figures 1A, 2A, during the first 5 weeks of first study phase, the monkeys were treated with different microbicide gels and challenged with SHIV-1157ipd3N4. The plasma viral loads during and after SHIV challenge were determined. Viral blips were only observed after the fourth and fifth challenge in 2 monkeys in SFT group, and 2 small blips were observed in HEC control group after SHIV challenges, indicating no productive infection was established during the first study phase, which was further confirmed by the sero-negative data (data not shown). We further examined CD4+ T cells and observed more stabilized CD4+ T-cell counts in the SFT group (Fig. 2B). Altogether, these data showed that there are no significant differences in protective efficacy or infection among different microbicide-treated groups, and the repeated mucosal challenges are unlikely to cause productive SHIV infection.
After SIVmac239 challenge, all monkeys were productively infected and reached their viral load peaks at 8 logs 2 weeks later. No significant differences were observed for their viral load peaks; however, the SFT group seems to have somewhat lower set-point viral loads (week 43) compared with other groups (Fig. 2A).
Macaques Pretreated With SFT Showed Significantly Higher T-Cell Responses After SIVmac239 Challenge
To further understand why the SFT-treated macaques showed higher survival, SIV-specific T-cell responses against SIVmac239 Gag, Nef, and Vpx were measured by ICS and IFN-γ ELISPOT at week 52. The data showed that, compared to MVC and TFV–treated groups, macaques pretreated with SFT mounted higher specific T-cell responses, especially against SIV Gag and Nef, and showed statistically significant differences between the SFT-treated group and the MVC-treated group in both ICS and ELISPOT (Figs. 3A, B).
Less Immune Genes Were Upregulated in SFT Pretreated Group Before SIVmac239 Challenge
To understand why SFT pretreated monkeys had better prognosis after SIV challenge, we performed transcriptomic analyses on whole blood from 8 monkeys before SIVmac239 challenge, which were challenged intravenously. As shown in Figure 4 (only ≥3-folds upregulated or downregulated genes relative to their expression in HEC group were included into this analyses), most genes are similarly either upregulated (upper half panel of genes) or downregulated (bottom half panel of genes) among different microbicide-treated groups. Interestingly, featured transcriptomic imprints were also observed (Fig. 4; see Table S2, Supplemental Digital Content, https://links.lww.com/QAI/A752). A panel of genes as listed in Box 1, including CD19, BCL11A, TLR10, and TNFRSF10D, is mainly upregulated in TFV-treated group, whereas several IFN-stimulating genes as listed in Box 2, including OASL, ISG15, DDX58, MX1, MX2, and IFI44, are more profoundly upregulated in MVC-treated group. In contrast, all those genes are downregulated in SFT-treated monkeys.
Previous studies indicated that ARV preexposure prophylaxis held promise to improve HIV prevention for MSM.29,30 A major advantage of ARV gel is that when applied to the mucosal site for potential virus exposure, ARV could rapidly reach tissues with much higher concentrations than those achieved by oral dosing and minimizes systemic drug toxicity.19,27,31–34 Importantly, this strategy is highly acceptable among populations at high HIV infection risk,35 including MSM and transgender women. However, it remains unknown whether the high concentration of ARV will alter the mucosal microimmune environment and thereby influence the immune responses to pathogens, including systemic immune responses to pathogens.
To test this concept, we designed a 2-phased study. In the first phase, we used the repeated–low-dose challenge model that has been suggested to better mimic natural transmission36,37 and allows repeated coexistence of microbicides and pathogens, and thereby amplify the potential effects of microbicides on the immune responses. Three ARV drugs, including the promising TFV, were formulated into gels as microbicides, and the process of pretreatment of microbicide and SHIV challenge was repeated for 5 times. Their influences on the immune system and disease progress were then assessed in the second phase, in which we used a high-dose pathogenic SIVmac239 (1 × 105 TCID50) to challenge all monkeys, which allows detecting of differences in disease progress and survival.
Our data showed that the repeated low-dose challenges did not result in a productive infection, which is suggested by undetectable viral loads through different time points before SIVmac239 challenge, although a few viral blips was observed. However, this may result in the coexistence of microbicides and pathogens, even a transient replicative pathogen (viral blip). A similar situation could be occurring in the clinic during the administration of microbicide, and therefore the influence of microbicides on the mucosal immune environment and immune responses should be taken into account when developing them.
In the second phase of our study, we rechallenged the monkeys with a high dose of SIVmac239. A high-dose challenge model was aimed to manifest the imprint from microbicide on immune system that could thereafter influence the disease progress and even survival. Surprisingly, we observed that the different microbicide-treated groups experienced a significant difference in survival. All monkeys in both TFV and MVC gel–treated groups rapidly progressed to death within 95 weeks, and in contrast, all monkeys in SFT gel–treated group survived significantly longer, and only one monkey died at week 95, suggesting that different treatments of microbicides may exert significant influences on the survival during subsequent infection. Interestingly, 2 monkeys experienced a rapid death in the control group treated with HEC gel and the remaining 2 monkeys survived throughout the observation. Similar to our control group, Suh et al38 found that 67% control macaques (4 of 6) died within 50 weeks after SIVmac239 challenge, and Westmoreland et al39 observed that 40% (4 of 10) monkeys progressed to AIDS within 200 days after challenge with SIVmac239. Taken together, these data indicate that microbicides, such as formulated by MVC and TFV in this study, might exert a negative influence on the survival. Overall, these data informed a very important primary observation that it is possible that microbicides could exert significant influence on the survival during subsequent SIV/HIV infection, and thereby alert us that the safety concern for testing microbicides may need to be revised to accommodate this observation. A further study is required to draw a solid conclusion with an increased sample size in clinical trials or animal study as only 4 monkeys in each group were used in this study.
As we proposed that the coexistence of microbicides and pathogens may influence the pathogen-specific immune responses, we next tested this concept in those monkeys. To simplify the model, we used SIV as our challenge virus and thereby neutralizing antibodies will be unlikely to play an important role in this circumstance because SHIV was used during the first phase of this study. T-cell immune responses were quantified with 2 assays: IFN-gamma–based ICS and ELISPOT. Importantly, monkeys in the SFT gel–treated group developed more significant T-cell immune responses against SIV Gag and Nef than those of monkeys in MVC and TFV gel–treated groups. It remains unknown how SFT could elicit higher SIV-specific T-cell responses than the MVC and TFV groups; 1 possibility is that SFT may have adjuvant effect and induced better memory T-cell immune responses at the mucosal site during SHIV challenge, which create an opportunity for the coexistence of SFT and SHIV and thereby help SHIV elicit SIV-specific T cells. Altogether, these data implicate that the microbicides might exert influences on the survival through regulating the T-cell immune responses during the coexistence with challenged SHIV viruses.
To determine whether the application of microbicide could imprint the immune system, we performed transcriptomic analyses on whole blood before challenge. Our data demonstrated that the major pattern is similar among 3 different groups; interestingly, we did observe difference in transcriptomic level among 3 groups. The most significant imprint is identified in MVC-treated monkeys, and several important IFN-stimulating genes were upregulated, such as OASL, ISG15, MX1, MX2, and IFI44; meanwhile, monkeys from TFV-treated group also showed that several immune genes were upregulated. In contrast, all those genes were downregulated in SFT-treated group. Overall, our data indicate that the application of microbicides is likely to imprint the immune system; the preactivation of immune system before challenge may be associated with a rapid disease progress.
Altogether, this study informed a preliminary but important observation on the influence of microbicides on the survival during subsequent SIV infection. A detailed study to further decipher how the influence is generated will be necessary. Meanwhile, the observations in this study suggest that the long-term immune safety concern for microbicides, which refers to the potential effect of microbicide on the immune responses that could cause the long-lasting damage on the subsequent disease progress or survival, should also be considered in the effort to develop effective microbicides.
The authors thank Zhidong Hu, Xiaonan Ren, and Xiangqing Ding for their kind help performing the experiments and thank Chenli Qiu and Jun Sun for their support with flow cytometry. The authors also thank Dr Michael Piatak Jr. and Dr Jeffrey D. Lifson for their kind gift on the plasmids for RNA standard preparation and for providing the protocols for RNA viral load quantification.
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