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Plasmodium falciparum malaria contributes to a significant global disease burden. Circumsporozoite protein (CSP), the most abundant sporozoite stage antigen, is a prime vaccine candidate. Inhibitory monoclonal antibodies (mAbs) against CSP map to either a short junctional sequence or the central (NPNA)n repeat region. We compared in vitro and in vivo activities of six CSP-specific mAbs derived from human recipients of a recombinant CSP vaccine RTS,S/AS01 (mAbs 317 and 311); an irradiated whole sporozoite vaccine PfSPZ (mAbs CIS43 and MGG4); or individuals exposed to malaria (mAbs 580 and 663). RTS,S mAb 317 that specifically binds the (NPNA)n epitope, had the highest affinity and it elicited the best sterile protection in mice. The most potent inhibitor of sporozoite invasion in vitro was mAb CIS43 which shows dual-specific binding to the junctional sequence and (NPNA)n. In vivo mouse protection was associated with the mAb reactivity to the NANPx6 peptide, the in vitro inhibition of sporozoite invasion activity, and kinetic parameters measured using intact mAbs or their Fab fragments. Buried surface area between mAb and its target epitope was also associated with in vivo protection. Association and disconnects between in vitro and in vivo readouts has important implications for the design and down-selection of the next generation of CSP based interventions.

Significance RTS,S/AS01 is a circumsporozoite protein (CSP)-based malaria vaccine that confers partial protection against malaria in endemic areas. Recent reports have elucidated structures of monoclonal antibodies that bind to the central (NPNA) repeat region of CSP and that inhibit parasite invasion. Antigen configuration and copy number of CSP repeats displayed on a tobacco mosaic virus (TMV) particle platform were studied. A TMV vaccine containing CSP repeats displayed as a loop induced 10× better antibody titer than a nearly full-length CSP in mice. In rhesus model, this translated to a 5× improvement in titer. Rhesus antibodies potently inhibited parasite invasion up to 11 mo after vaccination. An optimized epitope-focused, repeat-only CSP vaccine may be sufficient or better than the existing CSP vaccines., Plasmodium falciparum vaccine RTS,S/AS01 is based on the major NPNA repeat and the C-terminal region of the circumsporozoite protein (CSP). RTS,S-induced NPNA-specific antibody titer and avidity have been associated with high-level protection in naïve subjects, but efficacy and longevity in target populations is relatively low. In an effort to improve upon RTS,S, a minimal repeat-only, epitope-focused, protective, malaria vaccine was designed. Repeat antigen copy number and flexibility was optimized using the tobacco mosaic virus (TMV) display platform. Comparing antigenicity of TMV displaying 3 to 20 copies of NPNA revealed that low copy number can reduce the abundance of low-affinity monoclonal antibody (mAb) epitopes while retaining high-affinity mAb epitopes. TMV presentation improved titer and avidity of repeat-specific Abs compared to a nearly full-length protein vaccine (FL-CSP). NPNAx5 antigen displayed as a loop on the TMV particle was found to be most optimal and its efficacy could be further augmented by combination with a human-use adjuvant ALFQ that contains immune-stimulators. These data were confirmed in rhesus macaques where a low dose of TMV-NPNAx5 elicited Abs that persisted at functional levels for up to 11 mo. We show here a complex association between NPNA copy number, flexibility, antigenicity, immunogenicity, and efficacy of CSP-based vaccines. We hypothesize that designing minimal epitope CSP vaccines could confer better and more durable protection against malaria. Preclinical data presented here supports the evaluation of TMV-NPNAx5/ALFQ in human trials.

Exploration of animal models leads to discoveries that can reveal candidate biomarkers for translation to human populations. Herein, a model of hepatocarcinogenesis and protection was used in which rats treated with aflatoxin (AFB1 ) daily for 28 days (200 µg/kg BW) developed tumors compared with rats completely protected from tumors by concurrent administration of the chemoprotective agent, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im). Differential expression of miRNAs in tumors (AFB1 ) and nontumor (AFB1 + CDDO-Im) bearing livers and their levels in sera over the life-course of the animals was determined. miRNA transcriptome analysis identified 17 miRNAs significantly upregulated at greater than five-fold in the tumors. The ten most dysregulated miRNAs judged by fold-change and biological significance were selected for further study, including liver-specific miR-122-5p. Validation of sequencing results by real-time PCR confirmed the upregulation of the majority of these miRNAs in tumors, including miR-182, as well as miR-224-5p as the most dysregulated of these miRNAs (over 400-fold). The longitudinal analysis of levels of miR-182 in sera demonstrated significant and persistent increases (5.13-fold; 95% CI: 4.59-5.70). The increase in miR-182 was detected months before any clinical symptoms were present in the animals. By the terminal time point of the study, in addition to elevated levels of serum miR-182, serum miR-122-5p was also found to be increased (>1.5-fold) in animals that developed hepatocarcinomas. Thus, using the data from an unbiased discovery approach of the tissue findings, serum miR-182 was found to track across the complex, multistage process of hepatocarcinogenesis opening an opportunity for translation to human populations.

Aflatoxin B1 (AFB1) is a potent human and animal hepatocarcinogen. To investigate the effects of aflatoxin on miRNA expression during the initiation phase of carcinogenesis, next-generation sequencing was used to analyze liver tissues from F344 rats exposed to 200 µg/kg per day AFB1 for 4 weeks. A panel of miRNAs was identified that was upregulated with AFB1 treatment compared to controls: rno-miR-434-3p, rno-miR-411-5p, rno-miR-221-3p, rno-miR-127-3p, rno-miR-205, rno-miR-429, rno-miR-34a-5p, rno-miR-181c-3p, rno-miR-200b-3p, and rno-miR-541-5p. Analysis of rat livers exposed to AFB1 plus the chemopreventive triterpenoid CDDO-Im revealed a striking abrogation of this upregulation. These changes were validated by real-time PCR. We also explored the temporal variation in expression of the candidate miRNAs during the 4-week dosing period. Most of the candidate miRNAs were upregulated at week 1 and increased for the duration of AFB1 dosing over the 4-week period. Treatment with CDDO-Im ameliorated these effects at all time points. All candidate miRNAs were detectable in serum from aflatoxin treated animals; however, there was no significant difference in expression for 7 of the 11 miRNAs examined. Exposure to AFB1 upregulated miR-122-5p (5 fold), 34a-5p (13 fold), and 181c-3p (170 fold) compared with controls. The findings from this study give insight into epigenetic changes induced by aflatoxin taking place during the initial step of carcinogenesis. This article is protected by copyright. All rights reserved

Aflatoxin B1 (AFB1) is a potent human and animal hepatocarcinogen. To investigate the effects of aflatoxin on miRNA expression during the initiation phase of carcinogenesis, next-generation sequencing was used to analyze liver tissues from F344 rats exposed to 200 μg/kg per day AFB1 for 4 weeks. A panel of miRNAs was identified that was upregulated with AFB1 treatment compared to controls: rno-miR-434-3p, rno-miR-411-5p, rno-miR-221-3p, rno-miR-127-3p, rno-miR-205, rno-miR-429, rno-miR-34a-5p, rno-miR-181c-3p, rno-miR-200b-3p, and rno-miR-541-5p. Analysis of rat livers exposed to AFB1 plus the chemopreventive triterpenoid CDDO-Im revealed a striking abrogation of this upregulation. These changes were validated by real-time PCR. We also explored the temporal variation in expression of the candidate miRNAs during the 4-week dosing period. Most of the candidate miRNAs were upregulated at week 1 and increased for the duration of AFB1 dosing over the 4-week period. Treatment with CDDO-Im ameliorated these effects at all time points. All candidate miRNAs were detectable in serum from aflatoxin treated animals; however, there was no significant difference in expression for 7 of the 11 miRNAs examined. Exposure to AFB1 upregulated miR-122-5p (5 fold), 34a-5p (13 fold), and 181c-3p (170 fold) compared with controls. The findings from this study give insight into epigenetic changes induced by aflatoxin taking place during the initial step of carcinogenesis.

Hepatocellular carcinoma (HCC) continues to be a major cause of cancer death globally, with aflatoxin B1 (AFB1) as a prevalent risk factor in low- and middle-income countries. MicroRNAs have been shown to be differentially expressed in HCC and may serve as predictive biomarkers. In this study, we analyzed tumor and non-tumor tissue from rats dosed with AFB1 (200 µg/kg BW) for 28 consecutive days that received vehicle only or AFB1 plus the chemopreventive agent CDDO-Im (30 µmol), and control animals (Johnson et al., CaPR, 2014). Total RNA was isolated from tumor or non-tumor tissue at the time of sacrifice and sequenced. MicroRNA transcriptomic analysis revealed 17 miRNAs significantly upregulated (> 5 fold) in tumors compared to non-tumor tissue. The top ten dysregulated miRNAs determined by fold change and biological significance were selected for further investigation: rno-miR- 205, 200b-3p, 182, 429, 31a-5p, 10b-5p, 141-3p, 132-3p, 802-5p. Validation of sequencing results by quantitative PCR (qPCR) confirmed the upregulation of the majority of candidate miRNAs in tumors and rno-miR-224-5p as the most dysregulated miRNA (over 400 fold). We also examined the levels of these candidates in terminal sera of the same animals by qPCR. Circulating miRs-224-5p, 182, and 122-5p were increased (> 1.5 fold) in animals diagnosed with HCC compared to untreated animals and those previously dosed with AFB1 plus CDDO-Im. Analysis of tracking of serum miR-182 by generalized estimating equations (GEE) revealed significantly increased levels (5 fold; CI: 2.17- 2.50) in animals that developed HCC. This sustained increase in serum miR-182 is seen months before any tumors or other symptoms are present, and highlights this miRNA as a potential predictive biomarker in AFB1-induced HCC. Supported by T32ES007141-31A1 and CA197222. Citation Format: Merricka C. Livingstone, Natalie M. Johnson, Bill D. Roebuck, Thomas W. Kensler, John D. Groopman. Dysregulated microRNAs in aflatoxin-induced hepatocellular carcinoma: Serum miR-182 as a potential predictive biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5401.

Liver cancer is the second leading cause of cancer death worldwide: risk factors include the viruses, HBV and HCV, and the environmental carcinogen aflatoxin B1 (AFB1). Chemoprevention strategies that activate genes controlled by the KEAP1-NRF2 pathway, such as CDDO-Im, afford complete protection against AFB1-induced hepatocarcinogenesis in rats. The opportunity to deploy these types of agents in high-risk populations would be advanced by the validation of biomarkers reflecting their efficacy. MicroRNAs (miRNAs) affecting gene expression are important in normal physiology and have been shown to be frequently dysregulated in cancer. It is our hypothesis that specific rat liver miRNAs are biomarkers that temporally track with AFB1-induced liver cancer, and are modulated in animals that receive complete protection by CDDO-Im. MiRNAs were isolated from archived liver tissue of rats (Johnson et al., CaPR, 2014) that were dosed with AFB1 (200 μg) for 28 consecutive days. Two additional groups received either vehicle only or AFB1 + CDDO-Im (30 μmol). After RNA isolation (miRCURY tissue, Exiqon), samples were profiled by RNA sequencing (Illumina). Fourteen miRNAs were selected based on dynamic range and level of detection for validation by RT-qPCR (TaqMan microRNA assay). MiRNA profiling from animals at the end of the carcinogenic 28-day dosing regimen revealed an increased total number of miRNAs due to AFB1 exposure alone (n≈500) compared to control. Those miRNAs displaying a greater than 10 fold increase in expression between control and AFB1 treatment groups were examined in detail: 541-5p, 34a-5p, 127-3p, 205, 434-3p, 429, 411-5p, 181c-3p, 200b-3p, 221-3p. RNA-seq data showed that these were all upregulated in the AFB1-treated samples. Co-treatment with CDDO-Im shifted expression levels back to control. The miRNAs 192-3p, 92a-3p, 26b-3p, and 375-3p were expressed consistently in all treatment groups and were selected for normalization. Expression levels of the 14 candidate miRNAs were then examined in liver samples obtained from rats after 7, 14 and 21 doses AFB1 and compared to levels determined at week 4 (28 doses AFB1). Quantitative analysis demonstrated an increase in expression of the panel of miRNAs due to AFB1 exposure over the dosing period, potentially indicating a role in early carcinogenesis. MicroRNA expression showed a varying trend over time in animals treated with AFB1 plus CDDO-Im. Three invariant miRNAs (miR-375-3p, 92a-3p, 192-3p) showed little change during the 28-day exposure period. In conclusion, we have identified a panel of miRNAs that are upregulated and track with AFB1 exposure. These changes are abrogated by treatment with CDDO-Im and thus reflect the protective efficacy of the intervention. Supported by T32ES007141-31A1 and CA197222. Citation Format: Merricka C. Livingstone, Bill D. Roebuck, Natalie M. Johnson, Thomas W. Kensler, John D. Groopman. Temporal trends in microRNAs during subchronic aflatoxin dosing and modulation by the chemopreventive oleane triterpenoid, CDDO-Im. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1083.