Development and Characterization of an HPV Type-16 Specific

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Development and characterization of an HPV Type-16 specific modified DNA aptamer for the improvement of potency assays Jeremiah James Trausch, Mary Shank-Retzlaff, and Thorsten Verch Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04852 • Publication Date (Web): 24 Feb 2017 Downloaded from http://pubs.acs.org on February 26, 2017

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Analytical Chemistry

Development and characterization of an HPV Type-16 specific modified DNA aptamer for the improvement of potency assays

Jeremiah J. Trausch1, Mary Shank-Retzlaff, Thorsten Verch

Department of Vaccine Analytical Development, MRL, Merck & Co., Inc., West Point, PA USA

1

Corresponding author:

Phone: (215)-652-1363 Email: [email protected]

To whom correspondence should be addressed: Jeremiah J. Trausch, MRL, Department of Vaccine Analytical Development, 770 Sumneytown Pike, West Point, Pennsylvania 19486, USA; Phone: (215)-652-1363; Email: [email protected]

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ABSTRACT Measuring vaccine potency is critical for vaccine release and is often accomplished using antibody-based ELISAs. Antibodies can be associated with significant drawbacks that are often overlooked including lot-to-lot variability, problems with cell-line maintenance, limited stability, high cost, and long discovery lead times. Here, we address many of these issues through the development of an aptamer, known as a slow off-rate modified DNA aptamer (SOMAmer®), which targets a vaccine antigen in the human papillomavirus (HPV) vaccine Gardasil®. The aptamer, termed HPV-07, was selected to bind the Type 16 virus-like-particle (VLP) formed by the self-assembling capsid protein L1. It is capable of binding with high sensitivity (EC50 of 0.1 to 0.4 µg/mL depending on assay format) while strongly discriminating against other VLP types. The aptamer competes for binding with the neutralizing antibody H16.V5, indicating at least partial recognition of a neutralizing and clinically-relevant epitope. This makes it a useful reagent for measuring both potency and stability. When used in an ELISA format, the aptamer displays both high precision (intermediate precision of 6.3%) and a large linear range spanning from 25% to 200% of a typical formulation. To further exploit the advantages of aptamers, a simplified mix and read assay was also developed. This assay format offers significant time and resource reductions compared to a traditional ELISA. These results show aptamers are suitable reagents for biological potency assays and we expect their implementation could improve upon current assay formats. INTRODUCTION A key step in the development of any vaccine or biotherapeutic product is the generation of a suitable potency test. A well designed potency assay can be used to ensure efficacy, monitor product stability, and confirm lot-to-lot consistency. For biologics, the potency test may consist of a combination of a binding assay such as an enzyme-linked immunosorbent assay (ELISA) and a cell-based functional assay. For viral vaccines, potency often requires replication


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of the virus or cell entry. Therefore, a plaque or cell-based assay is typically used to monitor potency. For subunit and inactivated viral vaccines, the role of the antigen is to present one or more immunologically active epitopes to the immune system, with the goal of eliciting protective antibodies in the patient. In these cases, the potency test will typically consistent of an ELISA, rate nephelometry, competitive immunoassay, or other in vitro assay. Regardless of the assay format, the assay suitability and long-term robustness of any in vitro potency assay are largely dependent on the critical reagents used. Currently, antibodies are by far the most common choice for antigen-specific reagents. Antibodies offer high affinity and specificity but are associated with significant challenges. For example, antibody supplies can be hard to maintain over the multi-decade lifetime of a vaccine. They often require the maintenance of a cell line that can become compromised1 leading to complete loss of the reagent and the need to develop, validate and bridge to a new assay. During regular cell-based production, antibodies can exhibit large lot-to-lot variability that may undermine assay performance2. Also, the traditional antibody discovery process is extremely time consuming and difficult to control. Significant resources are typically required to generate a diverse panel of antibodies and to screen for attributes of interest. To address some of the issues with antibodies, we sought to explore the feasibility of using aptamers as affinity reagents in potency assays. Aptamers are nucleic acid based affinity reagents with similar binding properties as antibodies3, 4. Aptamers are made of single-stranded DNA or RNA and are capable of recognizing diverse targets such as small-molecules, proteins, or polysaccharides3. They are developed very differently than antibodies through a process called SELEX (systematic evolution of ligands by exponential enrichment)5, 6. This is entirely an in vitro process that uses a random library of nucleic acids to select sequences that show affinity to the target of choice. Aptamers offer several key advantages over antibodies in potency assays. First, because no animals are required for reagent generation, aptamers can provide significant cost 3 ACS Paragon Plus Environment


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savings and are compliant with recent recommendations from regulatory agencies and others to minimize the use of animals during product development and testing7. Also, the specificity of aptamers can be selected for and/or biased in the discovery process under controlled conditions. This may be critical when developing aptamers for use in a multi-valent vaccine consisting of distinct, but very similar antigens. Compared to antibodies, the aptamer generation/screening process is far more rapid. Commercially available libraries of random sequences can be readily purchased. SELEX screening can occur in as little as 8 weeks with the final material selected and scaled within only few months. In contrast, the production, isolation, and scale up of a monoclonal antibody typically requires around 9-12 months. Long-term, aptamers are expected to be significantly easier to resupply and maintain. The aptamers are chemically synthesized, therefore it is unnecessary to maintain a parental cell bank. Only the aptamer sequence is required to reproduce the material. In vitro synthesis provides high purity8, 9 and a consistent, and simple resupply. Additionally, aptamers can be extensively characterized using sequencing, high-performance liquid chromatography (HPLC), or mass spec to verify lot-to-lot consistency9. The same tools can be used to monitor the stability of the aptamer during storage, meeting modern regulatory requirements to establish data-driven expiry dating for all critical reagents. Further, aptamers are very stable (especially DNA or modified RNA)10. This lowers concerns over maintaining a strict cold chain during shipping and storage. Aptamers fold reversibly11, so even if the reagent is denatured it is not necessarily compromised. Aptamers can be readily modified during the synthesis process. For example, the introduction of a biotin tag or addition of a variety of fluorophores is very straightforward, making aptamers compatible with a broad range of analytical techniques including traditional ELISA formats12, fluorescence polarization methods13, analytical arrays14, surface plasmon resonance15 techniques and many others. 4 ACS Paragon Plus Environment

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To test the feasibility of using aptamers as an alternative to traditional antibody reagents in a vaccine potency assay, we used the recombinant vaccines Gardasil® and Gardasil®9 as model systems. Gardasil® is a licensed, quadrivalent-valent vaccine for the prevention of human papillomavirus (HPV)16-18. Among other diseases, HPV causes almost all cases of cervical cancer which is the fourth most common cancer in women16, 19, 20. Gardasil®9, the second generation vaccine, is a nonavalent formulation that protects against 90% of cervical cancers caused by HPV21. For each type, the vaccine contains a specific L1 protein that self-assembles into a virus-like-particle (VLP)22. Specificity is a significant challenge for a reagent used to determine potency in a multivalent vaccine. For this work, we selected an aptamer against the Type 16 VLP. We required the aptamer to bind Type 16 VLP but discriminate against other VLPs present within either the quadrivalent or nonavalent formulations. In a traditional reagent development track, antibodies would be generated against the Type 16 VLP and screened for characteristics such as specificity post discovery. For aptamers, specificity can be designed into the in vitro selection process through the addition of competitors3. This approach has been shown in the literature to generate aptamers that discriminated between proteins that differ by a single amino-acid23. The aptamer discovered in this study, HPV-07, specifically binds Type 16 VLP and discriminates against all other types tested. Although this aptamer was slightly less sensitive than previously developed antibodies, it was fit-for-purpose. Based on competition studies, HPV-07 binds to a neutralizing epitope and is sensitive to conformational changes, making it suitable for use in a stability assay. We established an aptamer-based assay which met the standard requirements for precision and linearity in a format mimicking a previously developed antibody-based ELISA. To move beyond a standard ELISA format, we also developed a mixand-read assay that would dramatically reduce the complexity of the current format. This work builds a promising foundation for using aptamers to monitor vaccine potency.



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EXPERIMENTAL SECTION Aptamer generation: The generation of a modified DNA aptamer against the HPV Type 16 VLP was commissioned through SomaLogic (Boulder, CO). The target, Type 16 VLP, was biotinylated using EZ-Link™ Sulfo-NHS-LC-Biotin (Thermo 21327) in 50 mM MES pH 6.2, 500 mM NaCl, 0.02% PS80 with a molar ratio of 5:1 (biotin to L1 protein). The library for the SELEX experiment contained a hydrophobic amino acid-like residue from every C5 uridine in the sequence24. Details of the SomaLogic selection have been previously published25. Briefly, the library was incubated with the target attached to a magnetic bead. A kinetic challenge was applied after round 1 that significantly diluted the complex prior to capture. In later rounds this dilution also contained 10 mM dextran sulfate25. After round one, a competition was introduced in order to ensure specificity for Type 16 VLP in the presence of other vaccine types. Unlabeled VLP Types 6, 11, 18, 31, 33, 55, and 58 were used as competitors within the binding phase of selection so that only sequences that bound to the biotinylated Type 16 VLP were selected for. Sequences were eluted from the protein using sodium perchlorate. Sequences were amplified into natural DNA and then converted back into modified sequences using a primer extension step. This pool was then used in further rounds of selection. Resulting aptamers or SOMAmers® (slow off-rate modified aptamers) contained a biotin at the 5’ end. All results were compared to a negative control (Neg Ctr) aptamer that was a SOMAmer® of similar chemical composition selected to bind a different protein target. ELISA. All ELISAs were run at a 100 µL assay volume, and each incubation was carried out at room temperature without shaking. The assay buffer was 50 mM MES at pH 6.2, 500 mM NaCl, 0.02% PS80, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA at pH 8.0, 0.1 mg/mL Salmon Sperm DNA, and 1% BSA. Plates were washed between each step three times with 1X TBS. Capture monoclonal antibody (mAb) at 10 nM was adsorbed to a 96-well black maxisorp plate (NUNC #437111), incubated for 2 hours, and decanted. When the aptamer was used as a


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capture reagent, 100 nM streptavidin (Thermo #21122) was adsorbed to the plate prior to the capture aptamer. Plates were then blocked with assay buffer for 1 hour. To streptavidin coated wells, 10 nM aptamer was added for 1 hour and blocked with assay buffer containing 1 mM biotin. Assay buffer was then added to all wells, and the VLP was added to the first column at 10 µg/mL. This solution was titrated in two-fold steps across the plate. Plates were incubated for 1.5 hours. Adjuvanted HPV samples were desorbed as previously described26. Briefly, 50 µL of the VLP was mixed with 50 µL of 2X citrate-phosphate buffer (60 mM sodium phosphate, 100 mM sodium citrate, 1 M NaCl, and 0.4% Tween 80, pH 6.75). The solution was then incubated overnight. In all ELISAs, 10 nM detection reagent (aptamers or antibodies as indicated) was added and incubated for 1 hour. Anti-species antibody-alkaline phosphatase conjugate (for antibody detection, Jackson ImmunoResearch, 115-055-207) or streptavidin-alkaline phosphate conjugate (for aptamer detection, Pierce, 21324) were then added at a 1:6000 dilution and incubated for 1 hour. Fluorescent signals were developed using 4-methylumbelliferyl (4-MUPs, ViroLabs XPHOS-100) for 45 min. Signals were measured using a SpectraMax M2E reader (Ex:360 nm, Em:450 nm). Data were fit using the four parameter logistic non-linear regression model in Prism 6 (GraphPad, La Jolla, CA). In vitro relative potency (IVRP) was calculated by dividing reference EC50 by sample EC50. For precision testing the ELISA was run 4 consecutive days. Each day 3 plates were run and on each plate, 1 reference and 3 samples were tested. The reference was a Type 16 VLP monovalent sample while the samples were quadrivalent, containing Type 6, 11, 16, and 18 in equivalent concentration. The in vitro relative potency (IVRP) was calculated for each sample and compared to determine precision. Surface plasmon resonance (SPR). Aptamer experiments were performed on a Biacore™ 3000 (GE Healthcare, Chicago, IL) using a streptavidin-coated chip (BR100398). 7 ACS Paragon Plus Environment


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Biotinylated aptamers were immobilized by flowing a 1 µM solution of aptamer in running buffer (50 mM MES at pH 6.2, 500 mM NaCl, 0.02% PS80, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA at pH 8.0, 0.1 mg/mL Salmon sperm DNA) over the flow cell at a flow rate of 5 µL/min for 20 min. A negative control (Neg Ctr) aptamer was bound to flow cell 1 and HPV-07 was bound to flow cell 2. Approximately 1000 RUs of each aptamer were immobilized on each surface after washing with 10 mM NaOH. Data were generated by injecting VLP (40 µL of 20 µg/mL or titrated) in running buffer over flow cell 1 and 2, setup in series at a flow rate of 10 µL/min for 4 or 8 min. For samples that were blocked with an mAb, the VLP was incubated for 1 hour with 50 µg/mL mAb prior to injection. Dissociation was monitored at a flow rate of 10 µL/min for 4 or 8 min. The surface was regenerated by injecting 10 µL of 10 mM NaOH at a flow rate of 10 µL/min. The signal generated by flow cell 1 was subtracted from flow cell 2 and subtracted form a buffer only run with BIAevalution 3.2 software. Antibody experiments were performed with the mAb covalently attachment to a CM5 chip (BR100399). The chip was activated with a 1:1 mixture of 0.1 M EDC and 0.1 M NHS. A negative control mAb was bound to flow cell 1 and H16.V5 mAb was bound to flow cell 2 at 10 µg/mL in 10 mM sodium acetate pH 4.0 at a follow rate of 5 µL/min for 8 min. The reaction was quenched with 1 M ethanolamine pH 8.0. VLP binding was monitored similarly to the aptamer experiments but now using 5 mM NaOH for regeneration and a buffer that did not contain the Salmon sperm DNA blocker. AlphaLISA. An AlphaLISA27 was developed to detect the Type 16 VLP using the same assay buffer as the ELISA. The final assay format was initiated with the addition of 16 µL of 45 nM aptamer to a 96-well half area plate (PerkinElmer, 6005560). This was mixed with 16 µL of a solution containing 60 µg/mL streptavidin-coated donor beads (PerkinElmer, 6760002b) and 15 µg/mL streptavidin-coated acceptor beads (PerkinElmer, AL125C). A 6 µg/mL solution of Type 16 VLP was titrated using a 1.8-fold dilution in a dilution plate to make a 12 point series. Then, 16 µL of this solution was added to the plate containing the donor and acceptor beads and 8 ACS Paragon Plus Environment

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mixed by pipetting. The plate was incubated at room temperature while shaking at 650 rpm for 90 min. The luminescence of the plate was read using an EnVision plate reader (PerkinElmer, Waltham, MA). Donor beads were excited at 680 nM while chemiluminescent signal was observed at 615 nm.

RESULTS AND DISCUSSION Aptamer characterization and specificity. The generation of modified DNA aptamers that target the virus-like particle of HPV was carried out by SomaLogic Inc. The SELEX experiment targeted Type 16 VLP while Types 6, 11, 18, 31, 33, 55, and 58 were used as competitors to promote specificity. The pool was sequenced and ten aptamers were synthesized. These representative aptamers were chosen to span distinct phylogenetic families within the sequence pool. The ten aptamers were evaluated in an ELISA (sometimes termed ELASA or enzyme-linked apta-sorbent assay12). The VLP was captured using the H16.V5 mAb28 and detected using the aptamers. About half of the aptamers showed a strong interaction with Type 16 VLP, with HPV-07 displaying the most sensitive EC50 (Figure 1A). Therefore, HPV07 was used exclusively in all subsequent work. Because the objective was to monitor Type 16 VLP potency in a multivalent matrix, detection of Type 16 by the aptamer was evaluated with and without competing types (Figure 1B). The aptamer displayed a strong response even when added in a solution that contained a mixture of Types 6, 11, 18, 31, 55, and 58 (10 µg/mL of each). In fact, the sensitivity was virtually identical to a solution that contained no competitor. A similar result was observed when the assay was reversed to use HPV-07 as a capture reagent (Figure 1C). Aptamer specificity was also tested by SPR. HPV-07 was attached to a streptavidinlabeled chip and specific VLP types were injected. Only Type 16 VLP showed an interaction with the aptamer (Figure 1D). Many types displayed a negative signal suggesting the VLPs had



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more of an interaction with the negative control surface, i.e. an aptamer of similar chemical composition developed against a different protein target, than the aptamer HPV-07. All data suggest the aptamer to be specific for Type 16 VLP with no cross-reactivity against other VLP types.

Comparison between the aptamer and a monoclonal antibody. Antibodies developed against the HPV Type 16 VLP have been used successfully to determine vaccine potency29. First, the HPV-07 aptamer and the neutralizing mAb, H16.V528, were compared by SPR for their ability to bind the VLP passed over the chip. The aptamer showed an equilibrium KD of 400 ± 30 pM VLP or ~8 µg/mL (Figure 2A and 2C). The mAb displayed a slightly lower KD of 230 ± 10 pM VLP or ~ 4.6 µg/mL (Figure 2B and 2C). No dissociation of the VLP was observed with either the aptamer or mAb (Figure 2A and 2B). Second, the sensitivity of the aptamer and mAb was compared by ELISA. In both cases, the VLP was captured with the non-neutralizing H16.J4 mAb28. Subsequent detection by neutralizing mAb H16.V5 was very sensitive with an EC50 of 32 ± 1 ng/mL (Figure 2D) while the aptamer displayed an EC50 of 350 ± 10 ng/mL (Figure 2D) Although the KD of the antibody is only about 2-fold lower, it is about 10-fold more sensitive than the aptamer in the ELISA. Note that comparing absolute values of affinity to assay sensitivity is misleading due to the differences in the measurement setup. Affinity was measured by SPR with the reagent bound to a surface whereas in ELISA the reagent was in solution. A possible reason for the lower EC50 of the antibody could be its bivalency compared to the monovalent aptamer. Thus when added in equal molar amounts, the antibody has double the number of binding regions than the aptamer. Another possible reason could be a more complete coating of the VLP by the antibody31. The negative charge of the aptamer could cause repulsion if a large number are bound densely to the surface. Although the aptamer EC50 is 10 ACS Paragon Plus Environment

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higher than the antibody, the EC50 was still ~200-fold lower than typical vaccine formulations which would be sufficient for most potency applications. While an additional affinity maturation selection potentially could increase sensitivity further32, aptamer performance was fit for purpose in this case. Stability Indication. It is important that a reagent measuring potency is sensitive to the stability of a vaccine antigen. The mAb H16.V5 has been shown to be stability-indicating for Type 16 VLP thermal stress26. Therefore, we evaluated aptamers for monitoring stability of the stressed VLP compared to the mAb H16.V5 (Figure 2E). Samples of Type 16 VLP were incubated at 64 °C, a condition that has previously been shown to destabilize the VLPs26. Both aluminum adjuvanted and non-adjuvanted samples were stressed. The samples were subsequently tested using an ELISA-type assay with an mAb against a different epitope as the capture reagent and either aptamer HPV-07 or the mAb H16.V5 as the detection reagent. The aptamer showed a drastic reduction in potency for the non-adjuvanted samples within a few minutes at 64 ºC. In contrast, samples protected by adjuvant exhibited a much smaller loss of potency with either HPV-07 or H16.V5. For the non-adjuvanted samples, the degradation kinetics and degree of degradation appear similar based on visual inspection of the data. For the adjuvanted sample, HPV-07 may be slightly more sensitive to degradation. Aptamer competition with a neutralizing antibody. When measuring potency it is not sufficient to use a reagent that binds to any epitope on the target. The reagent should, if possible, bind to a clinically-relevant epitope. Ideally this will be the epitope that the human immune system will develop neutralizing antibodies against. Previous literature reports suggest H16.V528 recognizes a clinical relevant epitope. VLPs that lack the H16.V5 epitope are less immunogenic in mice33, and H16.V5 can block the reactivity of human sera containing antiType 16 VLP antibodies34. To test if the HPV-07 aptamer recognizes the same critical epitope we carried out a competition ELISA. For the first experiment, the VLP was captured with H16.J4 and aptamer 11 ACS Paragon Plus Environment


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was added in the presence of different amounts of neutralizing mAb H16.V528 as competitor. H16.V5 blocked the ability of the aptamer to recognize/bind the VLP (Figure 3A, blue) in a concentration dependent manner. Thus, the aptamer cannot bind the VLP when neutralizing H16.V5 is bound. On the other hand, when the VLP was captured with H16.V5 and coated with the competing, non-neutralizing, mAb H16.J428, the aptamer was not blocked from recognizing the VLP (Figure 3A, green). In the final competition experiment, the non-neutralizing H16.J4 was used to capture VLP followed by coating of the immobilized VLP with aptamer. Then, neutralizing antibody H16.V5 was used for detection. In this case, no competition was observed (Figure 3A, magenta). Most likely the stronger affinity and potentially larger epitope of the mAb compared to the aptamer still allowed for mAb binding.

Competition between the aptamer and the

neutralizing mAb was also tested by SPR. When, aptamer HPV-07 was immobilized on a streptavidin chip followed by VLP injection, a strong binding response was measured (Figure 3B). When the VLP was coated with neutralizing mAb H16.V5 prior to injection, no binding was observed on the SPR chip. Alternatively, coating the VLP with non-neutralizing mAb H16.J4 still allowed for a significant interaction with the aptamer although reduced binding was observed. A possible explanation for the less than 2-fold reduction in binding could be the steric hindrance of mAbs bound to the surface of the VLP reducing epitope accessibility for the aptamer. Competition in both the ELISA and the SPR experiments indicates that the HPV-07 aptamer binds to a similar or closely related epitope as the neutralizing mAb H16.V5. Epitopemapping by deuterium exchange could provide additional conclusive data but faced technical challenges so far. No interaction was observed for the aptamer binding either mAb alone (data not shown).

Precision and linearity testing. Method validation is an important assessment to confirm assay performance and is a regulatory expectation for potency assays35, 36. While 12 ACS Paragon Plus Environment

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validation includes other parameters, we compared the precision and linearity of the HPV-07 aptamer to the antibody H16.V5 as key performance parameters. As noted above, specificity was assessed separately. Precision was determined for an ELISA using the H16.J4 mAb for capture and the HPV-07 aptamer for detection (Figure 4A). The intra-plate precision showed a %CV ranging from 1.0-7.3%. The intra-day precision had a range of 4.8-8.6%. Finally, intermediate precision across all runs was 6.3% CV. Assay imprecision was fit-for-purpose and lower than the previously determined estimates for an ELISA using the H16.V5 mAb. Assay linearity, i.e. the ability of the assay to measure changes in antigen concentration that are linearly proportional over a specific range, was tested using a reference and a mock multivalent-vaccine sample. Samples were diluted to 25%-200% of a normal VLP formulated concentration and then tested as if they were formulated at 100%. The ELISA showed a strong linear relationship between measured relative potency and the nominal VLP concentration (Figure 4B). The assay was also highly accurate when using HPV-07 in this format. In both the precision and linearity assessment, the aptamer was able to recognize Type 16 VLP in the sample accurately with respect Type 16 VLP within the reference (Figure 4A, 4B). Development of a mix-and-read aptamer assay. ELISAs are time intensive and potentially variable due to their complexity, in particular when dealing with multivalent vaccines. As an example, the original ELISA potency assay that was validated for Gardasil® required 3 days per run due to long incubations required for alum dissolution and sample capture. Global vaccine release often requires the potency assay be transferred to laboratories employing analysts with varying levels of scientific and technical training, thereby significantly compounding the chance of errors. Therefore, a simple no wash, mix-and-read assay format is highly desirable. AlphaLISA® technology has been well established in a number of biomarker assays and offers a homogenous format 27. Briefly, an antibody/aptamer-analyte sandwich is assembled 13 ACS Paragon Plus Environment


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connecting donor and acceptor beads(250-300 nm in diameter). Signal is generated by first exciting the donor beads at a wavelength of 680 nm. This will cause donor beads to create singlet oxygen from ambient oxygen using photosensitizing agent (phthalocyanine)37. The singlet oxygen can travel up to 200 nm from the bead. If an acceptor bead is pulled within this radius via an ELISA-type sandwich, the compound thioxene will react with the oxygen and create light37. This energy is passed to anthracene and final to rubrene that ultimately releases light at a wavelength of ~615 nm37. Fundamentally, the AlphaLISA® is an assay format that can monitor molecular interactions through physical proximity similar to fluorescence resonance energy transfer (FRET). In this case, a FRET assay could be more challenging and less sensitive because the VLPs are approximately 60 nm in diameter and FRET can only interpret distances less than about 9 nm. The repetitive nature of the epitope and the singular biotinylation of the aptamers allowed us to develop an assay that sandwiches our antigen with a single reagent, i.e. aptamer HPV-07 (Figure 5A). The assay uses donor and acceptor beads that are both functionalized with streptavidin. Because the beads may bind many aptamers and the VLPs have many epitopes, it is possible for beads to bind multiple VLPs or VLPs to bind multiple beads, potentially enhancing the sensitivity. The format was first optimized by titrating the aptamer concentration (from 0 to 300 nM) against the bead concentration (4:1 donor to acceptor ratio from 2.5:0.625 to 80:20 µg/mL). For each point a 0 and 2.5 µg/mL HPV concentration was tested. Maximal signal to background ratios were found at 45 nM aptamer and 60:15 µg/mL donor and acceptor beads (data not shown). Changing bead ratios, the order of addition, or the incubation times had insignificant effects. A negative control aptamer (Neg Ctr) evolved to a different protein target showed no response with increasing concentrations of VLP (Figure 5B). The optimized assay required only three solutions (biotin-labeled aptamer, mix of donor and acceptor beads, and the Type 16 VLP analyte). Reagents were mixed, incubated for 90 14 ACS Paragon Plus Environment

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min, and read on a plate reader. The assay had only a 48 µL total volume and took less than 2 hours to complete, representing a significant improvement to the previously reported format29. In addition, the AlphaLISA format exhibited a three-fold improvement in EC50 compared to ELISA (106± 4 ng/mL) and a much higher signal-to-background ratio (>300, Figure 5B). This assay format would be more challenging to carry out with antibodies due to the multiplicity of the label. Labeling of mAbs is often done with a NHS ester labeling reaction that could label any amine group on the mAb. This makes it very hard to control the degree of biotinylation, and many mAbs end up with multiple biotins following to a Poisson distribution38. In an assay using antibodies with multiple biotins, the two different SA-coated beads could be linked simply with antibodies in the absence of the HPV analyte. The assay could be done with different donor and acceptor bead types but this would complicate the sandwich and, in our experiences, lead to problems such as hook effects. Conversely, because the aptamer is synthesized with only one biotin on the 5’ end there is no chance of cross-linking of beads in the absence of analyte. An assessment of the precision and assay linearity was carried out for the aptamerbased AlphaLISA format. The intra-plate precision showed a %CV ranging from 2.4-16.7%. The intra-day precision had a range of 11.2-19.0%. Finally, intermediate precision across all runs was 16.6% CV (Figure 5C). Strong linearity was observed with moderately higher error than the ELISA (Figure 5D). Despite increased variability, both the precision and linearity tests showed high accuracy. The imprecision can largely be attributed to inconsistencies in the upper asymptote. Although the precision is higher in this assay format, we suspect this could be improved dramatically through further assay optimization, replication, plate layout randomization, and/or automation39, 40.

CONCLUSION



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In this study, we explored the feasibility of using aptamers as affinity reagents in an HPV potency assay. We generated an aptamer, HPV-07, that has many of the attributes of a high quality reagent such as (a) high affinity, (b) high specificity, (c) stability indication, (d) competition with a neutralizing reagent, (e) precision, (f) accuracy, (g) and a large linear range in standard assay conditions. In particular, specificity is critical when determining the potency of a multivalent vaccine such as Gardasil® 9 since competing types can be a major challenge. Achieving such specificity has been a problem for aptamers in the past3, 41 but the competitive SELEX procedure done here generated highly selective reagents. Simply replacing antibodies with aptamers in an ELISA potency assay would have notable benefits but may fall short of the true potential of aptamers. In this study, we also exploited the control and ease in which aptamers are modified to create a mix-and-read assay that contains only three solutions, no wash steps, and takes less than 2 hours to complete. Other assay strategies could leverage aptamers to create an even superior assay. Ideally, an assay would measure the potency off all antigens in a multi-valent vaccine simultaneously. One could imagine a multiplex aptamer assay where each type-specific aptamer would be synthesized with a barcode (unique sequence identifier). This barcode could then be detected similarly to a microarray annealing to specific sequences. Similar diagnostic assays have been developed for aptamer multiplexing in highly heterogeneous samples14. The data presented in this paper creates a foundation to develop aptamer-based mix-and-read or multiplexed vaccine potency assays.

CONFLICT OF INTEREST DISCLOSURE All authors are employees of Merck & Co, Inc., Kenilworth, NJ USA. Gardasil® and Gardasil®9 are HPV vaccine produced by Merck & Co., Inc., Kenilworth, NJ USA.


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Figure 1. Initial characterization and specificity assessment of aptamers. (A) Sandwich ELISA comparing different aptamers ability to detect Type 16 VLP. (B) Comparing the HPV-07 aptamers ability to detect Type 16 VLP in the absence and presence of competitors (Type 6, 11, 18, 31, 55, and 58 each at 10 µg/mL). (C) Comparing the HPV-07 aptamers ability to capture Type 16 VLP in the absence and presence of competitors (Type 6, 11, 18, 31, 55, and 58 each at 10 µg/mL). (D) SPR analysis of different HPV types binding to the HPV-07 aptamer 18 ACS Paragon Plus Environment

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immobilized on a streptavidin chip. The vertical dotted lines indicate the start of the injection and the dissociation phases.

Figure 2. Comparing the HPV-07 aptamer to H16.V5 antibody. (A) ELISA where Type 16 VLP was captured using H16.J4 mAb and detected using either HPV-07 or H16.V5. (B) Ability of the aptamer and antibody to monitor stability. In vitro relative potency (IVRP) was determined for a destabilized native or adjuvanted VLP.

Figure 3. HPV-07 aptamer competing with the neutralizing antibody H16.V5. (A) Competition ELISA where the VLP was held slightly above the EC50 and a competing reagent was titrated prior to detection. Antibody H16.V5 was use for capture in lieu of H16.J4 when H16.J4 was the completion reagent, green. (B) SPR analysis of uncoated and antibody-coated Type 16 VLPs binding to HPV-07 immobilized on a streptavidin-labeled chip. All data are shown in triplicate.

Figure 4. Aptamer precision and linearity testing. (A) Box plot of IVRP values determined by ELISA run with 3 replicates on 3 plates over 4 days. Upper and lower line represent maximum and minimum. Upper and lower boxes represent third and first quartile. The center line represents the median. (B). Linearity testing with samples diluted over a 25% to 200% range standard formulation. X-axis represents the mock vaccine concentration prior to diluting to the titration starting point.

Figure 5. Aptamer based mix-and-read assay. (A) Cartoon of the AlphaLISA setup using streptavidin coated donor and acceptor beads bound to the HPV-07 aptamer. When an aptamer-mediated sandwich is formed the acceptor bead can receive oxygen generated by the 19 ACS Paragon Plus Environment


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excited donor bead to create chemiluminescent signal. Figure is not to scale (beads ≈ 250-350 nm, Type 16 VLP42 ≈ 60 nm, aptamer ≈ 18 kDa). True stoichiometry of aptamers per bead or aptamers per VLP are unknown (B) Titration of Type 16 VLP in the mix-and read format. (C) Box plot of IVRP values determined by AlphaLISA run with 3 replicates on 3 plates over 4 days. Upper and lower line represent maximum and minimum. Upper and lower boxes represent third and first quartile. The center line represents the median. (D) Linearity testing for AlphaLISA format with samples diluted over a 25% to 200% range standard formulation. X-axis represents the mock vaccine concentration prior to diluting to the titration starting point.


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Figure 1. Initial characteriza 177x124mm (300 x 300 DPI)

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Figure 2. Comparing the HPV-07 177x116mm (300 x 300 DPI)


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Figure 3. HPV-07 aptamer compe 84x116mm (300 x 300 DPI)



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Figure 4. Aptamer precision an 84x49mm (300 x 300 DPI)


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Figure 5. Aptamer based mix-an 84x172mm (300 x 300 DPI)



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Abstract Graphic 84x47mm (300 x 300 DPI)


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Development and Characterization of an HPV Type-16 Specific

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