This study was funded by a National Health and Medical Research Council Project Grant to JRM and CJT (ID 1163732) and in part by the Victorian Government&#39;s Operational Infrastructure Support Program. SB is supported by a joint Baker Heart and Diabetes Institute-La Trobe University Doctoral Scholarship. KLW is supported by The Shine On Foundation and a Future Leader Fellowship from the National Heart Foundation of Australia (ID 102539). JRM is supported by a National Health and Medical Research Council Senior Research Fellowship (ID 1078985).

All aspects of animal care and experimentation were conducted following Florey Institute of Neuroscience and Mental Health guidelines and the Australian Code for the Care and Use of Animals for Scientific Purposes following Reference25. 1.5-3-year-old Merino ewes were used for the study. A schematic overview of the assay protocol is provided in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_up...

This report describes a colorimetric assay that assesses the extent of AAV neutralization in a given serum sample by evaluating a chromogenic reaction corresponding to the degree of in vitro viral transduction. The development of the protocol was based on the known chromogenic reaction between the enzyme alkaline phosphatase and NBT/BCIP, which has been widely utilized as a staining tool for the detection of protein targets in applications such as immunohistochemistry and as a reporter tool for evaluating viral ...

Adeno-associated viruses (AAV) are increasingly used as vectors for the delivery of gene therapies to trial treatments for various health conditions that impact the cardiovascular, pulmonary, circulatory, ocular, and central nervous systems1,2,3,4,5. The popularity of AAV vectors as a leading gene therapy platform stems from their positive safety profile, long-term transgene expression, and wide-ranging tissue-specific tropisms1,6. Successful outcomes in animal studies have paved the way for over fifty AAV gene therapy clinical trials that have successfully reached their efficacy endpoints7, as well as the release of the first commercially available AAV gene therapy drug approved by the US Food and Drug Administration8. Following initial successes, AAV has continued to gain traction in the basic and clinical research sectors as a vector of choice and is currently the only in vivo gene therapy approved for clinical use in the US and Europe9. Nonetheless, the presence of pre-existing neutralizing antibodies (NAbs) against AAV vector capsids remains a hindrance to both preclinical research and the efficacy of clinical trials. NAbs are present in both na&#239;ve human and animal populations and inhibit gene transduction following in vivo administration of an AAV vector1. AAV seropositivity is an exclusion criterion for most gene therapy trials, and therefore preliminary screening for host immunity is crucial in both the laboratory and the clinic. Establishing an assay that can detect the presence of NAbs against AAV is an essential step in the pipeline of any AAV gene therapy-based research project. This report focuses on AAV6 which has been of interest to researchers due to its efficient and selective transduction in striated muscle (heart and skeletal muscle)1,10,11,12. Gene therapy is considered a promising strategy for targeting the heart because it is difficult to specifically target the heart without invasive open-heart procedures.
Neutralizing activity is usually determined using either a cell-based in vitro or in vivo transduction inhibition assay. In vivo NAb assays usually involve administering serum from a test subject (e.g., human or large animal) into mice, followed by an AAV with a reporter gene, followed by testing for the expression of the reporter gene or corresponding antigen. In vitro assays determine NAb titers by incubating serum or plasma from a human or large animal in serial dilutions with a recombinant AAV (rAAV) that expresses a reporter gene. Cells are infected with the serum/virus mixture, and the extent to which the reporter gene expression is inhibited is assessed compared with controls. In vitro assays are widely used for NAb screening due to their comparatively lower cost, rapidity in testing, and greater capacity for standardization and validation13,14 compared with in vivo assays. In vivo assays are often reported to have greater sensitivity15,16, but the same claim has been made concerning in vitro assays14,17.
To date, in vitro NAb assays have mainly used luminescence (luciferase) as the reporter gene to detect neutralization. Although a light-based method has merit in many contexts, a colorimetric/chromogenic NAb assay may be advantageous in some circumstances. Colorimetric assays to assess neutralization have been successfully employed for other viruses such as influenza and adenovirus18,19. Their attractiveness stems from their simplicity, lower cost, and the requirement for only everyday laboratory apparatus and tools20. NAb assays that use a luminescence-based reporter gene require costly substrate kits, a luminometer, and corresponding software for analysis21. This colorimetric assay has the advantage of only requiring a light microscope and a very cheap substrate. Reporting of the sensitivity of colorimetric versus luminescent assays has yielded conflicting results. One study suggested luminescence-based ELISA assays display greater sensitivity and comparable reproducibility to colorimetric assays22, while another found colorimetric-based ELISA assays to confer greater sensitivity23. Here, a detailed protocol for an in vitro NAb assay against AAV that utilizes the chromogenic reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and a nitro blue tetrazolium /5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) substrate is provided. This step-by-step protocol was developed based on a previous report that utilized an hPLAP (human placental alkaline phosphatase) reporter gene (AAV6-hPLAP) to detect neutralizing activity against AAV24. This assay is cost-effective, time-efficient, easy to set up, and requires minimal technical skills, laboratory equipment, and reagents. Moreover, the simplicity of this assay gives it the potential to be optimized for broad applications across different types of cells, tissues, or viral serotypes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin/EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tube</td>
			<td>Falcon</td>
			<td>14-432-22</td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>75 cm2 square flasks</td>
			<td>Falcon</td>
			<td>353136</td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>96 well flat bottomed plate</td>
			<td>Falcon</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>AAV6-CMV-hPLAP Vector</td>
			<td></td>
			<td></td>
			<td>Muscle Research &#38; Therapeutics Lab (University of Melbourne, Australia) AAV6-CMV-hPLAP can be provided upon request.</td>
		</tr>
		<tr>
			<td>Aluminium foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-AAV6 (intact particle) mouse monoclonal antibody, (ADK6)</td>
			<td>PROGEN</td>
			<td>610159</td>
			<td>Positive control monoclonal antibody</td>
		</tr>
		<tr>
			<td>BCIP/NBT</td>
			<td>SIGMAFAST</td>
			<td>B5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell and tissue culture safety cabinet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic Pipette</td>
			<td></td>
			<td></td>
			<td>5 &#38; 10 mL stripette inserts</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10099-141</td>
			<td></td>
		</tr>
		<tr>
			<td>Haemocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High glucose Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965118</td>
			<td></td>
		</tr>
		<tr>
			<td>HT1080 cells</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td></td>
			<td></td>
			<td>Freely available: https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td></td>
			<td></td>
			<td>37 &#176;C, 5% CO2</td>
		</tr>
		<tr>
			<td>Light microscope with camera</td>
			<td></td>
			<td></td>
			<td>Capable of taking photos with a 4x objective lens</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td></td>
			<td></td>
			<td>For a 65 &#176;C incubation</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>MERCK</td>
			<td>30525-89-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes and tips</td>
			<td></td>
			<td></td>
			<td>20 &#956;L, 200 &#956;L &#38; 1 mL single pipettes and tips &#38; 200 &#956;L multichannel pipette</td>
		</tr>
		<tr>
			<td>Stericup quick release filter</td>
			<td>Millipore</td>
			<td>S2GPU10RE</td>
			<td>Used for combining media reagents</td>
		</tr>
		<tr>
			<td>Trypan blue solution</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>VACUETTE TUBE 8 ml CAT Serum Separator Clot Activator</td>
			<td>Greiner BIO-ONE</td>
			<td>455071</td>
			<td>Used for serum collection &#38; processing from sheep</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a step-by-step method to detect neutralizing antibodies against aav using a colorimetric cell-based assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

Transduction assay to establish the optimal viral dosage for plate coverage 
HT1080 cells, a well-established fibrosarcoma cell line, were selected for this assay. A concentration of 1 x 104 HT1080 cells/well provided ~50% cell confluency in each well of a 96-well plate. To determine the optimal viral concentration for the assay, an rAAV encoding an hPLAP (human placental alkaline phosphatase) reporter gene (AAV6-hPLAP)31 was added in triplicate at a range of conce...

Watch this Scientific Journal Video about A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay at JoVE.com

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...

Preexisting antibodies that neutralize AAV pose a barrier to the advancement of AAV gene therapies. This assay is a screening tool to detect neutralizing antibodies against AAV. The main advantage is that it's cost-effective, time efficient, easy to set up, and requires minimal technical skills, lab equipment, and reagents.Start plating HT1080 cells on day one by diluting the cells to a concentration of 1 x 10 to the fifth cells per milliliter in pre-warmed complete DMEM media. Then seed 100 microliters of cells per well into clear 96-well flat bottomed plates to the concentration of 1 x 10 to the fourth cells per well. Incubate the plate at 37 degrees Celsius with 5%carbon dioxide overnight for 16 to 22 hours.On day two, generate serial dilutions of the serum samples of interest in 1.5 milliliter microcentrifuge tubes using pre-warmed complete DMEM. To each tube with diluted serum samples, add 66 microliters of the 7.5x10 to the six viral genome per microliter virus working solution. Mix the virus serum dilution by pipetting and then place the tubes containing the virus serum mixtures in an incubator at 37 degree Celsius with 5%carbon dioxide for 30 minutes to allow potential neutralization to occur.After 30 minutes, pipette 100 microliters of the virus serum mixture to each well on the 96-well plate containing 1x10 to the fourth cells per well, then wrap the plate in a foil to place in an incubator at 37 degree Celsius with 5%carbon dioxide overnight for 16 to 24 hours. On day three, aspirate the media from the wells of the 96-well plate using a fume hood vacuum without disrupting the adhered cells and add 50 microliters of 4%paraformaldehyde to each well. After wrapping the plate in foil, leave it for 10 minutes at room temperature.Later, wash and aspirate the cells twice with 200 microliters of PBS at room temperature. After the second wash, pipette 200 microliters of pre-warmed PBS into each well and wrap the plate in foil followed by incubation at 65 degree Celsius for 90 minutes to denature endogenous alkaline phosphatase activity, following incubation at 50 microliters of the freshly prepared dissolved BCIP/NBT into each well and incubate the wrapped plates at room temperature for two to 24 hours. Later, take photos of each well using a 4X objective lens in a light microscope camera, ensuring the consistent use of the same exposure, white balancing, and light settings for all assays.To analyze the image in ImageJ, select the file and click Open. For the colored images, convert to gray scale by selecting the Image, Type, and 8-bit tabs in order. Then go to the Image tab and select Adjust and Threshold to alter the threshold until all colored areas are colored and red but the background is not.click on Analyze and Set Measurements and tick the check boxes for Area, Area fraction, Limit to threshold, and Display label before hitting OK.To determine the signal reading of a given well, click on Analyze and Measure so that the percent area column of the popup window displays the signal reading. The study established the optimal viral dosage by adding the AAV6-hPLAP reporter gene in the cells at a range of concentrations of the viral genome. A multiplicity of infection of 15, 000 conferred 36%plate coloration and was selected as the optimal viral dosage.The representative analysis displays the correlation between coloration and multiplicities of infection. The highest tested concentration that did not affect the linear correlation between coloration and viral concentration was obtained. The study of neutralizing activity on an anti-AAV6 monoclonal antibody against adeno-associate virus or AAV6 at log 10 dilution showed 50%inhibition of AAV6 transduction or TI50 at a concentration of 10 nanograms per milliliter.In the neutralizing antibody or NAb assay, the degree of AAV neutralization varied within the naive sample population with TI50 titre values ranging from 1/2 to 1/80. The assay results indicated that AAV inhibition TI50 titre values before AAV administration ranged from 1/4 to 1/80. After AAV administration, the TI50 titre increased to between 1/2000 and greater than 1/32, 000, demonstrating a clear contrast in titre values between pre-and post-AAV administration.When taking photos of the wells, it is important to consistently use the same microscope settings throughout the assay and to also make sure that the photos are taken directly in the center of the wells.

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Biology

4.2K Views.  Baker Heart and Diabetes Institute. Gli anticorpi preesistenti che neutralizzano l'AAV rappresentano una barriera al progresso delle terapie geniche AAV. Questo test è uno strumento di screening per rilevare gli anticorpi neutralizzanti contro l'AAV. Il vantaggio principale è che è conveniente, efficiente in termini di tempo, facile da configurare e richiede competenze tecniche minime, attrezzature di laboratorio e reagenti.Iniziare a placcare le cellule HT1080 il primo giorno diluendo le cellule ad una concentrazione...