We thank Chiron Behring and Bharat Biotech International for providing Particles for Humanity&#160;with the RABV antigen. We would also like to acknowledge Charles Rupprecht, VMD, MS, PhD., for his invaluable guidance and technical contributions. The authors would like to thank the generosity of Dr. Rebecca Richards-Kortum for allowing the use of her SciFLEXARRAYER S3 picoliter dispensing apparatus and Dr. Chelsey Smith&#39;s instruction on using the device. We also acknowledge the University of Massachusetts Chan Medical School for generating microscopy images of the rabies antigen.&#160;Finally, we thank Don Chickering and Erin Euliano for reviewing the document before submission. This work was supported by a grant (INV-004360) from the Bill and Melinda Gates Foundation.

<ol>
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A., Veena, V., Hanumanthaiah, A. N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compliance+to+anti-rabies+vaccination+in+post-exposure+prophylaxis.">Compliance to anti-rabies vaccination in post-exposure prophylaxis.</a> Indian Journal of Public Health. 59 (1), 58-60 (2015).</li><li>Cleland, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-administration+vaccines:+controlled-release+technology+to+mimic+repeated+immunizations.">Single-administration vaccines: controlled-release technology to mimic repeated immunizations.</a> Trends in Biotechnology. 17 (1), 25-29 (1999).</li><li>Yeh, M. K., Coombes, A. G. A., Jenkins, P. G., Davis, S. 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A., Bronshtein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabies+vaccine+preserved+by+vaporization+is+thermostable+and+immunogenic.">Rabies vaccine preserved by vaporization is thermostable and immunogenic.</a> Vaccine. 33 (19), 2203-2206 (2015).</li><li>McHugh, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+fillable+microparticles+and+other+complex+3D+microstructures.">Fabrication of fillable microparticles and other complex 3D microstructures.</a> Science. 357 (6356), 1138-1142 (2017).</li><li>Sanchez, M. E. 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Dr. McHugh has a patent filed based on the encompassed work and is currently a consultant for Particles for Humanity. Mr. Graf has a patent filed based on the encompassed work. Dr. Kadasia,&#160;Mr. Yang, and Ms. Brady are part of Particles for Humanity, a public benefit corporation that licensed a similar microparticle vaccine delivery technology from MIT during this work.

It is possible to alter particle geometry for specific needs; however, for cylindrical structures, the authors recommend maintaining a 5:4:1 ratio of the height:diameter:wall thickness described in the protocol. This aspect ratio ensures that enough PLGA material is present to seal the particles and remain mechanically robust enough for handling. Particle dimensions and shapes can easily be altered during the CAD process, enabling a myriad of geometries to be generated. Combining the flexibility of the CAD with 3D printing enables the rapid iteration of microparticle designs. Although this protocol uses a multi-photon 3D printer, any 3D printer with specifications capable of printing the microstructure dimensions in an appropriate material can be used to generate the initial master mold. Further, photolithography has previously been used to make similar structures in arrays much larger than those produced in this protocol; however, the labor, delay of ordering custom-made photomasks, and equipment accessibility would slow the iterative design process16. Finally, master mold generation can be outsourced to fee-for-service companies if in-house master mold fabrication is not feasible. Regardless of the 3D printer or method used to generate the master molds, the adhesion of the print to the substrate is critical for downstream steps. Specifically, if adhesion is inadequate during PDMS mold generation, printed particles will remain lodged in the PDMS mold, requiring manual removal of the printed particles and destruction of the master mold.
Particle filling is another critical aspect to consider. Microparticles have limited filling capacities, so filtration is used not only to concentrate the RABV antigen but also to remove stock excipients that would otherwise occupy a large portion of the microparticle core volume. However, given the large size of the RABV antigen (approximately 60 nm by 180 nm)17, it is possible to partially pellet out the antigen during the centrifugation steps. For this reason, it is important to resuspend the antigen by pipetting or vortexing after centrifugation to achieve a high recovery of the RABV antigen. A highly concentrated solution is ideal for dispensing, because it reduces the dispensing cycles and thereby limits antigen degradation during filling. However, viscosity is a major limitation of piezoelectric dispensing robots forming a stable drop, so dispensing a very high-concentration solution may not be possible or advisable. Diluting the filling solution is the easiest way to achieve a stable drop formation, but antigen stability over the additional filling cycles needed to achieve the desired loading and the longer amount of time required to fill particles should be considered.
Limitations 
This method requires highly specialized equipment to produce the initial molds and a specialized filling instrument for microparticle production. Although the need for a 3D printer with a printing resolution capable of generating the initial master molds can be subverted by a fee-for-service approach, accessibility to a piezoelectric dispensing robot is limiting. Procurement of a piezoelectric dispensing robot requires a significant initial upfront investment, often in the range of $80,000 to $200,000, depending on the brand, throughput, and capabilities. Although several other filling methods are potential alternatives, these methods have not been validated using the RABV antigen12.
Future applications 
A substantial proportion of encapsulated RABV antigen remained stable through the sealing process. In theory, by incorporating this antigen into particles composed of different types of PLGA that mimic the administration timeline of post-exposure prophylaxis treatment, all doses could be administered in a single injection. Eliminating the need for repeat hospital visits to administer additional doses will enhance patient compliance, resulting in better treatment outcomes. Further, having demonstrated the ability to retain the ELISA-reactivity of the highly complex inactivated rabies virus, it is likely that other antigens, including subunit vaccines, would be compatible with this encapsulation method. Using other prophylactic antigens with PULSED microparticles could save millions of lives in LMICs by increasing the vaccination rates of under-vaccinated populations. To accomplish this, however, vaccines must remain stable through not only encapsulation but also release, which may be challenging since the payload will be subjected to elevated temperatures and a potentially acidic microenvironment due to body heat and PLGA degradation products18. Future work will pursue stabilizing strategies of the antigen through release, which would open up the potential for a single-injection vaccination platform that is broadly applicable to prevent many infectious diseases.

Vaccination is an extremely effective healthcare tool, having prevented more than 37 million deaths between 2000 and 20191. Despite this effectiveness, vaccine-preventable diseases continue to pose a significant risk to global health, especially in low- and middle-income countries (LMICs) where high rates of un- and under-vaccination contribute to 1.5 million vaccine-preventable deaths annually2. Rabies is no exception to these disparities. Despite its status as the most deadly disease known to humankind, being almost universally fatal, rabies is fully treatable and is classified as eradicated in many high-income countries. Instead, the burden of rabies is disproportionately borne by people living in parts of Asia and Africa, where the disease has devastating outcomes on humans and livestock3,4.
Vaccination is critical to managing the global impact of rabies5. The cost of vaccination prohibits widespread implementation of pre-exposure prophylaxis (PrEP), considering the overall low incidence of the disease. Furthermore, in LMICs, the utility of post-exposure prophylaxis (PEP) is limited by socioeconomic pressures on patients seeking healthcare. Logistical factors, such as travel distance to healthcare access points, lost wages while obtaining treatment, the cost of treatment, appointments interfering with daily activities, and forgetfulness, result in PEP adherence rates as low as 60%6,7. This high patient attrition rate presents an opportunity for refining approaches to address gaps in rabies vaccination in order to combat the disease.
Single-injection (SI) vaccination systems that control the release of antigens have been explored as ways to obtain full immunization in one injection. Eliminating the need for multiple visits to a healthcare provider mitigates the burdens that prevent individuals from seeking adequate care. To achieve SI vaccination, an antigen is typically encapsulated within a biodegradable polymeric matrix that often takes the form of injectable microparticles. Once injected, the polymer degrades and releases the sequestered antigen. To date, two primary release strategies have been pursued to achieve SI vaccination. In one approach, the antigen is released continuously over an extended period of time. Although intended to enhance the immunogenicity of a single injection, it is unclear if this approach is sufficient to elicit a protective immune response against the rabies virus (RABV) in humans8. In the other, the antigen is released after a predetermined delay to mimic a conventional and proven prime-boost vaccine regimen. Spray drying and emulsion/solvent evaporation-based microparticle fabrication methods exhibit the former strategy, and have been used to successfully encapsulate both model vaccines9&#160;and highly stable antigens, such as tetanus toxoid10. However, these encapsulation methods involve stressors, including heat, solvent interaction, and physical forces, that can denature antigens11.
Particles Uniformly Liquified and Sealed to Encapsulate Drugs (PULSED) is a recently developed fabrication method that can be employed to encapsulate biologics in biodegradable microparticles. Micromolding is used to generate particles that are filled with a liquid payload and heated to allow the polymer to reflow and fully encapsulate the central depot of cargo within a contiguous layer of the biodegradable polymer. This microstructure results in the pulsatile release of the payload, after a duration that is dependent on the degradation rate of the polymeric shell12. This manuscript demonstrates the encapsulation of inactivated RABV within microparticles composed of poly(lactic-co-glycolic acid) (PLGA), a biodegradable polymer used in many FDA-approved formulations13, using the PULSED fabrication method to encapsulate stable RABV antigen as evaluated by an enzyme-linked immunosorbent assay (ELISA). By combining PLGA particles with different molecular weight and/or end groups, this approach has the potential to mimic the current rabies vaccination time course following a single injection.

<table><tbody><tr><td>0.22 &amp;micro;m PES filter</td><td>Cole-Parmer+B4B2:B63</td><td>04396-26</td><td></td></tr><tr><td>0.25 mm Shims</td><td>McMaster Carr</td><td>98090A935</td><td></td></tr><tr><td>0.75 inch Binder Clips</td><td>Staples</td><td>480114</td><td></td></tr><tr><td>10 mL Syringe</td><td>Becton, Dickinson and Company</td><td>309604</td><td></td></tr><tr><td>10 mL&amp;nbsp;Sterile Polystyrene Disposable Serological Pipets with Magnifier Stripe</td><td>Fisherbrand</td><td>13-678-11E</td><td></td></tr><tr><td>101.6 mm C-Clamp</td><td>Amazon</td><td>PT-SD-CP01A</td><td>Black handle will eventually fall off. Use pliers to adjust once this happens.</td></tr><tr><td>19 G needle</td><td>EXCELINT</td><td>26438</td><td></td></tr><tr><td>25 mL&amp;nbsp;Sterile Polystyrene Disposable Serological Pipets with Magnifier Stripe</td><td>Fisherbrand</td><td>13-678-11</td><td></td></tr><tr><td>3-(Trimethoxysilyl) Propyl Methacrylate&amp;nbsp;</td><td>Millipore Sigma</td><td>M6514-25ML</td><td></td></tr><tr><td>5 mL&amp;nbsp;Sterile Polystyrene Disposable Serological Pipets with Magnifier Stripe</td><td>Eppendorf</td><td>22431081</td><td></td></tr><tr><td>50 mL Centrifuge Tubes</td><td>Corning&amp;nbsp;</td><td>352098</td><td></td></tr><tr><td>50 mL&amp;nbsp;Sterile Polystyrene Disposable Serological Pipets with Magnifier Stripe</td><td>Fisherbrand</td><td>13-678-11F</td><td></td></tr><tr><td>Acetone</td><td>Fisher</td><td>AC268310010</td><td></td></tr><tr><td>Aluminum Block</td><td>McMaster Carr</td><td>9057K175</td><td></td></tr><tr><td>Aluminum Foil</td><td>VWR</td><td>89079-069</td><td></td></tr><tr><td>Amicon Ultra 0.5 mL Centrifugal Filters, 100 kDa</td><td>Millipore Sigma</td><td>C82301</td><td></td></tr><tr><td>Anti-Rabies Virus Antibody, Serum Free Antibody, clone 1112-1, 100</td><td>Fisherbrand</td><td>13-678-11D</td><td></td></tr><tr><td>Anti-Rabies Virus Mouse Monoclonal Antibody, Clone D1-25, biotinylated</td><td>Fisherbrand</td><td>14-388-100</td><td></td></tr><tr><td>Carboxymethyl Cellulose</td><td>Tokyo Chemical Industries</td><td>C0045</td><td></td></tr><tr><td>ClipTip 300, Filter, Racked</td><td>Fisherbrand</td><td>13-678-11</td><td></td></tr><tr><td>Costar 0.65 mL Low Binding Snap Cap Microcentrifuge Tube</td><td>Corning&amp;nbsp;</td><td>3206</td><td></td></tr><tr><td>Costar 1.7 mL Low Binding Snap Cap Microcentrifuge Tube</td><td>Corning&amp;nbsp;</td><td>3207</td><td></td></tr><tr><td>Describe</td><td>Nanoscribe</td><td></td><td>Software used to define the printing parameters for Nanoscribe 3D printer is step 1.2.&lt;br/&gt; Software provided with the printer.</td></tr><tr><td>Desiccator</td><td>Fisher Scientific</td><td>10529901</td><td>Or equivalent</td></tr><tr><td>Double-Sided Tape</td><td>Staples</td><td>649280</td><td></td></tr><tr><td>DPBS (10x), No Calcium, No Magnesium</td><td>Gibco</td><td>14200075</td><td></td></tr><tr><td>Ethanol</td><td>VWR</td><td>89370-084</td><td></td></tr><tr><td>F1-ClipTip Multichannel Pipettes, 30 to 300 &amp;micro;L</td><td>Fisherbrand</td><td>13-678-11E</td><td></td></tr><tr><td>Fisherbrand SureOne Aerosol Barrier Pipette Tips, 0.1 &amp;ndash; 10 &amp;micro;L</td><td>Fisherbrand</td><td>13-678-11F</td><td></td></tr><tr><td>Fisherbrand SureOne Aerosol Barrier Pipette Tips, 100 &amp;ndash; 1000 &amp;micro;L</td><td>Fisherbrand</td><td>03-448-17</td><td></td></tr><tr><td>Fisherbrand SureOne Aerosol Barrier Pipette Tips, 2 &amp;ndash; 20 &amp;micro;L</td><td>Fisherbrand</td><td>FB14955202</td><td></td></tr><tr><td>Fisherbrand SureOne Aerosol Barrier Pipette Tips, 20 &amp;ndash; 200 &amp;micro;L</td><td>Fisherbrand</td><td>13-374-10</td><td></td></tr><tr><td>Fisherbrand&amp;nbsp;Elite Pipette Kit</td><td>Fisherbrand</td><td>05-408-137</td><td></td></tr><tr><td>Fisherbrand&amp;nbsp;Pipet Controller&amp;nbsp;</td><td>Fisherbrand</td><td>FB14955202</td><td></td></tr><tr><td>Glass Petri Dish, 90 mm</td><td>VWR</td><td>470313-346</td><td></td></tr><tr><td>Glass Slides</td><td>Globe Scientific</td><td>1380-10</td><td></td></tr><tr><td>Helicon Focus 8</td><td>HeliconSoft</td><td></td><td>Software used to focus stack images</td></tr><tr><td>IP-Q Resin</td><td>Nanoscribe</td><td></td><td>Printer resin is compatable with the 10x lens and is used for printing large microstructures on the Nanoscribe Photonic Professional GT2</td></tr><tr><td>Lascar EL-USB-TC-LCD Thermocouple</td><td>Amazon</td><td>5053485896236</td><td>Or equivalent</td></tr><tr><td>Microscope Slide Box</td><td>Millipore Sigma</td><td>Z374385-1EA</td><td>Or equivalent</td></tr><tr><td>Nanoscribe Photonic Professional GT2 with 10X Objective</td><td>Nanoscribe</td><td></td><td></td></tr><tr><td>NanoWrite</td><td>Nanoscribe</td><td></td><td>Software used to interface with nanoscrive 3D printer.&lt;br/&gt; Software provided with printer.</td></tr><tr><td>Nunc MaxiSorp Flat-Bottom 96-well Plate</td><td>Invitrogen</td><td>44-2404-21</td><td></td></tr><tr><td>OPD Substrate Tablets (o-Phenylenediamine Dihydrochloride)</td><td>Fisherbrand</td><td>02-707-432</td><td></td></tr><tr><td>Parafilm M Wrapping Film, 4 in.</td><td>Fisherbrand</td><td>13-374-10</td><td></td></tr><tr><td>PDC 60 with Type 3 Coating</td><td>Scienion</td><td>P-2020</td><td></td></tr><tr><td>PDMS Particle Molds</td><td>Rice University</td><td>n/a</td><td>N/A- Particles are 400 &amp;mu;m in diameter with a wall thickness of 100 &amp;mu;m, and a height of 500 &amp;mu;m, resulting in an inner diameter of 200 &amp;mu;m. The arrays are 14 x 22 particles spaced 600 &amp;mu;m apart from each other. 4- and 5-point stars are used as fiducials, positioned 600 &amp;mu;m to the right and left of the top right and top left particles on the array.</td></tr><tr><td>Petri Dish</td><td>Fisher Scientific</td><td>08-757-100D</td><td></td></tr><tr><td>Pierce Stable Peroxide Substrate Buffer (10x)</td><td>Fisherbrand</td><td>02-707-430</td><td></td></tr><tr><td>Plastic Cups</td><td>Fisher Scientific</td><td>S04170</td><td></td></tr><tr><td>PLGA Film, 502H</td><td>Sigma</td><td>502H: 719897-1G</td><td></td></tr><tr><td>Propylene Glycol Monomethyl Ether Acetate</td><td>Millipore Sigma</td><td>484431</td><td></td></tr><tr><td>Rabies Antigen</td><td>Chiron Behring and Bharat Biotech International</td><td></td><td>Material was acquired by entering into a materials transfer agreement with the company.</td></tr><tr><td>Razor Blades</td><td>VWR</td><td>55411-050</td><td></td></tr><tr><td>Scalpel</td><td>VWR</td><td>21899-530 and 76457-512</td><td></td></tr><tr><td>SciFLEXARRAYER S3 with PCD 60</td><td>Scienion</td><td></td><td>Or equivalent</td></tr><tr><td>Sealing Tape for 96-Well Plates</td><td>Thermo Scientific</td><td>15036</td><td></td></tr><tr><td>Silicon Wafer</td><td>University Wafer</td><td>1025</td><td></td></tr><tr><td>Spring Clamps</td><td>IRWIN</td><td>VGP58100</td><td></td></tr><tr><td>Stainless Steel Block</td><td>McMaster Carr</td><td>9083K12</td><td></td></tr><tr><td>Streptavidin&amp;minus;Peroxidase Polymer, Ultrasensitive</td><td>Fisherbrand</td><td>02-707-404</td><td></td></tr><tr><td>Sylgard 184</td><td>DOW</td><td>2646340</td><td></td></tr><tr><td>Teflon Sheet</td><td>McMaster Carr</td><td>9266K12</td><td>Used to make PLGA films. Must be cut into appropriately sized pieces.</td></tr><tr><td>Teflon Sheet, 0.8 mm-thick</td><td>McMaster Carr</td><td>9266K81</td><td></td></tr><tr><td>Trichloro(1H, 1H, 2H, 2H-Perfluorooctyl) Silane</td><td>Sigma</td><td>448931-10G</td><td></td></tr><tr><td>Tweezers&amp;nbsp;</td><td>Pixnor</td><td>ESD-16</td><td></td></tr><tr><td>UltraPure Distilled Water</td><td>Fisher Scientific</td><td>10977015</td><td></td></tr><tr><td>UV Oven, CL-1000S UV Crosslinker</td><td>UVP</td><td>95-0174-01</td><td>Or equivalent</td></tr><tr><td>Vacuum Desiccator</td><td>Bel-Art</td><td>F420100000</td><td>Note you will need two of these. One will be used exclusively to pre-treat samples with trichloro(1H, 1H, 2H, 2H-perfluorooctyl) silane to prevent contamination.</td></tr><tr><td>Vacuum Oven Capable of Reaching 120 &amp;deg;C</td><td>VWR</td><td>97027-664</td><td>Or equivalent</td></tr><tr><td>Vacuum, CRVpro4&amp;nbsp;</td><td>Welch</td><td>3041-01</td><td>Or equivalent</td></tr><tr><td>Wooden Tongue Depressors</td><td>Electron Microscopy Sciences</td><td>72320</td><td></td></tr></tbody></table>

fabrication of pulsatile polymeric microparticles encapsulating rabies antigen

The current guidelines for rabies post-exposure prophylaxis require multiple injections administered over several weeks. This can be disproportionately burdensome to those living in low- and middle-income countries (LMICs), where the majority of deadly exposures to rabies occur. Different drug delivery strategies have been explored to condense vaccine regimens to a single injection by encapsulating antigens into polymeric particles. However, harsh stressors during the encapsulation process can cause denaturation of the encapsulated antigen. This article describes a method for encapsulating the rabies virus (RABV) antigen into polymeric microparticles that exhibit tunable pulsatile release. This method, termed Particles Uniformly Liquified and Sealed to Encapsulate Drugs (PULSED), generates microparticles using soft lithography to create inverse polydimethylsiloxane (PDMS) molds from a multi-photon, 3D-printed master mold. Poly(lactic-co-glycolic acid) (PLGA) films are then compression-molded into the PDMS molds to generate open-faced cylinders that are filled with concentrated RABV using a piezoelectric dispensing robot. These microstructures are then sealed by heating the top of the particles, allowing the material to flow and form a continuous, nonporous polymeric barrier. Post-fabrication, an enzyme-linked immunosorbent assay (ELISA) specific to the detection of intact trimeric rabies virus glycoprotein is used to confirm the high recovery of immunogenic antigen from the microparticles.

Particle sealing and filling are two of the most critical steps in this protocol. Particles were filled with fluorescein sodium salt to demonstrate ideal filling and some common errors. Fluorescein sodium salt was used in lieu of the RABV antigen for ease of visualization. During filling, it is important to dispense solution into the bottom of particle cores, then allow enough time for the solvent/water to evaporate. Once complete, a depot of the solute remains in the bottom of the particle core (Figure 1A). Once filled, it is critical to seal the particles correctly. Figure 1B demonstrates several outcomes (successful and unsuccessful) of the sealing process. After 12 s of sealing time, a distinct pathway remains from the particle center to the outside of the particle, demonstrating a particle that is not entirely sealed. Conversely, if left to seal for 36 s, the PLGA almost entirely melts, resulting in a microstructure with a shallow profile. The Ideal morphology can be visualized when particles are sealed for 18 and 24 s, as they contain cargo entirely encapsulated by the polymer while maintaining a particle structure. Figure 1C demonstrates several potential outcomes after filling and sealing. During dispensing, if the solvent does not reach the particle bottom, it leaves solute dried in the middle of the particle core (incorrectly filled); although these particles may still seal, the poor loading of cargo can limit loading efficiency. If particles are filled with too much cargo (overfilled), the sealing process is inhibited, as the cargo prevents PLGA from flowing over the opening. When correctly filled and sealed, particles of this geometry are small enough to fit easily inside a 19 G needle. Further, 10 particles consistently flowed through a 19 G needle (100% &#177; 0%) when injected with a viscous solution such as 2% carboxymethyl cellulose (Supplementary Figure 7).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65147/65147fig01.jpg" /> 
Figure 1: Common problems with the filling and sealing process. (A) Images show the evaporation of solvent after one filling cycle, where particles are loaded with 6 nL of 100 mg/mL fluorescein sodium salt dissolved in water. (B) Representative images of 502H PLGA particles removed from the sealing process at 0, 12, 18, 24, and 36 s. (C) Different outcomes of the sealing process when particles are filled correctly, incorrectly, or overfilled. Images are generated by focus stacking multiple images and merging them using focus stacking software. Scale bar = 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65147/65147fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Processing the antigen through a spin filter prior to loading it into the particles is important for two reasons. First, the centrifugation serves to remove stock excipients in the vaccine solution, that can limit particle loading capacity while retaining the RABV antigen. The current protocol purifies the antigen by approximately 50-fold. Second, the antigen is also concentrated during this process. Figure 2A shows a micrograph of intact RABV virions in the concentrated antigen sample. This antigen is approximately 4.4-fold more concentrated than the starting stock solution (Figure 2B). The amount of antigen initially loaded into the centrifugal spin filters can be altered to modulate the final fold concentration achieved. For example, loading 40 &#181;L of stock antigen results in an approximate 1.75-fold concentration. Supplementary Figure 8 demonstrates the importance of vortexing (step 5.15) in the concentration process. Neglecting to vortex or improperly vortexing the samples limits the concentration process.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65147/65147fig02.jpg" /> 
Figure 2: Antigen concentration. The concentration of antigen by centrifugal filtration shown by transmission electron microscopy (A) and confirmed by ELISA (B). Error bars indicate the standard deviation. Statistical analysis is done using Tukey&#39;s multiple comparison tests with one-way ANOVA. **p &#60; 0.01, ****p &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/65147/65147fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 3A depicts concentrated antigen filled into unsealed and sealed particles. Although a significant amount of antigen is loaded into the particles (0.0469 &#177; 0.0086 IU), this material comprises &#60;90% of the particle capacity, leaving ample space for the loading of additional antigen. Interestingly, unsealed particles only contain 0.0396 &#177; 0.0077 IU, comprising only 85% &#177; 16% of the total amount loaded. Although a statistically insignificant loss, some of the RABV antigen may have denatured during repeated rehydration and drying in the filling process. After sealing, 69% &#177; 5% of the antigen remains encapsulated in a bioactive form. Although this suggests significant loss occurs during the sealing process due to thermal stress, most of the inactivated viral antigen remains intact (Figure 3B). Co-encapsulation of stabilizing excipients along with the antigen is one possible strategy to further increase antigen stability throughout the fabrication process, and has previously been successful with other inactivated virus antigens14,15.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65147/65147fig03.jpg" /> 
Figure 3: Bioactive RABV antigen after particle fabrication. (A) Images show unsealed and sealed particles containing the RABV antigen. (B) Antigen stability through the particle fabrication process (n = 4). Loading control is generated by dispensing the antigen directly into the solution. Error bars indicate the standard deviation. Scale bar = 200 &#181;m. Statistical analysis is done using Tukey&#39;s multiple comparison tests with one-way ANOVA. *p &#60; 0.05. Images are generated by focus stacking multiple images and merging them using focus stacking software. <a href="https://www.jove.com/files/ftp_upload/65147/65147fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Particle, fiducial, and array dimensions. The figure shows the geometric properties of the four-pointed star fiducial (A), the cylindrical microparticle (B), the five-pointed star fiducial (C), and an array of particles with fiducials (D) displayed in the CAD software. <a href="https://www.jove.com/files/ftp_upload/65147/Figure S1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: This figure shows a cross-section of the structure placed into the oven to cure PDMS molds. Arrows indicate where binder clamps are applied. <a href="https://www.jove.com/files/ftp_upload/65147/Figure S2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: Proper technique to efficiently recover concentrated antigen. At the end of the first spin, the concentrated sample circled in red (A) is retained in the filter, while the filtrate is collected in the bottom of the collection tube (larger outer tube). To resuspend the pelleted antigen, the centrifugal filter unit is capped using the collection tube (B) in preparation for vortexing. When vortexing, the tip of the tube is kept in contact with the vortex pad and the cap end rotated around while maintaining a 45&#176; angle with the vortex pad (C). <a href="https://www.jove.com/files/ftp_upload/65147/Figure S3.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 4: Pre-run programming piezoelectric dispenser. (A) Set up the &#34;Find Target Reference Points&#34; by navigating from the &#34;Main tab&#34; to the Robot Setup &#62; Miscellaneous &#62;Div. Function &#62; Find Target Reference Points. Use the buttons highlighted in blue to set the fiduciary marks. First, select Learn Template and draw a box around the fiduciary mark of interest, the verify the template accuracy by clicking on Search Template, and save the template. Do this for the four- and five-pointed stars, and save the file names according to the instructions under Use Two Different Template Images. Next, ensure all the parameters in the black boxes match. Load the four-pointed star fiducial using the Load Template (green box). Save the &#34;Find Target Reference Points&#34; program by selecting Task List (orange box). (B) Set up the Run by navigating to the Robot Setup &#62; Tasks tab, then load the sequence of tasks shown in the blue box by adding tasks from the Task List (black box) using the Task in Run selections (green box). Finally, save the task (orange box). (C) Set up the &#34;Target Substrate&#34; by navigating to the Robot Setup &#62; Target Substrate, then add a target (blue box). (D) Enter the parameters shown here and select Save (blue box). <a href="https://www.jove.com/files/ftp_upload/65147/Figure S4.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 5: Loading antigen and calibrating dispending alignment. (A) Navigate to the Nozzle Setup &#62; Do Task tab and aspirate 10 &#181;L of the antigen into the dispensing tip by selecting TakeProbe10 uL (black box) and clicking on DO (black box). (B) This opens a separate window. Select the well the concentrated antigen was loaded into and select OK (blue box). (C) After the antigen has been aspirated, wash the tip by selecting the blue box, and repeat this wash two more times. Select the camera (black box) and determine the drop volume by selecting drop volume (green box). Ensure a stable drop is forming with a volume standard deviation (%) &#60;2 (orange box). (D) Following these steps will open up the Snap Drop Cam; select Image (blue box) in the drop-down menu and select Nozzle Head Camera Wizard. This opens a new window. Perform the next sequence of steps quickly. (E) Ensure the target made in Supplementary Figure 4 is loaded, then select Move To Target (blue box). Adjust the drops to 15 and select Spot (black box). Once spotted, immediately select Move (green box). Ensure Auto Find is selected and delete particle size = 12, then click on Start (orange boxes). If the auto-detection fails, repeat this process after moving to a different area on the slide (purple box). <a href="https://www.jove.com/files/ftp_upload/65147/Figure S5.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 6: Programing spotting array and beginning the run. (A) Navigate to the Target Setup &#62; Target, then fill in the parameters shown in the blue box. (B) Next, navigate to the &#34;Field Setup tab&#34; and enter 20 into the no. of drops field (blue box), select a well (black box), then select targets/particles to dispense into (green box). By selecting a different well and reselecting the targets/particles, additional filling cycles are performed during a single run. (C) On the main screen, ensure the run and target created in Supplementary Figure 5 are selected. (D) Navigate to the &#34;Run tab&#34; and select Start Run (blue box). <a href="https://www.jove.com/files/ftp_upload/65147/Figure S6.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 7: Microparticle injectability. (A-C) Focus-stacked stereoscope images of microparticles filled with fluorescein sodium salt and sealed in a 19 G needle. (D) A total of 10 particles were injected through a 19 G needle using a 2% carboxymethyl cellulose solution (n = 8). Scale bar = 1 mm. Error bars indicate standard deviation. <a href="https://www.jove.com/files/ftp_upload/65147/Figure S7.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 8: Potential issues with antigen concentration. The red line indicates the expected fold increase in concentration. Error bars indicate standard deviation. Statistical analysis was done using Tukey&#39;s multiple comparison tests with one-way ANOVA. *** p &#60; 0.001, ****p &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/65147/Figure S8_Revised.pdf" target="_blank">Please click here to download this File.</a>
Supplementary File 1: Preparation of buffers and solutions for RABV ELISA. <a href="https://www.jove.com/files/ftp_upload/65147/Supplementary File 1.docx" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 1: STL file containing particle array. <a href="https://www.jove.com/files/ftp_upload/65147/Supplemental File 1.STL" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 2: STL file containing geometry for the custom slide holder used for sealing particles. <a href="https://www.jove.com/files/ftp_upload/65147/Supplemental File 2.STL" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen at JoVE.com

Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen

This method describes the encapsulation of the rabies antigen into biodegradable polymeric microparticles with structural and material properties that enable pulsatile release after a predetermined delay. Enzyme-linked immunosorbent assay (ELISA) assessment of the antigen retrieved from the particle core confirms the presence of intact trimeric rabies virus glycoprotein through particle fabrication.

The current guidelines for rabies post-exposure prophylaxis require multiple injections administered over several weeks. This can be disproportionately ...

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Research

JoVE Journal

Bioengineering

1.1K Views.  Rice University. Het belang van deze aanpak is dat het kan worden gebruikt om vaccins met meerdere doses in één injectie toe te dienen, waardoor mogelijk miljoenen levens worden gered, vooral in lage- en middeninkomenslanden waar pasgeborenen niet worden geïmmuniseerd vanwege toegangsproblemen. Deze fabricagemethode zorgt ervoor dat het rabiësvaccin zijn oorspronkelijke immuniteitverlenende vorm behoudt wanneer het wordt ingekapseld in microdeeltjes waarvan de afgifte de huidige vaccinatieschema's naboots...

Video: Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen

Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular substrates offer key advantages over cell-based aAPC, including precise control of signal presentation parameters and physical properties of the aAPC surface to modulate its interactions with T cells. aAPC constructed from anisotropic particles, particularly ellipsoidal particles, have been shown to be more effective than their spherical counterparts at stimulating T cells due to increased binding and larger surface area available for T cell contact, as well as reduced nonspecific uptake and enhanced pharmacokinetic properties. Despite increased interest in anisotropic particles, even widely accepted methods of generating anisotropic particles such as thin-film stretching can be challenging to implement and use reproducibly.
To this end, we describe a protocol for the rapid, standardized fabrication of biodegradable anisotropic particle-based aAPC with tunable size, shape, and signal presentation for T cell expansion ex vivo or in vivo, along with methods to characterize their size, morphology, and surface protein content, and to assess their functionality. This approach to fabricating anisotropic aAPC is scalable and reproducible, making it ideal for generating aAPC for &#34;off-the-shelf&#34; immunotherapies.

Here, we present a protocol to quickly and reproducibly generate biologically inspired, biodegradable articifical antigen presenting cells (aAPC) with tunable size, shape, and surface protein presentation for T cell expansion ex vivo or in vivo.

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular ...

Environment

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

Immunology and Infection

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Synthesis and Characterization of mRNA-Loaded Poly(Beta Aminoesters) Nanoparticles for Vaccination Purposes

Vaccination has been one of the major successes of modern society and is indispensable in controlling and preventing disease. Traditional vaccines were composed of entire or fractions of the infectious agent. However, challenges remain, and new vaccine technologies are mandatory. In this context, the use of mRNA for immunizing purposes has shown an enhanced performance, as demonstrated by the speedy approval of two mRNA vaccines preventing SARS-CoV-2 infection. Beyond success in preventing viral infections, mRNA vaccines can also be used for therapeutic cancer applications.
Nevertheless, the instability of mRNA and its fast clearance from the body due to the presence of nucleases makes its naked delivery not possible. In this context, nanomedicines, and specifically polymeric nanoparticles, are critical mRNA delivery systems. Thus, the aim of this article is to describe the protocol for the formulation and test of an mRNA vaccine candidate based on the proprietary polymeric nanoparticles. The synthesis and chemical characterization of the poly(beta aminoesters) polymers used, their complexation with mRNA to form nanoparticles, and their lyophilization methodology will be discussed here. This is a crucial step for decreasing storage and distribution costs. Finally, the required tests to demonstrate their capacity to in vitro transfect and mature model dendritic cells will be indicated. This protocol will benefit the scientific community working on vaccination because of its high versatility that enables these vaccines to prevent or cure a wide variety of diseases.

Synthesis and Characterization of mRNA-Loaded PolyBeta Aminoesters Nanoparticles for Vaccination Purposes

Here, a simple protocol is presented for producing mRNA nanoparticles based on poly(beta aminoester) polymers, easy to be tailored by changing the encapsulated mRNA. The workflow for synthesizing the polymers, the nanoparticles, and their in vitro essential characterization are also described. A proof-of-concept regarding immunization is also added.

Vaccination has been one of the major successes of modern society and is indispensable in controlling and preventing disease. Traditional vaccines were ...

Field Postmortem Rabies Rapid Immunochromatographic Diagnostic Test for Resource-Limited Settings with Further Molecular Applications

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, animals suspected as rabies-positive must be submitted to a postmortem confirmation using classical or molecular laboratory methods. However, most endemic areas are in low- and middle-income countries where animal rabies diagnosis is restricted to central veterinary laboratories. Poor availability of surveillance infrastructure leads to serious disease underreporting from remote areas. Several diagnostic protocols requiring low technical expertise have been recently developed, providing opportunity to establish rabies diagnosis in decentralized laboratories. We present here a complete protocol for field postmortem diagnosis of animal rabies using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to the final interpretation. We complete the protocol by describing a further use of the device for molecular analysis and viral genotyping. RIDT easily detects rabies virus and other lyssaviruses in brain samples. The principle of such tests is simple: brain material is applied on a test strip where gold conjugated antibodies bind specifically to rabies antigens. The antigen-antibody complexes bind further to fixed antibodies on the test line, resulting in a clearly visible purple line. The virus is inactivated in the test strip, but viral RNA can be subsequently extracted. This allows the test strip, rather than the infectious brain sample, to be safely and easily sent to an equipped laboratory for confirmation and molecular typing. Based on a modification of the manufacturer&#39;s protocol, we found increased test sensitivity, reaching 98% compared to the gold standard reference method, the direct immunofluorescence antibody test. The advantages of the test are numerous: rapid, easy-to-use, low cost and no requirement for laboratory infrastructure, such as microscopy or cold-chain compliance. RIDTs represent a useful alternative for areas where reference diagnostic methods are not available.

We present a complete protocol for postmortem diagnosis of animal rabies under field conditions using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to final interpretation. We also describe further applications using the device for molecular analysis and viral genotyping.

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...

Quick and Early Detection of Rabies Antibodies to Evaluate the Immune Response in a Patient

Detection of Rabies IgG and IgM Antibodies Using the Rabies Indirect Fluorescent Antibody Test

The rabies indirect fluorescent antibody (IFA) test was developed to detect various rabies-specific antibody isotypes in sera or cerebral spinal fluid. This test provides rapid results and can be used to detect rabies antibodies in several different scenarios. The rabies IFA test is especially useful for the quick and early detection of antibodies to evaluate the immune response in a patient who has developed rabies. Although other methods for antemortem rabies diagnosis take precedence, this test may be utilized to demonstrate recent rabies virus exposure through antibody detection. The IFA test does not provide a virus-neutralizing antibody (VNA) titer, but the pre-exposure prophylaxis (PrEP) response can be evaluated through positive or negative antibody presence. This test can be utilized in various situations and can provide results for a number of different targets. In this study, we used several paired serum samples from individuals who received PrEP and demonstrated their rabies antibody presence over time using the IFA test.

Author Spotlight: Rabies-Specific Antibody Isotypes Detection in Sera or Cerebral Spinal Fluid Using an IFA Test 

The aim of this manuscript is to examine the use of the rabies indirect fluorescent antibody test for the detection of rabies-specific IgG and IgM antibodies.

The rabies indirect fluorescent antibody (IFA) test was developed to detect various rabies-specific antibody isotypes in sera or cerebral spinal fluid. ...

A Nanopore-Based Genomic Approach to Control Rabies in Low-Income Countries

Whole Genome Sequencing for Rapid Characterization of Rabies Virus Using Nanopore Technology

Genomic data can be used to track the transmission and geographic spread of infectious diseases. However, the sequencing capacity required for genomic surveillance remains limited in many low- and middle-income countries (LMICs), where dog-mediated rabies and/or rabies transmitted by wildlife such as vampire bats pose major public health and economic concerns. We present here a rapid and affordable sample-to-sequence-to-interpretation workflow using nanopore technology. Protocols for sample collection and the diagnosis of rabies are briefly described, followed by details of the optimized whole genome sequencing workflow, including primer design and optimization for multiplex polymerase chain reaction (PCR), a modified, low-cost sequencing library preparation, sequencing with live and offline base calling, genetic lineage designation, and phylogenetic analysis. Implementation of the workflow is demonstrated, and critical steps are highlighted for local deployment, such as pipeline validation, primer optimization, inclusion of negative controls, and the use of publicly available data and genomic tools (GLUE, MADDOG) for classification and placement within regional and global phylogenies. The turnaround time for the workflow is 2-3 days, and the cost ranges from $25 per sample for a 96 sample run to $80 per sample for a 12 sample run. We conclude that setting up rabies virus genomic surveillance in LMICs is feasible and can support progress toward the global goal of zero dog-mediated human rabies deaths by 2030, as well as enhanced monitoring of wildlife rabies spread. Moreover, the platform can be adapted for other pathogens, helping to build a versatile genomic capacity that contributes to epidemic and pandemic preparedness.

Author Spotlight: A Cost-Effective Genomic Workflow for Advancing Rabies Control in Resource-Limited Settings 

Here, we present a rapid and cost-effective workflow for characterizing rabies virus (RABV) genomes using nanopore technology. The workflow is intended to support genomics-informed surveillance at a local level, providing information on circulating RABV lineages and their placement within regional phylogenies to guide rabies control measures.

Genomic data can be used to track the transmission and geographic spread of infectious diseases. However, the sequencing capacity required for genomic ...

Biology

Formulating mRNA-Loaded Nanoparticles for Vaccination Purposes

Source: Fornaguera, C. et al., <a href="https://app.jove.com/v/62889">Synthesis and Characterization of mRNA-Loaded Poly(Beta Aminoesters) Nanoparticles for Vaccination Purposes.</a>&#160;J. Vis. Exp. (2021)
This video demonstrates a method for generating an mRNA vaccine candidate using polymeric nanoparticles. The cationic polymer interacts with negatively charged mRNA, forming polyplexes. The formation of polyplexes stabilizes the mRNA, making it a potential candidate for future mRNA vaccines in immunotherapeutic applications.

1. Synthesis of poly(beta amino esters)&#160;(pBAE) polymer with end oligopeptides (OM-pBAE)

	Polymerization of C6-pBAE
	
		Add 5-amino-1-pentanol (38 ...

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Immunology

Immunotherapy

Immunogenics and Vaccine Development

Using a Sandwich ELISA to Determine the Immunogenic Glycoprotein Content in a Vaccine

Source: Jallet, C. et al., <a href="https://app.jove.com/v/59641">In Vitro ELISA Test to Evaluate Rabies Vaccine Potency.</a>&#160;J. Vis. Exp. (2020)
This video showcases an indirect enzyme-linked immunosorbent assay or ELISA sandwich immunocapture assay designed to assess immunogenic glycoprotein levels in vaccines. The assay plate, containing glycoprotein-specific antibodies, is incubated with serially diluted vaccine samples, including reference and test vaccines, followed by immunoassay to quantify glycoprotein content in the test vaccine.

1. Security precautions
NOTE: This method is applicable to both live rabies virus (RABV) and inactivated vaccine.

	Use good Laboratory Practice and ...

Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen

Samenvatting

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