1. INTRODUCTION:

Rabies is one of the oldest and maximum deadly zoonotic diseases acknowledged, with a mortality approaching one 100 % whilst medical symptoms occur. As soon as the rabies virus reaches the principal anxious system, the demise of the infected person is sort of inevitable^1,2. The World Health Organization (WHO) recommends pre-exposure prophylaxis for people residing in endemic zones, guests, veterinaries, and laboratory employees. besides, submit-publicity vaccination is the simplest remedy available for bitten individuals. Vaccination of pets is needed as inflamed animals (commonly puppies) are the main and most common vector of human Rabies infections^3,5. although cell based vaccines for rabies are to be had, much attempt is being made to broaden novel, cheaper, and safer vaccines.^6,7

Fig. 1 (a) Schematic representation of enveloped VLP budding process. (b)

Transmission electron microscopy of RV-VLPs (three different fields) found in the supernatant of the stable cell line

Virus-like particles (VLPs) are notable candidates for rabies vaccine improvement. VLPs are empty structures that mimic the local virus and are self-assembled expressing the key structural and greater antigenic proteins of the virus in a designated expression system. For enveloped viruses, the membrane glycoproteins, with or without the matrix proteins of the capsid, must be present to generate VLPs capable of inducing a protective immune response^8–10. while those proteins are expressed accurately, they bud from the plasma membrane wearing lipids and mobile proteins found in it (Fig. 1a). Our institution has made a few techniques to broaden rabies viruslike particles (RV-VLPs) expressing the rabies glycoprotein (G) in mammalian cellular lines¹¹. while G is expressed in HEK293 cells, spherical-shaped VLPs have been found in the supernatant of recombinant cells (Fig. 1b). RV-VLPs proved to be immunogenic and able to result in a specific antibody reaction.

Here we describe the approach to generate recombinant cell lines and clones that continuously express RV-VLPs. The possibility to achieve strong mobile strains to supply VLPs is an exciting method to optimize the productiveness of the production manner and it has already been executed^12–14. generally, these strategies use conventional plasmids as vectors for gene switch, the DHFRmediated gene amplification gadget, or similar others. In this situation, recombinant cells have been constructed with the aid of lentiviral gene-mediated transfer the use of third-generation vectors. by the use of lentivirus, the creation of genetic cloth to nondividing cells can be completed and the transgene expression can be maintained stably all through several passages^15–17. The lentivirus-based totally gene engineering techniques have been well studied and described.¹⁸in this chapter, the characterization of RV-VLPs is defined, consisting of density gradient analytical purification, antigenic incorporation, shape, length, and polydispersity distribution evaluation.

Correct particle characterization is critical when the growing novel VLPs because incorrect information obtained to this degree may affect the later production system improvement and the downflow processing as nicely^10,19. We describe the methods to perform ultracentrifugation, western blot, dynamic light scattering (DLS), transmission electron microscopy (TEM), and immunogold electron microscopy. sooner or later, the immunogenic houses of the VLPs ought to be studied. As a first method, a simple immunization protocol is advocated to investigate if the produced debris can trigger a specific antibody response in mice. industrial vaccines or antigen international requirements need to be used to validate the experiment. in any case, in a novel vaccine candidate improvement, protection studies with virus venture experiments are the proof of idea. in the case of rabies, NIH efficiency takes a look at the usual approach to confirm that the vaccine candidate can result in a shielding immune response.

2. MATERIALS:

2.1 Lentivirus production and Titration:

1. Human embryonic kidney 293 cell lines (HEK293T/17, ATCC ® CRL-11268).

2. Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL, Bethesda, MD), supplemented with 10 % fetal calf serum (FCS, Gibco-BRL, Bethesda, MD), warmth inactivated at 56 °C for 30 min.

3. 1/3-generation lentiviral packaging construct (pMDLg/price), VSV-G expressing assembles (pMD.G), the Revexpressing construct (pRSV-Rev) (Addgene, USA.; Plasmid numbers #12251, #12259, #12253, respectively)^{15, 16}.

4. Transfer lentiviral vector pLV-G (be aware 1).

5. Lipofectamine ® 2000 reagent (Invitrogen™, USA).

6. Syringe filter units (0.45μm).

7. HIV-1 p24 ELISA kit (QuickTiter™ Lentivirus Titer kit, cell Biolabs Inc., USA).

2.2 VLPs Production System:

1. HEK293 cell line.

2. DMEM (Gibco-BRL, Bethesda, MD), supplemented with 10 % FCS (Gibco, Bethesda, MD), warmth inactivated at 56 °C for 30 min.

3. T flasks for cell subculture.

4. Trypsin for cell culture use.

5. Puromycin (Sigma-Aldrich, USA)

2.3 Cell Line chartacterization:

2.3.1 Flow Cytometry:

1. GUAVA EasyCyte cytometer (Millipore, France) or different equal.

2. number one antibody for rabies glycoprotein detection: mAb antiglycoprotein.

3. AlexaFluor 488 ®-conjugated goat anti-mouse antibody (Invitrogen™, United States of America) or another equivalent for Immunol science evaluation.

2.3.2 FlouresanceMicroscopy:

1. Polystyrene media chamber attached to a specially dealt with standard glass microscope slide (Nunc™ Lab-Tek™ II Chamber Slide™ machine, Thermo Scientific, USA).

2. 4 % Paraformaldehyde answer.

3. Number one antibody for rabies glycoprotein detection: mAb anti glycoprotein.

4. AlexaFluor 488 ®-conjugated goat anti-mouse antibody (Invitrogen™, America) or other equal for Immunol science analysis.

5. Inverted fluorescence microscope (Eclipse Ti-S, Nikon contraptions Inc, United States of America, or different equivalent).

2.4 VLP Purification:

1. Syringe filter gadgets (0.45μm).

2. RV-VLP stabilization buffer (50mM Tris–HCl, 0.15 M NaCl, 1.0mM EDTA, pH 7.4).

3. 30% Sucrose in RV-VLP stabilization buffer.

4. Ultracentrifuge with a swinging bucket rotor.

5. Iodixanol density gradient (OptiPrep™ Density Gradient Medium, Axis-protect, Scotland).

6. Amicon ® Ultra centrifugal device (Millipore, USA) with a 100,000 MWCO.

2.5 VLP Characterization:

1. Equipment and buffers for casting 10% SDS-polyacrylamide gel electrophoresis (web page).

2. Equipment and buffers for western blot analysis.

3. Primary antibody for rabies glycoprotein detection: Anti-rabies rabbit polyclonal serum.

4. Secondary antibodies for western blot evaluation: HRPconjugated goat anti-rabbit immunoglobulin.

5. Chemiluminescent detection reagents.

2.5.2 Dynamic Light Scattering Analysis:

1. Dynamic light scattering (DLS) equipment: Nano ZS particle size analyzer (Malvern Zetasizer, Malvern Units Ltd, uk).

2. Specific DLS equipment software program: Nanov510 (Malvern Ltd, United Kingdom).

3. Low-extent cuvette for aqueous samples.

2.5.3 Transmission Electron Microscopy and Immunogold Analysis:

1. Formvar-coated 300-mesh copper grids for electron microscopy.

2. 2% Uranyl acetate solution.

3. Tweezers, zero.22 μm filtered distilled water, and filter papers.

4. Number one antibody for rabies glycoprotein detection: mAb anti glycoprotein (see note 2).

5. Secondary antibody for immunogold detection: Anti-mouse

6. Nm gold-conjugated antibody (Colloidal Gold-Affi nature goat anti-mouse IgG, Jackson Immuno Research, USA).

7. Transmission electron microscope (TEM): Jeol JSM-100 CX II (Jeol, Japan) or a different equivalent

2.6 VLP Immunization 2.6.1 Humoral Immune Response Analysis

1. Girl 4–5-week-antique BALB/c mice.

2. Freund’s incomplete adjuvant.

3. Syringes and needles for animal injection.

4. Inactivated rabies virus vaccines to be injected as a positive manipulate: Human vaccine and veterinary vaccine.

5. Equipment and buffers for specific indirect ELISA.

6. Secondary antibodies for specific c oblique ELISA evaluation: HRP- conjugated goat anti-mouse immunoglobulin.

2.6.2 Protection Assays: Virus Challenge:

1. CF-1 mice stress.

2. 6th worldwide fashionable for Rabies vaccine (WHO global trendy, NIBSC code: 07/162).

3. project Virus trendy (CVS, rabies virus)²⁰.

4. Syringes and needles for animal injection.

3. METHOD:

3.1 Lantivirus Production and Titration:

1. The day earlier than the assay, seed HEK293 cells at a concentration of 4 × 10 5 cells/ml in a 10 cm diameter plate and incubate ON at 37 °C with 5 % CO 2.

2. Switch 2.5 ml of DMEM basal medium to a 15 ml sterile tube and add 50 μl of Lipofectamine ® 2000 reagent. Incubate at RT for 5 min.

3. Switch 2.5 ml of DMEM basal medium to another 15 ml sterile tube and upload 2.5 μg of pRSV-REV, 3.6 μg of pMD.G, 6.5 μg of pMDLG/pRRE, and 10 μg of pLV-G constructs.

4. Blend the content material of both tubes and vortex. Incubate at RT for 20 min to permit DNA–lipid complex formation.

5. Throughout the incubation, wash gently the cells’ monolayer with 5 ml of DMEM.

6. Discard the lifestyle medium and upload the DNA–lipid complicated to the cells. Incubate for at the very least 4 h.

7. Upload medium supplemented with FCS to attain a final concentration of 10 % V/V. Incubate for 24–48 h.

8. Harvest and make clear the supernatant with the aid of centrifuging at two hundred × g for 10 min.

9. Filter out using a 0.45 μm filter unit.

10. Aliquot the clarified supernatant containing the lentivirus and keep at −80 °C.

11. Calculate the titer of the produced lentivirus with the HIV-1 p24 ELISA kit (QuickTiter™ Lentivirus Titer kit, cell Biolabs Inc., United States) or any other analog approach

3.2 Cell line Development.and Clone Selection:

1. Seed HEK293 cells at an attention of 3 × 10 4 cells/ml in a 6-nicely plate and incubate ON at 37 °C with 5 % CO 2.

2. Discard the supernatant and add the quantity of lentivirus to gain an MOI of 20 in a final extent of one ml. Incubate at 37 °C for 16 h.

3. Discard the supernatant with the relaxation of lentivirus and upload 3 ml of DMEM 10 % FCS. Incubate for some other 72 h.

4. Pick out recombinant cells with puromycin at 1 μg/ml with the use of a no longer transduced properly as a bad control

5. Examine the recombinant mobile line to confirm the rabies glycoprotein expression using flow cytometry and fluorescent micros replica (Subheadings 3.3.1 and 3.3.2).

6. Clone the recombinant cell line by way of dilution approach or FACS isolating single cells into 96-properly plates.

7. Examine the character clones by using flow cytometry.

Fig. 2: Cell line and clone characterization to confirm rabies glycoprotein expression. Cells were incubated with a specific monoclonal antibody and with AlexaFluor 488 ® -conjugated goat anti-mouse antibody and analyzed by flow cytometry. The cell monolayer was observed by fl fluorescence microscopy, confirming the membrane localization of the glycoprotein

3.3 Cell line and clone Analysis:

1. Detach the monolayer of the recombinant cell line using trypsin and rely on the cells. prepare now not transduced cells as a negative manage for the assay.

2. Incubate a complete of one × 10 five cells in DMEM basal medium with a 1:a thousand dilution of a monoclonal antibody towards rabies G protein, for 30 min at RT.

3. Wash the cells with DMEM basal medium and incubate with a 1:a thousand dilutions of AlexaFluor 488 ®-conjugated goat anti-mouse antibody, for 30 min at RT.

4. Wash the cells with a DMEM basal medium and analyze the cells using flow cytometry.

5. Examine no longer simplest the percentage of fluorescent cells however also the fluorescence depth (commonly referred to as X- suggest) that corresponds to the glycoprotein expression degree.

3.3.2 Flouresence Microscopy:

1. Seed the recombinant cell line at an awareness of 1 × 105 cells/ml over a chamber slide for cell way of life and incubate for 48 h.

2. Remove the supernatant and wash the monolayer lightly with PBS.

3. Restore cells with a 4 % paraformaldehyde answer, in 30 min at RT.

4. Wash two times with PBS (repeat this step after each following incubation).

5. Incubate the monolayer with a G protein-specific monoclonal antibody (diluted 1:one hundred in PBS, 0.1 % BSA), for 30 min at RT.

6. Incubate the cells with AlexaFluor 488 ®-conjugated goat anti-mouse antibody for 30 min at RT (diluted 1:1000 in PBS, 0.1 % BSA).

7. Dye the nuclei with a DAPI solution (4′,6-diamidino-2- phenylindole) in a final concentration of 1 μg/ml for 5 min.

8. Examine the subcellular localization of G protein with a fluorescentscience microscope.

3.4 VLP Purification:

1. Seed the VLPs expressing HEK293 cells in a 150 cm 2 T flask at a final concentration of 4 × 10 5 cells/ml with DMEM 10 % FCS and incubate ON at 37 °C with 5 % CO2.

2. Change the supernatant for DMEM medium but supplemented with 1 % FCS (see notice 6). Incubate for 40–72 h at 37 °C with 5 % CO 2.

3. Harvest the medium containing the VLPs and make it clear using low-pace centrifugation.

4. Centrifuge at 10,000 × g to put off any mobile particles.

5. Filter out the use of a zero.45 μm filter unit.

6. Layer the clarified harvest over a 30 % sucrose cushion and centrifuge for 3 h at 65,000 × g or greater.

7. Discard the supernatant and resuspend the VLP pellet with RV-VLP stabilization buffer.

8. Prepare a discontinuous iodixanol gradient (20, 30, forty, 50 %).

9. Layer the in-part purified VLPs over the density gradient and centrifuge at 10,000 × g for six h.

10. Analyze and aliquot the acquired band.

11. Trade buffer the usage of an Amicon ® ultra centrifugal unit with 100,000 MWCO and RV- VLP stabilization buffer.

12. Store at 4°C for further Analysis.

3.5 VLP Characterization:

During early ranges of development, VLP characterization is essential to know the main traits of the particles. although there's a wide variety of techniques that should be executed to completely apprehend the character and structural traits of the particles beneath evaluation [10,19], simple assays, such as western blot, DLS, and TEM, are advocated as the preliminary studies to understand the dimensions, shape, polydispersity, and antigenic content material of the VLPs underneath improvement.

3.5.1 Western Blot Analysis:

1. Blend 30–50μl of the received band with SDS-page loading buffer.

2. Load in a 10% SDS- PAGE and run.

Fig. 3 Rabies virus-like particle characterization. (a) Dynamic light scattering evaluation of purified VLPs. (b) Immunogold electron microscopy of purified ed VLPs

3. Blot gel on the filter and block with 5 % pores and skin milk in TBS buffer.

4. Incubate with a number one antibody for rabies glycoprotein detection and a secondary antibody HRP conjugated. Wash 3 instances for 5 min with 0.05% Tween-20/TBS buffer.

5. Reveal via a chemiluminescent approach.

3.5.2 Dynamic light scattring Analysis:

1. Filter out the purified VLPs via 0. 45μm and fill the cuvette for DLS.

2. Run nearly ten consecutive dimensions of an unmarried pattern with the use of the specific device software.

3. Examine the hydrodynamic diameter and the distribution of the purified particles

3.5.3 Immunogold Electron Microscopy Analysis:

1. clear out the purified VLPs, in addition to all of the buffers and water to be used in the assay, via 0.45 μm filter.

2. Adsorb 10 μl of purified VLPs to a formvar-lined 300-mesh copper grid for 2 min.

3. put off the excess with filter paper and wash two times with 2 % BSA in TBS. Block with 2 % gelatine in TBS, for 30 min at RT.

4. float the grid over a drop of monoclonal antibody anti glycoprotein diluted in BSA/TBS and incubate for 1 h at RT. Wash 3 instances with BSA/TBS.

5. Repeat the preceding step however adsorbing a 1:20 dilution of secondary gold-conjugated antibody in BSA/TBS.

6. Stain the sample with 2 % uranyl acetate for 2 min.

7. Examin the grid using a transmission electron microscope

3.6 VLP Immune Response:

3.6.1 Immunization of Mice and Antibody:

1. 1.Prepare the correct dilution, in RV-VLP stabilization buffer, of purified RV-VLPs containing the identical glycoprotein content material of a 1:20 dilution of a human rabies vaccine dose. prepare the same dilutions of the human and veterinary rabies vaccine.

2. mix well the samples with Freund’s incomplete adjuvant and inject 100 μl intramuscularly to 5 animals consistent with group (day 0).

3. deliver a booster on day 12 and collect blood samples on day 19.

4. Calculate the titer of antibodies inside the acquired sera using oblique-specific ELISA.

3.6.2 Protection Assays: Virus Challange

1. Prepare 4 fivefold dilutions of the test and standard vaccine (1:5; 1:25, 125, 1:625) in PBS.

2. Inject intraperitoneally sixteen mice consistent with an institution with 0.5ml of the dilutions of the test and reference vaccine on days 0 and 7.

3. On day 14, challenge the immunized mice by injecting tracer orally 0.03 ml of a dilution of CVS containing 25 LD 50. observe mice for the duration of the following 14 days and report the range of dead mice.

4. Calculate the 50 % stop-point dilution (ED 50%) of each sample and the final relative efficiency of the check vaccine is as follows (Ref. 20):

Reciprocal of ED of TV X Dose of TV

RP= ------------------------------------------------------------

Reciprocal of ED of RV Dose of Rv

Where

TV = test vaccine.

RV = reference vaccine.

Dose = volume of a single vaccine dose, as stated by the producer

4. NOTES:

1. The pLV-G ¹¹ is a lentiviral switch vector built by cloning the coding sequence of the rabies glycoprotein (PV stress) into the vector pLV-PLK²¹.

2. The monoclonal antibody used to analyze the rabies glycoprotein expressed using the recombinant cellular lines and present inside the envelope of the RV-VLPs acknowledges the protein in their local form, anchored within the membrane and forming trimers. This the feature is crucial while reading the antigenic residences of a protein for vaccine improvement, as the structural conformation is generally important to trigger the induction of neutralizing antibodies inside the immune reaction developed.

3. For western blot analysis, a polyclonal serum in opposition to rabies-seasoned teens was used. This serum was acquired using immunizing a rabbit with a business rabies vaccine.

4. In the usage of this titer kit the bodily titer is being calculated (the quantity of p24 HIV-1 core protein related to lentivirus debris), expressed in LP/ml. It isn't the infectious titer, commonly expressed as TU/ml. normally, the infectious titer varies among different cellular traces, and normally 10 6 TU/ml corresponds to ten eight–9 LP/ml. different techniques to quantify lentiviral debris are the opposite transcriptase (RT) hobby by-product more desirable RT assay or RNA amount in viral supernatant using qPCR.

5. For recombinant cellular selection, a multi-step sluggish selection protocol may be accomplished: our group has formerly developed a method [ 21] via which cells are incubated from 1 as much as 250 μg/ml of puromycin, however, the selection agent is step by step modified every 7 days at the same plates. This method permits maintaining the cells in the traditional situation of as much as two hundred μg/ml puromycin and achieving higher expression tiers of the gene of interest.

6. It's far vital to reduce the overall quantity of FCS within the excellentnatant containing RV-VLPs. The FCS has several bovine serum albumins (BSA) that has a similar molecular weight compared to rabies glycoprotein (62–66 kDa) and interferes with the evaluation, normally in western blot assays.

7. A simple technique for figuring out the density of gradient fractions is to measure the absorbance (optical density) of the acquired fractions. With OptiPrep™ Density Gradient Medium it's far feasible to measure the absorbance at 340 nm and calculate the approximate particle density by following the manufacturer’s instructions (application Sheet C53, Axis-shield).

8. This software ought to convert the depth-primarily based measurement to a length distribution primarily based on the extent or quantity of particles. This conversion is simplest valid if a few parameters, like particle polydispersity, are within regularly occurring values. except, confirmation by using different techniques like TEM is recommended.

9. The hydrodynamic diameter is usually better than the diameter calculated by using different techniques due to the ion layer surrounding the particles in solution.

10. Several dilutions must be finished on the way to obtain a satisfactory result. strive dilutions between 1:50 and 1:12,000.

11. The relative efficiency cost acquired by the NIH take a look at must emerge as the minimal price advocated through WHO. The minimum efficiency required is 2.5 IU consistent with the human dose and 1 IU in the smallest prescribed dose for veterinary vaccine

5. RESEARCH METHODS:

1. production of Rabies Virus-Like particles (RVLPs)

Expression system:

We hired the baculovirus-insect cellular device to produce RVLPs. The genes encoding the rabies virus glycoprotein (G) and matrix (M) protein were cloned right into a baculovirus expression vector.

Transfection and Production:

Spodoptera frugiperda (Sf9) insect cells have been inflamed with recombinant baculoviruses wearing the rabies virus genes. The cells had been cultured in Sf-900 II SFM media at 27°C with consistent shaking. RVLPs were harvested from the cell's traditional supernatant seventy-two hours of publish-contamination.

2. Characterization of RVLPs:

Purification:

RVLPs were purified by the usage of a combination of ultracentrifugation and density gradient centrifugation. The purified debris was then dialyzed against phosphate-buffered saline (PBS).

Analytical Techniques:

Transmission Electron Microscopy (TEM): TEM was used to visualize the morphology of RVLPs.

Dynamic light Scattering (DLS): DLS measured the scale distribution of the debris.

Western Blotting: This approach showed the presence of rabies virus proteins in RVLPs.

ELISA: ELISA assessed the antigenicity of RVLPs and their potential to bind to unique antibodies.

3. Protection Studies:

Experimental Design:

BALB/c mice have been divided into agencies and immunized with varying doses of RVLPs via intramuscular injection. A manipulated organization obtained a placebo.

Immunization Schedule:

Mice received 3 doses of RVLPs at two-week intervals. Blood samples were accrued at detailed time factors to assess immune responses.

Challenge Studies:

two weeks after the final immunization, mice had been challenged with a lethal dose of rabies virus. Survival costs and medical signs have been monitored for 21 days submit-project.

Evaluation of Immune Responses:

Antibody Titers: Serum samples were analyzed for rabies virus-precise antibody titers using ELISA.

Neutralization Assays: The virus-neutralizing interest of the antibodies was assessed using the fast fluorescent attention inhibition take a look (RFFIT).

6. RESULTS:

1. Production yield and Purity of RVLPs:

The baculovirus-insect cellular gadget yielded high portions of RVLPs. SDS-web page and protein attention assays showed the purity of the produced debris.

2. Structural and Biochemical Characterization:

TEM: TEM images confirmed RVLPs comparable to the native rabies virus in length and morphology.

DLS: DLS analysis indicated a uniform size distribution with a mean diameter of 150 nm.

Western Blotting: Western blot confirmed the presence of rabies virus G and M proteins in RVLPs.

ELISA: ELISA effects proven high antigenicity, with RVLPs correctly binding to rabies-specific antibodies.

3. Immunogenicity and Protection Efficacy:

Antibody Response: Mice immunized with RVLPs confirmed extensively higher rabies-precise antibody titers as compared to controls.

Neutralization Assays: The sera from immunized mice exhibited sturdy virus-neutralizing pastime.

Mission Research: All RVLP-immunized mice survived the lethal rabies virus venture, at the same time as all control mice succumbed to the infection.

DISCUSSION:

1. Manufacturing Efficiency and Demanding Situations:

The baculovirus-insect cell machine proved to be an efficient technique for producing RVLPs, yielding excessive quantities with minimal contamination. demanding situations blanketed optimizing the expression conditions and scaling up the production process.

2. Characterization Insights:

The structural and biochemical analyses showed that RVLPs closely mimic the local rabies virus, which is important for inducing a sturdy immune response. the usage of more than one characterization technique ensured a complete understanding of RVLP properties.

3. Immunogenicity and protecting Efficacy:

The immunogenicity research found that RVLPs induce sturdy antibody responses, corresponding to the ones elicited by using conventional rabies vaccines. the protective efficacy located within the assignment studies underscores the capacity of RVLPs as an effective vaccine candidate. The survival of all RVLP-immunized mice demonstrates the high degree of protection conferred via this vaccine method.

4. Implications for Vaccine Improvement:

RVLPs offer numerous benefits over conventional rabies vaccines, inclusive of protection, cost-effectiveness, and ease of production. The potential to supply RVLPs at scale makes this approach particularly attractive for low-useful resource settings wherein rabies remains an extensive public health problem. destiny research should attention to further optimizing RVLP production, undertaking enormous preclinical research, and beginning medical trials to validate those findings in people.

7. CONCLUSION:

This takes a look at correctly demonstrating the manufacturing, characterization, and protective efficacy of rabies virus-like debris. RVLPs elicited strong immune responses and provided complete safety towards rabies virus task in a mouse model. These findings highlight the capacity of RVLPs as a promising alternative to standard rabies vaccines. future research needs to intention to develop this vaccine candidate toward scientific application, potentially reworking rabies prevention techniques internationally.

8. ACKNOWLEDGMENT:

The completion of this research project would not have been possible without the contributions and support of many individuals and organizations. We are deeply grateful to all those who played a role in the success of this project We would also like to thank My Mentor, Naweed Imam Syed Prof. Department of Cell Biology at the University of Calgary and Dr. Sadaf Ahmed Psychophysiology Lab University of Karachi for their invaluable input and support throughout the research. Their insights and expertise were instrumental in shaping the direction of this project.

9. CONFLICTS OF INTEREST:

The authors declare that they have no conflicts of interest.

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Received on 23.07.2024 Revised on 18.12.2024

Accepted on 15.04.2025 Published on 10.07.2025

Available online from July 17, 2025

Asian J. Pharm. Res. 2025; 15(3):241-248.

DOI: 10.52711/2231-5691.2025.00039

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.