Neutralizing Antibody Titer Test of Ebola Recombinant Protein Vaccine and Gene Vector Vaccine pVR-GP-FC

Objective In previous studies, we immunized mice with Ebola recombinant protein vaccine and gene vector vaccine. Both stimulated high levels of humoral immunity. In this work, we constructed a pseudovirus containing Ebola membrane proteins to verify whether the two immunization strategies can induce neutralizing antibodies in mice. Methods A pseudovirus containing an Ebola virus membrane protein based on the HIV-1 viral gene sequence was constructed and evaluated using a known neutralizing antibody. The titer of the neutralizing antibody in the sera of mice immunized with the recombinant protein and the gene vector vaccine was examined using a neutralization test. Results Ebola pseudovirus was successfully prepared and applied for neutralizing antibody detection. Immunological experiments showed that recombinant protein GP-Fc and gene vaccine pVR-modGP-Fc had good immunogenicity. The titer of the bound antibody in the serum after 8 weeks of immunization in mice was more than 1：105, and the recombinant protein induced greater humoral immunity. The results of the neutralization test based on the Ebola pseudovirus system demonstrated that both vaccines induced production of protective antibodies, while the gene vaccine induced a higher titer of neutralizing antibodies. Conclusion An Ebola pseudovirus detection system was successfully established and used to evaluate two Ebola vaccines. Both produced good immunogenicity. The findings lay the foundation for the development of new Ebola vaccines and screening for neutralizing monoclonal antibodies.

Protection Efficacy of Recombinant Infectious Laryngotracheitis Vaccine in Chicks

A commercial recombinant fowl pox vectored infectious laryngotracheitis （ILT） vaccine was examined its protection efficacy in one-day-old chicks by subcutaneous route with various dose-strength. The treatment groups I-III birds were vaccinated with a full, 1/2 and 1/3 dose of recommendation dose, respectively. Thereafter, at 6 wk post vaccination, they were challenged with 5x labeled dose of live ILT vaccine. Protection efficacy of the vaccine was compared among groups based on antibody production and eye lesion score. The results of eye lesion score showed that there was a significant difference between control and treatment groups, whereas the difference was not found among the treatment groups. For seroconversion, there was no significant difference between birds in group I and II. However, higher number of seroconversion birds in groups I and II than group III was observed （P 〈 0.05）. It was concluded that a half dose of recommendation dose had the same protection efficacy as a full dose of recombinant YP-LT vaccine.

Synthesis, Characterization, and Immunological Properties of LPS-Based Vaccines Composed of O-Polysaccharides Conjugated with Recombinant Exoprotein A from Pseudomonas aeruginosa

Pseudomonas aeruginosa remains one of the major pathogens affecting immunocompromised patients. LPS-based monovalent (MV) and polyvalent (PV) conjugate vaccines were prepared from the most prevalent strains of P. aeruginosa International Antigenic Typing Scheme (IATS) 6, 10, 11 and 20 to evaluate their immunogenicity and protective capacities from infection by the pathogens. Conjugation of the O-polysaccharide (O-PS) antigens of P. aeruginosa strains to the common immunogenic recombinant Exotoxin A (rEPA) supports the multi-antigenic approach for the development of a vaccine that provides cross protection against various strains of the pathogen. The O-PSs were indirectly conjugated through adipic acid dihydrazide (ADH) to rEPA by carbodiimidemediated condensation reaction. Mice were immunized with the conjugates emulsified with monophosphoryl lipid A (MPL) or Freund's adjuvant compared with conjugates without adjuvant, unconjugated mixture of rEPA and O-PS emulsified with MPL, and sterile saline. The MV and PV vaccines emulsified with MPL adjuvant elicited the highest anti-O-PS IgM and IgG antibodies. Immunization of mice with MV vaccines derived from IATS 10, 11, and 20, emulsified with MPL adjuvant provided a high level of protection against the homologous bacterial strain. Similarly, high protection was obtained when mice were immunized using PV and challenged separately with bacterial strains 10, 11, and 20, but lower protection against the IATS 6 strain. Also, high cross protection of MV vaccine derived from O-PS of IATS 10 and 20 was obtained against P. aeruginosa IATS 11 strain. The in vivo protection correlated with the level of anti-O-PS IgG in the mice serum.

Construction of Recombinant Modified Vaccinia Ankara(MVA) Expressing Hepatitis B Virus Surface Antigen

The T lymphocyte response has been shown to be the determinant in the clearance of many viral infections. Hence, therapeutic vaccine candidates against HBV are designed to enhance this response of the immune system. Vaccinia virus vector-based vaccines have been proposed as excellent candidates to elicit long-term and strong T lymphocyte mediated immune responses. In this study, the recombinant MVA expressing HBV surface antigen has been constructed, which can elicit a potent T cell mediated response. The ELISA results for the surface protein in the medium of the recombinant MVA, strongly indicate that the recombinant virus has been successfully obtained.

Immunization of Male Mice with a New Recombinant GnRH Fusion Protein Reduces the Testicular Function

The objective of the present study was to evaluate the effect of an immunogenic maltose-binding protein-gonadotropin releasing hormone （GnRH-6-MBP） using genetic engineering. The synthetic mammalian tandem repeated GnRH hexamer gene was inserted into the expression plasmid pMAL-c2x. Recombinant GnRH-6-MBP protein was over- expressed in E.coli strain BL21. Amylose resin with affinity chromatograph was used to purify target protein. The reactiongenicity of fusion protein was identified by indirect enzyme linked immunosorbent assay （ELISA）, and the antigenicity and biological effects of GnRH-6-MBP were tested in mice. In the experiment, 20 male Kunming white mice of 20 d old were randomly divided into treatment and control group. Ten mice were immunized with 100 μg GnRH-6-MBP administered subcutaneously （s.c.） thrice at 2-week intervals with GnRH-6-MBP. Mice were sacrificed after 3 weeks following the booster injection, the testis was removed, weighed and measured, and the histological structure was observed. The reactiongenicity of fusion protein to GnRH antibody was much higher than the control. Active immunization against GnRH-6-MBP reduced remarkably （P 〈 0.01） the length and weight of the testis, and shortened the girth and width of the testis （P 〈 0.05）, and suppressed testicular spermatogenesis compared to the control mice. These results indicate that the recombinant GnRH-6-MBP acted as a strong immunogen and caused atrophy of the testis.

Oral Immunization of Mice With Vaccine of Attenuated Salmonella typhimurium Expressing Helicobacter pylori Urease B Subunit

Objective To prepare the live recombinant vaccine of attenuated Salmonella typhimurium SL3261 expressing Helicobacterpylori （H. pylori） B subunit （UreB） and to determine whether it could be used as an oral vaccine against H. pylori infection. Methods Using genomic DNA of H. pylori Sydney strain （SS1） as template, the H. pylori UreB gene fragment was amplified by PCR and subcloned into the expression vector pTC01. The recombinant plasmid pTC01-UreB was then transferred into LBS000 to obtain modified forms, and further conversed into the attenuated Salmonella typhimurium SL3261 to obtain recombinant SL3261/pCT01-UreB as an oral immunization reagent, which was then used to orally immunize Balb/c mice twice at a three-week interval. Twelve weeks later, anti-UreB IgA antibodies in intestinal fluid and IgG antibodies in sera were determined by ELISA. The relating data in control groups （including body weight, gastric inflammation, etc.） were also collected. Results The sequencing analysis showed that the UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB gene. The restriction enzyme digestion revealed that the correct pTC01-UreB was obtained. SDS-PAGE and Western blot showed that a 61KD protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H, pylori UreB antiserum and was absent in the control containing only Salmonella typhimurium SL3261 strain. The multiple oral immunization with SL3261/pTC01-UreB could significantly induce H. pylori specific mucosal IgA response as well as serum IgG responses. IFN-T and IL-10 levels were significantly increased in SL3261/pTC01-UreB group, and no obvious side effect and change in gastric inflammation were observed. Conclusion The attenuated vaccine of Salmonella typhimurium expressing H. pylori UreB can be used as an oral vaccine against H. pylori infection.

Developments of Subunit and VLP Vaccines Against Influenza A Virus

Influenza virus is a continuous and severe global threat to mankind. The continuously re-emerging disease gives rise to thousands of deaths and enormous economic losses each year, which emphasizes the urgency and necessity to develop high-quality influenza vaccines in a safer, more efficient and economic way. The influenza subunit and VLP vaccines, taking the advantage of recombinant DNA technologies and expression system platforms, can be produced in such an ideal way. This review summarized the recent advancements in the research and development of influenza subunit and VLP vaccines based on the recombinant expression of hemagglutinin antigen (HA), neuraminidase antigen (NA), Matrix 2 protein (M2) and nucleocapsid protein (NP). It would help to get insight into the current stage of influenza vaccines, and suggest the future design and development of novel influenza vaccines.

The preparation and application of N-terminal 57 amino acid protein of the follicle-stimulating hormone receptor as a candidate male contraceptive vaccine

Follicle-stimulating hormone receptor （FSHR）, which is expressed only on Sertoli cells and plays a key role in spermatogenesis, has been paid attention for its potential in male contraception vaccine research and development. This study introduces a method for the preparation and purification of human FSHR 57-amino acid protein （FSHR-57aa） as well as determination of its immunogenicity and antifertility effect. A recombinant pET-28a（＋）-FSHR-57aa plasmid was constructed and expressed in Escherichia coil strain BL21 StarTM （DE3） and the FSHR-57aa protein was separated and collected by cutting the gel and recovering activity by efficient refolding dialysis. The protein was identified by Western blot and high-performance liquid chromatography analysis with a band of nearly 7 kDa and a purity of 97.4%. Male monkeys were immunized with rhFSHR-57aa protein and a gradual rising of specific serum IgG antibody was found which reached a plateau on day 112 （16 weeks） after the first immunization. After mating of one male with three female monkeys, the pregnancy rate of those mated with males immunized against FSHR-57aa was significantly decreased while the serum hormone levels of testosterone and estradiol were not disturbed in the control or the FSHR-57aa groups. By evaluating pathological changes in testicular histology, we found that the blood-testis barrier remained intact, in spite of some small damage to Sertoli cells. In conclusion, our study demonstrates that the rhFSHR-57aa protein might be a feasible male contraceptive which could affect sperm production without disturbing hormone levels.

Hepatitis E virus chimeric DNA vaccine elicits immunologic response in mice

AIM： To construct the plasmid pcHEV23 containing fragments of HEV ORF2 and ORF3 chimeric gene and to assess its ability to elicit specific immunologic response in mice. METHODS： The gene encoding the structural protein of HEV ORF2 fragment and full-length ORF3 was amplified by PCR. The PCR products were cloned into an eucaryotic expression plasmid pcDNA3. The resulting plasmid pcHEV23 was used as a DNA vaccine to inoculate BALB/c mice intramuscularly thrice at a dose of 100 or 200 ug. Mice injected with empty pcDNA3 DNA or saline served as control and then specific immune responses in the mice were detected. RESULTS： After 2-3 times of inoculation, all mice injected with pcHEV23 had anti-HEV IgG seroconversion and specific T lymphocyte proliferation. The lymphocyte stimulation index in the group immunized with pcHEV23 （3.1＋0.49） was higher than that in the control group （0.787±0.12, P〈0.01）. None in the control group had a detectable level of anti-HEV IgG. CONCLUSION： DNA vaccine containing HEV ORF2 and ORF3 chimeric gene can successfully induce specific humoral and cellular immune response in mice.

Immunogenicity of Lyophilized MVA Vaccine for HIV-1 in Mice Model

Highly attenuated modified vaccinia Ankara（MVA） is sensitive to repeat freeze-thaw cycle and easy to lose activity. In order to make the activity of MVA vaccine remain stable during its manufacturing, storage, and administration, the lyophilization as a good option could be resorted to; through screening, the right stabilizer composition and its production procedure were obtained. The final moisture content of freezing-dried recombinant MVA-HIV vaccine was lower than 3%. It can be reconstituted quickly and shows regular physical appearance and stable potency. In vivo functional experiment, mice were divided randomly into the liquid vaccination group, the lyophilized vaccination group, and the control group. Having been DNA vaccine priming, the mice were boosted with a dose of 10^7 pfu MVA- HIV vaccine, which produced indistinguishable antibody titer and cytotoxic T-lymphocyte（CTL） level compared with those of liquid vaccination group （ P 〉 0.05 ）. These results demonstrate that lyophilized MVA vaccine can induce high immunogenicity in mice.

Application Progress of Recombinant Attenuated Listeria monocytogenes in Tumor Immunotherapy

Much progress of application of bacterial vaccine in treatment and prevention of tumor was acquired,which showed broad prospect in clinical study of animals and humans. Listeria monocytogenes( L. monocytogenes) was considered much important by virtue of its special characteristic of biology and immunology.L. monocytogenes was ingested by professional or part-time phagocytes,survived and proliferated in the phagocytes under synergism of toxic factor secreted by itself,meanwhile,the cellular and humoral immune response was induced. Antigenic gene of specific tumor was loaded in the attenuated L. monocytogenes,which can enhance immune response of host cells. Effective cell targeted to enter tumor tissue and acted on tumor cells to induce apoptosis of tumor cells. Tumor degenerated not easy to reappear. Therefore,recombinant attenuated L. monocytogenes was a safe and effective anti-cancer vaccine vector. Now the work of researchers mainly focuses on solving practical problem in clinical application. Biological characteristics of L. monocytogenes,feasibility and superiority of L. monocytogenes as targeted vaccine vector,problem and prospect of L. monocytogenes in clinical application of anti-tumor were reviewed in this paper.

Immunogenicity and protective efficacy of recombinant M2e.Hsp70c(Hsp70_(359–610)) fusion protein against influenza virus infection in mice

New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70(mHsp70) is known to cultivate the function of immunogenic antigen-presenting cells, stimulate a strong cytotoxic T lymphocyte(CTL) response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2(M2e), was fused to the C-terminus of Mycobacterium tuberculosis Hsp70(Hsp70c), to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2 e.Hsp70c(Hsp70359–610). And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2 e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.

Research Progress on a SARS-CoV-2 Vaccine in China

Although many countries have controlled the pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through strict management, there are still many countries with record-breaking numbers of new cases. Therefore, it is very important to develop a vaccine that can cause wide cross reactivity in clinical trials. At present, more than 90 vaccines are entering clinical trials and progressing smoothly, including inactivated vaccines, adenovirus-vectored vaccines and other types of vaccines. Here, we review and summarize the efficacy and potential threats of a SARS-CoV-2 vaccine. We reviewed whole-virus vaccines, adenovirus-subunit vaccines and recombinant protein vaccines and discussed the positive and negative consequences of a SARS-CoV-2 vaccine. However, there are still heated debates on the mechanism, effectiveness, and breadth of protection. In conclusion, this study can predict the risk of new coronavirus outbreaks in the future by discussing the research and development status of new coronavirus vaccines in China and other countries. Looking to the future, it is important to mine the large amount of data generated in clinical trials of universal new coronavirus vaccines to ensure that these vaccine programs are equally useful in the face of new coronavirus mutations.

CONSECUTIVE IMMUNIZATION WITH RECOMBINANT FOWLPOX VIRUS AND PLASMID DNA FOR ENHANCING CELLULAR AND HUMORAL IMMUNITY

Objective: To investigate the influence of consecutive immunization on cellular and humoral immunity in mice. Methods: We evaluated a consecutive immunization strategy of priming with recombinant fowlpox virus vUTALG and boosting with plasmid DNA pcDNAG encoding HIV-1 capsid protein Gag. Results: In immunized mice, the number of CD 4 + T cells from splenic lymphocytes increased significantly and the proliferation response of splenocytes to ConA and LPS elevated markedly and HIV-1-specific antibody response could be induced. Conclusion: Consecutive immunization could increase cellular and humoral immunity responses in mice.

The development of a recombinant hybrid protein of Plasmodium falciparum and analysis of its antigenicity and protectivity

A hybrid gene encoding several putative protective epitopes from erythrocytic stage antigens (MSA1,MSA2 and RESA) of P. falciparum as well as exgenous T cell enhancer epitopes from interleukin-1 and tetanus toxin was synthesized chemically. This gene (named HGFC) was cloned and connected with another hybrid gene (HPFA) synthesized previously to make a bigger hybrid gene (HGFCAC). HGFC and HGFCAC were cloned in an expression vector pWR450-1 and transformed into E. Coli JM109. The engineered bacteria could express fusion proteins with molecular weights of 65 and 77 kDa after inducing with isopropylthio-β-D-galactoside (IPTG). The expression rate was about 35% of total bacterial proteins. The expressed products showed sepcific immunological reaction with rabbit antibodies against P. falciparum peptide Glu-Glu-Asn-Val-Glu-His-Asp-Ala (EENVEHDA)by Western blotting. The fusion proteins were pruified by precipitation with amonium sulfate. gel filtration and ion-exchange chromatography and the purity was 82%. The purified protein reacted specifically with mouse immune serum against falciparum blood stage antigens by dot enzyme-linked immunosorbent assay (dot-ELISA).The fusion protein was emulsified with Freund's complete adjuvant (FCA) and used to immunize rabbits. The immune serum can recognize P. falciparum erythrocytic stage antigens of Fcc-1/HN strain and Yunnan strain and had weak cross reaction with P. vivax,but had no reaction with P. cynomlogi and P. berghei antigens. The protective effect of the antibody was tested by in vitro inhibition test to cultured falciparum parasites. Preliminary results indicated that the immune sera could effectively reduce the invasion rates of the parasites to red blood cells and inhibit the growth of the in vitro cultured falciparum parasites. The inhibitory capacity of the immune sera to parasite invasion is enhanced as the amount of the sera increases and the incubation time of the sera with the parasites is prolonged.It was shown that after 72 h incubation at 20% concentration with the parasites, the serum can suppress the multiplication of P.falciparum growth in vitro to a level of 80%.The immune sera caused dispersion of the parasite cytoplasm,atrophy of parasites,agglutination of free merozoites and degeneration of schizonts.

Humoral Immune Response of Inactivated Bivalent <i>Leptospira</i>Vaccine among Dogs in Tiruchirappalli, Tamilnadu, India

Leptospirosis has been recognized as the disease of the dogs. The prevention of canine leptospirosis can block the transmission of the etiological agent to humans. Commercial vaccines prevent not only clinical leptospirosis but also the renal carrier state in dogs. Thus in this present investigation, the humoral immune response of the vaccinated dogs with the multivalent vaccine (Megavac-6) was analyzed. Enzyme linked immunosorbent assay (ELISA) with preparations of 2 whole cell lysate, 2 leptospiral LPS, 4 purified recombinant proteins were used to test sera from 30 dogs vaccinated with MEGAVAC-6, a commercial vaccine practiced by the animal husbandary Departments in Tamilnadu. All 30 sera were positive by ELISA with whole cell lysate of Canicola and Icterohaemorrhagiae, and ELISA with Canicola LPS, Icterohaemorrhagiae LPS;antibody titres ranged from 20 to ≥10,240. Although there was less frequent reactivity of sera with recombinant antigens, antibodies to LipL32 and LigA were detected in 25 (83.3%) serum samples. Less frequent reactivity was noted when recombinant GroEl, LK73.5 antigens were included separately in ELISAs. The highest MAT titre was observed against the serovar Canicola and Icterohaemorrhagiae. The more reactivity against LPS may be due to the dominance of serovar specific leptospiral LPS, which may be the dominant immunogenic antigen in inactivated bivalent leptospiral vaccines. Eventhough LPS is the dominant antigen. It is serovars specific and hence recommends the incorporation of the locally circulating serovars in vaccine for its efficient use. The efficiency at this point can be increased by the use of subunit vaccines rather than recombinant proteins as such. The present study has proposed certain epitopes with increased antigenic potency.

Protection by Recombinant Newcastle Disease Viruses (NDV) Expressing the Glycoprotein (G) of Avian Metapneumovirus (aMPV) Subtype A or B against Challenge with Virulent NDV and aMPV

Avian metapneumovirus (aMPV) and Newcastle disease virus (NDV) are threatening avian pathogens that can cause serious respiratory diseases in poultry worldwide. Vaccination, combined with strict biosecurity practices, has been the recommendation for controlling these diseases in the field. In the present study, we generated NDV LaSota vaccine strain-based recombinant viruses expressing the glycoprotein (G) of aMPV, subtype A or B, using reverse genetics technology. These recombinant viruses, rLS/aMPV-A G and rLS/aMPV-B G, were characterized in cell cultures and evaluated in turkeys as bivalent, next-generation vaccines. The results showed that these recombinant vaccine candi-dates were slightly attenuated in vivo, yet maintained similar growth dynamics, cytopathic effects, and virus titers in vitro when compared to the parental LaSota virus. The expression of the aMPV G protein in recombinant virus-infected cells was detected by immunofluorescence. Vaccination of turkeys with rLS/aMPV-A G or rLS/aMPV-B G conferred complete protection against velogenic NDV, CA02 strain challenge and partial protection against homologous patho-genic aMPV challenge. These results suggest that the LaSota recombinant virus is a safe and effective vaccine vector and expression of the G protein alone is not sufficient to provide full protection against aMPV-A or -B infections. Ex-pression of other aMPV-A or -B virus immunogenic protein(s) individually or in conjunction with the G protein may be necessary to induce stronger and more protective immunity against aMPV diseases.

In the era of rapid mRNA-based vaccines:Why is there no effective hepatitis C virus vaccine yet?

Hepatitis C virus(HCV)is responsible for no less than 71 million people chronically infected and is one of the most frequent indications for liver transplanta-tion worldwide.Despite direct-acting antiviral therapies fuel optimism in controlling HCV infections,there are several obstacles regarding treatment accessibility and reinfection continues to remain a possibility.Indeed,the majority of new HCV infections in developed countries occur in people who inject drugs and are more plausible to get reinfected.To achieve global epidemic control of this virus the development of an effective prophylactic or therapeutic vaccine becomes a must.The coronavirus disease 19(COVID-19)pandemic led to auspicious vaccine development against severe acute respiratory syndrome coronavirus-2(SARSCoV-2)virus,which has renewed interest on fighting HCV epidemic with vaccination.The aim of this review is to highlight the current situation of HCV vaccine candidates designed to prevent and/or to reduce HCV infectious cases and their complications.We will emphasize on some of the crossroads encountered during vaccine development against this insidious virus,together with some key aspects of HCV immunology which have,so far,ham-pered the progress in this area.The main focus will be on nucleic acid-based as well as recombinant viral vector-based vaccine candidates as the most novel vaccine approaches,some of which have been recently and successfully employed for SARS-CoV-2 vaccines.Finally,some ideas will be presented on which methods to explore for the design of live-attenuated vaccines against HCV.

Design of live-attenuated animal vaccines based on pseudorabies virus platform

Pseudorabies virus(PRV)is a double-stranded DNA virus with a genome approximating 150 kb in size.PRV contains many non-essential genes that can be replaced with genes encoding heterogenous antigens without affecting viral propagation.With the ability to induce cellular,humoral and mucosal immune responses in the host,PRV is considered to be an ideal and potential live vector for generation of animal vaccines.In this review,we summarize the advances in attenuated recombinant PRVs and design of PRV-based live vaccines as well as the challenge of vaccine application.

Pilot-Scale Production of Lyophilized Inactivated Rabies Vaccine Candidate in Vero Cells under Fully Animal Component-Free Conditions Using Microcarrier Technology and Laboratory Animal Trials

The upstream process was carried out in an animal component-free medium on Cytodex 1 microcarriers. Recombinant trypsin is a non-animal derived protease used as an alternative to animal-derived trypsin. To inactivate recombinant trypsin, a soybean trypsin inhibitor (STI) should be added to the medium. A protocol was first tested in T-flasks and then passaged to 500 mL and 3 L spinner flasks. Cell detachment was completed in 10 - 12 min, and 0.4 g/L STI was added to a 3L spinner, and cells were transferred into a 30 L stirred tank bioreactor. On day 5, the cell density had reached its maximum (around 1.8 × 106 cells/mL). At an MOI of 0.3 with serum-free medium conditions, cell infection yielded a maximal rabies virus titer of 1.82 × 107 FFU/mL at 5 days. All cell culture conditions and virus growth kinetics in serum-free media were investigated. In conclusion, Vero cells were grown on Cytodex 1 with serum-free media and a high amount of rabies virus was obtained. A mouse challenge was used to determine the immune response to an inactivated rabies virus vaccine candidate. Also, we evaluated inactive rabies vaccine candidate safety, and immunogenicity in mice, sheep, horses, and cattle. We found that no horses, sheep, or cattle who were given vaccine IM at 3.2 IU/dose exhibited any clinical sign of disease and all developed high VNA titers (up to 10.03 IU/mL) by 3 - 4 WPI. After the accelerated stability studies, the lyophilized inactivated rabies vaccine candidate showed enough antigenic potency (2.6 IU/mL) in the mouse challenge test. Also, 18-month long-term stability studies showed enough immune response (1.93 IU/mL) on day 14. The activity of the vaccine candidate showed a good immune response and safety criteria that meet WHO requirements. This is the first pilot-scale mammalian cell-based viral rabies vaccine production study in Türkiye that used microcarriers.