Since the RVNA titers progressively fall in the absence of subsequent antigenic exposures, a higher level of RVNA titer produced following immunization might confer longer protection against rabies, before it trails off to levels below 0

Since the RVNA titers progressively fall in the absence of subsequent antigenic exposures, a higher level of RVNA titer produced following immunization might confer longer protection against rabies, before it trails off to levels below 0.5 IU/mL. antibody (RVNA) titers in the immune sera were evaluated on days 14, 28, and 90 by rapid fluorescent focus inhibition test. Finally, an intracerebral challenge study using a challenge virus standard strain of rabies virus was done to evaluate the protective efficacy of the formulations. Results Protective levels of RVNA titer (0.5 IU/mL) were observed by day 14 in animals immunized with pIRES-Rgp and its dendriplex. Notably, PETIM-pIRES-Rgp produced 4.5-fold higher RVNA titers Povidone iodine compared to pIRES-Rgp at this time point. All mice immunized with the PETIM-pIRES-Rgp survived the intracerebral rabies virus challenge, compared with 60% in the group which received pIRES-Rgp. Conclusion Our results suggest that nanoformulation with PETIM dendrimer can produce an earlier onset of a high-titered protective antibody response to a plasmid-based rabies vaccine. PETIM dendriplexing appears to be an efficacious nonviral delivery strategy to enhance genetic vaccination. in the family Rhabdoviridae. The presence of huge numbers of diverse sylvatic animal reservoirs almost precludes the possibility of rabies eradication. However, human infections are entirely preventable by timely prophylaxis and efficacious vaccination of companion animals (especially dogs) as the major vectors of the disease in Asia and Africa.2 Vaccination of 70% of the canine population can drastically reduce human rabies in developing countries.3 However, this faces several challenges C difficult access to potent cell culture vaccines (costly and requiring cold chain and annual boosters), logistic issues, and suboptimal immune responses in animals in field conditions, to cite a few.3 Plasmid-based vaccination has been explored as an alternative strategy to cell culture-based rabies vaccines for animal prophylaxis.4,5 Deoxyribonucleic acid (DNA) vaccines are cheaper and easy to develop, stable at ambient temperature, able to induce prolonged cellular and humoral immune responses, and suitable for vaccination of pups. However, despite their exhibited feasibility in several animal models, inefficient cellular delivery and poor immunogenicity remain major drawbacks, restricting their applicability in field-level immunization of animals. Much of the current research to improve the efficacy of plasmid vaccines is focused on two areas: molecular adjuvanting and novel delivery systems.6C11 A group of nanosized polymeric molecules called dendrimers has recently been reported to be a Povidone iodine promising candidate in drug and gene delivery applications. These are globular, nanosized, hyperbranched polymeric molecules with precise molecular architecture and highly adaptable surface chemistries.12 The most well-known and Povidone iodine commercially available dendrimers include the poly(amido amine) and poly(propylene imine) groups. Poly(ether imine) (PETIM) dendrimer, a novel dendrimer developed at the Indian Institute of Science, Bangalore, India, was reported to have a low cytotoxicity, and we have earlier reported its utility as an in vitro gene delivery vehicle.13C15 In this study we employed the plasmid construct pIRES-Rgp and its dendriplex with PETIM for immunization of Swiss albino mice and compared their immunogenicity and protective efficacy. Our preliminary findings indicate that PETIM-dendriplexing can enhance the systemic stability and cellular uptake of the complexed plasmid, thus favoring an enhanced immune response and increased efficacy. Materials and methods Plasmids The bicistronic eukaryotic expression vector pIRES was a gracious gift from Dr Praveen K Gupta, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India. The plasmid pBacPak-GRC9 made up of the full-length rabies virus glycoprotein of a street virus isolate (human) was earlier produced in the Department of Neurovirology, National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore, India, as part of another project. EndoFree Plasmid Purification Giga Kit (QIAGEN, Dsseldorf, Germany) was used for the large-scale, endotoxin-free purification of the plasmid construct, as per manufacturer instructions. Enzymes and reagents TurboFect in vitro transfection reagent (catalog number R0531) and polymerase chain reaction reagents were obtained from Fermentas (Thermo Fischer Scientific, Waltham, MA, USA). The Rabbit Polyclonal to MAP4K6 restriction enzymes NheI (catalog number R0131) and EcoRI (catalog number R0101S) were purchased from New England BioLabs Ltd. (Ipswich, MA, USA). The primers used in the study were designed using nucleotide sequences available in GenBank, using PRIMEGENS v2.0.5 software (Digital Biology Laboratory, University of Missouri-Columbia, Columbia, MO, USA), and synthesized at Eurofins Genomics India Pvt Ltd., Bangalore, India. Antibodies and antibody conjugates The murine monoclonal antibody Povidone iodine to rabies virus glycoprotein used in the study was produced earlier in the Department of Neurovirology, NIMHANS.16 Goat anti-mouse immunoglobulin G (IgG)-fluorescein isothiocyanate (FITC) (catalog number 621120380011730).