Recombinant Rabies Virus With The Glycoprotein Fused With A DC-binding Peptide Is An Efficancious Rabies Vaccine

Rabies is a zoonotic viral disease that causes more than 59,000 human deaths annually all over the world.Its causative pathogen,rabies virus(RABV),is a neurotropic virus,consisting of five genes nucleoprotein(N),phosphoprotein(P),matrix protein(M),glycoprotein(G)and the viral RNA polymerase(L).From the site of entry,RABV moves fast along the peripheral nervous system and reach the central nervous system(CNS)eventually.It is almost a death sentence once clinical signs appear.Although rabies is fatal,it can be prevented by appropriate vaccination in humans and animals.After immunization,the immune system will be activated and antibodies produced to neutralize the virus.Since the first introduction in 1885,vaccination has become the most effective way to protect people from rabies.Millions of people are vaccinated globally and it is estimated that this saves more than 250,000 people from dying of rabies every year.Our previous studies demonstrated that recruiting and/or activating dendritic cells could enhance the immunogenicity of recombinant rabies viruses.Several cytokines or chemokines have been demonstrated to be capable of enhancing DC activation when overexpressed by recombinant RABV.However,over-expression of these cytokines or chemokines may provide other functions,even some side effects,beyond DC activation.It thus needs further investigation if solely increasing the binding of r RABV to DCs is sufficient to enhance the RABV immunogenicity.In this study,the r RABV LBNSE with the glycoprotein(G)fused with a small DCbinding peptide(designated as r LBNSE-DCBp)or a negative control peptide(designated as r LBNSE-DCCp)fused to the glycoprotein(G)were constructed and rescued.Multi-step growth curves on BSR and NA cells showed that insertion of DCBp or DCCp into G did not affect the viral replication in vitro,and the insertion of DCBp or DCCp did not affect the G protein expression,which is confirmed by western blot.In addition,the pathogenicity of the r RABVs was evaluated by measuring the mouse body weight changes after inoculation with each r RABVs through intracranial(i.c.)route.Results indicate that the insertion of DCBp or DCCp did not affect the viral pathogenicity in mice.As to immunogenicity,significantly more activated DCs were detected in r LBNSE-DCBp-immunized mice than those immunized with r LBNSE or r LBNSE-DCCp.Subsequently,significantly more generation of Tfh and GC B cells were observed in r LBNSE-DCBp immunized mice than those in r LBNSE or r LBNSE-DCCp-immunized mice.In addition,significantly higher levels of virus neutralizing antibodies(VNAs)were observed in mice immunized with live or inactivated r LBNSE-DCBp than those immunized with live or inactivated r LBNSE or r LBNSE-DCCp,resulting in a better protection of LBNSE-DCBp immunized mice against the lethal challenge.In summary,r LBNSE-DCBp could induce a robust VNA response by enhancing the DC binding and activation of DCs,and subsequently TFH and GC B cell generation after vaccination,leading to a better protection against lethal virus challenge,which indicates that r LBNSE-DCBp has the potential to be exploited as a safe and efficacious rabies vaccine.