Corinne Jallet

Immunization of dogs with a DNA vaccine induces protection against rabies virus

Rabies is a fatal encephalomyelitis which is transmitted to man, mostly by dogs in developing countries. This zoonosis can be prevented by vaccination of humans before or after exposure. However, a more radical approach is possible, involving the elimination of the principal vector/reservoir by vaccinating dogs. The vaccine must be effective, safe and inexpensive. Mass production of plasmids is possible and DNA-based immunization with a plasmid encoding the antigen responsible for inducing protection seems to be more cost-effective than classical techniques involving cell culture. Beagles were immunized by intramuscular (i.m.) injection with a plasmid encoding the rabies virus (PV strain) glycoprotein. Neutralizing antibodies against both wild-type rabies virus and European Bat Lyssaviruses (EBL1 and EBL2) were detected after a single injection and a boost, but levels of neutralizing antibodies against EBL1 were low. Moreover, all vaccinated dogs were protected against a lethal challenge with a wild-type dog rabies strain. This is one of the first studies to demonstrate that dogs can be protected by DNA vaccines, and opens important perspectives for rabies control.

Peptides that mimic the amino-terminal end of the rabies virus phosphoprotein have antiviral activity.

ABSTRACT We wanted to develop a therapeutic approach against rabies disease by targeting the lyssavirus transcription/replication complex. Because this complex (nucleoprotein N-RNA template processed by the L polymerase and its cofactor, the phosphoprotein P) is similar to that of other negative-strand RNA viruses, we aimed to design broad-spectrum antiviral drugs that could be used as a complement to postexposure vaccination and immunotherapy. Recent progress in understanding the structure/function of the rabies virus P, N, and L proteins predicts that the amino-terminal end of P is an excellent target for destabilizing the replication complex because it interacts with both L (for positioning onto the N-RNA template) and N (for keeping N soluble, as needed for viral RNA encapsidation). Thus, peptides mimicking various lengths of the amino-terminal end of P have been evaluated, as follows: (i) for binding properties to the N-P-L partners by the two-hybrid method; (ii) for their capacity to inhibit the transcription/replication of a rabies virus minigenome encoding luciferase in BHK-21-T7 cells; and (iii) for their capacity to inhibit rabies virus infection of BHK-21-T7 cells and of two derivatives of the neuronal SK-N-SH cell line. Peptides P60 and P57 (the first 60 and first 57 NH2 residues of P, respectively) exhibited a rapid, strong, and long-lasting inhibitory potential on luciferase expression (>95% from 24 h to 55 h). P42 was less efficient in its inhibition level (75% for 18 to 30 h) and duration (40% after 48 h). The most promising peptides were synthesized in tandem with the Tat sequence, allowing cell penetration. Their inhibitory effects were observed on BHK-21-T7 cells infected with rabies virus and Lagos bat virus but not with vesicular stomatitis virus. In neuronal cells, a significant inhibition of both nucleocapsid inclusions and rabies virus release was observed.

Interaction of lyssaviruses with the low-affinity nerve-growth factor receptor p75NTR.

The low-affinity nerve-growth factor receptor p75NTR interacts in vitro with the rabies virus (RV) glycoprotein and serves as a receptor for RV. The Lyssavirus genus comprises seven genotypes (GTs) of rabies and rabies-related viruses. The ability of p75NTR to interact with the glycoprotein of representative lyssaviruses from each GT was investigated. This investigation was based on a specific binding assay between BSR cells infected with a lyssavirus and Spodoptera frugiperda (Sf21) cells expressing p75NTR on the cell surface. A specific interaction was observed with the glycoprotein of GT 1 RV (challenge virus standard or Pasteur virus strains) as well as wild-type RV and the glycoprotein of GT 6 European bat lyssavirus type 2. In contrast, no interaction was detected with the glycoprotein of lyssaviruses of GTs 2-5 and 7. Therefore, p75NTR is only a receptor for some lyssavirus glycoproteins, indicating that the other GTs must use an alternative specific receptor.

Antiviral Drug Discovery Strategy Using Combinatorial Libraries of Structurally Constrained Peptides

ABSTRACT We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies virus and rabies-related viruses) as models. Based on the mimicry of natural bioactive peptides, two genetically encoded combinatorial peptide libraries composed of intrinsically constrained peptides (coactamers) were designed. Proteomic knowledge concerning the functional network of interactions in the lyssavirus transcription-replication complex highlights the phosphoprotein (P) as a prime target for inhibitors of viral replication. We present an integrated, sequential drug discovery process for selection of peptides with antiviral activity directed against the P. Our approach combines (i) an exhaustive two-hybrid selection of peptides binding two phylogenetically divergent lyssavirus Ps, (ii) a functional analysis of protein interaction inhibition in a viral reverse genetic assay, coupled with a physical analysis of viral nucleoprotein-P complex by protein chip mass spectrometry, and (iii) an assay for inhibition of lyssavirus infection in mammalian cells. The validity of this strategy was demonstrated by the identification of four peptides exhibiting an efficient antiviral activity. Our work highlights the importance of P as a target in anti-rabies virus drug discovery. Furthermore, the screening strategy and the coactamer libraries presented in this report could be considered, respectively, a general target validation strategy and a potential source of biologically active peptides which could also help to design pharmacologically active peptide-mimicking molecules. The strategy described here is easily applicable to other pathogens.

Inactivated rabies vaccine control and release:use of an ELISA method

Quality control of human rabies vaccines performed by National Control Laboratories (NCLs) prior to marketing vaccines batches requires in vivo and in vitro potency assays as requested by the relevant European Pharmacopoeia monographs, OMCLs guidelines and WHO technical recommendations. The aim of the present study was to check the suitability of an enzyme-linked immunosorbent assay (ELISA) using a virus neutralizing monoclonal antibody, directed to the rabies virus glycoprotein, to monitor the consistency of the lot to lot rabies vaccines production. Furthermore, this work was implemented to establish in house specifications for the glycoprotein content.

Is there an advantage to including the nucleoprotein in a rabies glycoprotein subunit vaccine

The PV rabies (genotype 1) G and N proteins were produced by recombinant baculoviruses in insect cells. We tested the ability of recombinant antigens to synergistically induce an immune response and, particularly, to broaden the spectrum of Lyssavirus-neutralizing antibodies produced. Cell-free preparations of recombinant proteins caused an immune response. Recombinant rabies G protein (RRG) from infected cell extract or supernatant induced virus neutralizing antibodies (VNAb) against rabies (CVS virus (genotype 1) and in a less extent against European Bat Lyssavirus-1 (EBL-1:genotype 5) Recombinant rabies N protein (RRN) induced antibodies that reacted with the rabies virus ribonucleoprotein (RNP) and primed mice for both the production of VNAb induced by inactivated and purified rabies virus and the protection conferred by RNP. RRN also had an adjuvant effect on VNAb production induced by RRG when the two recombinant proteins were physically associated either encapsulated in liposomes or subjected to ultrasound treatment. However, there was no increase in production of VNAb directed against EBL-1 although classical vaccines (genotype 1) induce partial protection against this virus. thus, beside its adjuvant effect there is some doubt as to whether including rabies N protein in a rabies subunit vaccine containing the recombinant G protein would be advantageous.

Canine adenoviruses elicit both humoral and cell-mediated immune responses against rabies following immunisation of sheep

Safe and efficient vaccination is important for rabies prevention in domestic animals. Replicative vectors expressing the rabies virus glycoprotein, derived from canine adenovirus have been reported to be promising vaccines in various animal models. In this paper we compare the potential of a replicative and a non-replicative vector, both based on canine adenovirus type 2 and expressing the rabies glycoprotein. Upon inoculation in sheep, immune responses against the rabies virus protein elicited by recombinant vectors were monitored. All immunised sheep produced a rapid and potent neutralizing antibody response against rabies virus after a single inoculation of either replicative or non-replicative recombinant canine adenovirus type 2. In addition, the non-replicative vector expressing the rabies glycoprotein stimulated antigen-specific CD4(+) and CD8(+) lymphocyte proliferation as well as IFN-γ production. These results suggest that vectors derived from canine adenovirus 2 could be considered for the development of promising vaccines in the ruminant species.

A relevant in vitro ELISA test in alternative to the in vivo NIH test for human rabies vaccine batch release

To assess the quality of vaccine batches before release, international regulation requires the control of potency of each lot of human rabies vaccines by the in vivo NIH challenge test. Meanwhile, the 3Rs strategy for animal testing encourages the replacement of the in vivo potency test by an in vitro assay. Consequently, since more than 10 years, an ELISA method has been implemented by ANSM in parallel to the NIH test for rabies vaccines lots. It consists in the evaluation of the glycoprotein content using a monoclonal antibody recognizing the trimeric native form of the glycoprotein. This ELISA method is able 1) to monitor the consistency of production with a similar profile than the NIH; 2) to detect a low quantity of glycoprotein in vaccines and 3) to agree with the manufacturers NIH results by declaring a non compliant batch. This ELISA which characterizes the immunogenic form of the glycoprotein formulated in vaccines seems to be relevant to replace the NIH test and is a promising candidate to be standardized by a collaborative study.

Discovery of hantavirus circulating among Rattus rattus in French Mayotte island, Indian Ocean

Hantaviruses are emerging zoonotic viruses that cause human diseases. In this study, sera from 642 mammals from La Réunion and Mayotte islands (Indian Ocean) were screened for the presence of hantaviruses by molecular analysis. None of the mammals from La Réunion island was positive, but hantavirus genomic RNA was discovered in 29/160 (18 %) Rattus rattus from Mayotte island. The nucleoprotein coding region was sequenced from the liver and spleen of all positive individuals allowing epidemiological and intra-strain variability analyses. Phylogenetic analysis based on complete coding genomic sequences showed that this Murinae-associated hantavirus is a new variant of Thailand virus. Further studies are needed to investigate hantaviruses in rodent hosts and in Haemorrhagic Fever with Renal Syndrome (HFRS) human cases.

Rabies Therapeutics: Development of Anti-Viral Approaches

Although rabies is reputed to be 100% fatal once the symptomatic phase is reached, in recent years an increased interest has arisen for therapeutic approaches. The “Milwaukee” protocol successfully applied to a rabid patient in 2005 has revitalized interest (although highly criticized by some investigators) in the rabies community. Most approaches still remain at a very early and conceptual stage of development. Rabies and its long incubation period is obviously an excellent target to develop an antiviral, even if its status as a neglected disease and its neurotropism are two economical and physiological obstacles, respectively, from this perspective. This review presents the empirical arsenal of substances having shown “some” anti-rabies viral (RABV) activity, then describes two complementary strategies that are currently elaborated for the rationale development of antivirals against RABV and other lyssaviruses: (1) a cognitive approach by destabilization of the known functional interactions between viral components, and (2) a random approach by high-throughput screening of highly diverse libraries of molecules using functional tests.