Chokri Bahloul

Evidence of two Lyssavirus phylogroups with distinct pathogenicity and immunogenicity.

ABSTRACT The genetic diversity of representative members of theLyssavirus genus (rabies and rabies-related viruses) was evaluated using the gene encoding the transmembrane glycoprotein involved in the virus-host interaction, immunogenicity, and pathogenicity. Phylogenetic analysis distinguished seven genotypes, which could be divided into two major phylogroups having the highest bootstrap values. Phylogroup I comprises the worldwide genotype 1 (classic Rabies virus), theEuropean bat lyssavirus (EBL) genotypes 5 (EBL1) and 6 (EBL2), the African genotype 4 (Duvenhage virus), and theAustralian bat lyssavirus genotype 7. Phylogroup II comprises the divergent African genotypes 2 (Lagos bat virus) and 3 (Mokola virus). We studied immunogenic and pathogenic properties to investigate the biological significance of this phylogenetic grouping. Viruses from phylogroup I (Rabies virus and EBL1) were found to be pathogenic for mice when injected by the intracerebral or the intramuscular route, whereas viruses from phylogroup II (Mokola and Lagos bat viruses) were only pathogenic by the intracerebral route. We showed that the glycoprotein R333 residue essential for virulence was naturally replaced by a D333 in the phylogroup II viruses, likely resulting in their attenuated pathogenicity. Moreover, cross-neutralization distinguished the same phylogroups. Within each phylogroup, the amino acid sequence of the glycoprotein ectodomain was at least 74% identical, and antiglycoprotein virus-neutralizing antibodies displayed cross-neutralization. Between phylogroups, the identity was less than 64.5% and the cross-neutralization was absent, explaining why the classical rabies vaccines (phylogroup I) cannot protect against lyssaviruses from phylogroup II. Our tree-axial analysis divided lyssaviruses into two phylogroups that more closely reflect their biological characteristics than previous serotypes and genotypes.

Antigenic and genetic divergence of rabies viruses from bat species indigenous to Canada

Antigenic characterisation of over 350 chiropteran rabies viruses of the Americas, especially from species reported rabid in Canada, distinguished 13 viral types. In close accord with this classification, nucleotide sequencing of representative isolates, at both the N and G loci, identified four principal phylogenetic groups (I-IV), sub-groups of which circulated in particular bat species. Amongst the North American bat viruses, there was a notable division between group I specimens associated with colonial, non-migratory bats (Myotis sp. and Eptesicus fuscus) and those of group II harbored by solitary, migratory species (Lasiurus sp. and Lasionycteris noctivagans). Certain species of Myotis were clearly identified as rabies reservoirs, an observation often obscured previously by their frequent infection by viral variants of other chiroptera. An additional group (III) apparently circulates in E. fuscus, whilst viruses harbored by both insectivorous and haematophagus bats of Latin America clustered to a separate clade (group IV). Comparison of the predicted N and G proteins of these viruses with those of strains of terrestrial mammals indicated a similarity in structural organisation regardless of host species lifestyle. Finally, these sequences permitted examination of the evolutionary relationship of American bat rabies viruses within the Lyssavirus genus.

DNA-based immunization for exploring the enlargement of immunological cross-reactivity against the lyssaviruses

DNA-based immunization was used for studying the cross-reactivity of lyssavirus neutralizing antibodies and for exploring the induction of a wider range of protection against lyssaviruses. In order to immunize mice with homogeneous and chimeric genes of glycoproteins (G) from two divergent lyssaviruses, we used for the first time a new plasmid (pCI-neo) known to be a highly efficient vector for in vitro expression. The homogeneous plasmids pGPV and pGMok encoded the Pasteur virus (PV: genotype 1-GT-) and Mokola virus (Mok: GT 3) G, respectively. The chimeric pGMokPV encoded the NH2 part of GMok and the COOH part of GPV. These plasmids elicited full protection against intracerebral challenges with various lyssaviruses and a range of antigen-specific and non-specific immune responses. Virus neutralizing antibody (VNAb) levels were dose dependent and a single intramuscular (i.m.) injection of plasmids was sufficient to induce continuous high levels of VNAb. Production of antigen-specific T helper (Th), cytotoxic T cells (Tc) and non-specific natural killer cells was observed. Cross-reactivity studies showed that VNAb are obtained by immunizing with: (i) pGPV against GT 1 (classical rabies), GT 4 (Duvenhage: Duv), GT 5 (European Bat Lyssavirus: EBL-1) and GT 6 (European Bat Lyssavirus: EBL-2); (ii) pGMok against GT 2 (Lagos Bat: LB) and GT 3 (Mokola: Mok); (iii) pGMokPV against all GTs except GT 4 which is weakly neutralized. Therefore, the DNA-based immunization with the chimeric pGMokPV, could be very interesting to enlarge protection to all the lyssaviruses. According to the cross-reactivity of VNAb induced by the G genes, the lyssavirus GTs could be classified into two groups: the first including GT 1, 4, 5 and 6; the second including GT 2 and 3.

Molecular characterization of canine parvovirus-2 variants circulating in Tunisia

Canine parvovirus type 2 (CPV2) emerged in 1978 as a highly contagious and very serious disease in dogs. The characterization of CPV2 antigenic types is exclusively based on the identification of the amino acid residue at position 426 of the capsid protein VP2. Currently, three antigenic types CPV-2a (asparagine N426), CPV-2b (aspartic acid D426) and CPV-2c (glutamic acid E426) are circulating worldwide. In Tunisia, despite the fact that many clinical and few serological investigations clearly indicate that CPV is widespread and of major concerns in the local dog population, no molecular and antigenic type characterization of circulating variants has been carried out. This investigation showed that most of clinically presumed CPV infections were confirmed by classical or real-time PCR. When no real-time PCR facilities were affordable, classical PCR as reported here in association with restriction fragment length polymorphism (RFLP) with MboI and MboII can be very useful for screening and diagnosing CPV infections. A total of 50 variants were characterized by sequencing and an almost even representation of the different antigenic types, including CPV-2c and slightly more type 2b, were evidenced. Characterization of the Tunisian variants by MGB probe assays as reported was inefficient for most of CPV-2a variants because of their typical nucleotide mutation C1269. Phylogenetic analysis showed that the Tunisian variants underwent evolution for a relatively long period of time inside the country. The analysis also showed some crossings of the different antigenic types, leaving both genotypic and phenotypic characteristic mutations.

Post-exposure therapy in mice against experimental rabies: a single injection of DNA vaccine is as effective as five injections of cell culture-derived vaccine.

Two rabies post-exposure therapies were comparatively evaluated: BALB/c mice were challenged at day 0 with rabies virus and then received either a single dose of rabies DNA vaccine administered at day 0, or five doses of cell culture-derived rabies vaccine administered at days 0, 3, 7, 15 and 28. Both regimens, rapidly triggered protective levels of neutralizing antibodies against rabies virus in vaccinated mice. In addition, one injection of DNA vaccine protected 53% of the challenged mice, compared to 40% of mice protected after five injections of cell culture-derived vaccine. We conclude that rabies post-exposure vaccination in BALB/c mice, based on a single administration of rabies DNA vaccine might be at least as effective as five injections of cell culture-derived vaccine.

DNA based vaccination with a cocktail of plasmids encoding immunodominant Leishmania (Leishmania) major antigens confers full protection in BALB/c mice

Despite the lack of effective vaccines against parasitic diseases, the prospects of developing a vaccine against leishmaniasis are still high. With this objective, we have tested four DNA based candidate vaccines encoding to immunodominant leishmania antigens (LACKp24, TSA, LmSTI1 and CPa). These candidates have been previously reported as capable of eliciting at least partial protections in the BALB/c mice model of experimental cutaneous leishmaniasis. When tested under similar experimental conditions, all of them were able to induce similar partial protective effects, but none could induce a full protection. In order to improve the level of protection we have explored the approach of DNA based vaccination with different cocktails of plasmids encoding to the different immunodominant Leishmania antigens. A substantial increase of protection was achieved when the cocktail is composed of all of the four antigens; however, no full protection was achieved when mice were challenged with a high dose of parasite in their hind footpad. The full protection was only achieved after a challenge with a low parasitic dose in the dermis of the ear. It was difficult to determine clear protection correlates, other than the mixture of immunogens induced specific Th1 immune responses against each component. Therefore, such an association of antigens increased the number of targeted epitopes by the immune system with the prospects that the responses are at least additive if not synergistic. Even though, any extrapolation of this approach when applied to other animal or human models is rather hazardous, it undoubtedly increases the hopes of developing an effective leishmania vaccine.

Comparative evaluation of specific ELISA and RFFIT antibody assays in the assessment of dog immunity against rabies

Two techniques are currently used to evaluate the humoral immune responses to rabies vaccination: ELISA, which detects binding antibodies to viral antigens and the WHO reference rapid fluorescent focus inhibition test (RFFIT), which assays in vitro virus-neutralizing antibodies. In this study, we have comparatively evaluated antibody responses of dogs reared either in an experimental kennel or living in field conditions after vaccination with a cell culture-derived rabies vaccine. In experimental conditions, both ELISA and RFFIT techniques were well correlated. However, in field conditions, they yielded discrepant results particularly in evaluating the residual rabies immunity before vaccine administration and in identifying seroconverted dogs. After rabies vaccination in field conditions, while similar antibody titres and seroconversion rates were obtained using either technique, the discrimination of a given dog according to the seroconversion threshold depended on the assay. We concluded, that whereas in experimental conditions, ELISA and RFFIT were well correlated, in field conditions ELISA yielded upper estimates. Consequently, RFFIT, although a cumbersome test, should continue to be considered as the reference rabies antibody assay technique. A seroconversion threshold of 0.5 IU/ml should be cautiously considered and a higher threshold (1 IU/ml) could be more appropriate in the evaluation of rabies immunity in the field in order to marginalize the interfering factors.

Evaluation of Mass Vaccination Campaign Coverage Against Rabies in Dogs in Tunisia

In Tunisia, rabies continues to be considered as a serious public health concern. Very costly mass vaccination of dogs against rabies and expensive post‐exposure prophylaxis are prerequisites to maintain a low level of human rabies cases. In Tunisia, the implementation of mass vaccination campaigns at the national level has undoubtedly contributed to the drop of rabies endemicity, but the overall outcome is rather suboptimal. In this investigation, we wanted to estimate the extent of the vaccination coverage in dogs in three Governorates (Manouba, Kassrine and Mednine), by collecting data through questionnaires and interviews relevant to 1470 owned dogs. When the campaign is correctly applied, as in Manouba, almost all the targeted dog population can be reached by parenteral vaccination and an almost elimination of the disease can be evidenced. However, in Kasserine and especially in Medenine, where the vaccination coverage is lower than 31%, a reservoir for the disease can be maintained. To match the official figures of vaccination coverage, we should update the statistics of the size of dog population which seems to be bigger than what is assumed. In addition, we wanted to assess the level of involvement of the local population with the vaccination campaigns by marking vaccinated dogs with necklaces and establishing later on the vaccination coverage accordingly. The highest levels of vaccination coverage can be reached in the rural regions. Therefore, the low vaccination coverage in rural areas, reported at the national level, is more attributable to the lack of human and financial resources to reach remote regions. We think that rabies control programmes in Tunisia can be successful if vaccination coverage can reach 70% in all parts of the country. The achieved vaccination coverage should be estimated after random visits in many parts of the country and by checking valid vaccination certificates.

Design of different strategies of multivalent DNA-based vaccination against rabies and canine distemper in mice and dogs

BackgroundDuring the vaccination campaigns, puppies younger than 3 months old are not targeted and remain unvaccinated for at least the first year of their lives. Almost half of the reported rabid dogs are 6 months or younger. Hence, we should recommend the vaccination against rabies of young puppies. Unfortunately, owing to the exposure of puppies to infections with either canine parvovirus (CPV) or distemper virus (CDV) after the intervention of the vaccinators, owners are reluctant to vaccinate puppies against rabies. Therefore, it is necessary to include the CPV and CDV valences in the vaccine against rabies. Multivalent DNA-based vaccination in dogs, including rabies and distemper valences, could help in raising vaccine coverage.MethodsWe have designed monovalent and multivalent DNA-based vaccine candidates for in vitro and in vivo assays. These plasmids encode to the rabies virus glycoprotein and/or the canine distemper virus hemagglutinin. The first strategy of multivalent DNA-based vaccination is by mixing plasmids encoding to a single antigen each. The second is by simply fusing the genes of the antigens together. The third is by adding the foot and mouth disease virus (FMDV) 2A oligopeptide gene into the antigen genes. The last strategy is by the design and use of a bicistronic plasmid with an “Internal Ribosome Entry Site” (IRES) domain.ResultsThe monovalent construct against canine distemper was efficiently validated by inducing higher humoral immune responses compared to cell-culture-derived vaccine both in mice and dogs. All multivalent plasmids efficiently expressed both valences after in vitro transfection of BHK-21 cells. In BALB/c mice, the bicistronic IRES-dependant construct was the most efficient inducer of virus-neutralizing antibodies against both valences. It was able to induce better humoral immune responses compared to the administration of either cell-culture-derived vaccines or monovalent plasmids. The FMDV 2A was also efficient in the design of multivalent plasmids.ConclusionsIn a single shot, the design of efficient multivalent plasmids will be very beneficial for DNA-based vaccination against numerous diseases.

Toward a DNA Vaccine Against Lyssa Viruses

Classical rabies vaccine strains (genotype 1) protect inefficiently against rabies-related lyssaviruses (other genotypes) and in particular against the Mokola virus (genotype 3). DNA vaccine modern approach was used to produce Mokola valency. Purified Plasmid vector was injected into each anterior tibialis muscle of BALB/c mice. It appeared that the Mokola DNA vaccine has induced a sustained level of VNAb, a T helper cell response and confered protection against Mokola virus challenge. Therefore, it seems that the DNA vaccine approach is a very promising technology, which needs further investigations for a possible application.