Use of mouse anti-rabies monoclonal antibodies in postexposure treatment of rabies.

Immunization of mice and hamsters with a cocktail of mouse MAbs specific for rabies virus nucleocapsid protein and glycoprotein protected animals not only when challenged with a lethal dose of rabies virus after immunization, but also in post-exposure situations. Hamsters treated with the MAb cocktail 3 h after virus inoculation were completely protected from lethal rabies virus infection, and 80% of the animals survived when the MAb cocktail was given 36 h after virus challenge. The potential usefulness of this MAb cocktail for the postexposure treatment of human rabies is discussed.

Nonpermissive infection of lymphoblastoid cells by vesicular stomatitis virus. II. Effect on viral morphogenesis.

The human B-lymphoblastoid cell line Raji is nonpermissive for infection by vesicular stomatitis virus (VSV). The VSV particles released from Raji cells display a more heterogeneous distribution in equilibrium sucrose density gradients than particles released from BHK cells. The particles released from Raji cells contain approximately one-half to one-third as much viral matrix protein, relative to the nucleocapsid protein, as is normal. They also contain a higher proportion of the unglycosylated form of the G protein. The particles released from Raji cells are unstable and many disintegrate in the growth medium. Most of them deform when subjected to ultracentrifugation prior to fixation. The ratio of plaque-forming units to physical particles is much lower for the virions released from Raji cells.

Field evaluation of a dog owner, participation-based, bait delivery system for the oral immunization of dogs against rabies in Tunisia.

We evaluated a dog owner, participation-based, bait delivery system for the oral immunization of dogs against rabies. In a field study in a semirural area of northern Tunisia, dog owners were asked to come to temporary bait delivery sites. A total of 314 baits were given to 178 dog owners in four sites. The experimental baits used consisted of a freeze-dried core unit containing sulfadimethoxine (SDM) as a biological marker and an aromatized paraffin envelope. No vaccine was used. Preliminary tests had shown that by using a rapid commercial card test, positive SDM serum levels were detected in more than 95% of dogs up to two days after bait ingestion. During the two days following bait delivery, we visited more than 95% of all households in the study area and took blood samples from as many owned dogs as possible. Unconsumed baits were recovered and human contacts with the bait matrix were recorded. The campaign required 7.6 person-min per bait and 13.5 person-min per dog owner for providing baits, gloves, and instructions. The estimated average cost effectiveness ratio per dog accepting a bait was 1.7 US dollars. From the indications given by the dog owners and the results of the SDM test, it was concluded that 85-90% of the owned dogs in the study area had consumed a bait at least partially. Of 314 baits delivered, 78.7% were fully consumed by dogs and 4.1% were recovered during the household survey. The remaining baits (17.2%) that were not recovered were either not consumed or only partially consumed by the target dogs (3.7 baits per 100 inhabitants). These baits probably remained within the highly populated areas and were potentially accessible to other domestic animals and other nontarget species, including humans. Twenty-five unprotected human contacts with baits were recorded (1.7% of all inhabitants). Our study has demonstrated the potential of dog owner based bait delivery. This technique is simple and efficient, particularly if the human population is accustomed to mass immunization in defined centers. Before applying this method on a large scale with live vaccine loaded baits, further studies should focus on minimizing the number of human contacts with the vaccine bait, systematizing contact identification and establishing structures in ensuring proper treatment if exposure to vaccine should occur.

SAG-2 oral rabies vaccine.

The live modified rabies virus vaccine strain SAG-2 was selected from SADBerne in a two step process employing anti-rabies glycoprotein monoclonal antibodies. The first two nucleotides coding for the amino acid in position 333 of the rabies glycoprotein are mutated. Arginine at position 333, which is associated with rabies pathogenicity, was substituted first by lysine and then by glutamic acid. The two nucleotide differences at position 333 in SAG-2 to any of six possible arginine triplets translated into excellent genetic stability and apathogenicity for adult mice, foxes, cats and dogs. The vaccination of foxes and dogs by the oral route provided protection against a lethal challenge with rabies virus.

Acceptance of candidate baits by domestic dogs for delivery of oral rabies vaccines.

Protocols for evaluating oral rabies vaccine baits for domestic dogs were field tested in central Mexico, after which dog-food manufacturers and suppliers to the pet-food industry were advised as to potential ingredients for use in prototype dog baits. Bait-preference trials in which confined dogs were used were then undertaken, followed by field tests of free-ranging farmer-owned dogs in three towns in the Nile River Delta region of Egypt. Both confined and free-ranging dogs showed strong preferences for certain baits or bait coatings (poultry, beef tallow, cheese, egg and a proprietary product). Fish-meal polymer baits, widely used for wildlife species, were less preferred. In Egypt, a commercial dog-food-meal bait coated with beef tallow and dry cheese, was consumed at a rate approaching that of a chicken-head bait. The percentage baits that were actually eaten after they had been offered to dogs, ranged from 71-96% for household dogs tested in Mexico, 65-91% for confined dogs (beagles and mixed breeds) tested in the United States, and 32-88% for farmer-owned dogs tested in Egypt.

Effectiveness of SAG1 oral vaccine for the long-term protection of red foxes (Vulpes vulpes) against rabies.

Three groups of 10 foxes were vaccinated by the direct oral instillation of 2 ml of SAG1 rabies virus vaccine containing 10(6) MICLD50 (10(7) TCID50/ml) infectious viral particles/ml. SAG1 is a natural variant of the attenuated rabies vaccine strain SAD Bern and was selected in the presence of monoclonal antibodies. The strain is devoid of residual pathogenicity for the fox and the highly susceptible adult laboratory mouse by the oral, intramuscular and intracerebral routes. The foxes were challenged six, 12 and 18 months later with a virulent vulpine street rabies virus (GS 7-1-1). They all survived, whereas seven of eight unvaccinated control foxes died.

Efficacy and bait acceptance of vaccinia vectored rabies glycoprotein vaccine in captive foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides) and dogs (Canis familiaris).

The red fox, dog, and raccoon dog are known to play a major role in the global epidemiology of rabies. These three canid species were used to compare the appetency and efficacy of two commercial bait formats, each containing a single dose of vaccinia-rabies glycoprotein (V-RG) vaccine. Square and rectangular RABORAL V-RG baits were fed to individual caged animal, and results were evaluated using three parameters: bait consumption, induction of rabies virus neutralizing antibodies and protection after a virulent rabies challenge. The rectangular and square RABORAL V-RG baits were found to deliver the oral rabies vaccine in a similar manner to all three species resulting in acceptable seroconversion and effective protection levels after the rabies challenge. Appetency of each bait type was measured by bait consumption and found to be similar for both RABORAL V-RG bait formats in the fox and dog. The square RABORAL V-RG bait, however, was consumed more effectively than the rectangular RABORAL V-RG bait by the raccoon dog.

Inhibition of immune responses against rabies virus by monoclonal antibodies directed against rabies virus antigens.

Treatment of mice with a cocktail of murine anti-rabies monoclonal antibodies (mAb-C) interfered with the ability of these animals to mount a virus-neutralizing antibody response to rabies vaccine. Administered mAb-C did not affect the induction of rabies virus-specific T-helper cells. The magnitude of the inhibition of rabies virus-specific B-cell response was dependent on the concentration of the mAb-C and the duration of the mAb-mediated interference was inversely proportional to the biological half-life of the mAb. As long as the serum titres were above a critical threshold, the suppression could not be overcome even by multiple vaccinations. Since injection of mice with immunocomplexes consisting of inactivated rabies virus and mAb rendered the animals non-responsive to a subsequent vaccination with inactivated rabies virus, it is concluded that the mAb-induced suppression might be caused by the formation of antigen-antibody complexes which exert a negative signalling effect to premature B cells.

Innocuity studies of SAG-2 oral rabies vaccine in various Zimbabwean wild non-target species.

The SAG-2 modified live rabies vaccine was tested for innocuity when administered by the oral route in several potential wild non-target bait-consuming species, as follows: ten chacma baboons (Papio ursinus), six African civets (Civettictis civetta), six slender mongooses (Galerella sanguinea), six honey badgers (Mellivora capensis), six large-spotted genets (Genetta tigrina), 39 multi-mammate mice (Mastomys natalensis), 26 bushveld gerbils (Tatera leucogaster) and six pied crows (Corvus albus). At least 9.0 log10 median tissue culture infectious doses (TCID50), given in a volume of 1 ml, was administered orally to each of the animals, except the rodents which received 8.0 log10 TCID50, given in 0.1 ml. All the animals were observed for not < 90 days for signs of vaccine-induced rabies. Most of the species were also tested for vaccine virus replication in the oral cavity and persistent virus infection in the brain, salivary gland and tonsil. None of the animals died of rabies and no persistent infection was found. Rabies virus which was pathologically and serotypically indistinguishable from the vaccinal strain was isolated from the saliva of one genet 1 day after vaccine administration. From this study it was concluded that SAG-2 rabies vaccine would be safe for use in most situations where oral vaccination campaigns for jackals are required in Zimbabwe.

Efficacy of SAG-2 oral rabies vaccine in two species of jackal (Canis adustus and Canis mesomelas).

Trials were carried out to test the efficacy of SAG-2 oral rabies vaccine in two species of jackals, namely the side-striped jackal (C. adustus) and the black-backed jackal (C. mesomelas). The first trial tested the efficacy of SAG-2 when given by direct oral administration at doses of 6.5 and 7.5 log10 median tissue culture infectious doses (TCID50). One side-striped jackal which had received the higher dose did not seroconvert and succumbed to challenge, while all other jackals seroconverted and resisted a lethal challenge. The second trial tested the efficacy in side-striped jackals only of the SAG-2 vaccine when given within chicken head baits. A volume of 1.8 ml of vaccine fluid with titers of 7.0 or 8.0 log10 TCID50/ml were placed into blisters which were stapled under the skin of the chicken heads. All jackals (5/5) which received 8.0 log10 TCID50/ml and 3 of 5 which received 7.0 log10 TCID50/ml seroconverted and resisted lethal challenge. A third trial tested the rate of vaccine virus titer loss in chicken head baits placed under field conditions. Titer loss was marked in baits which were not protected from direct sunshine, whereas under vegetation cover approximately log10 TCID50/ml was lost every 3 days. Hence, it was concluded that a bait vaccine virus titer of 8.0 log10 TCID50/ml will be sufficient to immunize wild jackal populations if enough baits can be consumed by jackals within 3 days. This conclusion needs to be tested through the use of oral vaccine in field trials.

Rabies virus nucleoprotein expressed in and purified from insect cells is efficacious as a vaccine.

A cDNA copy of the RNA gene that encodes the nucleoprotein N of rabies virus Evelyn-Rokitnicki-Abelseth strain was cloned into baculovirus. The recombinant baculovirus expressed the N protein abundantly in Spodoptera frugiperda cells. The N protein was extracted from infected Spodoptera frugiperda cells and purified to near homogeneity by affinity chromatography. The purified N protein reacted with 31 of 32 monoclonal antibodies that recognize native rabies virus ribonucleoprotein. Like the ribonucleoprotein, the purified N protein was a major antigen capable of inducing virus-specific helper T cells. Priming of mice with the purified N protein prior to a booster inoculation with inactivated Evelyn-Rokitnicki-Abelseth virus vaccine resulted in a 20-fold increase in the production of virus-neutralizing antibodies. After immunization with the purified N protein, mice developed a strong anti-ribonucleoprotein antibody response and were protected against a lethal challenge of rabies virus. These data indicate that the N protein expressed in insect cells is antigenically and immunogenically comparable to the authentic rabies virus ribonucleoprotein and therefore represents a potential source of an effective and economical vaccine for large-scale immunization of humans and animals against rabies.

Immunogenicity, efficacy and safety of an oral rabies vaccine (SAG-2) in dogs.

A study of immunogenicity and efficacy of Street Alabama Gif (SAG-2) attenuated rabies virus vaccine in laboratory beagles was conducted. Four groups of ten dogs each received either 1.0 ml of SAG-2 orally on the tongue or 1.5 ml in baits. On day 180 postvaccination, all dogs were challenged with a street rabies virus. The antibody response in groups that received the vaccine directly on the tongue was higher than in those vaccinated with baits, but the difference between groups was not statistically significant. All vaccinated dogs survived, whereas 80% of controls died of rabies. Our findings demonstrate that the SAG-2 is a safe and effective vaccine for oral immunization of canines.

Safety study of the SAG2 rabies virus mutant in several non-target species with a view to its future use for the immunization of foxes in Europe.

The safety of the SAG2 virus, a low virulence mutant of the SAD strain, was investigated in ten species of mammals and seven species of birds liable to consume vaccine baits. These species are the western hedgehog (Erinaceus europaeus), the meadow vole (Microtus arvalis), the bank vole (Clethrionomys glareolus), the water vole (Arvicola terrestris), the field mouse (Apodemus flavicollis or A. sylvaticus), the Norway rat (Rattus norvegicus), the european badger (Meles meles), the domestic ferret (Mustela putorius furo), the wild boar (Sus scrofa), the domestic goat (Capra hircus), the carrion crow (Corvus corone), the rook (Corvus frugilegus), the buzzard (Buteo buteo), the red kite (Milvus milvus), the tawny owl (Strix aluco), the long-eared owl (Asio otus) and the barn owl (Tyto alba). The vaccine was administered orally to each species, by an intramuscular (i.m.) route to the rodents and ferret, and by an intracerebral route to the field mouse. No pathogenicity was observed in the 169 animals vaccinated throughout an observation period of over 30 days. After euthanasia, no rabies virus could be detected either in the brain or in the salivary glands of any of the animals. The SAG2 virus administered orally, triggered a specific seroconversion in the field mouse, wild boar, ferret and most of the raptors. Following administration by the i.m. route, specific antibody titres were observed in most of the rodents, as well as in the ferrets.

Safety evaluation of the SAG2 rabies virus mutant in Tunisian dogs and several non-target species.

The safety of the SAG2 rabies virus, a highly attenuated mutant of the SAD strain intended to vaccinate dogs by the oral route, was evaluated in local Tunisian dogs and in five other local species likely to consume vaccine baits. These species were the domestic cat (Felis catus), the jackal (Canis aureus), the jerboa (Jaculus orientalis), the merion (Meriones sp.) and the gerbil (Gerbillus campestris). The vaccine was administered orally to 21 dogs, 11 cats and eight jackals and orally or intramuscularly to 62 wild rodents of the above-mentioned species. Seven dogs, one cat, five jackals all juvenile and with poor health status) and two rodents died for intercurrent causes. The others were observed for 60-180 days. No animal showed any rabies symptom. Seroneutralizing antibodies were observed in all experimental groups, only after vaccination, with the highest rate being observed in jackals and rodents. The rabies virus was detected in the oral cavity of three cats 6 h after oral instillation, but was not isolated later either in saliva or in salivary glands. Tissue samples (brain and salivary glands) from dead or euthanized animals were examined for the rabies virus antigen by a fluorescent antibody test. No rabies antigen was detected. These trials confirm the safety of the SAG2 strain on the Tunisian species already demonstrated by other authors on many other target and non target species.

Field evaluation of two bait delivery systems for the oral immunization of dogs against rabies in Tunisia.

Two bait delivery systems for the oral immunization of dogs against rabies were tested in small scale field trials in a semi-rural area in Tunisia: bait delivery to owned dogs during door to door visits of households (door to door baiting) and distribution of baits on transect lines (transect line baiting). A prototype bait (DBL2) configured for industrial production and containing either sulfadimethoxine (SDM) as a systemic marker or Rhodamine B as a topical marker was used. The overall proportion of dogs which took a bait and presented topical marker staining after door to door baiting was 59.1%. The total time and costs spent per bait accepting dog averaged 34 person minutes and US$4, respectively. Unconsummated baits were readily recovered. No unprotected human contacts with baits were recorded. Door to door baiting is a very specific but time-consuming method that enables a safe administration of vaccine baits to owned dogs. For transect line baiting, baits were distributed at a density of ca 3000 baits per km2 along double transect lines. Baits were recovered after 20 h. According to the proportion of SDM positive serum samples, 24.1% of owned dogs in the baiting area had consumed baits. Of all owned and ownerless dogs, presumably free-roaming during transect line baiting, > 40% had consumed baits. The total time and costs spent per bait accepting dog averaged 48 person minutes and ca US$20, respectively. The household census revealed 32 direct human contacts with the bait matrix which corresponds to 1.4% of inhabitants. Placing baits on transect lines gives the possibility to vaccinate dogs not accessible by vaccination systems which base on dog owner participation. However, the method is not specific, less safe than other systems, not easily accepted by the human population, and costly.