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Alternatives for potency testing of veterinary vaccines: rabies vaccine as a model.

Optimum information about potency of a vaccine is achieved by a protection test on the target animal species. However, application of this procedure to routine batch control requires a great number of target animals. In order to reduce the number of such animals, laboratory animal tests or in vitro tests have to be established. These tests must be correlated to target animal protection tests allowing the estimation of the vaccine potency with the same accuracy. With respect to testing the potency of rabies vaccines, alternatives are discussed, i.e. the mouse protection test, induction of antibodies in mice and in vitro tests.

Animals↗

[Potency testing of veterinary vaccines, rabies vaccines as an example]

Vaccines which are produced commercially should be controlled with respect to their efficacy (potency) and their "harmlessness" to the recipient animal. The basis for any potency test is the protection test in the target animal species. When a vaccine has been shown to be capable of protecting a particular species from a certain disease, alternative methods for potency testing often can be developed. The justification of the latter is that experiments which analyze the protection are cost-intensive, require a large number of animals and give only a moderate degree of reproducibility. Methods such as protection experiments in laboratory animals, the induction of the substances responsible for protection (such as antibodies) in target or laboratory animals as well as with in vitro tissue culture systems, or the quantitative determination of the immunogens (the components of a vaccine which should be responsible for protection) may be used only if their results correlate with those obtained from protection experiments in the target animal species. Rabies glycoprotein has been implicated as the main agent responsible for the induction of protection against rabies. The higher the content of glycoprotein in a rabies vaccine, the greater will be the potential of the vaccine to induce protection against the disease. Analyses of the rabies glycoprotein content in vaccines can make use of a number of methods which have proven acceptable (the immunodiffusion test, the antibody binding assay and the ELISA). However, in veterinary medicine most vaccines are associated with an adjuvant - a substance which should amplify the immune response of an animal against a vaccine. Since the adjuvants and vaccines in such preparations are inseparable, the presence of an adjuvant in a vaccine reduces the applicability of many in vitro methods. Thus, the above mentioned methods and possibilities for their adaptation to the analysis of adjuvanted vaccines will be discussed.

Journal Article↗

Genetic engineering of live rabies vaccines.

Rabies virus is not a single entity but consists of a wide array of variants that are each associated with different host species. These viruses differ greatly in the antigenic makeup of their G proteins, the primary determinant of pathogenicity and major inducer of protective immunity. Due to this diversity, existing rabies vaccines have largely been targeted to individual animal species. In this report, a novel approach to the development of rabies vaccines using genetically modified, reverse-engineered live attenuated rabies viruses is described. This approach entails the engineering of vaccine rabies virus containing G proteins from virulent strains and modification of the G protein to further reduce pathogenicity. Strategies employed included exchange of the arginine at position 333 for glutamine and modification of the cytoplasmic domain. The recombinant viruses obtained were non-neuroinvasive when administered via a peripheral route. The ability to confer protective immunity depended largely upon conservation of the G protein antigenic structure between the vaccine and challenge virus, as well as on the route of immunization.

Animals↗

Gray fox response to baits and attractants for oral rabies vaccination.

Rabies is a widespread zoonosis that recently reached epidemic proportions in gray foxes (Urocyon cinereoargenteus) in central Texas. The objectives of this study were to determine bait and attractant preferences among captive gray foxes, to determine the behavioral responses of gray foxes to selected bait-attractant combinations, and to evaluate baits as a delivery mechanism of oral rabies vaccines. Trials were conducted to determine bait preferences of captive gray foxes to selected baits and attractants. Tested baits consisted of a polymer-bound cube made of either dog food meal or fish meal, a polymer-bound cylinder made of dog food meal, and a wax-lard cake that was enhanced with marshmallow or chicken flavoring. Attractants were additives to baits that exuded sweet, sulfurous, fruity, fatty, cheesy, honey, and fishy odors and flavors. Captive gray foxes (n = 31) exhibited a preference for marshmallow wax cakes and polymer dog food baits with a lard interior and granulated sugar exterior. However, gray foxes exhibited chewing behaviors consistent with ingesting an oral vaccine only with the wax cake baits.

Administration, Oral↗

Controversies in rabies vaccination.

Rabies is a cent per cent fatal disease and there should not be any controversy in giving rabies vaccine to the victims. WHO has fixed schedules for doses for both pre and post-exposure in different category of cases, which also help us to avoid all controversies. But controversies arise in five main areas, which are related to the strategies of rabies prevention. These are: (i) Replacing use of NTV by MTCV. (ii) Intradermal schedule of MTCV, in place of Essen protocol of 5 i.m. doses to reduce the cost. (iii) Acceptability and inclusion of pre-exposure doses of MTCV in the immunization schedule of children as additional vaccine (iv) Schedule for re-exposure in already post-exposure vaccinated cases and schedule for exposure in pre-exposure vaccinated cases. (v) Uses of RIG in WHO category III cases. If these controversial issues are considered scientifically, rabies prophylaxis will see the light of success.

Adolescent↗

Treatment of patients bitten by rabid or suspected rabid wolves with inactivated tissue culture rabies vaccine and rabies gammaglobulin.

During 1972-1976 46 persons in 9 foci were bitten by wolves. 39 of them were immunized with antirabies gammaglobulin and tissue culture rabies vaccine; 7 received culture vaccine only. Rabies in wolves was confirmed clinically or in the laboratory in 8 foci. Bites of dangerous localization: face, head or fingers of the hands, predominantly multiple, were noted in 25 humans; 5 of them were young, 7 to 16 years old. Antirabies gammaglobulin was given to 9 people, predominantly in the dose of 0,5 ml per kg of weight, once on the 1st day after exposure (381-538 IU per kg of weight) to 14 people, once on the 2nd day (706-773 IU) to 3 people, twice on the 2nd and 3rd to 3 people, once on the 3rd and 5th day to 10 people, twice on the 2nd and 3rd day or on the 5th day after exposure. Vaccination course was started 24 hours after administration of gammaglobulin and predominantly in the dose of 5 ml; it lasted for 25 days and was followed by 3 booster injections on the 10th, 20th and 30th day. Titres of virus neutralizing antibody were tested in dynamics in 39 people immunized with gammaglobulin and tissue culture vaccine. Antirabies gammaglobulin induced some inhibitory effect, but 2-3 booster injections of the tissue culture rabies vaccine completely compensated this effect. During the observation period of 10 months to 5 years all the exposed people remained healthy.

Adolescent↗

Intradermal immunization with human diploid cell rabies vaccine: serological and clinical responses of immunized persons to intradermal booster vaccination.

Rabies antibody titers ranged from 0-9.3 IU/mL in 117 human volunteers one year after intradermal vaccination with one or two doses of human diploid cell rabies vaccine (HDCV). At that time, each volunteer received one 0.1-mL booster dose of HDCV intradermally. All 117 volunteers showed good anamnestic responses, with antibody titers rising to 0.5-54.3 IU/mL within seven days of booster injection. Vaccine safety was good; only minor reactions were experienced, all of which resolved spontaneously.

Drug Administration Schedule↗

Large-scale eradication of rabies using recombinant vaccinia-rabies vaccine.

Rabies infection of domestic and wild animals is a serious problem throughout the world. The major disease vector in Europe is the red fox (Vulpes vulpes) and rabies control has focused on vaccinating and/or culling foxes. Culling has not been effective, and the distribution of five vaccine baits is the only appropriate method for the vaccination of wild foxes. Although some European countries have conducted field vaccination campaigns using attenuated rabies virus strains, their use has not been extensively approved because they retain pathogenicity for rodents and can revert to virulence. These strains cannot be used in North America because they are pathogenic for the striped skunk (Mephitis mephitis) and are ineffective in the raccoon (Procyon lotor). We have constructed a recombinant vaccinia virus, VVTGgRAB, expressing the surface glycoprotein (G) of rabies virus (ERA strain). The recombinant was a highly effective vaccine in experimental animals, in captive foxes and in raccoons. We report here the results of a large-scale campaign of fox vaccination in a 2,200 km2 region of southern Belgium, an area in which rabies is prevalent. After distribution, 81% of foxes inspected were positive for tetracycline, a biomarker included in the vaccine bait and, other than one rabid fox detected close to the periphery of the treated area, no case of rabies, either in foxes or in domestic livestock, has been reported in the area.

Animals↗

Rhesus diploid rabies vaccine (adsorbed), a new rabies vaccine. Results of initial clinical studies of preexposure vaccination.

To meet the need for a safe, efficacious, and low-cost rabies vaccination program, the Michigan Department of Public Health developed a new rabies vaccine: rhesus diploid rabies vaccine, adsorbed (RDRV). Initial clinical studies were conducted in 534 volunteers using preexposure protocols consisting of two injections of RDRV given 1, 2, or 4 weeks apart. This new rabies vaccine induced an excellent rabies virus antibody response two to three weeks after vaccination: antibody levels were superior to those reported after duck embryo rabies vaccine and were similar to those reported with human diploid rabies vaccine. In addition, vaccination with RDRV was associated with an acceptable level of local and constitutional symptoms.

Adolescent↗

Rhesus diploid rabies vaccine (adsorbed), a new rabies vaccine. II. Results of clinical studies simulating prophylactic therapy for rabies exposure.

Rhesus diploid-cell-strain rabies vaccine (RDRV) (adsorbed) is a new rabies vaccine intended for use in man. Sixty volunteers were given five doses of RDRV at 0, 3, 7, 14, and 28 days to simulate prophylactic treatment of persons exposed to a rabid animal. Thirty-five volunteers were given commercial high-titer rabies immune globulin, 20 IU/kg, before the first dose of RDRV, and 25 were given RDRV without prior rabies immune globulin. Antibody responses at 14, 28, and 42 days were comparable with those reported with human diploid rabies vaccine. Simulated postexposure prophylactic treatment with RDRV was associated with acceptable levels of local and constitutional symptoms.

Adsorption↗

The abbreviated 2-1-1 schedule of purified chick embryo cell rabies vaccination for rabies postexposure treatment.

During August 1988 to January 1990, the immunogenicity and safety of purified chick embryo cell rabies vaccine (PCEC) given by the conventional and abbreviated regimens in 82 vaccinees moderately to severely exposed to laboratory proven rabid animals were studied. The 16 vaccinees received PCEC six doses as conventional schedule on days 0, 3, 7, 14, 28 and 90, the 11 vaccinees received six doses of PCEC plus human rabies immune globulin (HRIG) on day 0. The 29 vaccinees received an abbreviated schedule of PCEC as two doses on day 0, one dose each on days 7 and 21 and the 26 cases received PCEC abbreviated schedule plus HRIG on day 0. The kinetics of the neutralizing antibodies on days 0, 7, 14, 28, 56, 180 and 365 were studied for comparative purpose. All vaccinees had high antibody levels from day 14 which last longer than a year and were safe after one year follow up. The adverse reactions of the vaccine were mild and self-limited.

Adolescent↗

Zonal-centrifuged purified duck embryo cell culture rabies vaccine for human vaccination.

Rabies virus produced in duck embryo cell culture was concentrated from volumes of 14 to 30 liters to 400 to 800 ml by zonal centrifugation. Virus titers of peak fractions were from 100- to 1,000-fold greater than those of the starting material. Vaccines were prepared by combining fractions with peak virus titers and diluting back to 10 times concentration. The resulting beta-propiolactone-inactivated vaccines, when prepared as lyophilized vaccines with AlPO(4) adjuvant diluents, were low in protein nitrogen (0.01 mg/ml), and three of four lots passed the National Institutes of Health potency test when tested as equivalent to a standard 10% suspension of duck embryo or mouse brain tissue vaccine. These vaccines also induced good sero-conversion in adult rabbits after a single 1-ml dose of vaccine. Guinea pigs sensitized with zonal-centrifuged purified duck embryo vaccine (with AlPO(4) adjuvant) did not exhibit anaphylactic shock reactions when challenged with homologous vaccine. Also, no anaphylactic shock reactions were observed when guinea pigs were sensitized with either a 10% experimental duck embryo vaccine or cell culture vaccine and then challenged with the zonal-purified vaccine. However, guinea pigs sensitized with cell culture or zonal-purified vaccine and then challenged with the 10% experimental vaccine did show slight transitory congestion. The 10% experimental whole duck embryo vaccine was responsible for all observed anaphylactic shock reactions whether homologous or heterologous.

Adjuvants, Immunologic↗

Antibody response of patients after postexposure rabies vaccination with small intradermal doses of purified chick embryo cell vaccine or purified Vero cell rabies vaccine.

Although the introduction of tissue culture vaccines for rabies has dramatically improved the immunogenicity and safety of rabies vaccines, they are often prohibitively expensive for developing countries. To examine whether smaller doses of these vaccines could be used, we tested the safety and immunogenicity of purified chick embryo cell vaccine (PCECV) on 211 patients in Thailand with World Health Organization (WHO) category II and III exposures to rabies. The patients presented at two Thai hospitals and were randomized into three groups. Patients in Group 1 received 0.1 ml PCECV intradermally at two sites on days 0, 3, 7, and at one site on days 30 and 90. Group 2 was treated similarly, except that purified Vero cell rabies vaccine (PVRV) was used instead of PCECV. Group 3 received 1.0 ml PCECV intramuscularly on days 0, 3, 7, 14, 30 and 90. After 0, 3, 7, 14, 30 and 90 days serum was collected from the subjects and the geometric mean titres (GMTs) of rabies virus neutralizing antibody determined. After 14 days the GMT of 59 patients vaccinated intradermally with PCECV was equivalent to that of patients who received PVRV. Adverse reactions were more frequent in patients who received vaccines intradermally, indicating the reactions were associated with the route of injection, rather than the vaccine per se. We conclude that PCECV is a safe and highly immunogenic vaccine for postexposure rabies vaccination when administered intradermally in 0.1-ml doses using the two-site method ("2,2,2,0,1,1") recommended by WHO.

Adolescent↗

[Comparison of duck embryo vaccine and Hempt's vaccine for rabies vaccination (author's transl)].

For many years, Hempt's vaccine has been used in Germany for vaccination against rabies. In recent years the duck embryo vaccine has been gradually introduced in its place. From 1970 to 1975, 658 consultations were undertaken at the Hospital of the Tropical Institute at Hamburg because of the risk of rabies. 267 persons had to undergo vaccination, 159 of them being treated with Hempt's vaccine and 108 with duck embryo vaccine. Side effects of a more severe type were not seen in either group. Disadvantages of the duck embryo vaccine are that more individual inoculations are necessary, significantly more frequent and more persistent local and general vaccination reactions occur and that there is a delayed, possibly weaker formation of antibodies.

Animals↗

Three-year experience with 4-site intradermal booster vaccination with rabies vaccine for postexposure prophylaxis.

For booster vaccination of previously immunized persons with potential exposure to rabies, the World Health Organization recommends 2 doses of cell-culture vaccine administered intramuscularly or intradermally on days 0 and 3. We believe that four 0.1-mL intradermal booster doses given on a single day could be used at no risk to the recipient. We studied use of a single booster vaccination on day 0 followed by four 0.1-mL intradermal doses of cell-culture rabies vaccines, and we determined that this is a safe, convenient, and economical regimen for postexposure treatment of previously vaccinated individuals.

Adolescent↗