Purification of rabies virus from tissue culture

Summary A procedure for the purification of rabies virus grown in BHK-21 C 13 cells was developed that consisted of batch adsorption of the virus to aluminium phosphate gel equilibrated with 0.05m phosphate buffer at pH 7.1–7.4 and elution from the gel with 0.3m phosphate buffer at pH 8.0. On average 75 per cent of viral infectivity and 25 per cent of hemagglutinating activity were recovered. Purification factors of about 2000 were achieved as indicated the comparison of the protein contents of original and purified virus samples with their biological activities. On average purified preparations contained 10¹¹ LD₅₀, 2 × 10⁵ hemagglutinating units and 3×10⁴ complement fixing units per mg of protein. Immunization of rabbits with AlPO₄-purified virus produced potent rabies antisera which reacted with extracts of BHK-21 cells to a neglegible extent but not with bovine serum albumin. In CsCl gradients virus infectivity banded at densities of 1.22 and 1.20 g/ml.     

Thymus dependence of rabies vaccine.

Immune responses to rabies vaccine were compared with those to known thymus-dependent and thymus-independent antigens, in nude mice and their normal litter mates. No antibody response to, or protection against, challenge was observed in nude mice inoculated with either 1 or 2 doses of rabies vaccine. A single dose of the same vaccine induced a substantial antibody response and protected normal mice. These data indicate that the antigens in rabies vaccine that induce neutralizing antibody and protect mice are thymus-dependent.     

Properties of rabies vaccine freed of neuroallergenic factor from brain tissue

Fixed rabies virus in the form of infected sheep brain suspension was freed from approx. 80% of ballast proteins, inactivated by beta-propiolactone and lyophilized. The vaccine thus obtained was devoid of neuroallergenicity when tested on guinea pigs and was highly antigenic and immunogenic. The vaccine caused no generalized reactions in volunteers; local reactions were weak and of short duration. Antibody formation was intensive in all volunteers.     

Prophylactic immunization of humans against rabies by intradermal inoculation of human diploid cell culture vaccine.

The antirabies human diploid cell vaccine produced by 1'Institute Merieux, Lyon, France, was administered intradermally to 35 high-risk volunteers using 0.2-ml amounts and various immunization schedules. Three groups never before vaccinated against rabies developed virus-neutralizing antibodies, the titer of which was dose dependent. A single injection stimulated the formation of antibodies. Four inoculations induced the highest antibody levels and the longest persistence of antibody. The administration of a single intradermal booster inoculation was sufficient, even in the case of low-persisting antibody, to elicit a rapid increase of antibodies to high levels. A primary inoculation course of two injections induced a sufficient antibody level which, in case of exposure, could apparently be rapidly elevated by a 0.2-ml intradermal booster inoculation. Adverse side reactions were observed in 7 of 14 individuals after a 1- or 1.5-year intradermal booster inoculation. We therefore suggest that the intramuscular and subcutaneous routes continue to be used for primary vaccinations and that the highly effective intradermal route be restricted to booster inoculations. This is the first long term study of this vaccine and should be a guideline for the pre-exposure treatment of high-risk personnel.     

Development of a thermoresistant tissue culture rinderpest vaccine virus

The currently used Plowright's tissue culture rinderpest vaccine (RBOK strain) gives full protection and lifelong immunity, but it is highly thermolabile and requires maintenance of cold chain from vaccine production till delivery. Keeping in view the need for a thermostabile vaccine in tropical developing countries with limited refrigeration facilities, we passaged serially the RBOK strain of rinderpestvirus (RPV) at gradually elevated temperature up to 40 degrees C to obtain a thermoresistant RPV (TR-RPV) mutant. The thermoresistance (thermostability) and antigenicity of TR-RPV were compared with those of the vaccine virus by various methods, confirming the acquired properties. Thus, the infectivity titres of the TR-RPV mutant and vaccine virus were determined after incubation for various times at 37 degrees C. Regression analysis indicated that TR-RPV had a half-life of 1.81 hr and a degradation constant of 0.1656, while the parent vaccine virus had a half-life of 1.11 hr and a degradation constant of 0.2686. In capture ELISA with four different monoclonal antibodies (MAbs) to the N protein of RPV, TR-RPV showed a 10-fold higher reactivity with one MAb as compared to the vaccine virus. Although TR-RPV did react also with the other three MAbs, its reactivity was only 4-5 times higher than that of the vaccine virus. A treatment of the virus with Triton X-100 resulted in 2-4 times higher reactivity with the MAbs. The 35S-methionine-labeled vaccine virus-and TR-RPV-infected Vero cell lysates showed 6 polypeptide bands with identical pattern of migration in polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE). Radioimmunoprecipitation assay (RIPA) of the TR-RPV and vaccine virus with a rabbit anti-RPV immune serum (RHIS) and bovine anti-RPV hyperimmune serum (BHIS) showed the presence of four identical antigenic proteins, namely H, N, F and M, for both viruses. It can be concluded that TR-RPV has indeed retained the antigenic properties of the parental vaccine virus besides acquiring thermoresistance.     

Correlation of RNA secondary structure and attenuation of Sabin vaccine strains of poliovirus in tissue culture.

Part of the 5' noncoding regions of all three Sabin vaccine strains of poliovirus contains determinants of attenuation that are shown here to influence the ability of these strains to grow at elevated temperatures in BGM cells. The predicted RNA secondary structure of this region (nt 464-542 in P3/Sabin) suggests that both phenotypes are due to perturbation of base-paired stems. Ts phenotypes of site-directed mutants with defined changes in this region correlated well with predicted secondary structure stabilities. Reversal of base-pair orientation had little effect whereas stem disruption led to marked increases in temperature sensitivity. Phenotypic revertants of such viruses displayed mutations on either side of the stem. Mutations destabilizing stems led to intermediate phenotypes. These results provided evidence for the biological significance of the predicted RNA secondary structure.     

铝佐剂对实验狂犬病疫苗的影响

目的对狂犬病疫苗是否应该含有铝佐剂进行实验研究。方法通过检测疫苗抗体、效力（ＥＤ５０）及建立符合狂犬病现场实际的动物模型比较含铝佐剂和不含铝佐剂疫苗防治狂犬病的效果。结果免疫后第４天和第７天抗体滴度铝佐剂疫苗组明显低于不含铝佐剂疫苗组;在ＮＩＨ效力测定中,铝佐剂疫苗组的ＥＤ５０为９３１３２ｎｇ较疫苗组ＥＤ５０２２１ｎｇ少用抗原量一半左右,但该效力测定法不符合狂犬病现场实际;在符合现场实际的动物模型中,铝佐剂对狂犬病疫苗没有任何促进作用。结论在实验狂犬病中,铝佐剂不促进狂犬病疫苗防治狂犬病,而且有不利影响。     

原代沙鼠肾细胞流行性出血热双价疫苗临床观察与免疫学效果研究

目的对新近研制成功的沙鼠肾原代细胞培养的流行性出血热双价疫苗（汉滩型＋汉城型）进行Ⅱ期临床扩大观察，考核其对人体的安全性和中和抗体反应。方法将观察人群设在不同地区，分别选择中国南方和北方两个点，每个点接种５００人的双价疫苗，同时接种２５０人的商品化单价疫苗（汉滩型）作对照，观察接种对象的副反应程度和采集其血清样品测定荧光抗体和以蚀斑减少中和法测定中和抗体，考核疫苗效果。结果观察结果显示，在观察的２８０人中分别有７人呈现轻度局部副反应和３７．５℃以下低热的全身反应，总反应率分别为２．５％。ＩＦＡ抗体阳转率１００％，ＧＭＴ１∶６７。中和抗体阳转率汉滩型为８７．６％，汉城型为９６．３％（ｎ＝８１）。如果以同一份血清中任何单一血清型阳转来计算，阳转率为１００％。以同一份血清两个血清型均需阳转来计算，则阳转率为７５．３％（６１／８１）。结论经过Ⅱ期临床人体观察结果显示，该双价疫苗仅有轻度局部反应和良好的中和抗体应答。证明该疫苗对人体安全，具有较好的免疫原性     

Prolonging morbidity in rabid dogs by intrathecal injection of attenuated rabies vaccine.

Dogs vaccinated intrathecally with attenuated rabies vaccine developed antibodies that were detected in the cerebrospinal fluid, blood, and brain; dogs similarly vaccinated but with an inactivated vaccine developed no antibodies in the brain. When the attenuated vaccine was administered to rabid dogs, a prolongation of the morbidity period was noted and, in some dogs, recovery from the disease. Rhesus monkeys died when administered any of the available attenuated vaccines intrathecally, and further studies with that species could not be undertaken.