Dilution Of Vaccine Research Articles

Spotted Fever Vaccine; Potency Assay by Direct Challenge of Vaccinated Mice with Toxin of Rickettsia Rickettsii

Summary A mouse protection test, devised for the assay of Rocky Mountain spotted fever vaccine, determined the minimal quantity of vaccine capable of protecting mice against 2 LD50 toxic doses of Rickettsia rickettsii. This technique, which has several advantages over the standard guinea pig potency test, gave relatively consistent results. Thus, potency values were usually reproducible within 0.5 log dilution of vaccine, but variations as great as 0.8 log were observed. Several samples of yolk-sac products and tick-tissue (Dermacentor andersoni) vaccines which varied from 1 to 14 years in age exhibited mouse protection titers of from 1.0 to 2.2 logs. The minimal doses of vaccine required to protect guinea pigs and mice against challenge with R. rickettsii were similar. An antitoxin test similar to the standard method for potency assay of typhus vaccine was also studied. Minimal doses of spotted fever vaccine capable of stimulating a demonstrable amount of antitoxin in the serum of vaccinated guinea pigs were also capable of inducing resistance in these animals. Marked resistance was found to develop in both mice and guinea pigs by the 3rd day following vaccination. Serum antitoxin also developed rapidly in guinea pigs, reaching maximal titer by the 5th day after vaccination. When immunized guinea pigs were challenged with doses of R. rickettsii varying from 50 to 5000 ID50, the resulting vaccine protection titers were similar. However, a challenge dose of 5 ID50 resulted in a 4-fold increase in protective titer.

インフルエンザウイルスの精製に関する研究

In Japan, vaccination is established for the prevention of influenza. Our influenza virus vaccines contain 100 CCA per mililiter of each strain of A1, A2 and B type.According to author's experience, chief reactions to vaccines are redness, swelling and heat at the site of injection, which give discomfort to the recipients. While systemic reactions, such as the increase in body temperature are not common.To eliminate these reactions, we tried the purification of the viruses by the chromatographic method with brushite form of calcium phosphate, which was reported by Taverne et al in 1958. We repeated many experiments with 3 types of the viruses and obtained average recovery of 65.3%, ranging from 39.0% to 96.4%, in 7 experiments with A1 type, 79.4%, ranging from 55.6% to 89.3%, in 6 experiments with A2 type, and 73.8%, ranging from 52.7% to 87.5%, in 4 experiments with type B.By rechromatography, the recovery rate was as high as over 90%.Purified monovalent vaccines of type A and B viruses, each containing 300 CCA units, were examined for their safety and potency comparing with the non-purified, which were treated only once with Sharples centrifuge and resuspended in buffered physiological saline. In safety test conducted by the guinea pig method, we could obtain far better results from the purified ones.Potency test was conducted with mice, injecting 5 fold graded dilutions of vaccines intraperitoneally. They were bled after 2 weeks, blood collected, sera separated and used for neutralization test in embryonated eggs. The results showed that purified vaccines had potency not inferior to non-purified ones.The same vaccines were examined for reactions and antigenicity to human bodies. Four groups of recipients, each consisting of 20, were inoculated with one of four vaccines, purified or non purified vaccines of type A2 and B.Reactions (redness, swelling, heat at injection site and increase in body temperature etc.) were examined. Systemic reactions were weak in all vaccines. Redness began to appear 4 hours after inoculation and faded after 3 to 4 days. In the purified vaccine groups of both types, redness were 8.1mm at 24 hours after inoculation and 16.0mm at 48 hours by type B purified vaccine, wheras those of the non-purified of the same type were 60.2mm and 74.6mm. By A2 type purified vaccine, they were 7.2mm and 6.0mm, but non-purified control showed 23.5mm and 12.9mm.Swelling and heat at injection site were far less in the case of purified vaccines of both types compared to the non-purified.Antigenicity of vaccines was estimated by examining increase of HAI titer 4 weeks after vaccination. Purified vaccines of both types of A2 and B gave lower antigenicity to some extent. But, further large scale experiments may be necessary to confirm whether the purified vaccines are truly inferior or not.

Antibody Response to a Combined Living Attenuated Distemper/Hepatitis Vaccine

SUMMARY A trial with a combined living vaccine containing the Beckenham egg embryo-adapted strain of Onderstepoort attenuated canine distemper (CD.) virus and the Cornell strain of pig kidney-adapted canine contagious hepatitis (C.C.H.) virus is described. Effects of vaccination, development of antibodies, and resistance to challenge were studied in groups of dogs given undiluted and serial dilutions of vaccine. Neutralizing antibodies were demonstrated in many cases by the second week after inoculation. They reached a high level in dogs receiving the larger doses of vaccine. In-contact dogs remained clinically healthy, although the attenuated C.C.H. virus was evidently excreted, since these dogs all developed C.C.H. antibodies except the one in contact with the group receiving the highest dilution of vaccine. Whereas all dogs that developed specific neutralizing antibodies successfully resisted challenge, non-immunized dogs in the higher vaccine dilution groups and the challenge controls reacted with varying degrees of severity, and some died. The 50 per cent immunizing dose of the CD. component was about 1000 EID50, and of the C.C.H. component less than 2 TCID50. Histological examinations of livers and kidneys revealed no evidence of damage despite vaccination with large amounts of attenuated virus followed by challenge with virulent virus.

Potency of inactivated poliovirus vaccines, an in vitro method for assay of antibody combining capacity

Anin vitro method for determination of antigenic potency of inactivated poliovirus vaccines has been developed. Serial dilutions of vaccine and a fixed amount of live virus are allowed to combine firmly with antibody. By titrations for residual virus activity the dilution of vaccine is determined where neutralization is reduced to a certain limit. The reproducibility of the test results was good. A statistically significant correlation was demonstrated between results of thein vitro test and results obtained with the guinea pig test.

現行日本脳炎ワクチンの免疫効果に関する実験的研究 (第2報)

It was assumed that the Japanese encephalitis vaccine of mouse brain type might give rise preventive effect on the disease in nature in approximately 75 per cent. However, such a brain type vaccine was proved to contain possibly some factor to produce demyelinization following inoculation with the vaccine combined with Freund's adjuvant into guinea pigs. Accordingly the minimum requirement of the vaccine was revised to be prepared from the supernatant of 2 per cent emulsion of infected mouse brain following centrifugation at 3, 000 rpm for 30 min. Encephaligenic factor of such vaccine was remarkably minimized so that no more experimental demyelinization in guinea pig was encountered with this.Herewith an attempt was made to elucidate immune response following injection of formalin killed mouse brain type vaccine of G-1 strain which was prepared following the method of the minimum requirement. G-1 strain of Japanese encephalitis virus was isolated from an encephalitis patient by us in 1949 and had been passed through mouse brain roughly by 200 generations. To criticize immune response by the vaccine, the vaccine was diluted to 1:1, 1:16 and 1:128 or 1:1, 1:10, 1:100 and 1:1, 000 and a group of mice was immunized with each dilution. Those immunized mice were divided into subgroups, and challenged with a series of 10 fold dilution of G-1 strain intracerebrally at 2 and 3 weeks after the initial immunization. On the other hand, neutralization, complement-fixation (CF) and hemagglutination inhibition (HAI) tests were carried out with pooled mouse sera from each group at 2 and 3 weeks after the initial vaccination. By comparing protection index (PI) with neutralization index (NI), CF and HAI antibody titers in mice at 2 and 3 weeks after the immunization with each dilution of the vaccine, those may be said:1) The less PI and antibody titers were found in mice immunized with the higher dilution of vaccine at 2 weeks.2) There was no significant difference in immune responses of mice vaccinated with both original and 10 times diluted vaccines. The minimum effective dose of the vaccine in mice was computed to be between 10 and 100 times dilution of the vaccine.3) Comparing with immune responses each other in mice at 2 and 3 weeks, both PI and CF antibody titers reached their peaks at 2 weeks and fell down by 3 weeks, while both neutralizing and HAI antibody titers were found almost the same.From the foregoing results it may be suggested that the effect of vaccine should be criticized on the basis of results obtained in mice first immunized with dilutions of vaccine of some range and then challenged with a series of virus dilution.

Round Table Discussion

Specific Diagnostic Practice Chairman Collins-Williams: The specific diagnosis of the child with respiratory allergy comprises the following: (1) general pediatric care; (2) careful allergic history; (3) complete physical examination; (4) indicated laboratory procedures including nasal smears; (5) skin testing. Skin testing is necessary in order to help determine the specific etiologic allergens. The allergens tested with should include the common foods and the common inhalants, pollens and molds in the environment. Eosinophiles and Eosinophilia Eosinophilia occurs in a great many conditions other than allergy and these must be taken into account in interpreting differential blood counts. The nasal smear is a useful laboratory test in the diagnosis of nasal allergy. Eosinophiles may temporarily appear in considerable numbers in the nasal smear of the nonallergic child during recovery from a respiratory infection, but otherwise a predominance of eosinophiles in the nasal smear is diagnostic of nasal allergy. Office Treatment of Respiratory Allergy The office treatment of a child with respiratory allergy includes the following: (1) general pediatric care; (2) symptomatic relief of allergic symptoms; (3) removal of incriminated foods from the diet; (4) as complete an elimination of environmental allergens as possible; (5) specific desensitization. Specific Desensitization Dr. Ratner: The method of specific desensitization which we use in practice is as follows: The treatment extract consists of a 1:5,000 dilution of each of the inhalants, pollens and molds with which the patient gave a positive reaction. Each week the patient receives one injection from a tuberculin syringe containing 0.15 cc. of this treatment extract, 0.15 cc. of a 1:20 dilution of mixed bacterial vaccine, 0.15 cc. of a 1:20 dilution of pertussis vaccine, and 2 minims of adrenaline.

A Study of Influenza Virus Vaccines by a Serum-Virus Neutralization Test and by Active Immunization

Summary It required approximately 63 to 125 times more influenza virus vaccine to immunize mice actively against infection caused by intranasal instillation of influenza viruses, PR 8 and Weiss (type A), than it did to stimulate circulating antibodies which neutralized the same quantity of virus, the tests being carried out in identical mice and at the same time. For the Lee strain (type B), five times more vaccine was necessary to produce active immunity comparable to the protection afforded to mice against influenza virus neutralized by immune serum elicited by a small amount of vaccine. The dilution of vaccine which actively immunized mice against influenza virus stimulated the production of immune serum of higher titer than did the dilutions of vaccine which did not actively protect the mice. A measurement of the smallest amount of vaccine which would produce immune serum in mice capable of protecting other mice against approximately 103LD50 doses of virus was utilized to determine the potency of influenza virus vaccines.

Anti-Typhoid Vaccine

A MEMOIR “On the Standardisation of Anti-typhoid Vaccine,” by Captain George Lamb and Captain W. B. C. Forster, has just appeared (Scientific Memoirs of the Government of India, No. 21. Calcutta: The Government Printing Office, 1906. Pp. 15. Price 7d.). After reviewing the various methods which have been proposed for the standardisation of Wright's anti-typhoid vaccine, Captains Lamb and Forster come to the conclusion that the virulence of the organisms used in the preparation of the vaccines must be taken into account. Since it appears that virulence is in direct proportion to the number, or avidity for immune body, of the receptors, an estimation of these latter in any vaccine will take cognisance of the virulence of the organism from which it was prepared. Admitting this as a basis, the method of standardisation suggested by Captains Lamb and Forster is to estimate what dilution of the various vaccines when mixed in equal parts with serum is able to remove completely the bactericidal power of that serum; in other words, to determine in what dilution of vaccine the receptors completely neutralise the amboceptor content of the serum. This is carried out by preparing a number of different dilutions of the vaccine, which are each mixed with the same amount (100 c.cm.) of fresh goat serum, and left in contact for an hour at 37° C. At the end of this period a small quantum of living typhoid culture is added to each tube, the several tubes are incubated for about twenty-four hours, and then sterile broth is added to each tube in order to ascertain whether the bacilli have been killed or no, and in this way various vaccines may be compared. The memoir must be consulted for the details of the method.