Dilution Of Vaccine Research Articles

Evaluation of the sensitivity of PCR methods for the detection of extraneous agents and comparison with in vivo testing

In this study the sensitivity of polymerase chain reaction (PCR) methods for the detection of Newcastle disease virus (NDV), avian reovirus (ARV), avian influenza virus (AIV) and avian infectious bronchitis virus (IBV) was compared to the sensitivity of the corresponding serological tests described in the European Pharmacopoeia (Ph. Eur.). For this purpose, serial 10-fold dilutions of the respective inactivated vaccines were prepared and groups of SPF chickens were vaccinated with a double dose of the vaccine dilutions. After a period of 21 days, the animals were revaccinated with a single dose. Two weeks later, serum samples from each animal were tested for antibodies using an Idexx enzyme linked immunosorbent assay (ELISA). In parallel, samples of the diluted vaccines were tested by PCR. It was found that the sensitivity of the four PCR tests is comparable to or even slightly better than that of the corresponding serological tests. Thus these PCR tests fulfil the sensitivity requirements of the Ph. Eur. and could be used as alternative tests for the detection of extraneous agents in final batches of inactivated vaccines.

Scientific importance of Serbian Pasteur institutes

Only 12 years after Paris, the first Pasteur Institute in Serbia and the Balkans was founded in Nis, in 1900. Its contribution to preventive medicine of Serbia was enormous, primarily in production of vaccine against variola and rabies. After the liberation in 1919, the Pasteur Institute was re-established and it continued its scientific work under the management of Gerasim Alivizatos, a Greek who had come to help Serbian people. He is the author of the so-called mixed method in rabies prophylaxis, in which he combined dilution of live vaccine with concentrated ether treated vaccine. He published his work on this method in the journal Deutschen Medicinische Wochenschrift in 1922. After 1928, the only active Pasteur Institute in the whole Kingdom of Serbs, Croats and Slovenes was the Pasteur Institute in Novi Sad, which has remained the central anti-rabies institution to the present day. Its first director, Dr. Adolf Hempt, born in Novi Sad in 1874, was the author of the world famous vaccine against rabies, which was named after him and was in use from 1925 to 1989. He was the first in the world to have made a completely inactive, i.e. dead vaccine, which was much safer for use, so many European countries such as Austria, Czechoslovakia, Germany, Hungary gradually accepted it under the name of Hempt, and from the Novi Sad Institute it was exported to some African countries as well. Dr Hempt published his first work on the new vaccine in the magazine Annales de l'Institut Pasteur in 1925 in French, and he also published a monograph in German by Bering Institute in 1938, nowadays ranked as a monograph of international importance.

English

  Oral polio vaccine (OPV) proved to be superior in administration eliminating the need of sterile syringes and making the vaccine more suitable for mass vaccination campaigns. Poliovirus is heat sensitive in nature, and thus OPV is stored at low temperature (frozen). The growth medium containing varying concentration of serum such as 6, 8, 10, 12, 14% were prepared. 10 ml of the above mentioned growth media containing different concentration of serum were added to different culture bottles. The culture flasks containing different volumes of growth medium with 10% serum concentration such as 8, 9, 10, 11 and 12 ml were added to a series of culture flasks. All the culture flasks were inoculated with the RD cells (10,000 cells/culture flask) and kept at 37°C. The most favoured serum concentration and volume for the growth of RD cells was found and used for testing the potency of vaccine. Vaccines from two manufacturers were kept at three different temperatures, 2-8 ± 0.5°C (refrigerator), 26 ± 0.5°C and 37 ± 0.5°C (Incubator). Cytopathic viruses were titrated by the determination of a tissue culture infectious dose50 (TCID50), vaccine dilutions were seeded in replicate onto cells in multiwell plates (usually 96 wells). After a suitable incubation period, wells were examined microscopically and scored as infected or not infected. The potency of vaccines was tested using the Karber’s Formula.   Key words: Oral polio vaccine, rhabdomyosarcoma, thermostability, potency.

Potency evaluation of rabies vaccine for human use: The impact of the reduction in the number of animals per dilution

In order to evaluate the effects of reducing the number of animals used in the NIH mouse protection test for potency determination of inactivated rabies vaccines for human use, a retrospective study of the results obtained in the Brazilian National Control Laboratory, Instituto Nacional de Controle de Qualidade em Saúde (INCQS), was performed, comprising 214 vaccine lots. The INCQS Standard Operating Procedure establishes the use of three vaccine dilutions and 18 animals per dilution, separated into two cages with 9 mice each. The results of the two cages of each dilution were considered as two different groups (C1 and C2), and therefore, for each vaccine lot, three results were obtained: one for the standard test (ST) with 18 mice, one using the C1 cages with 9 mice and another using the C2 cages with 9 mice. The results were evaluated as repeated measures of the same method on the same samples. In this study, the effects of the reduction in: (a) the measurement error and its association with the size of measurement, (b) the agreement between the results using the concordance coefficient of correlation, (c) the agreement of categorized results as “Pass” or “Fail” using the Kappa index, (d) the precision of potency determinations using the 95% confidence interval and (e) the incidence of statistically invalid assays due to non-linearity and non-parallelism were evaluated. It was concluded that the results from the NIH mouse protection test using 9 mice per dilution are in good agreement with the results obtained using 18 mice per dilution. Therefore, nine animals per dilution is a suitable number to meet the statistical requirement for valid assays.

Stabilization of Live Mycoplasma gallisepticum Vaccines During Vaccination with Second-Generation Spray-Vac Vaccine Stabilizer

ABSTRACT Dilution and application of live Mycoplasma gallisepticum vaccines without the use of vaccine-stabilizing compounds may lead to significant loss of vaccine viability and loss of vaccine efficacy. Vaccine viability may decrease because of osmotic lysis of the mycoplasma as well as the presence of free chlorine or other detrimental chemicals in the water. Second-generation Spray-Vac vaccine stabilizer was developed and shown to maintain live Mycoplasma gallisepticum vaccine viability during exposure to free chlorine while protecting from the other factors that appear to decrease vaccine survival in solution. Increased vaccine survival in solution should lead to increased survival of the vaccine during vaccination. Field trial results demonstrate that second-generation Spray-Vac vaccine stabilizer yields excellent results without the need for distilled water or other vaccine-stabilizing compounds.

Correlation of Clostridium botulinum type C antitoxin titers in mink and guinea pigs to protection against type C intoxication in mink

In USA, the potency of commercial vaccines containing Clostridium botulinum type C toxoid is determined by a mink vaccination-challenge assay outlined in the Code of Federal Regulations, Title 9, Part 113.110. A more humane potency test is desired, and this study provides preliminary data in support of a serological assay that correlates post-vaccination antitoxin titers of guinea pigs to vaccine efficacy in mink. Mink and guinea pigs were injected with varying dilutions of a vaccine containing C. botulinum type C toxoid. Blood samples were collected from each animal prior to challenging the mink with type C toxin. Serum antitoxin titers of mink and guinea pigs were measured by a mouse protection test, and the results were compared to the outcome of the toxin challenge in mink. A dose-dependent antitoxin response was observed in guinea pigs vaccinated with the critical dilutions of vaccine bracketing the minimum protective dose in mink. These preliminary data suggest that it may be possible to correlate post-vaccination antitoxin titers in guinea pigs to vaccine efficacy in mink. This correlation could be used as the basis for a more humane potency test for C. botulinum type C toxoids.

Antibody Responses to Vaccinia Membrane Proteins after Smallpox Vaccination

Vaccinia virus (VV) membrane proteins are candidates for orthopoxvirus subunit vaccines and potential targets for therapeutic antibodies. Human antibody responses to these proteins after VV vaccination have not been well characterized. Pre- and postvaccination (day 26-30) serum specimens from 80 VV vaccine recipients were examined for immunoglobulin G antibodies specific for B5, A33, A27, and L1 by enzyme-linked immunosorbent assay (ELISA). Responses were compared between vaccinia-naive and previously vaccinated (nonnaive) recipients and between nonnaive recipients of undiluted or 1 : 10 diluted vaccine. VV vaccination elicited anti-A33 and anti-A27 antibodies in nearly all vaccinia-naive subjects (100% and 93%, respectively). Preexisting antibodies were commonly detected in nonnaive subjects (for anti-B5, 68%; for anti-A33, 59%; for anti-A27, 38%; and for anti-L1, 10%). Anti-B5 antibodies were strongly boosted by undiluted vaccine (geometric mean titer [GMT], 151 vs. 1010 for pre- vs. postvaccination; P<.001), whereas anti-L1 antibody responses were less robust (detection rate, 31%; GMT, 75) in nonnaive subjects. Diluted vaccine elicited antibody responses that were similar to those elicited by undiluted vaccine. Vaccination with VV elicits long-lived specific antibody responses directed against VV membrane proteins that vary by previous vaccination status but not with respect to 10-fold dilution of vaccine. B5, A33, and A27 should be considered for inclusion in future human orthopoxvirus subunit vaccines.

Cell-Mediated Immune Responses to Smallpox Vaccination

We report that vaccine dilution (1:1 or 1:10) and previous vaccinia virus vaccination status had no significant effect on cell-mediated immune responses (i.e., the immediate vaccinia virus-specific gamma interferon-producing T-cell response measured by enzyme-linked immunospot assay) 1 month after smallpox vaccination (Lancy-Vaxina; Berna Biotech, Switzerland).

Clinical Responses to Smallpox Vaccine in Vaccinia‐Naive and Previously Vaccinated Populations: Undiluted and Diluted Lancy‐Vaxina Vaccine in a Single‐Blind, Randomized, Prospective Trial

We conducted a single-blind, randomized trial of 2 dilutions (1:1 or 1:10) of Lancy-Vaxina vaccine (Berna Biotech) in vaccinia-naive persons (n=36) and persons previously vaccinated >25 years ago (n=76). All vaccinees responded successfully to the vaccination. There were no significant differences in the size of the skin lesions, the number of adverse events, the amount of viral shedding, or the level of antibody responses between the undiluted (n=56) and diluted (n = 56) vaccine groups. Compared with vaccinia-naive persons, previously vaccinated persons exhibited significantly smaller and more rapidly evolving skin lesions and fewer adverse events. Previously vaccinated persons had significantly higher neutralizing antibody levels before the administration of the study vaccine than vaccinia-naive persons, and viral shedding from lesions in previously vaccinated persons was lower and diminished more rapidly than from lesions in vaccinia-naive persons.

Smallpox Vaccination Does Not Elevate Systemic Levels of Prothrombotic Proteins Associated with Ischemic Cardiac Events

During the recent smallpox vaccination campaigns, ischemic cardiac complications were observed after vaccination. To examine a possible association between the smallpox vaccine and postvaccination ischemic events, we investigated alterations in levels of prothrombotic proteins (plasminogen activator inhibitor type 1 [PAI-1] and soluble CD40 ligand [sCD40L]) in recently vaccinated individuals. Vaccinia-naive (cohort N; aged 18-32 years) and vaccinia-experienced (cohort E; aged 33-49 years) healthy adults were vaccinated with a 1 : 5 dilution of the Aventis Pasteur smallpox vaccine. Plasma levels of PAI-1 and sCD40L were measured in 30 subjects (cohort N, n=15; cohort E, n=15) at baseline and twice after vaccination (between days 7 and 9 and between days 26 and 30). Baseline mean PAI-1 levels significantly differed between cohorts N and E (P=.04). Within each exposure cohort, mean PAI-1 levels did not significantly change after vaccination. Baseline sCD40L levels did not differ between cohorts N and E. In cohort N, sCD40L levels significantly decreased after vaccination but returned to baseline levels within 1 month. Vaccination did not significantly alter levels of sCD40L in cohort E. Levels of PAI-1 and sCD40L did not significantly increase after smallpox vaccination. Vaccine-induced alterations in levels of these prothrombotic proteins do not appear to play a role in ischemic events observed after smallpox vaccination.

Reduction of Numbers of Animals Used in the Quality Control of Biologicals

Laboratory animals are used for the quality control of vaccines. In particular, the potency testing of batches of inactivated vaccine requires large numbers of animals. The possibilities for reduction have been evaluated, and the results are summarised in this paper. Several approaches were studied, including the retrospective analysis of test data, with the objectives of determining the minimum number of animals required per vaccine dilution group, and evaluating the feasibility of a single-dose potency test. Other studies focused on the development of serology-based models and the use of genetically uniform animals. Based on the outcome of these studies, a substantial reduction in the number of animals used for the potency testing of toxoid vaccines has been achieved or will be achieved in the near future. As reduction alternatives can generally be explored in a relatively simpler and less time-consuming way than replacement alternatives, more emphasis should be placed on reduction strategies than at present.

Rabies cell culture vaccines reconstituted and stored at 4 °C for 1 year prior to use protect mice against rabies virus

Human exposure to rabid dogs in developing countries is an ongoing problem that continues to demand effective, safe, and affordable post-exposure rabies vaccinations. Sheep and suckling mouse brain rabies vaccines used in developing countries are being replaced by expensive inactivated-virus cell culture vaccines. Human studies using cell culture vaccines have determined that cost is reduced and protection is maintained by injecting the unused portion of vaccines that have been reconstituted and stored refrigerated for 1 week. Here we determined whether reconstituted purified chick embryo cell and human diploid cell vaccine that had been stored at 4 °C for intervals up to 1 year elicit neutralizing antibody, and protect mice against rabies virus. Undiluted, or 1:5 and 1:25 dilutions of both vaccines injected immediately after reconstitution, or after reconstitution and storage at 4 °C for 1 week, 1 month, 3 months, 6 months or 1 year elicited high levels of neutralizing antibody and protected 100% of the mice injected with rabies virus.

Dose‐Dependent Neutralizing‐Antibody Responses to Vaccinia

To evaluate the humoral immune responses to smallpox-vaccine stocks currently available in the United States (Dryvax; Wyeth) and to generate data for comparison of responses to newly produced lots of smallpox vaccine, we evaluated dose-response effects, using undiluted and diluted smallpox vaccine. At 28 and 56 days after vaccination, serum samples were obtained from vaccinated subjects (N=674) who had participated in a randomized, single-blinded trial of an undiluted or a 1 : 5 or 1 : 10 dilution of smallpox vaccine and who subsequently were tested for plaque-reduction neutralizing-antibody titer. All subjects who developed a vesicle after vaccination also developed neutralizing antibodies by day 28. Subjects given either a 1 : 5 or 1 : 10 dilution of vaccinia had significantly higher neutralizing-antibody titers than did subjects given undiluted vaccine. Larger lesion size and fever after vaccination were associated with significantly higher neutralizing-antibody titers after vaccination.

A practical approach to immunization in atopic children.

A practical approach to immunization in atopic children.

Vaccination of Turkeys with an Avian Pneumovirus Isolate from the United States

Four-week-old poults obtained from avian pneumovirus (APV) antibody-free parents were vaccinated with different serial 10-fold dilutions of cell culture-propagated APV vaccine. The birds were vaccinated with 50 microl into each conjunctival space and nostril (total of 200 microl). Each poult of each group was vaccinated in groups that received doses of 4 x 10(4), 4 x 10(3), 4 x 10(2), 4 x 10(1), or 4 x 10(0) 50% tissue culture infective dose (TCID50) of APV vaccine, respectively. Respiratory signs were seen between 3 and 12 days postvaccination (PV) in the poults that were vaccinated with 4 x 10(4), 4 x 10(3), and 4 x 10(2) TCID50, respectively. In these groups, APV was detected from swabs collected at 5 days PV and seroconversion was detected at 2 wk PV. The groups that were originally vaccinated with 4 x 10(1) and 4 x 10(0) TCID50 developed mild clinical signs after vaccination, but neither virus nor antibody was detected PV. At 2 wk PV (6 wk of age), birds from each group, along with five unvaccinated controls, were challenged with APV. Upon challenge, the 4 x 10(4) and 4 x 10(3) TCID50 groups were protected against development of clinical signs and were resistant to reinfection. The group previously vaccinated with 4 x 10(2) TCID50 developed clinical signs after challenge that were considerably milder than those seen in the groups that had previously been vaccinated with lower doses or no virus. Even though 4 x 10(2) TCID50 vaccine dose administered by intranasal ocular route resulted in infection, incomplete protection resulted with this pivotal dose. Upon challenge, the 4 x 10(1) and 4 x 10(0) TCID50 groups exhibited milder disease signs than those seen in the challenged unvaccinated controls. In these groups, APV was detected in preparations of swabs collected at 5 days postchallenge (PC) and seroconversion was detected at 2 wk PC. These results indicate that the dose of APV vaccine that causes protection is higher than that required to produce infection.

In vitro correlate of immunity in a rabbit model of inhalational anthrax

A serological correlate of vaccine-induced immunity was identified in the rabbit model of inhalational anthrax. Animals were inoculated intramuscularly at 0 and 4 weeks with varying doses of Anthrax Vaccine Adsorbed (AVA) ranging from a human dose to a 1:256 dilution in phosphate-buffered saline (PBS). At 6 and 10 weeks, both the quantitative anti-protective antigen (PA) IgG ELISA and the toxin-neutralizing antibody (TNA) assays were used to measure antibody levels to PA. Rabbits were aerosol-challenged at 10 weeks with a lethal dose (84–133 LD 50) of Bacillus anthracis spores. All the rabbits that received the undiluted and 1:4 dilution of vaccine survived, whereas those receiving the higher dilutions of vaccine (1:16, 1:64 and 1:256) had deaths in their groups. Results showed that antibody levels to PA at both 6 and 10 weeks were significant ( P<0.0001) predictors of survival.

In vitro correlate of immunity in an animal model of inhalational anthrax

The incidence of anthrax in humans is extremely low. Human vaccine efficacy studies for inhalational anthrax cannot be conducted. The identification of a correlate of protection that predicts vaccine efficacy is crucial for determining the immune status of immunized humans. This surrogate marker of immunity can only be established by using an appropriate animal model. Numerous studies showed that protective antigen (PA) is the principle protective antigen in naturally- or vaccine-induced immunity. However, attempts to correlate the quantity of anti-PA antibodies with protective immunity in the guinea pig model for anthrax and various vaccine formulations have failed. In these studies, we used the licensed anthrax vaccine adsorbed (AVA) in rabbits. Groups of New Zealand white rabbits, 10 or 20 per group, were immunized intramuscularly (two doses, 4 weeks apart) with varying doses of AVA, ranging from a human dose to 1:256 dilution in sterile phosphate-buffered saline (PBS). Control rabbits received PBS/Alhydrogel according to the same schedule. Each rabbit was bled 2 weeks after the second dose, and antibody levels to PA measured by both the quantitative anti-PA IgG ELISA and the toxin-neutralizing antibody (TNA) assay. Rabbits were aerosol-challenged 10 weeks from day 0 with a lethal dose of Ames spores. All the rabbits that received the undiluted and 1:4 dilution of vaccine survived, whereas those receiving the higher dilutions of vaccine (1:16, 1:64 and 1:256) had deaths in their groups. All the controls died. Rabbit survival was compared with the antibody response. Statistical models were used to test for significance of the peak antibody responses to predict survival. Results showed that both the amount of anti-PA IgG and TNA titres present in the sera at the time of the peak antibody response were significant (P < 0.0001) predictors of survival. These results demonstrate that the humoral immune response to AVA can predict protection in the rabbit model of inhalational anthrax.

Observations on the Effect of the Diluent Used for Diluting Challenge Toxin in the Clostridium botulinumPotency Test

Clostridium botulinumvaccines were assayed for potency according to the method described in the European Pharmacopoeia. This method uses 20 white mice which are vaccinated with a dilution of vaccine in 0·9% saline. Twenty-one days later the vaccinated mice and ten unvaccinated control mice are challenged with C. botulinumtoxin and observed for 7 days. In this study the challenge toxin was diluted in two separate diluents, 0·9% saline and peptone broth. More reproducible results were achieved with peptone broth as the diluent.

Peste suína clássica: ação térmica da amostra chinesa Porto Alegre (CPA) em coelhos

Foram avaliadas a temperatura normal do coelho e sua reação térmica após inoculação com a amostra Chinesa Porto Alegre (CPA) do vírus da Peste Suína Clássica (PSC), sob três condições de temperatura ambiental. Os resultados mostraram que a temperatura normal do coelho aumenta com a elevação da temperatura ambiental (P&lt;0,001). As médias das temperaturas normais dos coelhos foram de 39,53°C; 39,65°C e 39,92°C, nas temperaturas ambientais de 12,82 ± 1,29°C; 20,65 ± 1,79°C e 28,24 ± 2,09°C respectivamente. Os coelhos inoculados tiveram seu pique térmico também aumentado com a elevação da temperatura ambiental (P&lt;0,001), mas não sofreram influência da diluição do vírus. As médias dos piques térmicos observados foram de 41,17°C; 41,24°C e 41,47°C. Para um intervalo de confiança de 95%, estabeleceram-se as temperaturas de 40,60°C; 40,67°C e 40,90°C como limites, a partir dos quais a reação térmica foi considerada específica, em cada uma das temperaturas ambientais utilizadas. A elevação térmica não sofreu influência da temperatura ambiental nem da diluição do vírus, apresentando uma média global de 1,59°C. Para um intervalo de confiança a nível de 95%, estabeleceu-se o limite de 1,06°C como o mínimo de elevação térmica para uma reação específica. O pique térmico iniciou a partir de 24h após inoculação. Seu início foi retardado com o aumento da diluição do vírus (P &lt; 0,001), mas não com a temperatura ambiental. A sensibilidade e especificidade da reação térmica foi avaliada pelo teste de desafio. A análise de regressão permitiu concluir que um pique térmico mínimo de 40,6°C, associado a uma elevação térmica mínima de 1,06°C, pode ser considerada reação específica dos coelhos inoculados com a amostra CPA.

Modulation of antiviral immune responses by exogenous cytokines: effects of tumour necrosis factor- , interleukin-1 , interleukin-2 and interferon- on the immunogenicity of an inactivated rabies vaccine

In vivo administration of exogenous cytokines may influence elicited immune responses, and hence may change the efficacy of a vaccine. We investigated the effects of tumour necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) on the immune response elicited by inactivated rabies virus vaccine in a mouse model. Each of the cytokines increased virus-specific IgG responses after primary and after secondary immunization. A single dose of 1.3 ng TNF-alpha or IL-1 alpha, when injected shortly before vaccination, only marginally stimulated resistance to challenge infection (four- and seven-fold, respectively) without enhancing virus neutralizing antibody (VNAb) responses. In contrast, a single injection of 10(3) units of IFN-gamma or five daily injections of 1.6 micrograms IL-2 increased vaccine dilutions protecting 50% of mice (PD50 values) 77- to 50-fold, respectively, with a concomitant enhancement of VNAb. At a 1:10,000 dilution of a standard inactivated rabies vaccine preparation both IFN-gamma and IL-2 increased protective immunity without enhancing VNAb responses; in non-vaccinated animals this treatment had no effect on resistance to challenge. Combined administration of IFN-gamma and IL-2 synergistically enhanced VNAb responses. In contrast to the other cytokines tested, IFN-gamma preferentially stimulated virus-specific IgG2a production. It also augmented the vaccine-induced priming of rabies virus-specific splenocyte proliferation. These results document that certain cytokines alone or in combination are potent immunological adjuvants which may direct and modulate immunization-induced antiviral immune responses.