Mechanisms of protective immunity against Aujeszky's disease virus

An understanding of the role that different types of immune effector mechanisms have in mediating protective immunity against virus diseases of swine is key to the development of effective vaccines for this species. The abundant literature available on the efficacy of vaccines against Aujeszky's disease virus (ADV) has provided evidence that inactivated ADV vaccines are not as effective as modified live virus (MLV) vaccines at stimulating protective immunity. The fact that similar titers of virus-neutralizing antibodies are present in pigs receiving either type of vaccine has fueled the speculation that cell-mediated immunity must therefore be responsible for mediating protection from disease. Certainly, the lesser ability of inactivated ADV vaccines to provide protective immunity must reflect the inability of this type of vaccine to induce sufficient levels of immune effector mechanism(s) important for protection. However, the exact nature of this deficit is unknown. We have examined this issue by measuring the serum titer of virus-neutralizing antibodies and the frequency of ADV-specific interferon-gamma (IFN-$\gamma$)-producing cells in the peripheral blood mononuclear cells of pigs after immunization with either inactivated or MLV ADV vaccines. We found that while both MLV and inactivated vaccines are able to induce similar levels of neutralizing antibodies, an inactivated vaccine is not as effective as a MLV vaccine at stimulating ADV-specific IFN-$\gamma$-producing cells. Indeed we found a correlation between the intensity of this response and the level of protective immunity. This correlation was further confirmed by the observation that pigs immunized with inactivated virus in combination with either human recombinant IL-12 or an oil-in-water adjuvant, develop an enhanced IFN-$\gamma$ response and level of protective immunity, as compared to pigs receiving the inactivated virus alone. In contrast, the titer of virus neutralizing antibodies produced in response to the inactivated vaccine was mildly affected by either of these adjuvants. The data obtained clearly show that there are differences in the quality and quantity of the immunity induced by a live versus an inactivated ADV vaccine. While inactivated commercial vaccines are equally efficient as a MLV vaccine at inducing humoral immunity, they only induce a weak and transient virus-specific IFN-$\gamma$ response. In contrast, a MLV vaccine induces a robust virus-specific IFN-$\gamma$ response. In all of the different ADV vaccine formulations tested in our experiments, a high level of protective immunity correlated with the presence of a strong IFN-$\gamma$ response, while the titer of virus-neutralizing antibodies did not. Although the results from this study do not rule out a role for humoral immunity in protection, they do suggest that cell-mediated immunity participate in providing a high degree of protective immunity. The data presented argue in favor of the notion that the level of cell-mediated immunity is a major factor in determining the level of protection from ADV-induced disease. At the very minimum, a strong IFN-$\gamma$ response in a pig is a good predictor that the animal has developed a strong protective immune response against this virus

Characteristics of the immune response of pigs to PRRS virus

Several studies were conducted to evaluate the characteristics of the immunity induced by infection with wild type PRRS virus or vaccination with a PRRS modified live virus (MLV) vaccine. By monitoring the kinetics of the immune response to virus we made several remarkable observations. As a result of either infection with wild type PRRS virus or vaccination with a PRRS MLV vaccine, an antibody response was readily detectable by ELISA within 2 weeks after the administration of virus. A striking observation resulting from this research is that the generation of immunity able to mediate virus-inhibitory function, i.e., virus neutralizing antibodies and interferon gamma (IFN-$\gamma$)-producing T cells, becomes detectable only several weeks after exposure of pigs to either wild type virus or a MLV vaccine. The presence of virus-specific IFN-$\gamma$-producing cells was not frankly detectable until 8 to 10 weeks after virus administration. However, even then the frequency was fairly low as compared to the frequency of virus-specific cells induced by an Aujeszky's disease virus (ADV) MLV vaccine. In animals inoculated with the wild-type PRRS virus, the frequency of PRRS virus-specific IFN-$\gamma$-producing cells increased gradually over time so that by 9-10 months after infection it was comparable to the frequency induced by the administration of an ADV MLV vaccine within 2-3 weeks. By 12 months after infection with wild-type PRRS virus the titer of antibodies against this virus declines significantly, becoming even undetectable by ELISA in at least 30% of the pigs. In contrast, the intensity and quality of the virus-specific IFN-$\gamma$ response increases in both quality and quantity. Of note was the observation that the IFN-$\gamma$ ELISPOT remained at the same level in all animals infected with the wild-type virus from 300 to 690 days post infection (latest time tested). In contrast, during this time the presence of anti-PRRS virus antibodies in the serum of these animals decreased, so that at 690 days after infection 80% of the animals became negative by PRRSV IDEXX ELISA test. This is despite the fact that the frequency of IFN-$\gamma$-producing cells remained at the same level as that of the measurement at 300 days post infection. Although the reason for the delayed PRRS virus-inhibitory immune response is unclear at this time, we have evidence indicating that accessory cell-derived cytokines such as IL-12 and IL-10 are able to enhance and suppress, respectively, the intensity of the virus-specific cellular immune response at least in vitro. The complexity of the regulation of the immune response of pigs to PRRS virus is clearly indicated by the concomitant gradual decline of humoral immunity while the cell-mediated immunity increases. The rational development of effective PRRS virus vaccines requires an understanding of the mechanisms that regulate the kinetics, quality and intensity of the humoral and cellular immunity against this virus. Given the economic losses attributed to this disease this information is urgently needed. Based on these observations, it seems reasonable to postulate that an intrinsic property of PRRS virus is responsible for the inefficient stimulation of potentially protective immunity at least in the short term, namely, virus-neutralizing antibodies and IFN-$\gamma$-producing T cells. Although the reason(s) behind the poor immunogenicity of PRRS virus are unknown, we are currently examining several possibilities. We believe that clarification of this issue is essential for the development and formulation of a highly immunogenic and effective PRRS vaccine

Effects of cytokines upon the immune response of swine to Aujeszky's disease virus

Aujeszky's disease is caused by suid herpesvirus 1 (SHV-1) a member of the alphaherpesvirinae. This large and complex virus has been the subject of many studies designed to identify successful vaccine formulations and many varied and efficacious vaccines have been described in the literature. SHV-1 infection can manifest as a broad range of clinical signs ranging from depression to death, and in many instances both the general health and immune status of the animal upon challenge determine the outcome. Interestingly, challenge with virulent SHV-1 can have significant effects upon the growth performance of pigs, apparent as a significant reduction in the accretion of body mass or even cachexia and emaciation. The change in weight seven days after challenge ($\Delta$G7) has been shown to be a quantitative indicator of the magnitude of protection induced by vaccination. Several investigators have used the $\Delta$G7 weight change to define a spectrum of vaccine ability to protect swine from infection. Many of the studies have detailed that modified live virus vaccines (MLV) offer the best protection, with some of the most successful ones preventing animals from showing any decrease in the rate of gain during the first seven days of infection. In contrast, many of the killed virus vaccines and sub-unit vaccines (live vectored or inactivated), while engendering a protective immune response, fail to prevent the $\Delta$G7 day weight loss as effectively as some MLV. This model of weight loss due to infection offers a unique model that is able to quantitatively differentiate the performance of vaccines and their delivery vehicles (live vectors, adjuvants, etc). We have recently started to use this model to investigate the role of cytokine therapy upon the types of immune response generated during vaccination. Primarily using RT-PCR strategies we have obtained cDNAs encoding the open reading frames of several porcine cytokines. Experiments using genetic immunization approaches had identified this technology as a viable delivery system for functional cDNAs. Consequently, we decided to investigate this strategy for both vaccination and co-administration of a cytokine cDNA. The model system we are using utilizes an immunization vector that expresses the envelope glycoprotein gC of SHV-1. In addition several genetic immunization vectors have been constructed to express the porcine cytokine cDNAs for IL-1b, IL-2, IL-4, IL-5, IL-6, IFN-$\gamma$ or GM-CSF. Some of these vectors express the cytokine alone, while others express them in a fusion with a truncated version of gC. All of these construct are currently being evaluated in vaccination trials of swine, using both in vitro analysis of immune responsiveness and challenge trials to determine the effects of these cytokines upon the porcine immune system. Groups of SHV-1 free piglets were immunized with purified plasmids encoding the cDNA for gC and/or a cDNA for a porcine cytokine. The developing SHV-1 specific immune response was monitored by in vitro assays measuring humoral response (ELISA and serum neutralization assays) and cell mediated immunity (IFN-$\gamma$ ELISPOT and lymphoproliferation assays). Four to six weeks after vaccination the animals were weighed and challenged intranasally with virulent SHV-1. Surviving animals were weighed again seven days later. All animals vaccinated with plasmids encoding the gC protein developed a specific immune response against SHV-1 as measured with the in vitro assays. The magnitude of these responses were not as great as those induced by commercial vaccines (killed or MLV) and consequently the degree of protection against $\Delta$G7 weight changes was not as great. Co-administration of cytokines intended to bias the immune response towards a TH-1 (increase in IFN-$\gamma$-secreting antigen-specific cells) or TH-2 (decrease in IFN-$\gamma$-secreting antigen-specific cells) did not have the expected effect. While several studies have shown that IFN-$\gamma$ can have small enhancing effects upon the IFN-$\gamma$ secreting antigen specific response, the inhibitory effect of IL-10 co-administration was not seen. Surprisingly, despite the inhibitory effect of porcine IL-10 on in vitro mitogen stimulated IFN-$\gamma$ secreting cells (unpublished data), co-administration of the plasmid encoding IL-10 had enhancing effects upon the induction the IFN-$\gamma$ secreting SHV-1 specific cell response in vivo. A precise interpretation of this effect after challenge was further complicated by the modulating effect of the cytokine plasmids alone upon the $\Delta$G7 weight change. These unexpected effects of cytokine co-administration are being further evaluated using both genetic immunization and recombinant SHV-1 vectors. In conclusion, the data collected to date has demonstrated that cytokines delivered with genetic immunization can have an effect upon the developing immune response, and that the characteristic TH-1 and TH-2 biases described in other species may not function similarly in swine

Avaliação microbiológica, histológica e imunológica de frangos de corte desafiados com Salmonella Enteritidis e Minnesota e tratados com ácidos orgânicos

Dois experimentos foram desenvolvidos para avaliar a eficiência de ácidos orgânicos frente a Salmonella enterica enterica sorovar Enteritidis (SE) e Minnesota (SM) em frangos. No primeiro experimento foram avaliados 3 tratamentos: T1 - ração adicionada de ácido orgânico, T2 - ração adicionada de ácido orgânico e ácido orgânico na água de bebida, T3 - grupo controle. Todos os animais foram inoculados com SE, via oral. A utilização de ácidos orgânicos na ração (T1) e na ração e na água (T2) diminuíram a excreção de Salmonella no papo e no ceco 7 dias pós inoculação com SE e houve redução de células CD3+ no jejuno dos frangos. No segundo experimento foram avaliados 4 tratamentos sendo T1 - controle, T2 - controle inoculado via oral com Salmonella Minnesota (SM), T3 - animais inoculados via oral com SM e ácidos orgânicos na ração e T4 - animais inoculados via oral com SM e ácidos orgânicos na ração e na água de bebida. Ácidos orgânicos a ração (T3) e na ração e na água (T4) reduziram a excreção de SM em papo de frangos de corte desafiados, 7 dias após inoculação. O uso de ácidos orgânicos na ração e na ração e na água foram mais eficientes em reduzir SE do que SM

Field evaluation of safety during gestation and horizontal spread of a recombinant differential bovine herpesvirus 1 (BoHV-1) vaccine Avaliação a campo da segurança para vacas prenhes e capacidade de disseminação horizontal de uma vacina diferencial recombinante contra o Herpes-vírus Bovino tipo 1 (BoHV-1)

Bovine herpesvirus type 1 (BoHV-1) is recognized as a major cause of respiratory, reproductive disease and abortion in cattle. Vaccination is widely applied to minimize losses induced by BoHV-1 infections; however, vaccination of dams during pregnancy with modified live virus (MLV) vaccines has been occasionally associated to abortions. We have previously reported the development of a BoHV-1 recombinant virus, constructed with basis on a Brazilian BoHV-1 (Franco et al. 2002a) from which the gene coding for glycoprotein E (gE) was deleted (gE-) by genetic manipulation. Such recombinant has been previously evaluated in its potential as a differential vaccine (gE- vaccine) that allows differentiation between vaccinated and infected animals. Here, in the first part of the present study, the safety of the gE- vaccine during pregnancy was evaluated by the intramuscular inoculation of 10(7.4) tissue culture 50 % infective doses (TCID50) of the virus into 22 pregnant dams (14 BoHV-1 seronegative; 8 seropositive), at different stages of gestation. Other 15 pregnant dams were kept as non-vaccinated controls. No abortions, stillbirths or fetal abnormalities were seen after vaccination. Seroconversion was observed in both groups of previously seronegative vaccinated animals. In the second part of the study, the potential of the gE- vaccine virus to spread among beef cattle under field conditions was examined. Four heifers were inoculated intranasally with a larger amount (10(7,6) TCID50) of the gE- vaccine (to increase chances of transmission) and mixed with other sixteen animals at the same age and body condition, in the same grazing area, at a population density equal to the average cattle farming density within the region (one cattle head per 10,000 m²), for 180 days. All animals were monitored daily for clinical signs. Serum samples were collected on days 0, 30, 60 and 180 post-vaccination. Seroconversion was observed only in vaccinated heifers. These results indicate that, under the conditions of the present study, the gE- vaccine virus did not cause any noticeable harmful effect on pregnant dams and on its offspring and did not spread horizontally among cattle.<br>Infecções pelo herpesvírus bovino tipo 1 (BoHV-1) são importantes causas de doença respiratória, reprodutiva e abortos em bovinos. A vacinação é freqüentemente empregada para minimizar as perdas produzidas pela infecção. Todavia, a imunização de vacas durante a prenhez com algumas vacinas contendo vírus vivo modificado (MLV) pode ocasionalmente causar abortos. Em trabalho prévio, nosso grupo desenvolveu uma vacina recombinante de BoHV-1 construída a partir de um isolado brasileiro de BoHV-1 (Franco et al., 2002a) do qual o gene que codifica para a glicoproteína E (gE) foi artificialmente deletado. Tal recombinante (gE-) vem sendo avaliado como vacina diferencial, isto é, capaz de permitir a diferenciação entre animais vacinados e infectados. No presente estudo, o potencial de disseminação do vírus recombinante foi avaliado em um rebanho de gado de corte, em condições de campo. Para tanto, a segurança da vacina gE- quando aplicada durante a prenhez foi avaliada pela inoculação intramuscular de 10(7,4) doses infectantes para 50% dos cultivos celulares (DICC50) do vírus em 22 fêmeas prenhes (14 previamente soronegativas e 8 previamente soropositivas para BoHV-1) em diferentes fases da gestação. Outras 15 vacas prenhes foram mantidas como controles não-vacinados. Não ocorreram abortos, natimortos ou anormalidades fetais em nenhum dos grupos. Soroconversão foi observada nas fêmeas vacinadas previamente soronegativas. Em um segundo experimento, 4 novilhas foram inoculadas pela via intranasal com 10(7,6) DICC50 do vírus recombinante, sendo mantidos em contato com 16 novilhas em uma área de campo, a uma densidade de 1 animal por hectare. Os animais foram monitorados quanto à presença de sinais clínicos; amostras de soro foram coletadas nos dias 0, 30, 60 e 180 após a vacinação. Soroconversão foi observada apenas nos animais vacinados e não nos contatos. Estes resultados indicam que, nas condições do presente estudo, a vacina gE- não tem efeitos deletérios para fêmeas gestantes nem para seus fetos e não se dissemina horizontalmente no rebanho