Adjuvant activity of multimolecular complexes based on Glycyrrhiza glabra saponins, lipids, and influenza virus glycoproteins.

Numerous studies have shown that immunostimulatory complexes containing Quil-A saponin and various antigens are effective in stimulating the immune response and can be used as vaccine preparations for animals and humans. However, Quil-A saponin possesses toxicity and haemolytic activity. In the present work, a saponin-containing preparation named "Glabilox" was isolated from the roots of a Glycyrrhiza glabra L. plant by high-performance liquid chromatography (HPLC). The results showed that Glabilox has no toxicity or haemolytic activity and can form stable immunostimulatory complexes. Subcutaneous immunization of mice with an immunostimulating complex containing Glabilox and H7N1 influenza virus antigens stimulated high levels of humoral and cellular immunity. Vaccination of chickens with the same immunostimulating complex protected 100% of the animals after experimental infection with a homologous virus. Comparative studies showed that the immunogenic and protective activity of immunostimulatory complexes containing Quil-A and immunostimulatory complexes containing Glabilox are comparable to each other. The results of these studies indicated that Glycyrrhiza glabra saponins show great promise as safe and effective adjuvants.

Serology and longevity of immunity against Echinococcus granulosus in sheep and llama induced by an oil-based EG95 vaccine.

An oil-based formulation of the EG95 vaccine to protect grazing animals against infection with Echinococcus granulosus was formulated in Argentina. The efficacy of the vaccine was monitored by serology in sheep and llama (Lama glama) and was compared to the serology in sheep previously published using a QuilA-adjuvanted vaccine. Long-term efficacy was also tested in sheep by challenging with E. granulosus eggs of the G1 strain 4 years after the beginning of the trial. The serological results for both sheep and llama were similar to those described previously, except that there was a more rapid response after the first vaccination. A third vaccination given after 1 year resulted in a transient boost in serology that lasted for about 12 months, which was similar to results previously described. Sheep challenged after 4 years with three vaccinations presented 84·2% reduction of live cysts counts compared with control group, and after a fourth vaccination prior to challenge, this reduction was 94·7%. The oil-based vaccine appeared to be bio-equivalent to the QuilA vaccine.

Dietary administration of saponin stimulates growth of the swimming crab Portunus trituberculatus and enhances its resistance against Vibrio alginolyticus infection.

The immunostimulatory role of dietary saponins on swimming crabs was investigated under field conditions. Portunus trituberculatus were fed diets enriched with Quillaja saponin (QS) at 150, 300 and 450 mg kg-1. They had an enhanced growth rate and increased resistance against Vibrio alginolyticus compared to crabs not fed with QS. Significant effects were observed on the average body weight, percentage weight gain and specific growth rate (p < 0.05). Total hemocyte and hyaline cell counts of P. trituberculatus fed with 300 and 450 mg kg-1 saponin in their diets significantly increased (p < 0.05) compared to the control. Phenoloxidase, superoxide dismutase, catalase and glutathione peroxidase activities significantly increased in response to the incorporation of QS in the diet. However, the respiratory burst activity did not increase significantly. The phagocytic activity was significantly enhanced at 300 and 450 mg kg-1 of saponin. QS diets can enhance growth of P. trituberculatus and its immune resistance against V. alginolyticus. Dietary administration of saponin may help to control diseases and improve production in the crab aquaculture industry.

Chitosan hydrogel vaccine generates protective CD8 T cell memory against mouse melanoma.

CD8(+) T cells are important in the control of viral infections and cancers because of their cytolytic activity. A vaccine able to generate these cells could be beneficial in the prevention or treatment of these diseases. Chitosan hydrogel is a promising vaccine formulation that has previously been shown to generate effector CD8(+) T cells in a mouse model. This vaccine promotes sustained release of antigen and adjuvant, which generates a robust effector response. For longer lasting immunity, a memory population of these CD8(+) T cells is required to control further disease. We found that vaccination with chitosan hydrogel or dendritic cells using ovalbumin protein as a model antigen and Quil-A adjuvant provided protection in a subcutaneous melanoma challenge 30 days later. Ovalbumin-specific memory CD8(+) T cells were detectable following vaccination with the chitosan hydrogel but not the dendritic cell vaccine and an in vivo cytotoxicity assay demonstrated specific lysis of target cells in chitosan hydrogel vaccinated mice but not those receiving dendritic cell vaccination. These results demonstrate that vaccination with chitosan hydrogel is equally effective as dendritic cell vaccination in tumour protection but has more readily detectable immune correlates of protection. This may be advantageous in predetermining protection in vaccinated individuals.

A Fasciola hepatica-derived fatty acid binding protein induces protection against schistosomiasis caused by Schistosoma bovis using the adjuvant adaptation (ADAD) vaccination system.

Several efforts have been made to identify anti-schistosomiasis vaccine candidates and new vaccination systems. The fatty acid binding protein (FAPB) has been shown to induce a high level of protection in trematode infection. The adjuvant adaptation (ADAD) vaccination system was used in this study, including recombinant FABP, a natural immunomodulator and saponins. Mice immunised with the ADAD system were able to up-regulate proinflammatory cytokines (IL-1 and IL-6) and induce high IgG2a levels. Moreover, there was a significant reduction in worm burden, egg liver and hepatic lesion in vaccinated mice in two independent experiments involving Schistosoma bovis infected mice. The foregoing data shows that ADAD system using FABP provide a good alternative for triggering an effective immune response against animal schistosomiasis.

Matrix M Adjuvanted H5N1 Vaccine Elicits Broadly Neutralizing Antibodies and Neuraminidase Inhibiting Antibodies in Humans That Correlate With In Vivo Protection.

The highly pathogenic avian influenza H5N1 viruses constantly evolve and give rise to novel variants that have caused widespread zoonotic outbreaks and sporadic human infections. Therefore, vaccines capable of eliciting broadly protective antibody responses are desired and under development. We here investigated the magnitude, kinetics and protective efficacy of the multi-faceted humoral immunity induced by vaccination in healthy adult volunteers with a Matrix M adjuvanted virosomal H5N1 vaccine. Vaccinees were given escalating doses of adjuvanted vaccine (1.5µg, 7.5µg, or 30µg), or a non-adjuvanted vaccine (30µg). An evaluation of sera from vaccinees against pseudotyped viruses covering all (sub)clades isolated from human H5N1 infections demonstrated that the adjuvanted vaccines (7.5µg and 30µg) could elicit rapid and robust increases of broadly cross-neutralizing antibodies against all clades. In addition, the adjuvanted vaccines also induced multifaceted antibody responses including hemagglutinin stalk domain specific, neuraminidase inhibiting, and antibody-dependent cellular cytotoxicity inducing antibodies. The lower adjuvanted dose (1.5µg) showed delayed kinetics, whilst the non-adjuvanted vaccine induced overall lower levels of antibody responses. Importantly, we demonstrate that human sera post vaccination with the adjuvanted (30µg) vaccine provided full protection against a lethal homologous virus challenge in mice. Of note, when combining our data from mice and humans we identified the neutralizing and neuraminidase inhibiting antibody titers as correlates of in vivo protection.

QuilA-Adjuvanted T. gondii Lysate Antigens Trigger Robust Antibody and IFNγ+ T Cell Responses in Pigs Leading to Reduction in Parasite DNA in Tissues Upon Challenge Infection.

Toxoplasma gondii is an intracellular parasite of all mammals and birds, responsible for toxoplasmosis. In healthy individuals T. gondii infections mostly remain asymptomatic, however this parasite causes severe morbidity and mortality in immunocompromised patients and congenital toxoplasmosis in pregnant women. The consumption of raw or undercooked pork is considered as an important risk factor to develop toxoplasmosis in humans. Since effective therapeutic interventions to treat toxoplasmosis are scarce, vaccination of meat producing animals may prevent T. gondii transmission to humans. Here, we evaluated the elicited immune responses and the efficacy of a potential vaccine candidate, generated by size fractionation of T. gondii lysate proteins, to reduce the parasite burden in tissues from experimentally T. gondii infected pigs as compared to vaccination with total lysate antigens (TLA). Our results show that both the vaccine candidate and the TLA immunization elicited strong serum IgG responses and elevated percentages of CD4+CD8+IFNγ+ T cells in T. gondii infected pigs. However, the TLA vaccine induced the strongest immune response and reduced the parasite DNA load below the detection limit in brain and skeletal muscle tissue in most animals. These findings might inform the development of novel vaccines to prevent T. gondii infections in livestock species and humans.

Co-administration of saponin quil A and PRRSV-1 modified-live virus vaccine up-regulates gene expression of type I interferon-regulated gene, type I and II interferon, and inflammatory cytokines and reduces viremia in response to PRRSV-2 challenge.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a devastating virus which suppresses the expression of type I and II interferons (IFNs) as well as several pro-inflammatory cytokines. Our previous study reported that saponin quil A had a potential to up-regulate the expression of type I IFN-regulated genes and type I and II IFNs in porcine peripheral blood mononuclear cells (PBMC) inoculated with PRRSV. The present study evaluated the immunostimulatory effect of quil A on potentiating cross protective immunity of PRRSV-1 modified-live virus (MLV) vaccine against PRRSV-2 challenge. Twenty-four 4-week-old PRRSV-seronegative pigs were divided into four groups of six pigs. Group 1 and group 2 pigs were vaccinated with PRRSV-1 MLV vaccine at 0 dpv (day post vaccination), and additionally group 2 pigs were injected intramuscularly with quil A at -1, 0, 1 dpv. Group 3 pigs were injected with PRRSV-1 MLV vaccine solvent at 0 dpv and served as challenge control, while group 4 pigs served as strict control. Group 1-3 pigs were challenged intranasally with PRRSV-2 at 28 dpv and immune and clinical parameters were observed from 0 until 49 dpv. Group 1 pigs showed significantly reduced PRRSV viremia, number of viremic pigs, and clinical scores, and significantly improved average daily weight gain (ADWG), compared to group 3 pigs. Group 2 pigs showed significantly increased mRNA expressions of interferon regulatory factor 3, 2'-5'-oligoadenylatesynthetase 1, osteopontin, IFNα, IFNß, IFNγ, interleukin-2 (IL-2), IL-13 and tumor necrosis factor alpha, compared to group 1 pigs. The animals demonstrated significantly reduced PRRSV viremia and number of viremic pigs, but did not demonstrate any further improved PRRSV-specific antibody levels, neutralizing antibody titers, rectal temperature, clinical scores, and ADWG as compared to group 1 pigs. Our findings suggest that quil A up-regulates type I IFN-regulated gene, type I and II IFNs, and inflammatory cytokine expressions which may contribute to further reducing PRRSV viremia and number of viremic pigs which were conferred by PRRSV-1 MLV vaccine. Our findings also suggest that quil A may serve as an effective immunostimulator for potentiating cell-mediated immune defense to PRRSV.

Leaf saponins of Quillaja brasiliensis enhance long-term specific immune responses and promote dose-sparing effect in BVDV experimental vaccines.

Saponin-based adjuvants are promising adjuvants that enhance both humoral and T-cell-mediated immunity. One of the most used natural products as vaccine adjuvants are Quillaja saponaria bark saponins and its fraction named Quil A®. Despite that, its use has been restricted for human use due to safety issues. As an alternative, our group has been studying the congener species Quillaja brasiliensis saponins and its performance as vaccine adjuvants, which have shown to trigger humoral and cellular immune responses comparable to Quil A® but with milder side effects. Here, we studied a semi purified aqueous extract (AE) and a previously little characterized saponin-enriched fraction (QB-80) from Q. brasiliensis as vaccine adjuvants and an inactivated virus (bovine viral diarrhea virus, BVDV) antigen co-formulated in experimental vaccines in mice model. For the first time, we show the spectra pattern of the Q. brasiliensis saponins by MALDI-TOF, a novel and cost-effective method that could be used to characterize different batches during saponins production. Both AE and QB-80 exhibited noteworthy chemical similarities to Quil A®. In addition, the haemolytic activity and toxicity were assessed, showing that both AE and QB-80 were less toxic than Quil A®. When subcutaneously inoculated in mice, both fractions promoted long-term strong antibody responses encompassing specific IgG1 and IgG2a, enhanced the avidity of IgG antibodies, induced a robust DTH reaction and significantly increased IFN-É£ production in T CD4+ and T CD8+ cells. Furthermore, we have proven herein that AE has the potential to promote dose-sparing, substantially reducing the dose of antigen required for the BVDV vaccines and still eliciting a mixed Th1/Th2 strong immune response. Based on these results, and considering that AE is a raw extract, easier and cheaper to produce than commercially available saponins, this product can be considered as candidate to be escalated from experimental to industrial uses.

Preliminary evaluation of a thermosensitive chitosan hydrogel for Echinococcus granulosus vaccine delivery.

The EG95 vaccine is effective in protecting grazing animals from infection with Echinococcus granulosus. Six male lambs were used in the study, two were each vaccinated subcutaneously with 50µg EG95/1mg Quil-A, two animals were each vaccinated with 50µg EG95/1mg Quil-A in 1% chitosan thermolabile gel subcutaneously, and two animals served as non-vaccinated controls. Two vaccinations were given at a 7 week interval. Two vaccinations induced a significantly higher antibody titre in the chitosan group compared with the Quil-A only group. The chitosan vaccine group also had a significantly higher antibody titre compared with a positive control sera from vaccinated and challenged sheep. Incorporating the EG95/Quil-A vaccine in a thermo-responsive chitosan sol-gel stimulated, after the second injection, a high level of antibody absorbance which remained high for at least one year. This response was significantly greater than the response to vaccine without the gel.

Effect of Chitosan and Liposome Nanoparticles as Adjuvant Codelivery on the Immunoglobulin G Subclass Distribution in a Mouse Model.

BACKGROUND: We investigate the immunogenic properties of chitosan and liposome nanoparticles as adjuvant codelivery against a commercial pneumococcal conjugate vaccine (PCV) in an animal model. METHODS: The chitosan and liposome nanoparticles were prepared by ionic gelation and dry methods, respectively. The PCV immunization was performed intradermally in the presence of adjuvants and booster injections which were given without an adjuvant. The Quil-A® was used as a control adjuvant. The ELISA was performed to measure the antibodies against pneumococcal type 14 polysaccharide (Pn14PS). RESULTS: The level of total antibodies against Pn14PS antigen was no different between the mouse groups with or without adjuvant codelivery. Codelivery of the PCV with chitosan nanoparticles as well as the Quil-A adjuvant elicited IgG1, IgG2a, IgG2b, and IgG3 antibodies. Meanwhile, codelivery of liposome nanoparticles elicited mainly IgG1 antibodies against the Pn14PS. CONCLUSIONS: The chitosan and liposome nanoparticles as adjuvant codelivery were successfully synthesized. These nanoparticles have different shapes in particle formation, liposome nanoparticle with their unilamellar shape and chitosan nanoparticles in large shape due to the aggregation of small-size particles. Codelivery of chitosan nanoparticles has more effect on the IgG subclass antibody production than that of liposome nanoparticles in a mouse model.

rROP2 from Toxoplasma gondii as a potential vaccine against oocyst shedding in domestic cats.

The aim of the present study was to evaluate oocyst shedding in cats immunized by nasal route with T. gondii proteins ROP2. Twelve short hair cats (Felis catus) were divided in three groups G1, G2 and G3 (n=4). Animals from G1 received 100 µg of rROP2 proteins plus 20 µg of Quil-A, G2 received 100 µg of BSA plus 20 µg of Quil-A, and the G3 only saline solution (control group). All treatments were done by intranasal route at days 0, 21, 42, and 63. The challenge was performed in all groups on day 70 with ≅ 800 tissue cysts of ME-49 strain by oral route. Animals from G1 shed less oocysts (86.7%) than control groups. ELISA was used to detect anti-rROP2 IgG and IgA, however, there were no correlation between number of oocyst shedding by either IgG or IgA antibody levels. In the present work, in spite of lesser oocysts production in immunized group than control groups, it was not possible to associate the use of rROP2 via nostrils with protection against oocyst shedding. For the future, the use of either other recombinant proteins or DNA vaccine, in combination with rROP2 could be tested to try improving the efficacy of this kind of vaccine.

Host protective ASP-based vaccine against the parasitic nematode Ostertagia ostertagi triggers NK cell activation and mixed IgG1-IgG2 response.

The mucus-dwelling parasite Ostertagia ostertagi is one of the most important gastrointestinal nematodes in cattle. Our group has previously demonstrated the protective capacity of a vaccine against this parasite based on a native activation-associated secreted protein ASP1 (nASP) in combination with the saponin adjuvant QuilA. The aim of the current study was to analyse the effect of both antigen and adjuvant on the cellular and humoral vaccine-induced immune responses by comparing the native ASP to a recombinant version expressed in Pichia pastoris (pASP) and replacing QuilA by Al(OH)3. Immunization of cattle with the protective nASP+QuilA vaccine was associated with antigen-induced proliferation of natural killer (NK) cells combined with IFN-γ secretion and the induction of a mixed IgG1/IgG2 antibody response. ASP-specific activation and proliferation of NK cells was also observed in mice following the same vaccination regime. Replacing QuilA by Al(OH)3 or nASP by pASP significantly decreased the capacity of the vaccines to trigger both NK cell activation and antibody responses and failed to induce protection against a challenge infection. Reduction of the structurally anchoring disulphide bonds of the nASP completely abolished its ability to induce NK cell activation and antibody responses, highlighting the importance of protein conformation for the immunostimulatory activity.

Characterization of the immune response and evaluation of the protective capacity of rSsnA against Streptococcus suis infection in pigs.

The efforts made to develop vaccines against Streptococcus suis have failed because of lack of common antigens cross-reactive against different serotypes of this species. The cell wall-anchored proteins can be good vaccine candidates due to their high expression and accessibility to antibodies, among these, a cell-wall protein, DNA-nuclease (SsnA), present in most of the S. suis serotypes and clinical isolates collected from infected pigs, was selected. An experimental challenge against S. suis serotype 2 in a pig model was used to validate the efficacy of recombinant SsnA combined with aluminium hydroxide plus Quil A as adjuvants, previously tested in mice by our research group with good results. In our study, clinical characteristics, bacterial load and spread, haematological and immunological parameters and the antibody response, including the opsonophagocytosis analysis of the sera were evaluated. Moreover the composition of peripheral blood leukocyte populations was studied in infected animals. The results show that the immunization of piglets with rSsnA elicits a significant humoral antibody response. However, the antibody response is not reflected in protection of pigs that are challenged with a virulent strain in our conventional vaccination model. Further studies are necessary to evaluate the use of rSsnA as a vaccine candidate for swine.

Quillaja brasiliensis saponins induce robust humoral and cellular responses in a bovine viral diarrhea virus vaccine in mice.

A saponin fraction extracted from Quillaja brasiliensis leaves (QB-90) and a semi-purified aqueous extract (AE) were evaluated as adjuvants in a bovine viral diarrhea virus (BVDV) vaccine in mice. Animals were immunized on days 0 and 14 with antigen plus either QB-90 or AE or an oil-adjuvanted vaccine. Two-weeks after boosting, antibodies were measured by ELISA; cellular immunity was evaluated by DTH, lymphoproliferation, cytokine release and single cell IFN-γ production. Serum anti-BVDV IgG, IgG1 and IgG2b were significantly increased in QB-90- and AE-adjuvanted vaccines. A robust DTH response, increased splenocyte proliferation, Th1-type cytokines and enhanced production of IFN-γ by CD4(+) and CD8(+) T lymphocytes were detected in mice that received QB-90-adjuvanted vaccine. The AE-adjuvanted preparation stimulated humoral responses but not cellular immune responses. These findings reveal that QB-90 is capable of stimulating both cellular and humoral immune responses when used as adjuvant.

rROP2 from Toxoplasma gondii as a potential vaccine against oocyst shedding in domestic cats / rROP2 de Toxoplasma gondii como potencial vacina contra a eliminação de oocistos em gatos domésticos

Abstract The aim of the present study was to evaluate oocyst shedding in cats immunized by nasal route with T. gondii proteins ROP2. Twelve short hair cats (Felis catus) were divided in three groups G1, G2 and G3 (n=4). Animals from G1 received 100 μg of rROP2 proteins plus 20 μg of Quil-A, G2 received 100 μg of BSA plus 20 μg of Quil-A, and the G3 only saline solution (control group). All treatments were done by intranasal route at days 0, 21, 42, and 63. The challenge was performed in all groups on day 70 with ≅ 800 tissue cysts of ME-49 strain by oral route. Animals from G1 shed less oocysts (86.7%) than control groups. ELISA was used to detect anti-rROP2 IgG and IgA, however, there were no correlation between number of oocyst shedding by either IgG or IgA antibody levels. In the present work, in spite of lesser oocysts production in immunized group than control groups, it was not possible to associate the use of rROP2 via nostrils with protection against oocyst shedding. For the future, the use of either other recombinant proteins or DNA vaccine, in combination with rROP2 could be tested to try improving the efficacy of this kind of vaccine.

Resumo O objetivo do presente estudo foi avaliar a eliminação de oocistos de Toxoplasma gondii em gatos imunizados pela via nasal com proteínas ROP2 de T. gondii. Doze gatos sem raça definida (Felis catus) foram divididos em três grupos experimentais G1, G2 e G3 (n = 4). Os animais do G1 receberam 100 μg de proteínas de rROP2 mais 20 μg de Quil-A, G2 recebeu 100 μg de albumina de soro bovino (BSA) junto com 20 μg de Quil-A, e o G3 recebeu apenas solução salina (grupo de controle). Todos os tratamentos foram realizados pela via intranasal nos dias 0, 21, 42 e 63. O desafio foi realizado em todos os grupos no dia 70 com aproximadamente 800 cistos de tecido da cepa ME-49 por via oral. Os animais de todos os grupos tiveram as suas fezes examinadas e o número de oocistos foi determinado durante 20 dias após o desafio. Os animais de G1 eliminaram menos oocistos (86,7%) do que os grupos controles. O ELISA foi utilizado para detectar IgG e IgA anti-rROP2, no entanto, não houve correlação entre o número de eliminhação de oocistos com os níveis de anticorpos IgG ou IgA. No presente trabalho, apesar da menor produção de oocistos no grupo imunizado (G1) em relação aos grupos controles (G2 e G3), não foi possível associar o uso de rROP2 pela via nasal com proteção contra eliminação de oocistos de T. gondii. Para o futuro, a utilização de outras proteínas recombinantes, ou mesmo vacina de DNA, em combinação com rROP2 poderia ser utilizada para tentar melhorar a eficácia deste tipo de vacina.