Saponins from Quillaja saponaria and Quillaja brasiliensis: Particular Chemical Characteristics and Biological Activities.

Quillaja saponaria Molina represents the main source of saponins for industrial applications. Q. saponaria triterpenoids have been studied for more than four decades and their relevance is due to their biological activities, especially as a vaccine adjuvant and immunostimulant, which have led to important research in the field of vaccine development. These saponins, alone or incorporated into immunostimulating complexes (ISCOMs), are able to modulate immunity by increasing antigen uptake, stimulating cytotoxic T lymphocyte production (Th1) and cytokines (Th2) in response to different antigens. Furthermore, antiviral, antifungal, antibacterial, antiparasitic, and antitumor activities are also reported as important biological properties of Quillaja triterpenoids. Recently, other saponins from Q. brasiliensis (A. St.-Hill. & Tul.) Mart. were successfully tested and showed similar chemical and biological properties to those of Q. saponaria barks. The aim of this manuscript is to summarize the current advances in phytochemical and pharmacological knowledge of saponins from Quillaja plants, including the particular chemical characteristics of these triterpenoids. The potential applications of Quillaja saponins to stimulate further drug discovery research will be provided.

Nanoparticulate Tubular Immunostimulating Complexes: Novel Formulation of Effective Adjuvants and Antigen Delivery Systems.

New generation vaccines, based on isolated antigens, are safer than traditional ones, comprising the whole pathogen. However, major part of purified antigens has weak immunogenicity. Therefore, elaboration of new adjuvants, more effective and safe, is an urgent problem of vaccinology. Tubular immunostimulating complexes (TI-complexes) are a new type of nanoparticulate antigen delivery systems with adjuvant activity. TI-complexes consist of cholesterol and compounds isolated from marine hydrobionts: cucumarioside A2-2 (CDA) from Cucumaria japonica and monogalactosyldiacylglycerol (MGDG) from marine algae or seagrass. These components were selected due to immunomodulatory and other biological activities. Glycolipid MGDG from marine macrophytes comprises a high level of polyunsaturated fatty acids (PUFAs), which demonstrate immunomodulatory properties. CDA is a well-characterized individual compound capable of forming stable complex with cholesterol. Such complexes do not possess hemolytic activity. Ultralow doses of cucumariosides stimulate cell as well as humoral immunity. Therefore, TI-complexes comprising biologically active components turned out to be more effective than the strongest adjuvants: immunostimulating complexes (ISCOMs) and complete Freund's adjuvant. In the present review, we discuss results published in series of our articles on elaboration, qualitative and quantitative composition, ultrastructure, and immunostimulating activity of TI-complexes. The review allows immersion in the history of creating TI-complexes.

An Updated Review of ISCOMSTM and ISCOMATRIXTM Vaccines.

The need for the improvement of vaccine potency as well as reducing toxicity in healthy recipients has motivated studies of the formulation of vaccines regarding control of how, when, and where antigens and adjuvants encounter immune cells and other cells/tissues following vaccine administration. Immunostimulatory complexes (ISCOMs) and ISCOMATRIXTM adjuvants are versatile and flexible systems with various phospholipids and saponin components. They are being used in novel vaccines for either infectious diseases or cancer. This article presents a brief review of the latest developments using such adjuvants and possible new applications.

Randomized controlled field trial to assess efficacy of a Moraxella bovis pilin-cytotoxin-Moraxella bovoculi cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis.

OBJECTIVE: To evaluate efficacy of a recombinant Moraxella bovis pilin-cytotoxin-Moraxella bovoculi cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK). ANIMALS: 107 beef steers. PROCEDURES: 2 groups of calves were inoculated SC with an immunostimulating complex (ISCOM) matrix adjuvant (control group; n = 54) or a recombinant M bovis pilin-cytotoxin-M bovoculi cytotoxin subunit antigen with the ISCOM matrix adjuvant (vaccine group; 53); calves received booster injections 21 days later. Calves were examined once weekly for 16 weeks. Investigators and herd managers were not aware of the inoculum administered to each calf throughout the trial. Primary outcome of interest was the cumulative proportion of calves that developed IBK. Serum samples were obtained before inoculation (day 0) and on days 42 and 112. Serum hemolysin-neutralizing titers against native M bovis and M bovoculi cytotoxin were determined. RESULTS: No difference was detected between groups for the cumulative proportion of calves that developed IBK at weeks 8 and 16 after inoculation. Non-IBK-affected calves in the vaccine group had a significantly higher fold change in serum hemolysin-neutralizing titer against native M bovoculi cytotoxin from day 0 to 42 compared to control calves. CONCLUSIONS AND CLINICAL RELEVANCE: The M bovis pilin-cytotoxin-M bovoculi cytotoxin subunit vaccine with the ISCOM matrix adjuvant was not effective at preventing naturally occurring IBK. It is likely that the incorporation of additional protective antigens in a recombinant Moraxella spp subunit vaccine will be required to yield a product that can be used for effective immunization of cattle against IBK.

Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis pilin-Moraxella bovis cytotoxin-ISCOM matrix adjuvanted vaccine.

To evaluate the efficacy of a recombinant Moraxella bovis pilin-M. bovis cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK; pinkeye), a randomized, blinded, controlled field trial was conducted during summer 2005 in a northern California herd of beef cattle. One hundred and one steers were vaccinated with ISCOM matrix (adjuvant control), recombinant M. bovis cytotoxin carboxy terminus+ISCOM matrix (MbxA), or recombinant M. bovis pilin-cytotoxin carboxy terminus+ISCOM matrix (pilin-MbxA); calves received secondary vaccinations 21 days later. Calves were examined once weekly for 18 weeks for the development of corneal ulcers associated with IBK. Overall, the pilin-MbxA vaccinated group had the lowest overall cumulative proportion of ulcerated calves. Calves that received MbxA, whether alone or with pilin had significantly higher M. bovis cytotoxin serum neutralizing titers as compared to control calves. Results of ocular cultures suggested that vaccination with an M. bovis antigen affected organism type isolated from an ulcer: M. bovis was cultured more often from the eyes of control calves than from the eyes of calves vaccinated with MbxA and pilin-MbxA. In addition, vaccination of calves with MbxA and pilin-MbxA resulted in a higher prevalence of Moraxella bovoculi sp. nov. in ocular cultures. While no significant difference was observed between a cytotoxin versus pilin+cytotoxin vaccine against IBK, the reduced cumulative proportion of IBK in the pilin-cytotoxin vaccinated calves suggests it may provide an advantage over a cytotoxin vaccine alone. Efficacy of an M. bovis vaccine may be reduced in herds where IBK is associated with M. bovoculi sp. nov.

[Adjuvants--essential components of new generation vaccines]. / Adiuwanty--niezbedne skladniki szczepionek nowej generacji.

Adjuvants are essential components of vaccines that augment an immunological reaction of organism. New vaccines based on recombinant proteins and DNA, are more save than traditional vaccines but they are less immunogenic. Therefore, there is an urgent need for the development of new, improved vaccine adjuvants. There are two classes of adjuvants: vaccine delivery systems (e.g. emulsions, microparticles, immune-stimulating complexes ISCOMs, liposomes) and immunostimulatory adjuvants (e.g. lipopolysaccharide, monophosphoryl lipid A, CpG DNA, or muramylpeptides). The discovery of more potent and safer adjuvants may allow to development better prophylactic and therapeutic vaccines against chronic infectious (e.g., HSV, HIV, HCV, HBV, HPV, or Helicobacter pylori) and noninfectious diseases as multiple sclerosis, insulin-dependent diabetes, rheumatoid arthritis, allergy and tumors (e.g., melanoma, breast, or colon cancer).

Safety and efficacy of immune-stimulating complex-based antigen delivery systems for neonatal immunisation against respiratory syncytial virus infection.

To protect against human respiratory syncytial virus (hRSV)-induced bronchiolitis in early infancy, vaccines need to be designed which are effective in the neonatal period. To test the safety and efficacy of adjuvants in neonatal mice, we injected hRSV surface proteins combined with immune-stimulating complexes (ISCOMs) prepared from fractions A, C or A + C of Quillaja saponins. All were well tolerated in adults, but A + C ISCOMS proved lethal in neonates; A or C fractions alone were well tolerated by neonates up to the adult dose. hRSV-ISCOM A induced antibody responses similar to combined fractions, and potent in vitro cytotoxic T cell responses. Adult-like in vitro cytotoxicity against hRSV-infected targets and precursor cytotoxic T cell frequencies were observed within one week of neonatal priming and hRSV-ISCOM A-primed neonates showed virtually complete protection against subsequent viral challenge. hRSV challenge was associated with some pulmonary eosinophilia in both age groups, with higher IL-4 production by lung CD4+ T cells in mice primed as neonates. This was, however, accompanied by only minor (approximately 10%) and transient illness and weight loss. Thus, the identification of hRSV antigen delivery systems with an age-appropriate adjuvanticity/reactogenicity balance may be feasible even in the vulnerable early-life period.

Immunostimulatory activity of cationic-lipid-nucleic-acid complexes against cancer.

Recent studies have highlighted the immunostimulatory nature of nucleic acids and the enhancement of such immunostimulation when nucleic acids are complexed to cationic liposomes to form cationic-lipid-nucleic-acid-complexes or lipoplexes. While such immunostimulation may have deleterious consequences for nucleic acid delivery, especially in the field of gene therapy, it may be harnessed for efficacious usage against the various forms of cancer.

Recent developments in adjuvants for vaccines against infectious diseases.

New generation vaccines, particularly those based on recombinant proteins and DNA, are likely to be less reactogenic than traditional vaccines, but are also less immunogenic. Therefore, there is an urgent need for the development of new and improved vaccine adjuvants. Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action; vaccine delivery systems and 'immunostimulatory adjuvants'. Vaccine delivery systems are generally particulate e.g. emulsions, microparticles, iscoms and liposomes, and mainly function to target associated antigens into antigen presenting cells (APC). In contrast, immunostimulatory adjuvants are predominantly derived from pathogens and often represent pathogen associated molecular patterns (PAMP) e.g. LPS, MPL, CpG DNA, which activate cells of the innate immune system. Once activated, cells of innate immunity drive and focus the acquired immune response. In some studies, delivery systems and immunostimulatory agents have been combined to prepare adjuvant delivery systems, which are designed for more effective delivery of the immunostimulatory adjuvant into APC. Recent progress in innate immunity is beginning to yield insight into the initiation of immune responses and the ways in which immunostimulatory adjuvants may enhance this process. However, a rational approach to the development of new and more effective vaccine adjuvants will require much further work to better define the mechanisms of action of existing adjuvants. The discovery of more potent adjuvants may allow the development of vaccines against infectious agents such as HIV which do not naturally elicit protective immunity. New adjuvants may also allow vaccines to be delivered mucosally.

Estudo aberto multicêntrico do uso de Broncho-Vaxom® no auxílio do tratamento e prevenção de infecções comunitárias do trato respiratório em crianças / An Open Multicenter Study Using Broncho-Vaxom® as an Adjunt Therapy for the Treatment a Prevention of Respiratory Community Aquired Infections in Children

Vários estudos controlados na Europa têm mostrado a eficácia do Broncho-Vaxom® (imunoestimulante oral contendo frações bacterianas) no auxílio ao tratamento e prevenção de infecções respiratórias em crianças. Entretanto, até o momento, não é do nosso conhecimento nenhuma publicação referente à sua utilização no tratamento destas afecções na população infantil em nosso meio. Objetivo: No presente estudo, os autores avaliam a eficácia e segurança do Broncho-Vaxom® no tratamento e prevenção de doenças respiratórias de 792 crianças com idade entre 6 meses e 12 anos, atendidas em vários centros médicos do Brasil com diagnóstico de otite média, sinusite, bronquite/pneumonia e faringotonsilites recorrentes. Material e método: Os pacientes foram avaliados através de critérios objetivos e subjetivos. O diagnóstico foi baseado em critérios pré-estabelecidos da história clínica e exame físico, e radiografia simples. Broncho-Vaxom® foi prescrito por 10 dias (3,5 mg/dia em uma tomada), e os pacientes foram reavaliados no 30°, 60° e 90° dias após o primeiro dia de medicação. Resultados: De forma geral, na maioria os pacientes apresentaram uma melhora acentuada dos quadros de infecção, com ausência de infecção neste período, e/ou aumentando o intervalo entre as infecções e/ou com diminuição da gravidade dos sintomas e/ou com redução do período de sintomatologia. Conclusão: Os efeitos adversos observados foram leves, na maioria relacionados com o sistema gastrointestinal (vômitos, náusea e diarréia) em 6% das crianças, sendo que em nenhuma uma delas o tratamento foi interrompido.

Controlled clinical studies in Europe have already shown-the efficacy of Broncho-Vaxom® (an oral immunomodulating Material lysate) for the treatment and prevention of bronchitis, sinusitis, pharyngitis and otitis media in children. However, it is not of our knowledge any studies of Broncho-Vaxom® in children with respiratory infections in our population. Aim: In order to validate, the efficacy and safety of Broncho-Vaxom® the authors evaluated the treatment of 792 children assisted in several medical centers in Brasil, ages 6 months to 12 years, referred for recurrent otitis media, recurrent sinusitis, recurrent bronchitis/pneumonia and recurrent pharyngitis/tonsilitis. Material and methods: The patients were evaluated both by subjetive and objective criteria. The diagnosis was based on a standardized history and physical examination and roentgenograms. Broncho-Vaxom® was prescribed for 10 days (3,5 mg/day once a day), for 3 consecutive months. They were reevaluated at 30, 60 and 90 days after the first day of medication. Results: Overall, most of the patients presented excellent and good results, with absence of infections in 90 days, and/or increasing the period between infections and/or reducing the gravity or the time lasting of infections. Conclusion: Overall, minor adverse reactions were observed in 6% dos patients (vomit, diarrhea, nausea). There was no report of interruption of the treatment.

Adjuvants and delivery systems for viral vaccines--mechanisms and potential.

Of the vaccines against viral diseases of man currently available, several are less than satisfactory, and the present surge of interest in improving such vaccines, and in developing new vaccines against viral diseases as yet unchallenged, has led to major developments in three areas. The capacity to identify the nature and form of antigenic epitopes in proteins allows the specific design of molecular entities to promote relevant and protective immune responses. Such entities, although ideal in terms of specificity and purity, may not achieve their goals through failure to reach relevant cells of the immune system due to simple dilution, elimination by host enzymes or lack of specific targeting. Concomitant with the above there has been development of a plethora of adjuvants aimed at enhancing immune responses to these 'new' immunogens, paralleled by an almost equally rapid increase in understanding the complex nature of the immune response, particularly with respect to antigen processing, the nature and role of cytokines and the importance of T-cell subsets in infection. These developments allow exploration of matching the properties and mechanistic action of a given adjuvant to a defined immune response. Adjuvants can be grouped according to their physical characteristics and mode of action. They include particulate adjuvants, oil and emulsifier-based adjuvants, those providing controlled antigen delivery, adjuvants based on specific targeting of antigen, and gel-type adjuvants. They may act non-specifically in promoting an immune response to an antigen through depot formation, or very specifically as in a "delivery system" where an antigen is linked to a cellular protein, targeted to a specific cell receptor. As adjuvant technology develops it is becoming increasingly clear that these differing approaches may be combined, and an adjuvant/delivery system designed, to provide slow release of a targeted antigen. The role of adjuvants in modern viral vaccine technology and their influence on the immune system are the subject of this review.

Humoral response to HSV-1 subunit vaccines--a statistical analysis.

Following primary infection with HSV, the virus becomes latent in the local sensory ganglia for the lifetime of the host. In some cases, periodic reactivation may occur due to various stimuli and cause a recrudescent lesion at or near the initial site of infection. As yet there is no suitable vaccine to prevent its spread within the human population. We investigated the potential of a large number of commercial and experimental adjuvant preparations to enhance the immunogenicity of an HSV-1 glycoprotein subunit vaccine. Evaluation was based on toxicity, total antibody titre, neutralizing antibody production and protection against lethal challenge. All adjuvants tested increased the titre of antigen specific total and neutralizing lg when compared to subunit vaccine alone, although functional neutralising antibody was only detected in some cases. Following challenge, a broad range of protective responses was noted but no correlation between antibody levels and protection was observed. The results emphasize the requirement of adjuvants when using subunit preparations as vaccine formulations and demonstrate that the magnitude and effectiveness of the induced immune response varies greatly with the choice of adjuvant.

Induction of protective immunity to Theileria annulata using two major merozoite surface antigens presented by different delivery systems.

Allelic forms (Tams1-1 and Tams1-2) of the major merozoite surface antigen gene of Theileria annulata have recently been expressed in Escherichia coli and in Salmonella typhimurium aroA vaccine strain SL3261. To test the potential of subunit vaccines against T. annulata infection, we immunized four groups of three calves with either recombinant (re-) (Tams1-1 and Tams1-2) proteins or naked DNA encoding these antigens. Group I was immunized intramuscularly with both re-proteins incorporated into immunostimulating complexes (ISCOMs). Group II was inoculated intramuscularly with naked plasmid DNA encoding Tams1-1 and Tams1-2. Groups III and IV received S. typhimurium SL3261 [pSTams1-1][pIP5] and SL3261 [pSTams1-2] [pIP5] subcutaneously and orally, respectively. A final group of three animals (Group V) served as an unimmunized control group. Four weeks after the last immunization all calves were challenged with a T. annulata stabilate generated from blood of an infected animal with 30% piroplasm parasitaemia. All calves vaccinated with ISCOMs proved to be protected from T. annulata infection and had generated antibodies against both re-(Tams1-1 and Tams1-2) at the time of challenge. In two of these animals the antibody had a surface binding profile by IFAT. Two of three calves immunized with naked DNA also proved to be protected, but none of the animals had generated any detectable antibodies against the recombinants. Salmonella-based delivery of the recombinants did not induce any protection; two of six animals died of theileriosis and there was no difference between subcutaneous or oral administration. These preliminary results show that re-(Tams1-1 and/or Tams1-2) may elicit protective immune responses in cattle, depending on the antigen delivery system.

Immunity against Yersinia enterocolitica by vaccination with Yersinia HSP60 immunostimulating complexes or Yersinia HSP60 plus interleukin-12.

Microbial heat shock proteins (HSP) are dominant antigens for the host immune response. Because of the high sequence homology between mammalian and microbial HSP, their value as component of a subunit vaccine has been the subject of controversy. Previous work from this laboratory, however, demonstrated for the first time that the adoptive transfer of HSP60-reactive CD4+ alphabeta T-cell clones confers protection against bacterial infection in mice but does not induce autoimmunity. In the present study, we have therefore evaluated the potential role of Yersinia HSP60 (Y-HSP60) as a vaccine in the Yersinia enterocolitica mouse infection model. For this purpose, immunostimulating complexes (ISCOM) which included Y-HSP60 were constructed. Parenteral administration of this vaccine induced high Y-HSP60-specific serum antibody responses as well as T-cell responses. This reaction was parallelled by immunity against a lethal challenge with Y. enterocolitica. In contrast, mucosal application of Y-HSP60-ISCOM failed to induce systemic Y-HSP60-specific T-cell responses and thus failed to induce immunity against yersiniae. Likewise, vaccination with purified recombinant Y-HSP60 induced antibody responses but only weak T-cell responses. Therefore, this vaccination protocol was not protective. However, when interleukin-12 was used as an adjuvant, purified Y-HSP60 induced significant Y-HSP60-specific T-cell responses and thus induced protection against subsequent challenge with yersiniae. These studies suggest that (i) microbial HSP might be promising candidates for the design of subunit vaccines and (ii) interleukin-12 is an efficient alternative adjuvant to ISCOM particles for induction of protective CD4 Th1-cell-dependent immune responses against bacterial pathogens.

Adjuvanticity of ISCOMs incorporating a T cell-reactive lipoprotein of the facultative intracellular pathogen Francisella tularensis.

Immunostimulating complexes (ISCOMs) are known to be highly effective adjuvants for envelope antigens of viral agents, but have not been evaluated for use with antigens of intracellular bacteria. Balb/c mice were subcutaneously immunized with ISCOMs into which the T cell-reactive membrane protein TUL4 of Francisella tularensis had been incorporated. Spleen cells from the immunized mice responded in vitro to TUL4 and to heat-killed F. tularensis live vaccine strain (LVS) with proliferation and production of gamma-interferon, whereas spleen cells from control mice immunized with TUL4 only did not respond to the antigens. When mice immunized with TUL4 ISCOMs were challenged with F. tularensis LVS, bacterial counts in spleen and liver were lower than in non-immunized mice. Again, TUL4 had no effect when used without ISCOMs. When proteins of a total membrane preparation of F. tularensis LVS were incorporated in ISCOMs and used for immunization, a decrease in bacterial counts was obtained which was similar in magnitude to that of TUL4 ISCOMs. Generally, the adjuvant effects demonstrated did not compare with the excellent protective effect of live tularaemia vaccine. Nonetheless, ISCOMs provide a means whereby protective antigens of F. tularensis can be tested.

Immune responses and protective effect in mice vaccinated orally with surface sporozoite protein of Eimeria falciformis in ISCOMs.

Immunostimulating complexes (ISCOMs) were built after treatment of a purified surface protein from Eimeria falciformis sporozoites with a palmitic acid derivation, leading to a high ratio (33-64%) of P27 incorporation in these cage-like structures. P27 kept its antigenicity after incorporation in ISCOMs, which induced, after iterative intubations by the oral route to groups of mice, a systemic IgG response, a local IgA response, and a local enhanced cellular response as demonstrated by lymphoproliferation of mesenteric lymph node cells upon in vitro stimulation with antigen. This immunization (120 micrograms in six oral doses at 2-day intervals) afforded mice a partial protection (60%) against a subsequent 400 oocyst challenge. The reduction in daily oocyst excretion was corroborated by significantly different weight losses between immunized and control mice on days 9 and 10 postinfection and the subsequent death of these control mice. These observations provide the first application of ISCOMs to parasitic intestinal diseases.