Into Immunostimulating Complexes Research Articles

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.

Single epitope mucosal vaccine delivered via immuno-stimulating complexes induces low level of immunity against simian-HIV

The difficulty in developing an effective vaccine to contain the HIV/AIDS epidemic coupled with the fact that primary HIV-1 infection typically occurs via mucosal sites has increased emphasis on vaccine approaches that protect at mucosal surfaces. In this study we employed HIV and simian-HIV (SHIV)-derived T helper (Th) and cytotoxic T lymphocyte (CTL) single epitopes incorporated into immuno-stimulating complexes (ISCOM) as a candidate immunogens. Immunized rhesus macaques ( Macaca mulatta) were challenged with CCR5-tropic SHIV SF162p4. On the day of challenge, low levels of virus-neutralizing antibodies (Ab) and CTLs were detected in ISCOM-immunized macaques. Greater than 10 5 viral RNA copies per ml of plasma in 2/5 immunized and 3/4 control macaques were detected within 3 weeks post-challenge. Depletion of CD4+ T cells from gut-associated lymphoid tissues (GALT) was observed by post-challenge day (PCD) 14 in all macaques regardless immunization. Nonetheless, lower viral loads and relatively better preservation of peripheral CD4+ T cells following the SHIV infection was observed in ISCOM-immunized macaques. We predict that if coadministered with additional epitopes and/or more efficacious mucosal delivery system or route, HIV/SIV-derived peptide vaccines may have potential to elicit heterologous protection.

Morphological and immunological characterization of immunostimulatory complexes based on glycoglycerolipids from Laminaria japonica.

Some physicochemical properties of glycoglycerolipids (monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol) from the sea algae Laminaria japonica, as well as their ability to become incorporate into immunostimulating complexes (ISCOMs), used as a delivery system of microbial and tumor antigens in vesicular form, were studied. These glycolipids were found to differ essentially in fatty acid composition, unsaturation index and thermotropic behavior. The possibility of ISCOM modification by embedding the glycolipids studied instead of a phospholipid component in vesicles was shown. A preliminary research of the immunogenicity of the pore-forming protein from Yersinia pseudotuberculosis in modified (by monogalactosyldiacylglycerol) and typical (egg phosphatidylcholine) ISCOMs did not reveal a significant enhancement of immune response in comparison with that of isolated protein.

Morphological and immunological characterization of immunostimulatory complexes based on glycoglycerolipids from Laminaria japonica.

Some physicochemical properties of glycoglycerolipids (monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol) from the sea algae Laminaria japonica, as well as their ability to become incorporate into immunostimulating complexes (ISCOMs), used as a delivery system of microbial and tumor antigens in vesicular form, were studied. These glycolipids were found to differ essentially in fatty acid composition, unsaturation index and thermotropic behavior. The possibility of ISCOM modification by embedding the glycolipids studied instead of a phospholipid component in vesicles was shown. A preliminary research of the immunogenicity of the pore-forming protein from Yersinia pseudotuberculosis in modified (by monogalactosyldiacylglycerol) and typical (egg phosphatidylcholine) ISCOMs did not reveal a significant enhancement of immune response in comparison with that of isolated protein.

A comparison of oral and parenteral routes for therapeutic vaccination with HSV-2 ISCOMs in mice; cytokine profiles, antibody responses and protection

It is likely that recurrent infections with HSV-2 (or HSV-1) are influenced by local levels of immunity at mucosal surfaces, when virus reactivated from the latent state is infecting mucosal epithelial cells. Increasing the levels of cellular and humoral immunity through immunisation and maintaining such increased levels, may reduce establishment and spread of reactivated virus at the local site, thereby ameliorating recurrent disease symptoms. The use of HSV-2 antigens incorporated into immunostimulating complexes (ISCOMs) for immunisation of mice previously infected with HSV-2 was investigated in the present study. Prophylactic administration of HSV-2 ISCOM vaccine to mice elicits local antibody detectable in nasal washings, serum antibody and the presence of cytokines IL-2, IFN-γ and IL-4 in supernatants from spleen cell cultures stimulated in vitro with HSV-2 antigens. Use of the same vaccine in mice infected previously with HSV-2, results in increased levels of total and subclass serum ELISA antibody and also increased levels of serum neutralising antibody. Treatment of HSV-2 infected mice with the HSV-2 ISCOM vaccine also induces higher levels of the cytokines IL-2, IFN-γ and IL-4, in in vitro stimulated spleen cell cultures. Challenge with a lethal dose of HSV-1 showed that mice previously infected with HSV-2 and subsequently given two doses of HSV-2 ISCOMs vaccine were protected.

Antibody responses, cytokine levels and protection of mice immunised with HSV-2 antigens formulated into NISV or ISCOM delivery systems

The immunogenicity of a type 2 herpes simplex virus (HSV-2) antigen preparation following its formulation into immunostimulating complexes (ISCOMs) or non-ionic surfactant vesicles (NISV) was investigated in a murine model. The immune responses induced by each formulation were characterised by antigen specific total and subclass serum responses, and by lymphocyte proliferation and cytokine (interleukin-2 (IL-2), interleukin-4 (IL-4) and interferon-gamma (IFN-γ)) production by in vitro restimulated spleen cells. The degree of protection afforded to mice by these various HSV-2 vaccine preparations against homologous (HSV-2) and heterologous (HSV-1) challenge infection was also determined. The findings suggest that formulation of the HSV-2 glycoprotein antigens with ISCOM or NISV delivery vehicles, and the methods used to prepare these formulations, influenced the immunogenicity of the final preparation. Higher IgG2a and neutralising antibody levels, IL-2 and IFN-γ levels and lymphoproliferative responses were noted in mice immunised with the HSV-2 ISCOM formulated vaccine preparation. Furthermore, although HSV-2 antigens formulated in dehydration–rehydration NISV, or entrapped in NISV by freeze–thawing at 30°C (HSV-2 NISV 30), also elicited relatively high antibody, IL-2 and IFN-γ levels and relatively high lymphoproliferative responses, formulation of HSV-2 antigens by freeze–thawing with NISV at 60°C (HSV-2 NISV 60) did not. There were no differences between any of the HSV-2 vaccine formulations in terms of IL-4 induction in in vitro stimulated spleen cell cultures. Almost complete protection against HSV-2 challenge was afforded by the HSV-2 ISCOM preparation, while partial protection against challenge infection was afforded by the HSV-2 NISV 30 vaccine formulation. The findings are discussed in relation to the nature of the immune mechanisms, particularly Th1- or Th2-like responses, that may be elicited by HSV-2 antigen preparations formulated into various delivery systems and the relevance of these immune responses to protection against HSV infection in the murine model.

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.

Antibody Responses of Chickens to Inoculation with Mycoplasma gallisepticum Membrane Proteins in Immunostimulating Complexes

Membrane proteins of Mycoplasma gallisepticum (MG) strain R were extracted with the detergent Mega-10 and incorporated into immunostimulating complexes (ISCOMs). A membrane protein of approximately 64 kD (p64) molecular weight was a major component of MG ISCOMs. Six-week-old specific-pathogen-free leghorn chickens were inoculated by various routes (subcutaneous; combined intranasal and eyedrop; and combined subcutaneous, intranasal, and eyedrop) with 10 micrograms MG proteins in ISCOMs, or inoculated subcutaneously with 10 micrograms MG proteins in Freund's adjuvant. Subcutaneous inoculation of MG ISCOMs, or MG Freund's adjuvant resulted in higher sero-positive rates (detected by enzyme-linked immunosorbent assay) in serum and respiratory tract washings, compared to combined routes of MG ISCOM inoculation. In chickens inoculated subcutaneously with MG ISCOMs antibodies were first detected at 31 days postinoculation (PI) and the sero-positive rate peaked at 56 days PI. Sero-positive rates started to decline at day 64 PI. In the Freund's adjuvant group, MG antibodies were first detected at day 21 PI, and the sero-positive rate peaked at day 39 PI and did not decline. MG antibodies were detected by ELISA in upper respiratory tract and tracheal washes from chickens inoculated subcutaneously with MG ISCOMs and MG Freund's adjuvant. Immunoblots to MG strain R whole cell proteins showed that respiratory tract washings and sera from chickens inoculated subcutaneously with MG ISCOMs contained immunoglobulins to MG proteins, with a prominent reaction to p64.

Linkage of a fusion peptide to a CTL epitope from the nucleoprotein of measles virus enables incorporation into ISCOMs and induction of CTL responses following intranasal immunization

The introduction of soluble protein antigens into the endogenous processing pathway is a prerequisite for the efficient induction of MHC class-1 restricted cytotoxic T-lymphocytes (CTLs). Antigens incorporated into immunostimulating complexes (ISCOMs) containing lipids and Quil-A are able to induce CD8 + CTL responses in vivo. Furthermore, lipopeptides have also been used to raise peptide-specific CTLs and bypass the requirement for the use of an adjuvant. Although conventional ISCOM technology is in general restricted to the use of hydrophobic proteins or fatty acid-derivitized proteins or peptides, we have demonstrated that the linkage of a conserved paramyxovirus fusion peptide to a CTL epitope NP29 (residues 281–290 of measles virus nucleoprotein) resulted in the incorporation of this hydrophilic CTL epitope into ISCOMs and the in vivo priming of peptide specific CTLs following intranasal immunization. In addition, the fusion peptide-CTL epitope chimera was able to efficiently sensitive P815 target cells for lysis by nucleoprotein specific CTLs induced following immunization of mice with recombinant RAd-68 adenovirus, suggesting the efficient introduction of the peptide into the class-1 restricted antigen processing pathway. Furthermore, immunization of mice with this fusion peptide chimera in saline was able to prime an NP29-specific CTL response despite the absence of adjuvant.

Stratégies de vaccination contre la péripneumonie contagieuse bovine en Afrique

Contagious bovine pleuropneumonia (CBPP) is regarded as the second most important disease of cattle in Africa. The disease was eradicated from Europe through drastic slaughter campaigns with quarantine and restriction of cattle movements. CBPP was mastered in Australia using these methods combined with vaccination. However, the disease remains endemic in Asia and Africa, where it inhibits livestock farming. In these continents, vaccination is the preferred means of control; the aim is to reduce incidence until complementary disease control measures can be applied. The success of a vaccination campaign depends on four main factors: good planning and good organisation; staff who are well-trained, fully equipped and highly motivated; high quality vaccines; good international co-operation. Vaccine strains recommended for use in Africa are strain T1/44 and its variant T1-SR. To improve the immunogenicity of these strains, the Pan African Rinderpest Campaign (PARC) secured financial support for research into immunostimulating complexes (ISCOM). It is hoped that this technology can improve vaccines, leading to effective eradication of the disease. In the meanwhile, systematic and repeated vaccination is the method of choice against CBPP in Africa.

Protection of mice against an influenza virus infection by oral vaccination with viral nucleoprotein incorporated into immunostimulating complexes.

Influenza A virus nucleoprotein (NP) was integrated into immunostimulating complexes (ISCOMs) after attachment of bacterial lipopolysaccharide to the antigen. Oral immunization with these NP-ISCOMs protected mice fully against an otherwise lethal challenge infection with an unrelated influenza virus subtype without the appearance of severe clinical signs or extensive pathological lesions in the lungs. Mice immunized with analogous bovine serum albumine-incorporated ISCOMs all died. After oral immunization, high titers of NP-specific antibodies, particularly IgA, could be detected in the bronchoalveolar fluid and in the blood serum. No cytotoxic lymphocytes could be demonstrated in the spleens or the lungs of vaccinated mice, and no anti-NP antibody-dependent cytolysis of infected host cells was mediated by complement or in the form of an antibody-dependent cell cytotoxicity. However, a vigorous delayed-type hypersensitivity reaction was produced after probing vaccinated animals with purified NP. No comparable protective immunity or antibody response was induced by a strictly intragastric administration of NP-ISCOMs. It appears, therefore, that the general and local immune response in the lungs was primarily stimulated through contact of NP-ISCOMs with the mucous membrane of the oro-pharyngeal cavity and that cytotoxic effects did not play a major role for the establishment of the protective immunity. Partial protection against a lethal challenge was observed in chickens immunized with NP-ISCOMs in the drinking water.

Induction of feline leukaemia virus-neutralizing antibodies by immunization with synthetic peptides derived from the FeLV env gene

The surface glycoprotein, gp70, of feline leukaemia virus (FeLV) contains sites which are important in inducing neutralizing antibodies. Using synthetic peptides corresponding to nucleotide sequence 729–957 (amino acid sequence 243–319) of FeLV-A/Glasgow-1, the antigenicity and immunogenicity of this part of the viral surface glycoprotein were investigated. The region contains two highly conserved domains separated by a variable region (VR4), when compared with FeLV of subgroups B and C, and an epitope known to be involved in virus neutralization is located in the N-terminal conserved domain. Five murine monoclonal antibodies generated by immunization with virus were found to be directed to this domain and four were virus-neutralizing. Polyclonal mouse, rabbit and cat anti-FeLV antisera, which were virus-neutralizing, were directed to B-cell epitopes in the peptides. To determine if those synthetic peptides could induce neutralizing antibodies, rabbits were immunized with the peptides, singly or in combination. Antibody responses were measured by ELISA for anti-peptide, anti-FeLV and anti-gp70 activity, by immunoblotting, by membrane immunofluorescence and by virus-neutralization tests. Virus-neutralizing antibodies were induced by FeLV gp70 peptides and there was a synergistic effect on antibody production when a combination of peptides covering amino acid sequence 243–319 of FeLV-A was used. In a second experiment, six rabbits and six cats were immunized with a combination of two peptides, which covered the above-defined FeLV gp70 sequence. Comparisons were made of the responses to these peptides incorporated into immunostimulating complexes (ISCOMs) via myristic acid tails, inoculated with ‘empty’ ISCOMs, or with Al(OH) 3. Complete Freund's adjuvant (CFA) had a very strong potentiating effect on the induction of antibodies and immunization with peptides incorporated into ISCOMs was superior to immunization using adjuvants other than CFA. It is very promising that not only in rabbits, but also in the natural host of FeLV, the cat, anti-FeLV gp70 (peptides) antibodies could be induced by FeLV gp70 peptides.

The IgA and subclass IgG responses and protection in mice immunised with influenza antigens administered as ISCOMS, with FCA, ALH or as infectious virus.

Comparative studies on the local IgA, and circulating IgG subclass antibody responses of mice to A/Sichuan/2/87 (H3N2) influenza virus surface antigens administered with different carrier or delivery systems by the parenteral route, were carried out. The results obtained were compared with the responses observed following live influenza virus infection, and the protection afforded to these animals by these various preparations determined. Infection with live virus elicited early and high levels of protection against homologous virus challenge and this correlated with both local IgA and circulating IgG2a antibody levels. When incorporated into immunostimulating complexes (ISCOMS), A/Sichuan surface antigens promoted high levels of local IgA and circulating IgG1 antibody, and achieved a more rapid and more solid immunity against homologous virus challenge infection, than that elicited by the same surface antigens administered alone or together with Freund's complete adjuvant or alhydrogel.