Synthesis and Evaluation of QS-7-Based Vaccine Adjuvants.

We have designed and synthesized two analogs (5 and 6) of QS-7, a natural saponin compound isolated from Quillaja saponaria (QS) Molina tree bark. The only structural difference between compound 5 and 6 is that 5 is acetylated at the 3- and 4-O positions of the quillaic acid C28 fucosyl unit while 6 is not. However, the two analogs show significantly different immunostimulant profiles. Compound 5 may potentiate a mixed Th1/Th2 (Th, T helper cells) immune response against the specific antigens while compound 6 may only induce a Th2-biased immunity. These results suggest that the 3- and/or 4-O acetyl groups of the fucosyl unit may play an important role in tuning the adjuvanticity of the QS-7 analogs, and compound 5 can serve as a structurally defined synthetic adjuvant when a mixed Th1/Th2 immune responses is desired.

Food-Grade Saponin Extract as an Emulsifier and Immunostimulant in Emulsion-Based Subunit Vaccine for Pigs.

Subunit vaccines consisting of highly purified antigens require the presence of adjuvants to create effective and long-lasting protective immunity. Advances on adjuvant research include designing combination adjuvants which incorporate two or more adjuvants to enhance vaccine efficacy. Previously, an oil-in-water emulsion adjuvant (OW-14) composed of mineral oil and an inexpensive gum Arabic emulsifier has been reported demonstrating enhanced and robust immune responses when used as an adjuvant in swine subunit vaccines. This study presents a modified version of OW-14 prepared with food-grade Quillaja saponin extract (OWq). In new OWq emulsion, saponin extract served as an emulsifier for stabilization of emulsion droplets and as an immunoactive compound. The use of saponins allowed to reduce the required amount of emulsifier in the original OW-14. However, emulsion stabilized with saponins demonstrated extended physical stability even at elevated temperature (37°C). The two-dose vaccination with a classical swine fever virus (CSFV) glycoprotein E2-based vaccine formulated with OWq produced higher levels of E2-specific IgG and virus neutralizing antibodies in pigs in contrast with animals that received the vaccine adjuvanted with oil only. In addition, new OWq adjuvant was safe to use in the vaccination of pigs.

Alternative inactivated poliovirus vaccines adjuvanted with Quillaja brasiliensis or Quil-a saponins are equally effective in inducing specific immune responses.

Inactivated polio vaccines (IPV) have an important role at the final stages of poliomyelitis eradication programs, reducing the risks associated with the use of attenuated polio vaccine (OPV). An affordable option to enhance vaccine immunogenicity and reduce costs of IPV may be the use of an effective and renewable adjuvant. In the present study, the adjuvant activity of aqueous extract (AE) and saponin fraction QB-90 from Quillaja brasiliensis using poliovirus antigen as model were analyzed and compared to a preparation adjuvanted with Quil-A, a well-known saponin-based commercial adjuvant. Experimental vaccines were prepared with viral antigen plus saline (control), Quil-A (50 µg), AE (400 µg) or QB-90 (50 µg). Sera from inoculated mice were collected at days 0, 28, 42 and 56 post-inoculation of the first dose of vaccine. Serum levels of specific IgG, IgG1 and IgG2a were significantly enhanced by AE, QB-90 and Quil-A compared to control group on day 56. The magnitude of enhancement was statistically equivalent for QB-90 and Quil-A. The cellular response was evaluated through DTH and analysis of IFN-γ and IL-2 mRNA levels using in vitro reestimulated splenocytes. Results indicated that AE and QB-90 were capable of stimulating the generation of Th1 cells against the administered antigen to the same extent as Quil-A. Mucosal immune response was enhanced by the vaccine adjuvanted with QB-90 as demonstrated by increases of specific IgA titers in bile, feces and vaginal washings, yielding comparable or higher titers than Quil-A. The results obtained indicate that saponins from Q. brasiliensis are potent adjuvants of specific cellular and humoral immune responses and represent a viable option to Quil-A.

ISCOMATRIX adjuvant combines immune activation with antigen delivery to dendritic cells in vivo leading to effective cross-priming of CD8+ T cells.

Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8α(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α(+) DCs in vivo for efficient induction of CTL responses.

ISCOMATRIX adjuvant induces efficient cross-presentation of tumor antigen by dendritic cells via rapid cytosolic antigen delivery and processing via tripeptidyl peptidase II.

Cancer vaccines aim to induce antitumor CTL responses, which require cross-presentation of tumor Ag to CTLs by dendritic cells (DCs). Adjuvants that facilitate cross-presentation of vaccine Ag are therefore key for inducing antitumor immunity. We previously reported that human DCs could not efficiently cross-present the full-length cancer/testis Ag NY-ESO-1 to CTL unless formulated as either an immune complex (NY-ESO-1/IC) or with ISCOMATRIX adjuvant. We now demonstrate that NY-ESO-1/ICs induce cross-presentation of HLA-A2- and HLA-Cw3-restricted epitopes via a proteasome-dependent pathway. In contrast, cross-presentation of NY-ESO-1/ISCOMATRIX vaccine was proteasome independent and required the cytosolic protease tripeptidyl peptidase II. Trafficking studies revealed that uptake of ICs and ISCOMATRIX vaccine by DCs occurred via endocytosis with delivery to lysosomes. Interestingly, ICs were retained in lysosomes, whereas ISCOMATRIX adjuvant induced rapid Ag translocation into the cytosol. Ag translocation was dependent on endosomal acidification and IL-4-driven differentiation of monocytes into DCs. This study demonstrates that Ag formulation determines Ag processing and supports a role for tripeptidyl peptidase II in cross-presentation of CTL epitopes restricted to diverse HLA alleles.

Synthetic studies of complex immunostimulants from Quillaja saponaria: synthesis of the potent clinical immunoadjuvant QS-21Aapi.

QS-21 is one of the most promising new adjuvants for immune response potentiation and dose-sparing in vaccine therapy given its exceedingly high level of potency and its favorable toxicity profile. Melanoma, breast cancer, small cell lung cancer, prostate cancer, HIV-1, and malaria are among the numerous maladies targeted in more than 80 recent and ongoing vaccine therapy clinical trials involving QS-21 as a critical adjuvant component for immune response augmentation. QS-21 is a natural product immunostimulatory adjuvant, eliciting both T-cell- and antibody-mediated immune responses with microgram doses. Herein is reported the synthesis of QS-21A(api) in a highly modular strategy, applying novel glycosylation methodologies to a convergent construction of the potent saponin immunostimulant. The chemical synthesis of QS-21 offers unique opportunities to probe its mode of biological action through the preparation of otherwise unattainable nonnatural saponin analogues.

The immunomodulating properties of human respiratory syncytial virus and immunostimulating complexes containing Quillaja saponin components QH-A, QH-C and ISCOPREP703.

A successful vaccine against human RSV (HRSV) is likely to induce a Th1 or a balanced Th1/TH2 cytokine response. We tested a panel of HRSV immunostimulating complexes (ISCOMs) containing different Quillaja saponin fractions (QH-A, QH-C, and 703: a mixture of 70% QH-A and 30% QH-C) with different immunological properties for their capacity of inducing innate and acquired immune responses. The HRSV 703 ISCOMs induced the strongest innate and acquired immune responses, followed by RSV QH-C and QH-A ISCOMs. All three formulations induced various degrees of Th1 bias response with prominent production of IFN-gamma being 10-50 times higher than that of IL-4 and IL-5. The HRSV specific IgG isotype profile correlated with the predominant secretion of Th1 cytokines, with strong induction of IgG2a antibodies. The 703 ISCOMs induced the most pronounced Th1 profile followed by QH-C and QH-A ISCOMs. The high incorporation of F protein in these ISCOMs compared to G protein combined with the Th1 biased nature of ISCOM are likely to be the causes to promote a Th1 type of profile. The prospect to formulate an RSV ISCOM formulation with an optimal Th1/Th2 balance is in reach particularly in view of the versatile properties of the ISCOM concept.

Pulcherrimasaponin, from the leaves of Calliandra pulcherrima, as adjuvant for immunization in the murine model of visceral leishmaniasis.

A novel triterpenoidal saponin, called pulcherrimasaponin (CP05), isolated from the leaves of Calliandra pulcherrima Benth. shows remarkable similarities to the previously described potent adjuvant, QS21 saponin (Quillaja saponaria Molina). On the basis of chemical and physicochemical evidence, its structure was established as [3beta,16alpha,28[2E,6S[2E,6S(2E,6S)]]]-olean-12-en-28-oic acid 3-[[O-alpha-l-arabinopyranosyl-(1-->2)-O-alpha-l-arabinopyranosyl-(1-->6)-2-(acetylamino)-2-deoxy-beta-d-glucopyranosyl]oxy]-16-hydroxy-O-beta-d-glucopyranosyl-(1-->3)-O-[O-beta-d-xylopyranosyl-(1-->3)-beta-d-xylopyranosyl-(1-->4)-O-6-deoxy-alpha-l-mannopyranosyl-(1-->2)-6-O-[6-[[2-O-2,6-dimethyl-1-oxo-6-(beta-d-xylopyranosyloxy)-2,7-octadienyl]-[(6-deoxy-beta-d-glucopyranosyl)oxy]-2,6-dimethyl-1-oxo-2,7-octadienyl]-beta-d-xylopyranosyl]oxy]-2,6-dimethyl-1-oxo-2,7-octadienyl]-beta-d-glucopyranosyl ester. In vivo toxicity assays disclosed similar and transitory local swelling and loss of hair but no lethality for mice. The haemolytic index was higher for QS21 (5 microg/ml) than for CP05 (13 microg/ml). Mouse vaccination with either CP05 or QS21 in combination with the fucose-mannose ligand (FML) antigen of Leishmania donovani showed anti-FML responses, significantly enhanced over the saponin and saline controls, in IgM, IgG, IgG1, IgG2a, IgG2b and IgG3. Antibody levels were similar for both vaccines in most subtypes. However, QS21-FML vaccine showed a 1.5 to 2.1 proportional increase over the CP05-FML vaccine in IgG, IgG2a and IgG3 responses. The delayed type of hypersensitivity against leishmanial antigen was impressively increased for CP05-FML and for QS21-FML-treated animals over controls (p<0.005). Enhancement was similar for both vaccines (p<0.05). The safety analysis and the effect on humoral and cellular immune responses demonstrated that the novel Calliandra pulcherrima Benth. CP05 saponin is a potential candidate for a vaccine adjuvant.

Effective immunotherapy against canine visceral leishmaniasis with the FML-vaccine.

The potential effect of the fucose mannose ligand (FML)-vaccine on immunotherapy of canine visceral leishmaniasis was assayed on five mongrel dogs experimentally infected with Leishmania donovani and on 21 Leishmania chagasi naturally infected dogs when seropositive to FML but completely asymptomatic. The clinical signs of the experimentally infected, symptomatic dogs only disappeared after the complete vaccination. Protection was obtained in 3/5 animals that remained asymptomatic, IDR positive and parasite free, 1 year after infection. Furthermore, the asymptomatic, FML-vaccine treated dogs showed stable anti-FML IgG1 levels, increasing IgG2 levels and 79-95% of positive DTH response, during the whole experiment. Twenty-two months after complete vaccination, no obits due to visceral leishmaniasis were recorded and 90% of these dogs were still asymptomatic, healthy and parasite free. On the other hand, 37% (17/46 dogs) kala-azar obits were recorded in a control group that received no treatment during the same period, and that was FML-seropositive and asymtpomatic at the beginning of the assay. Our results indicate that the FML-vaccine was effective in the immunotherapy against visceral leishmaniasis of asymptomatic infected dogs. Normal proportions of CD4 and CD21 lymphocytes were detected in PBMC by FACS analysis, in dogs submitted to immunotherapy, suggesting their non-infectious condition. All animals showed as well significantly increased percents of CD8 lymphocytes as expected for Quillaja saponin (QuilA) vaccine treatments.