Effects of N-acylation on the immune adjuvanticity of analogs of the Quillaja saponins derivative GPI-0100.

Modification of the 3-glucuronic acid (GlcA) residue from the Quillaja saponin (QS) adjuvants by N-acylation, yields derivatives with linear alkylamides that show structural and functional changes. Structural, since the relatively unreactive added hydrophobic alkyl chains may modify these glycosides' conformation and micellar structure. Functional, because altering the availability of proposed pharmacophores, like fucose (Fucp) and aldehyde groups, to interact with their cellular receptors, may change these glycosides' adjuvanticity. While deacylated QS (DS-QS) adjuvants bias the response toward a sole anti-inflammatory Th2 immunity against an antigen, their N-alkylated derivatives carrying octyl to dodecylamide residues, modify that response to a pro-inflammatory Th1 immunity. As shown by their IgG2a/IgG1 titer ratios, which are higher than those for Th2 immunity. A result of the fact that in mice, the IgG2a levels are dependent on the direct influence of secreted interferon-γ (IFN-γ), a crucial Th1 cytokine. But addition of the longer and more lipophilic tetradecylamide group, yields derivatives that like DS-QS induce Th2 immunity, as shown by their low IgG2a/IgG1 ratio. Results that imply that changes in these analogs' conformation and micellar structure, would affect the immunomodulatory properties or adjuvanticity of N-acylated DS-QS. Physical changes that may alter the availability of groups like Fucp, to bind to its presumed dendritic cells' lectin receptor DC-SIGN; an essential step in the stimulation of Th2 immunity. Structural properties that in an aqueous environment, would depend on these glycosides' balance of their hydrophilic and lipophilic moieties (HLB), and the interactions of the newly introduced alkyl chain with the native QS' lipophilic triterpene aglycone and hydrophilic oligosaccharide chains. A situation that would explain these new derivatives' qualitative and quantitative changes in adjuvanticity.

Elucidating the Mechanisms of Action of Saponin-Derived Adjuvants.

Numerous triterpenoid saponins are adjuvants that modify the activities of T cells and antigen-presenting cells, like dendritic cells (DCs). Saponins can induce either proinflammatory Th1/Th2 or sole anti-inflammatory Th2 immunities. Structure-activity relationships (SARs) have shown that imine-forming carbonyl groups are needed for T cell activation leading to induction of Th1/Th2 immunities. While saponins having different triterpenoid aglycons and oligosaccharide chains can activate DCs to induce Th1/Th2 immunoresponses, fucopyranosyl residues from their oligosaccharides by binding to the DC-SIGN receptor can bias DCs toward a sole Th2 immunity. Here we discuss the mechanisms of action of these saponins in view of new information, which may serve as a basis to design improved adjuvants and related drugs.

Effects of immunomodulators on the response induced by vaccines against autoimmune diseases.

A promising treatment for T-cell-mediated autoimmune diseases is the induction of immune tolerance by modulating the immune response against self-antigens, an objective that may be achieved by vaccination. There are two main types of vaccines currently under development. The tolerogenic vaccines, composed of proteins formed by a cytokine fused to a self-antigen, which usually induce tolerance by eliminating the T-cells that are immune reactive against the self-antigen. The immunogenic vaccines, comprised of a self-antigen plus a sole Th2 adjuvant either free or conjugated, that alleviate autoimmunity by switching the immune response against the self-antigen, from a damaging pro-inflammatory Th1/Th17 to an anti-inflammatory Th2 immunity. Another type of vaccines is the DNA vaccines, where cells transiently express the self-antigen encoded by DNA, which induces a Th2 immunity. Actually, DNA vaccines can benefit from the presence of an adjuvant that elicits a systemic sole Th2 immunity to enhance the initially weak immune response characteristic of these vaccines. While in the tolerogenic vaccines, cytokines are the endogenous immunomodulators, in the immunogenic vaccines, the adjuvants are exogenous agents that elicit Th2 immunity with a production of anti-inflammatory cytokines and antibodies against the self-antigen. Because the commonly used Th2 adjuvant alum, fails to induce an effective immunity in the elderly population, it is unlikely that it would be widely used. Another Th2 adjuvant, the oil/water emulsions mixed with the antigen, while effective in vaccines against infectious agents, due to potential aldehydes in their formulation may be not suitable for autoimmune vaccines. A unique compound is glatiramer, which seems to be both a random polypeptide antigen and an immune modulator that biases the response to Th2 immunity. Its mechanism of action seems to implicate binding to MHC-II, which alters the outcome of T-cell signaling, leading to anergy. Glatiramer, while effective in the treatment of multiple sclerosis has not shown efficacy in other autoimmune diseases. An important new group of promising sole Th2 adjuvants are the fucosylated glycans, which by binding to DC-SIGN bias dendritic cells to Th2 immunity while inhibiting Th1/Th7 immunities. These glycans are similar to those produced by parasitic helminths to prevent inflammatory responses by mammalian hosts. A novel group of sole Th2 adjuvants are some plant-derived fucosylated triterpene glycosides, which share the immune modulatory properties from the fucosylated glycans. These glycosides have also an aldehyde group that delivers an alternative co-stimulatory signal to T-cells, averting the anergy associated with aging due to the loss of the CD28 receptor on T-cells. Hence, the development of vaccines to treat and/or prevent autoimmune conditions and some proteopathies, will significantly benefit from the availability of new sole Th2 adjuvants that while inducing an anti-inflammatory immunity, they do not abrogate pro-inflammatory Th1/Th17 immunities.

Facing Alzheimer's disease in the developing countries / Enfrentando la enfermedad de Alzheimer en los países en desarrollo

Although the increase in the Alzheimer's disease (AD) in developed countries will be limited, in the developing countries of Latin America, Asia and Africa, it will be by the year 2050 three times that of the developed countries. That some correlations between AD and risk factors valid for developed countries are not as robust when compared to those in development, suggests differences in drug development. Due to the high cost of medicines from industrialized countries, it is doubtful that AD treatment would be a priority in the developing ones; thus, a better strategy would be to prevent or delay the disease. Of the options, the AD vaccine is the most favorable despite that all the attempts to develop it have failed, a result of products developed using inadequate information, which have fostered a negative opinion of this vaccine. Yet, the available information shows that these vaccines' antigens probably induced a detrimental immunity and that the inflammatory response elicited by some of them aggravated the onset of AD. Because of immunosenescence and the irreversible damage caused by AD, it is doubtful that vaccines would have practical value in treating advance disease. Yet, the available information provides basis for designing vaccines to prevent and/or retard this disease; information that has been ignored and the vaccines to prevent AD in fact discarded. Nonetheless, there is now a good understanding of the antigens needed to induce a protective immunity and the adjuvants required to stimulate production of protective antibodies while suppressing harmful inflammatory immune responses. Considering the limited choices that the developing countries have to control the AD epidemic, it would be sound if they collaborate and use the available knowledge to develop vaccines to prevent/delay AD. This development would yield both medical and economic benefits for Latin America, as well as other regions of the world.

Aunque el incremento de casos de enfermedad de Alzheimer (EA) en los países desarrollados será limitado, en los países en desarrollo de América Latina, Asia y África será en el año 2050 tres veces el de los países desarrollados. Que ciertas correlaciones entre la EA y factores de riesgo válidas para los países desarrollados no son tan categóricas cuando se aplican a los países en desarrollo, sugiere diferencias en el desarrollo de fármacos para esta enfermedad. Es improbable que, debido al costo de las medicinas de los países industrializados, el tratamiento de la EA sea una prioridad en los países subdesarrollados; por lo tanto, una estrategia más efectiva sería el prevenir o retrasar esta enfermedad. De las opciones, la vacuna contra la EA es la más favorable a pesar de que todos los intentos para producirla han fracasado, resultado de productos desarrollados usando información insuficiente; lo que ha fomentado una opinión negativa de esta vacuna en los círculos científicos. Sin embargo, la información disponible revela que los antígenos de esas vacunas posiblemente indujeron una inmunidad perjudicial y que la respuesta inflamatoria estimulada por varias de esas vacunas agravó el inicio de EA. Además, debido al daño irreversible causado por la EA y la inmunosenescencia, es poco probable que las vacunas terapéuticas tengan valor práctico en el tratamiento de la EA en estado avanzado. Sin embargo, esta información provee bases sólidas para diseñar nuevas vacunas para prevenir o retardar esta enfermedad; información que ha sido ignorada y las vacunas para prevenir la EA, de hecho descartadas. No obstante, ahora hay un mejor conocimiento de los inmunógenos necesarios para inducir una inmunidad protectora y de los adyuvantes requeridos para estimular la producción de anticuerpos protectores e inhibir la inmunidad inflamatoria perjudicial. Considerando las opciones limitadas que tienen los países en desarrollo para controlar la epidemia de EA, sería razonable si estos países colaboraran y usaran los conocimientos disponibles para desarrollar vacunas para prevenir/retrasar la EA. Un avance que produciría beneficios médicos y económicos para América Latina y otras regiones del mundo.

Rejecting the Alzheimer's disease vaccine development for the wrong reasons.

The development of amyloid ß (Aß) vaccines for Alzheimer's disease (AD) has consistently failed clinically, an outcome that is assumed to result from flaws in the proposed role of Aß as the crucial causative agent of this disease. This opinion resulted in this research approach being disregarded, yet, review of the development of these vaccines indicates that they are more suited to transgenic mice, which is unsurprising given that these animal models were used to determine the efficacy of these vaccines, and that the approach overlooked research findings relevant to AD vaccines. Hence, new strategies using new immunogens and anti-inflammatory adjuvants mimicking the natural protective immunity against AD should be implemented to develop effective preventive vaccines.

A retrospective analysis of the Alzheimer's disease vaccine progress - The critical need for new development strategies.

The promising results obtained with aducanumab and solanezumab against Alzheimer's disease (AD) strengthen the vaccine approach to prevent AD, despite of the many clinical setbacks. It has been problematic to use conjugated peptides with Th1/Th2 adjuvants to induce immune responses against conformational epitopes formed by Aß oligomers, which is critical to induce protective antibodies. Hence, vaccination should mimic natural immunity by using whole or if possible conjugated antigens, but biasing the response to Th2 with anti-inflammatory adjuvants. Also, selection of the carrier and cross-linking agents is important to prevent suppression of the immune response against the antigen. That certain compounds having phosphorylcholine or fucose induce a sole Th2 immunity would allow antigens with T-cell epitopes without inflammatory autoimmune reactions to be used. Another immunization method is DNA vaccines combined with antigenic ones, which favors the clonal selection and expansion of high affinity antibodies needed for immune protection, but this also requires Th2 immunity. Since AD transgenic mouse models have limited value for immunogen selection as shown by the clinical studies, screening may require the use of validated antibodies and biophysical methods to identify the antigens that would be most likely recognized by the human immune system and thus capable to stimulate a protective antibody response. To induce an anti-Alzheimer's disease protective immunity and prevent possible damage triggered by antigens having B-cell epitopes-only, whole antigens might be used; while inducing Th2 immunity with sole anti-inflammatory fucose-based adjuvants. This approach would avert a damaging systemic inflammatory immunity and the suppression of immunoresponse against the antigen because of carrier and cross-linkers; immune requirements that extend to DNA vaccines.

Is fucose the answer to the immunomodulatory paradox of Quillaja saponins?

Quillaja saponins, e.g. QS-21, are immunomodulating aldehyde-carrying triterpene glycosides, which depending on the acylation state of their single fucosyl residue (Fucp) induce either Th1/Th2 or Th2 immunity. Indeed, their changes in immunomodulation or adjuvanticity from Th1/Th2 to sole Th2 immunity, correlate with the presence of acylated and de-acylated Fucp residues, respectively. Thus, it is possible to infer that the single Fucp residue is responsible for the Th2 immunity biasing induced by de-acylated Q. saponins (QT-0101). That removal of the fucosylated oligosaccharide from de-acylated Q. saponins results once more in the induction of Th1/Th2 immunity supports the Fucp role in polarizing the response toward Th2 immunity. From structural and functional analogies with the helminths' fucosylated glycans, it is possible to infer that these saponins' Fucp must bind to the lectin DC-SIGN on dendritic cells (DC). This binding to DC-SIGN, a C-type lectin that shows significant pliability in its binding interactions, must result in polarization toward Th2 while inhibiting Th1 immunity. Apparently, acylation of the Fucp by large fatty acids sterically hinders this sugar from binding to DC-SIGN, preventing a biasing to Th2 immunity. Evidently, de-acylation of Q. saponins may negatively affect vaccines requiring Th1 immunity for immune protection, particularly those against pathogens that use DC-SIGN to infect DCs and modulate Th2 immunity. However, it could be valuable in vaccines that require a sole Th2 immunity, like those against proteinopathies, e.g. Alzheimer's disease. Hence, it would valuable to elucidate the possible interactions between DC-SIGN and the QT-0101 immunomodulator.

Alzheimer's disease vaccine development: A new strategy focusing on immune modulation.

Despite significant advances in the development of Alzheimer's disease (AD) vaccines effective in animal models, these prototypes have been clinically unsuccessful; apparently the result of using immunogens modified to prevent inflammation. Hence, a new paradigm is needed that uses entire AD-associated immunogens, a notion supported by recent successful passive immunotherapy results, with adjuvants that induce Th2-only while inhibiting without abrogating Th1 immunity. Here, we discuss the obstacles to AD vaccine development and Th2-adjuvants that by acting on dendritic and T cells, would elicit regardless of the antigen a safe and effective antibody response, while preventing damaging neuroinflammation and ameliorating immunosenescence.

Enfermedad de Alzheimer: hacia el diseño racional de una vacuna eficaz / Alzheimer's disease: toward the rational design of an effective vaccine

The promising clinical results with the human monoclonal antibodies aducanumab and solanezumab targeting β-amyloid in AlzheimerÆs disease treatment, confirm both the amyloid cascade hypothesis and protective natural immunity, while strengthening the immunotherapeutic approach. That aducanumab recognizes a conformational epitope formed by oligomers emphasizes the need for whole β-amyloid, not just its B-cell epitopes as have been the norm to avoid pro-inflammatory Th1-reactions.That truncated β-amyloid having N-terminal pyroglutamate is present only in diseased brain simples a new useful vaccine antigen. Another relevant antigen is the tau protein, which shows a close association and cooperativity with β-amyloid in exacerbating this disease. Hence, effective vaccines may be polyvalent, presenting to the immune system a number of antigens relevant to induce an immune response to prevent or slowdown the onset of this disease. The presence of both B and T cell epitopes in the antigens, require a sole Th2 immunity to avert brain inflammation; a task that cannot be attain with adjuvants that under any conditions induce Th1 and/or Th17 immunities. Hence, new vaccine adjuvants are need to safely induce Th2 while inhibiting Th1 immunity, an objective that can be achieved with certain fucosylated glycans or triterpene glycosides, which apparently bind to the DC-SIGN lectin on dendritic cells polarizing the immune response toward Th2 immunity. Because the triterpene glycosides have the pharmacophore needed to co-stimulate T cells, they may ameliorate the T-cell anergy associated with immunosenescence and responsible for poor vaccine efficacy in the elderly population, a critical issue for an AlzheimerÆs vaccine...

Los resultados prometedores en el tratamiento de la enfermedad de Alzheimer con Aducanumab y Solanezumab, anticuerpos monoclonales humanos contra β-amiloide, ratifican la hipótesis de la cascada del amiloide y la existencia de inmunidad natural contra Alzheimer, mientras refuerza el método inmunoterapéutico. Que Aducanumab reconoce un epίtopo conformacional formado por oligómeros, acentúa la necesidad del β-amiloide completo y no solo sus epίtopos de células B, como ha sido la norma para evitar reacciones pro-inflamatorias Th1. De que el β-amiloide truncado con piroglutamato en su extremo N-terminal se encuentra solo en cerebros enfermos, es un antígeno útil; otro antígeno importante es la proteína tau, que tiene una estrecha asociación y cooperatividad con β-amiloide en exacerbar esta enfermedad. Una vacuna eficaz puede ser polivalente para presentar al sistema inmunológico una variedad de antígenos importantes e inducir una respuesta para prevenir o retardar el comienzo de esta enfermedad. La necesidad de epítopos de células B y T en los antígenos, implica una inmunidad tipo Th2 para evitar inflamación del cerebro; objetivo que no se puede alcanzar con adyuvantes que inducen inmunidades Th1 y/o Th17. Consecuentemente, se necesitan nuevos adyuvantes de vacunas para inducir sin riesgos la inmunidad Th2 mientras se inhibe la Th1, objetivo que se puede lograr con ciertos glicanos fucosilados o glucósidos triterpénicos que se unen a la lectina DC-SIGN en células dendríticas, polarizando la respuesta hacia la inmunidad Th2. Como los glucósidos triterpénicos tienen el farmacóforo necesario para co-estimular las células T, podrán moderar la anergia de las células T asociada con immunosenescencia, responsable por la baja eficacia de las vacunas en la población anciana, materia critica para vacunas contra la enfermedad de Alzheimer...

New Th2 adjuvants for preventive and active immunotherapy of neurodegenerative proteinopathies.

Active immunotherapy of neurodegenerative proteinopathies, such as Alzheimer's disease, requires a Th2 antibody immune response with exclusion of damaging inflammatory Th1 immunity. Because these diseases affect the elderly whom, owing to immunosenescence, are frequently immune compromised, a successful therapy would require immune agonists capable of eliciting Th2 immunity solely while ameliorating the immune decline linked to aging; an objective hampered by the scarcity of Th2 immune agonists. The fact that some helminth-derived glycans and modified triterpene glycosides elicit Th2 immunity only, even when administered with antigens carrying T cell epitopes, presents new ways to improve the active immune therapy of proteinopathies. Of additional benefit is that these triterpene glycosides could amend some of the detrimental effects of the immunosenescence.

Effect of an immune enhancer, GPI-0100, on vaccination with live attenuated herpes simplex virus (HSV) type 2 or glycoprotein D on genital HSV-2 infections of guinea pigs.

These studies were performed to determine the effect of AD-472, an attenuated human herpes simplex virus (HSV) type 2 or HSV-2 glycoprotein D (gD) when combined with an adjuvant, GPI-0100, a semi-synthetic Quillaja Saponin analog in a genital HSV-2 infection in guinea pigs. While animals immunized with either vaccine had reduced clinical disease, GPI-0100 only improved the efficacy of gD and did not affect the efficacy of the live vaccine. Neither vaccine had any therapeutic effect if administered 24 h after viral infection.

Effect of immunization with herpes simplex virus type-1 (HSV-1) glycoprotein D (gD) plus the immune enhancer GPI-0100 on infection with HSV-1 or HSV-2.

These studies were performed to determine the effects of GPI-0100, a semi synthetic Quillaja Saponin analog, formulated with herpes simplex virus type-1 (HSV-1) glycoprotein D (gD) on immunity to HSV. SKH-1 hairless mice, used as a model of herpes labialis, inoculated with HSV-1 results in facial lesions, virus replication and mortality. Mortality rates, lesion scores and viral titers were significantly reduced in SKH-1 mice immunized with gD/GPI-0100 prior to cutaneous inoculation with HSV-1 and the protective effects were greater than those using the standard alum adjuvant. Genital HSV-2 infections in guinea pigs were also utilized to determine if gD combined with GPI-0100 was protective against infection, disease severity and viral shedding. Guinea pigs immunized with HSV-1 gD with or without GPI-0100 had significantly reduced area under the curve lesion scores, but infection rates and virus shedding was not altered. When Tween 40 was added to gD and GPI-0100, mean peak lesion scores were also significantly reduced. The results obtained in a genital HSV-2 infection of guinea pigs did not indicate enhanced protection or reduced virus shedding following immunization with GPI-0100 and gD. There was, however, a significant improvement in clinical herpetic genital disease with the combination of gD plus the immune enhancer GPI-0100.

Vaccine adjuvants: role and mechanisms of action in vaccine immunogenicity.

Inactivated vaccines require adjuvants to stimulate an immune response. The choice of adjuvant or immune enhancer determines whether the immune response is effective, ineffective or damaging. Accordingly, there is a need for new adjuvants that stimulate the appropriate immunity, for example, T cell immunity for intracellular pathogens and cancer vaccines. In several adjuvants, the identification of chemical groups that interact with specific cell toll-like receptors (innate immunity) or receptors for co-stimulatory ligands (adaptive immunity), has enabled the establishment of structure-function relationships that are useful in the design of new adjuvants. Because of the crucial immunomodulating role of adjuvants, sub-unit vaccine development will remain dependent on new adjuvants.

Effectiveness of the quillaja saponin semi-synthetic analog GPI-0100 in potentiating mucosal and systemic responses to recombinant HagB from Porphyromonas gingivalis.

The gram-negative, anaerobic bacterium Porphyromonas gingivalis, has been implicated in the etiology of adult periodontal disease. Among the potential virulence factors of this bacterium, the non-fimbrial adhesin hemagglutinin B (HagB) appears to be involved in the initial adherence of the bacteria to host tissue and the induction of anti-HagB antibody responses affords some protection from experimental alveolar bone loss. In the present study, we have investigated the ability of the quillaja saponin derivative GPI-0100 to act as an immunostimulant of responses to HagB following subcutaneous (s.c.) or intranasal (i.n.) immunization of mice. We have also compared the immunopotentiating ability of GPI-0100 with that of five other adjuvants. Evidence is provided that GPI-0100 was more effective than monophosphoryl lipid A and alum in inducing serum anti-HagB responses following s.c. immunization. A comparison of the responses induced following i.n. immunization with HagB and adjuvant revealed that the heat-labile toxin of Escherichia coli (LT) and the non-enzymatic mutant LT (E112K), followed by GPI-0100 potentiated higher serum and mucosal anti-HagB antibody responses, which in most cases were higher than those seen with the other adjuvants tested (i.e. monophosphoryl lipid A, alum and the B subunit of cholera toxin). Furthermore, a difference was seen in the nature of the serum IgG anti-HagB response based on the adjuvant used and route of immunization. These results demonstrate the effectiveness of GPI-0100 as both a systemic and mucosal adjuvant and support its potential use in the development of vaccines against periodontal, as well as other pathogens.

Fractionation, structural studies, and immunological characterization of the semi-synthetic Quillaja saponins derivative GPI-0100.

Unfractionated GPI-0100 (UFGPI-0100) containing semi-synthetic derivatives of deacylated Quillaja saponins (DS saponins) modified at the glucuronic acid residue was resolved by reverse phase low-pressure liquid chromatography (RP-LPLC) into two fractions, RP18-1 and RP18-2, with different compositions and adjuvanticity. The fraction RP18-1 contained DS saponin adducts of N-dicyclohexylurea, and stimulated Th2 immunity with production of IgG1, while the RP18-2 fraction contained the dodecylamide derivatives of DS saponins and stimulated Th1 immunity with production of IgG2a, IFN-gamma, IL-2, and CTL. The strong immune stimulatory properties of RP18-2, relative to RP18-1, and the formation of RP18-1/RP18-2 mixed micelles may account for the effective stimulation of Th1 immunity by UFGPI-0100. UFGPI-0100 was free of acylated quillaja saponin components, including the more stable QS-7.