As late as 1989, Janeway aptly called adjuvants: "the immunologist's dirty little secret". This statement reflected the ignorance on the mechanisms of action of most known adjuvants. Yet, rational vaccine design involves a logical choice of adjuvant based on a knowledge of their mode of action and their effects on product efficacy and safety. However, even today the key processes critical for immune induction in general and those evoked by vaccine adjuvants in particular are being disputed among immunologists. The 4 most important concepts likely to explain some of the mechanisms of vaccine adjuvants. They include:

• the geographical concept of immune reactivity
• the depot concept : formation of a deposit of Ag at the site of inoculation, with slow release; provision of a vehicle for transport of emulsified Ag throughout the lymphatic system to distant places, such as lymph nodes and spleen, where new foci of Ab formation can be established. The adjuvant emulsion may be widely disseminated in various organs, depending on the route of inoculation, with the development of focal granulomatous lesions at distal places.
• the hypothesis of pathogen-structure recognition : stimulation of macrophage activation and lymphocyte proliferation by nonspecifically enhancing the costimulatory signals (CDs and cytokines) provided by APC to T_h cell
• the damage/endogenous danger theory

• inducing T_h2-polarized immune response
• mineral compounds
• aluminium-adsorbed or aluminium-adjuvanted vaccines : despite conflicting results, they are universally used as adjuvants for the DPT vaccine. Type I hypersensitivity reactions following their administration have been reported.
• aluminum hydroxide gel (Alhydrogel^®) obtained by precipitation of aluminum hydroxide Al(OH)₃ under alkaline conditions (alum  = K₂Al₂(SO₄)₄ ^. 24H₂O) : the most widely used in vaccines for Homo sapiens together with MF59. It activates T_h2 lymphocytes and induces IgE and IgG₁ production.
• aluminium phosphate Al(PO₄)
• calcium phosphate Ca₃(PO₄)₂ is a normal constituent of the body and is better tolerated and absorbed than other adjuvants. It entraps Ags very efficiently and allows slow release. Additionally, it elicits high amounts of IgG Abs an much less of IgE Abs.
• magnesium hydroxide Mg(OH)₂
• cerium nitrate Ce(NO₃)₄
• zinc sulphate ZnSO₄
• colloidal iron hydroxide
• calcium chloride CaCl₂
• water-in-mineral oil emulsions
• Adjuvant 65 : trademark for a water-in-oil emulsion containing antigen in peanut oil with Arlacel A (mannide monooleate) and aluminum monostearate as the emulsifying agent
• incomplete Freund’s adjuvant (IFA) / Freund's incomplete adjuvant (FIA) (source : Statens Seruminstitut, Copenhagen, Denmark) : mixture of mineral oil (Marco 52) and emulsifier (Arlacel A [mannide monooleate], Drakeol 6VR, and Montanide ISA-51) as an emulsion of 85% mineral oil and 15% emulsifier. Used mainly for boosting after immunizing with CFA^ref. Freund’s adjuvants can be dangerous due to :
• immediate release of more than the tolerated amount of properly emulsified vaccine in sensitive persons
• breaking of the emulsion with the release of all or part of the full content of the allergen within a brief period of time
• type I hypersensitivity reactions
• long-term delayed reactions, including the development of nodules, cysts or sterile abscesses requiring surgical incision.
May cause granulomas and abscesses at the site of injection. Induces production of ascites in BALB/C mice when injected i.p. with antigen^ref. [Herbert, W. J., 1967, Methods for the preparation of water-in-oil, and multiple, emulsions for use as antigen adjuvants; and notes on their use in immunization procedures. Handbook of Experimental Immunology, D. M. Weir and Blackwell, Eds., 1207-1217]
• Montanide ISA 720 (source : Seppic) : a highly refined emulsifier from the mannide monooleate family in a natural metabolizable oil solution. The exact nature of the emulsifier and the metabolizable in MONTANIDE ISA 720 is proprietary, but can be disclosed under specific agreement. "Ready to use" oil for water-in-oil emulsion adjuvants. Final injection product usually 70% Montanide ISA 720 (0.5-1 mL injection volume). LD50 (mice) by i.p. route, > 22g/kg. LD50 (rats) by oral route, > 2 g/kg, Non irritating (skin) in rabbits, Slight irritancy (ocular) in rabbits. No abnormal toxicity in mice or guinea pigs. Acute toxicity by i.m. injection (rats), > 5g/kg. Pyrogen-free. Ames and Mouse Micronucleus test (Montanide 80) - no effect^{ref1, ref2}[Elliot, S. et al., 1994, Human compatible adjuvant induces protective cytotoxic T lymphocytes JIM peptide

vaccine - Proceedings in CHI Vaccines - New Technologies and Applications -Alexandria VA, March 21-23]
• Marcol 52:Montanide 888 6:1 or 9:1
• Montanide 80^® or Marcol 82^®
• Span Tween Marcol (STM)
• squalene-oil-water emulsion (see also Gulf War syndrome (GWS))
• MF59 : together with aluminum-based salts, this is the only other adjuvant approved for clinical use
Because of the difficulty of removing squalene-containing fingerprint oils from laboratory glassware, it is hard to know whether the squalene is truly present in some lots of the vaccine or is introduced by the testing process itself. DoD, the Food & Drug Administration (FDA), and several civilian advisory committees agree that squalene at such low levels has no adverse health consequences. In September 2000, DoD became aware of FDA test results finding trace amounts of squalene in 3 out of 3 US vaccines tested: tetanus, diphtheria, and anthrax. The level of squalene identified by the FDA test is so minute that it is likely the result of squalene in the oil of a fingerprint not completely cleaned from lab glassware. Before they go looking for squalene, lab workers have to use a chemical solvent such as hexane to completely remove their own squalene-containing fingerprint oils from lab glassware. When lab workers intentionally tested an extract of fingerprint oil, the squalene reading went off the chart. Before the FDA test results became known, Stanford Research International (SRI), under DoD contract, looked for squalene in anthrax vaccine. At the limit of detection of its test, 140 parts per billion, SRI found no squalene in several lots of anthrax vaccine. The FDA's test, which was developed later, is more sensitive. It is able to detect as little as 10 parts per billion. The FDA found squalene at 10 to 83 parts per billion in diphtheria toxoid, tetanus toxoid, and anthrax vaccine. The trace level of squalene found by the FDA in anthrax vaccine is less than the concentration naturally present in human blood (250 parts per billion)^{ref1, ref2, ref3}. After the FDA reported its results, DoD asked SRI to refine its assay. Using an improved method that could detect as little as one part per billion, SRI found no squalene in 32 out of 33 lots of anthrax vaccine tested (including lots in which FDA found low levels of squalene). In one lot, they found up to 9 parts per billion. At the initial limit of detection of its test, 140 parts per billion, SRI found no squalene in anthrax vaccine^ref. It was scientifically proper to say "no squalene was found to the limit of detection of the assay," which DoD officials sometimes oversimplified to say "there is no squalene present." Squalene is not added to anthrax vaccine or any US-licensed vaccine. The United Kingdom's Ministry of Defense arranged for an independent laboratory to test 11 lots of the British anthrax vaccine manufactured at Porton Down, as well as other vaccines. No squalene was detected in those lots of vaccine, with a limit of detection of 0.1 mg/ml (100 parts per billion)^ref. 4 independent civilian panels considered the February 2000 article by Asa and colleagues^ref and other allegations related to squalene and anti-squalene antibodies.
• when the Institute of Medicine (part of the National Academy of Sciences) Committee on Gulf War and Health (the "Sox committee") evaluated the 2000 Asa claims of anti-squalene antibodies in the blood of ill Gulf War veterans, it concluded that the paper contains shortcomings, some serious, that combine to invalidate the authors' conclusions. The report says: "The committee does not regard this study as providing evidence that the investigators have successfully measured antibodies to squalene"^ref.
• the civilian experts on the Armed Forces Epidemiological Board (AFEB) said in July 2000, "the research reported in this paper does not support this claim; it remains unclear if the assay actually measures antibodies to squalene, as the authors asserts"^ref. Regarding assertions that Service Members who received anthrax vaccination from the 5 lots cited in the FDA squalene tests experienced more or more severe adverse events after vaccination, the civilian physicians on the Anthrax Vaccine Expert Committee (AVEC) evaluated adverse events by lot and geographic location. They found no meaningful differences based on lot or on geographic location^{ref1, ref2}. Of note, the 5 lots cited in the FDA squalene tests were shipped to multiple DoD installations. In addition, Dover AFB received lots other than the 5 lots mentioned above. After the comprehensive review of anthrax vaccine safety by the National Academy of Sciences (the "Strom Committee," March 2002^ref), which included hearing from personnel, from Dover AFB and elsewhere, concerned that they suffered adverse events after anthrax vaccination, the civilian physicians and scientists concluded that "The [SRI] study report, dated 14 Aug 2001, found that one lot of over 30 lots tested contained measurable levels of squalene. 3 samples from that lot [FAV008] contained squalene at 7, 9, and approximately one parts per billion, respectively. Use of vaccine from that lot has not been associated with elevated rates of adverse events. Because the available data ... demonstrate that the presence of trace amounts of squalene is not associated with an increase in the rates of adverse events following vaccination with AVA, the committee concludes that further investigation of possible AVA contamination is not warranted at this time.".
• the 3rd study from this research effort, published in 2004, adapts the test described above, so that it could detect anti-squalene antibodies if present in human serum. Serum from 3 groups of people were tested: retired employees of the US Army Medical Research Institute of Infectious Diseases (average 68 years of age, 88% of whom received anthrax vaccine, mean = 26 doses per person), civilian volunteers of similar age from Frederick, Maryland (none of whom received anthrax vaccine), and random blood donors from Fort Knox, Kentucky (vaccination status unknown). This next study indicates that anti-squalene IgG are found in 7.5% of the vaccinated USAMRIID alumni, 15% of the unvaccinated Frederick civilians, and in 0% of the Fort Knox blood donors. Researchers found IgM in all 3 groups (37%, 32%, 19%). The researchers found that anti-squalene antibodies are more common with increasing age (a characteristic also found in mice). The presence of anti-squalene antibodies was unrelated to anthrax vaccination status. They concluded that anti-squalene antibodies occur naturally in humans^ref
• a panel of civilian physicians known as the Anthrax Vaccine Expert Committee (AVEC), selected by the Department of Health & Human Services reviewed all reports of adverse events after anthrax vaccination from 1998 to 2001^{ref1, ref2}. To evaluate assertions that Service Members who received anthrax vaccination from the 5 lots cited in the FDA squalene tests experienced more or more severe adverse events after vaccination, these civilian physicians evaluated adverse events by lot and geographic location. They found no meaningful differences based on lot or on geographic location.
• inducing T_h1-polarized immune response
• bacterial products :
• acting as agonists of TLRs on APCs (see also xenogenic TLR9 agonists used as immunostimulants) :
• mycobacterial products
• complete Freund’s adjuvant (CFA) / Freund's complete adjuvant (FCA) / CIA^ref(source : Statens Seruminstitut, Copenhagen, Denmark) consists of the same components of IFA but  with 500 µg heat-killed and dried Mycobacterium tuberculosis or Mycobacterium butyricum per mL of emulsifier mixture (main mycobacterial component is D-wax). It is an agonist of TLR2 on APCs : it induces IgG_2a Ab production without inducing IgE Ab production^ref. CFA is the most used priming adjuvant in experimental studies but it is not suited for human vaccination because it may cause granulomas and abscesses at the site of injection. May cause arthritis, amyloidosis and allergic reactions. Causes ascites, production in BALB/C mice when injected i.p. with antigen^ref. The ethics of using CFA in animals are at present disputed, due to the profile of severe side-effects. Store at 2-8° C. Do not freeze the final emulsion, as it is disrupted by freezing. [Herbert, W. J., 1967, Methods for the preparation of water-in-oil, and multiple, emulsions for use as antigen adjuvants; and notes on their use in immunization procedures. In: Handbook of Experimental Immunology, D. M. Weir and Blackwell, Eds., 1207-1217]
• live bacille Calmette-Guérin (BCG) (Tice BCG^®, Theracys^®) : an attenuated strain of Mycobacterium bovis used also for vaccination against Mycobacterium tuberculosis. The incidence of acute leukemia appeared to be decreased in children who received BCG vaccination. Prospective confirmatory trials were discouraged by the results of other retrospective trials that suggested that BCG vaccination in Puerto Rico was associated with a slight excess of lymphomas. BCG-activated killer (BAK) cells are natural killer (NK) lymphocytes stimulated by IFN-g and IL-2 produced by BCG-activated T_h1 lymphocytes. Local complications of BCG administration have included erythema, induration, pruritus, and ulceration at injection sites, associated with regional lymphadenopathy. Systemic reactions have been observed more frequently after intralesional (e.g. cancer) injection than after intradermal administration of the vaccine. Chills, fever, and malaise have been observed after repeated injections. Rarely, erythema nodosum, granulomatous hepatitis, anaphylaxis, and shock associated with disseminated intravascular coagulation (DIC) have been observed. Progressive BCG infection has occurred in a small number of profoundly immunosuppressed patients. When recognized, BCG infection has responded to treatment with multiple antituberculous drugs.
• methanol extraction residue (MER) of BCG tubercle bacillus
• muramyldipeptide (MDP) = N-acetyl-muramyl-L-Ala-D-isoGln. MDPs are potent pyrogens and their action is not completely understood. Following parenteral administration, MDP is cleared from the circulation within 60', an interval that does not permit systemic activation of macrophages. Greater antitumor activity has been obtained with muramyl tripeptide phosphatidylethanolamine (MTP-PE), a lipophilic derivative of water-soluble MDP that can associate more effectively with liposomes (L-MTP-PE).
• D-wax / tubercle bacillus wax : a high-molecular-weight phosphatidic glycolipid extracted from the cell walls of Mycobacterium tuberculosis, made up of arabinoglycans and mycolic and muramic acids. It is used as an adjuvant to enhance the immunogenicity of tuberculin preparations. It induces IgG₂.
• SRL172 : heat-killed Mycobacterium vaccae
• Mycobacterial Cell Wall-DNA Complex (MCC) (source : Bioniche Life Sciences)
• HIV gp120 DNA vaccine mixed with Ag85B DNA as an adjuvant induced HIV gp120-specific Th1 responses, as shown by delayed-type hypersensitivity, cytokine secretion, and increasing HIV-specific CTL responses. Moreover, these responses were enhanced in mice primed with Mycobacterium bovis bacillus Calmette-Guerin (BCG) before immunization of HIV DNA vaccine mixed with Ag85B DNA. Furthermore, these immunized mice showed substantial reduction of HIV gp120-expressing recombinant vaccinia virus titers compared with the titers in other experimental mice after recombinant vaccinia virus challenge. Because most humans have been sensitized by spontaneous infection or by vaccination with mycobacteria, these findings indicate that Ag85B is a promising adjuvant for enhancing CTL responses in a DNA vaccination strategy^ref.
• immunostimulatory (ISS) oligodeoxynucleotides (ODN)
• flagellin
• killed Bordetella pertussis : there are a number of admitted and well-describe reactions to pertussis toxin (PT), such as convulsion, infantile spasms, epilepsy, sudden infant death syndrome (SIDS), Reye's syndrome, Guillain-Barré syndrome, transverse myelitis and cerebral ataxia. Anyway chemically detoxified or genetically inactivated PT may not exhibit the adjuvant effects comparable to the native PT.
• Propionibacterium granulosum (a.k.a. Corynebacterium granulosum)-derived P40 component : it is a particulate fraction composed of the cell wall peptidoglycan associate with a glycoprotein. In animals, it displays a number of activities such as stimulation of the RES, enhancement of phagocytosis and activation of macrophages. P40 abolishes drug-induced immunosuppression and increase non-specific resistance to bacterial, viral, fungal and parasitic infections. It induces the formation of IL-2, TNF-a, IFN-a and IFN-g.
• killed Propionibacterium acnes(a.k.a. Corynebacterium parvum) (Coparvax^®)
• killed Listeria monocytogenes (KLM)
• Nocardia rubra cell wall skeletons (N-CWS) (Rubratin^®)
• lipopolysaccharide (LPS) increases the number of long-lived Ag-specific T cells, rescuing them from peripheral deletion. It is too toxic, even in minute doses, to be used as an adjuvant in humans
• very small size proteoliposomes (VSSP) : gangliosides incorporated into vesicles from Neisseria meningitidis. VSSP is a good alternative to the existing adjuvants for use in whole cells vaccines since it promotes 80% tumour rejection and growing delay in the CT26 and F3II tumour models respectively. Also VSSP induces activation of CTL responses to co-injected trimmed peptides and soluble proteins. This phenomena is facilitated by the cross-presentation of exogenous antigen and do not need cooperation of CD4 T cells for primary CD8 T cells expansion.
• liposome-Ag-nucleic acid complexes (LANAC) are particularly effective adjuvants for eliciting CD4⁺ and CD8⁺ T cell responses against peptide and protein Ags. Notably, LANAC containing TLR3 or TLR9 agonists effectively cross-primed CD8⁺ T cell responses against even low doses of protein Ags, and this effect was independent of CD4⁺ T cell help. Ag-specific CD8⁺ T cells elicited by LANAC adjuvants were functionally active and persisted for long periods of time in tissues. In a therapeutic tumor vaccine model, immunization with the melanoma peptide trp2 and LANAC adjuvant controlled the growth of established B16 melanoma tumors. In a prophylactic vaccine model, immunization with the Mycobacterium tuberculosis protein ESAT-6 with LANAC adjuvant elicited significant protective immunity against aerosol challenge with virulent M. tuberculosis. These results suggest that certain TLR agonists can be combined with cationic liposomes to produce uniquely effective vaccine adjuvants capable of eliciting strong T cell responses against protein and peptide Ags^ref
• NOD agonists that mimic the PGN moiety^ref
• NOD1 agonists : FK565 and FK156
• NOD2 agonists : muramyldipeptide (MDP)
• tetanospasmin from Clostridium tetani
• heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETECs) : mutants of LT have been generated that contain a single amino acid substitution within the A subunit and possess nondetectable levels of ADP-ribosyltransferase activity. The idea of using the Escherichia coli labile enterotoxin (LT) has been around for several decades, but was temporarily abandoned because it triggers a strong and dangerous immune reaction when it is added to the vaccine, swallowed or sprayed up the nose : applying it to the skin prompts a milder reaction.
• in vitro toxicity on Y1 adrenal cells is shown for LT wild type (LTwt), for the mutants generated by Chiron, which have modifications of the enzymatic active site of the A1 subunit (LTK63 and LTR72), and for an alternative mutant, LTR192G, which is modified in its ability to be enzimatically cleaved and activated by trypsin. LTK63 has no enzyme activity in the A1 subunit and shows no toxicity on Y1 cells, whereas LTR72 has residual enzyme activity (about 0.6% of LTwt) and shows residual toxicity, but is 100,000-fold less toxic than LTwt. By contrast, LTR192G shows only a marginal reduction in toxicity.
• similar results were obtained for in vivo toxicity in the standard rabbit ileal loop model, which shows significant fluid accumulation with LTwt. In contrast, LTK63 shows no fluid accumulation, even at the highest dose tested (1 mg), whereas LTR72 shows reduced fluid accumulation and the requirement for a higher dose to trigger this effect. LTR192G shows high levels of fluid accumulation, even at low doses, similar to LTwt, probably because there are alternative enzymes present that can cleave the molecule, in addition to trypsin^{ref1, ref2}

LT has been shown to penetrate intact skin and to activate adaptive immunity. A nontoxic mutant, nLT, and its B subunit (LTB), have been evaluated separately for their potential use as a tool for transcutaneous delivery of antigens for cancer immunotherapy. FITC-labeled nLT is taken up by human dendritic cells (hDC) in vitro and in mouse skin, and induces maturation and activation of hDC in vitro. hDC matured with nLT enhanced nonspecific melanoma antigen uptake and presentation to autologous CD8⁺ T cells. In mouse in vivo studies, nLT or LTB were applied on the skin either mixed with recombinant gp100 or genetically fused with a multiepitope polypeptide (MEP). Fused LTB-MEP induced antibody production that was dependent on LTB cell binding. LT derivatives may be useful for the transcutaneous delivery of tumor antigens for cancer immunotherapy^ref.
• B subunit of cholera exotoxin from Vibrio cholerae increases Ag capture by cells expressing the receptor GM₁ ganglioside (including M cells and enterocytes in gut lining epithelium => so it is extensively used for edible (oral) subunit vaccines
• diphtheria toxoid CRM₁₉₇ from Corynebacterium diphtheriae is an immunogenic carrier for carbohydrate antigens
• lentinan is a b(1->3) glucan with b(1->6) branches.
• virosomes are lipidic envelopes devoid of genetic information, but which retain the antigenic profile and fusogenic properties from their viral origin. Virosomes are versatile antigen carriers and can be engineered to perform various tasks in cancer immunotherapy. Preclinical data have fostered the development of innovative clinical protocols. Hence, immunopotentiating reconstituted influenza virosomes will be assessed in breast and melanoma immunotherapy, and may contribute to the development of clinically effective cancer vaccines and ultimately improve patient outcomes. The objective of this review is to provide an overview of the potential clinical applications of virosomes as innovative and potentially effective reagents in active specific cancer immunotherapy^ref
• fungal products :
• polysaccharide-K (PSK / PS-K) (Krestin^®) : a protein-bound polysaccharide from the Basidiomycetes Coriolus vesicolor that has been shown to exhibit antitumor activity against a variety of tumor cells such as gastric, colorectal, and lung cancers. The antitumor effects of PSK have been also considered to be caused by its activity as an immunomodulator. The precise mechanism at the molecular level remains unknown. Previously, we found that PSK can significantly inhibit the actin-activated Mg²⁺-ATPase activity of myosin from rabbit skeletal muscle
• b-glucans administered intravenously and orally promote tumor regression and survival by priming granulocyte and macrophage CR3 / iC3bR / CD11b/CD18 to trigger the cytotoxicity of tumor cells opsonized with iC3b via anti-tumor Abs. Despite evidence for priming of macrophage CR3 by oral b-glucan in vivo, the current study in C57BL/6 and BALB/c mice showed that granulocytes were the essential killer cells in mAb- and oral b-glucan-mediated tumor regression, because responses were absent in granulocyte-depleted mice. Among granulocytes, neutrophils were the major effector cells, because tumor regression did not occur when C5a-dependent chemotaxis was blocked with a C5aR antagonist, whereas tumor regression was normal in C3aR^-/- mice. Neutrophil recruitment by C5a in vivo required amplification via LTB₄, because both C5a-mediated leukocyte recruitment into the peritoneal cavity and tumor regression were suppressed in LTB₄-R-deficient (BLT₁^-/-) mice^ref. Tumor-bearing mice treated with a combination of b-glucan and an anti-tumor mAb show almost complete cessation of tumor growth. This activity evidently derives from a 25-kDa fragment of b-glucan released by macrophage processing of the parent polysaccharide. This fragment, but not parent b-glucan, binds to neutrophil CR3, induces CBRM 1/5 neoepitope expression, and elicits CR3-dependent cytotoxicity. These events require phosphorylation of the tyrosine kinase, Syk, and consequent PI3K activation because b-glucan-mediated CR3-dependent cytotoxicity is greatly decreased by inhibition of these signaling molecules. Thus, b-glucan enhances tumor killing through a cascade of events, including in vivo macrophage cleavage of the polysaccharide, dual CR3 ligation, and CR3-Syk-PI3K signaling. These results are important inasmuch as b-glucan, an agent without evident toxicity, may be used to amplify tumor cell killing and may open new opportunities in the immunotherapy of cancer^ref.
• vegetal products :
• saponins^ref isolated from the bark of Quillaja saponaria mollina tree (species native to Chile and Argentina) by aqueous extraction. Purified by normal phase and reverse phase chromatography^ref
• QS7 a 3,28-O-bisglycoside quillaic acid, with some differences being a higher degree of glycosylation and a considerably shorter fatty acyl unit in QS-7 => hydrophilic saponin with low lytic activity can stimulate MHC class I CTL responses although a higher minimum dose may be required for some antigens^ref
• QS21 (Stimulon™; source : Antigenics, Inc., New York, NY, USA; previously Aquila Biopharmaceuticals, Inc.) is a highly purified triterpene glycoside saponin that induces T_h1-polarized immune responses^{ref1, ref2, ref3} (Kensil, C. R., J. Y. Wu, and S. Soltysik. 1995. Structural and immunological characterization of the vaccine adjuvant QS-21, p. 525-541. In M. F. Powell, and M. J. Newman (ed.), Vaccine design: the subunit and adjuvant approach. Plenum Press, New York, N.Y.). Water soluble. No emulsification required. Can be used alone or combined with aluminum hydroxide adjuvant. Store solid QS-21 under low humidity conditions at -20° C. Protect from light. Optimum storage conditions are under evaluation. No apparent degradation under low humidity conditions after storage at 25° C for 3 years. Aqueous solutions are optimally stable between pH 5 to 7 and in micellar form. Solutions of QS-21 in 0.5 mg/mL solution may be stored in this pH range at 5° C for 2 or 3 years. QS-21 is less stable at lower concentrations; HPLC analysis is recommended for analysis of any new vaccine formulation. Protect from light. In aqueous solution, the fatty acid ester bond migrates between the 3 and 4 position on fucose, with the ester at the 4 position being favored. Both forms are active as adjuvants^ref. Primary degradation reaction is alkaline hydrolysis of the fatty acid ester bond at the 3 or 4 position on fucose. Due to alkaline-catalyzed degradation reaction, sterilization should be carried out by membrane filtration instead of autoclaving. QS-21 has been entered in a Phase III trial of a therapeutic melanoma vaccine at 100 µg QS-21 per dose. QS-21 has been evaluated in Phase I and II trials of 31 different vaccines over a QS-21 dose range of 25 to 100 µg. At present, over 1500 individuals have received QS-21 adjuvanted vaccines^ref. Shown to stimulate humoral immune responses in mice, including antigen-specific IgG₁, IgG_2b and IgG_2a titers. Most QS-21 formulations in mice have been administered by the subcutaneous or intramuscular route, but intranasal and oral administration have also been shown to be effective. Augments production of IgG responses to ganglioside antigen in melanoma vaccine in human Phase I clinical trials. Augments protective benefit of a recombinant malaria vaccine in human Phase I clinical trials. Shown also to stimulate CTL responses in mice^{ref1, ref2, ref3, ref4} [Kensil, C. R. et al., 1993, The use of Stimulon adjuvant to boost vaccine response. Vaccine Research, 2:273-281; Kensil, C. et al., 1995, Structural and immunological characterization of the vaccine adjuvant QS-21, in: Vaccine Design, M. F. Powell and M. J Newman (Eds.) Pharmaceutical Biotechnology Series, Plenum Publishing Corp., New York., pp. 525-541]


• QA-21 is a QS21-related adjuvant
• AS02 / SBAS2 adjuvant is a water-in-oil emulsion of monophosphoryl lipid A (MPL) and QS21 that induces T_h1-polarized immune responses
• 3-Q-desacyl-4'-monophosphoryl lipid A; 3D-MLA (MPL^TM) is composed of a series of 4'-monophosphoryl lipid A species that vary in the extent and position of fatty acid substitution. The hexaacyl structure shown below is the most highly acylated and most abundant component in MPLO. Species with five and four fatty acids are also present. All structures contribute to the adjuvant activity of MPLO. Derived from the lipopolysaccharide (LPS) of Salmonella minnesota R595. Obtained by treatment of LPS with mild acid and base hydrolytic conditions, and chromatographic purification of the resulting 3D-MLA. Used as a primary adjuvant in adjuvant formulations. Adjuvant activity is manifested either alone in aqueous solution with antigen, or in combination with particulate vehicles (e.g., oil-in-water emulsions). Activity may be enhanced by use of vehicle that enforces close association with antigen. Has been studied in human phase I/II clinical trials. Results to date indicate that MPLO is well tolerated at doses that exhibit beneficial immunostimulating activities. MPLO is pyrogenic at high doses [Rudbach, J. A. et al. Prophylactic use of monophosphoryl lipid A in patients at risk for sepsis, in: Bacterial Endotoxins: Basic Science to Anti-Sepsis Strategies - Proceedings of the International Conference on Endotoxins IV (J. Levin, A. Sturk, T. Van Der Poll, and S.J.H. Van Deventer, eds), John Wiley, New York; Thoelen, S. et al. Immunogenicity of a recombinant hepatitis B vaccine with monophosphoryl. lipid A

administered following various two-dose schedules, Abst. 340, 33rd Intersci. Conf. of Antimicrobial Agents and Chemother., Oct., p. 182 (1993).antiumoral ane · Van Damme, -P. et al S * ety, t d cellular immunify~ 6t recombinant hepatitis B vaccine with monophosphoryl lipid A in healthy volunteers, Abst. 667, 33rd Intersci. Conf. of Antimicrobial Agents and Chemother., Oct., p. 241 (1993)]^{ref1, ref2, ref3} [Ulrich, J. T. et al. The adjuvant activity of monophosphoryl hpid A in: Topics in Vaccine Adjuvant Research Spriggs, D.R. and Koff, W.C., Eds. CRC Press, Boston, MA, pp. 133-143 (1991); Ulrich, J. T. and Myers, K. R. Monophosphoryl. Lipid A as an adjuvant: Past experience and new directions, in: Vaccine Design M. F. Powell and M. J Newman (Eds.) Pharmaceutical Biotechnology Series, Plenum Publishing Corp., New York, 1994]
• immunostimulating complexes (ISCOMs) : relatively stable but non-covalently-bound complex of saponin adjuvant Quil-A from Quillaja saponaria mollina, cholesterol and amphipathic Ag in a molar ratio of approximately 1:1:1. ISCOMs have been shown to induce CTLs. Following oral administration, some types of CTLs were found in mesenteric lymph nodes and in the spleen, and specific IgA response could be induced. ISCOMs have only been used in veterinary vaccines, partly due to their haemolytic activity and some local reactions all reflecting the detergent activity of the Quil-A molecule. Saponin induces both apoptosis and necrosis : dendritic cells were shown to phagocytose both the antigen-saponin complexes and the saponin-induced dead cells^ref
• Quil-A : a complex but purified mixture of Quillaja saponins which are glycosides of quillaic acid and carbohydrates. The Higuchi formula of Quil A :


Quil-A is used in veterinary vaccines and for production of ISCOMs. The mixture contains fractions that bind to cholesterol, are adjuvant active, is hemolytic, and are able to form ISCOMs. Avoid inhalation and eye contact when handling Quil-A. Quil-A is highly irritating to mucosa. Quil-A contains hemolysing saponins. Quil A is not used in human trials because of overt toxicity. It is, however, used extensively in veterinary vaccines^{ref1, ref2, ref3, ref4, ref5} [Osterhaus, A. and Rimmelzwaan G. F., 1995, A novel generation of viral vaccines based on the ISCOM matrix, in: Vaccine Design, M. F. Powell and M. J Newman (Eds.) Pharmaceutical Biotechnology Series, Plenum Publishing Corp., New York; Campbell, J.B., 1995, Saponins, in: The Theory and Practical Application of Adjuvants, Stewart-Tull (Ed.), Wiley and Sons]
• a-GalCer is presented in a CD1D-dependent manner to NKT cells inducing T_h1 and T_h2 cytokines release.
• polycations :
• poly-L-Arg (pR)
• poly-L-Lys
• synthetic molecules :
• TLR7 agonists : epicutaneous (e.c.)(4) application of an ointment containing a CTL epitope and the TLR7 ligand imiquimod is highly effective in activating T cells in mice using TCR-transgenic CTL or in wild-type mice. Transcutaneous immunization-activated CTL mount a full-blown immune response against the target epitope characterized by proliferation, cytolytic activity, and the production of IFN-gamma that is completely restricted to the epitope used for vaccination^ref. Topical application of vaccines consisting of synthetic peptides formulated with imiquimod generates strong T cell responses that exhibit effective anti-tumor effects in a murine melanoma model system^ref. In comparison to microparticles containing only OVA, bulk cultures of bone marrow-derived plasmacytoid and myeloid dendritic cells produced more IL-12 and IFN-a when stimulated with microparticles containing OVA and poly-U. Subcutaneous injection of comicroencapsulated OVA and poly-U resulted in statistically elevated levels of serum anti-OVA IgG₁ (P<0.05 versus naive mice). Conversely, anti-OVA IgG₁ levels in C57 BL6 mice immunised with OVA loaded microparticles (without RNA) were statistically indifferent to naive animals. Furthermore, injection of coencapsulated OVA and poly-U resulted in greater numbers of OVA specific IFN-g secreting T-cells as compared with mice injected with OVA loaded microparticles. A similar trend was seen in mice immunised with OVA loaded microparticles decorated with CpG or solutions of admixed OVA and CpG. These data demonstrate, for the first time, that appropriately formulated ssRNA can act as a potent adjuvant and modulator of adaptive immunological responses^ref.
• TLR9 agonists : combination immunotherapy consisting of vaccination with a synthetic peptide corresponding to an immunodominant CTL epitope derived from TRP2 administered with CpG-ODN adjuvant and followed by systemic injection of anti-CTLA-4 antibodies increased the survival of mice against the poorly immunogenic B16 melanoma. Interestingly, whereas this combination therapy was effective when administered to tumor-bearing mice (therapeutic protocol), it had no significant effect when applied in the prophylactic mode (i.e., before the tumor challenge). Moreover, the antitumor effect of the combination immunotherapy required the participation of CD4⁺ and CD8⁺ T lymphocytes and was accompanied by the induction of antitumor CD4⁺ T-cell responses. The overall results suggest that peptide vaccination of tumor-bearing mice, applied in combination with a strong adjuvant and CTLA-4 blockade, is capable of eliciting durable antitumor T cell responses that provide survival benefit. These findings bear clinical significance for the design of peptide-based therapeutic vaccines for human cancer patients^ref.
• Trp-Lys-Tyr-Met-Val-d-Met (WKYMVm) is a synthetic peptide known to activate human neutrophils, monocytes and DCs, resulting in the enhancement of superoxide generation, bactericidal activity, chemotactic migration and survival. WKYMVm enhances the surface expression of CD80, but not that of CD40, CD86 and MHC class II, on mouse BMDCs which is one of the essential costimulatory signals for the induction of immune responses. Furthermore, when WKYMVm was codelivered with HIV, HBV and Influenza DNA vaccines, WKYMVm selectively enhanced the vaccine-induced CD8⁺ T cell responses in a dose-dependent manner, in terms of IFN-g secretion and cytolytic activity^ref.
• solid core nano-beads of narrowly defined size (0.04-0.05 mm) covalently conjugated to antigen localize to dendritic cells (DEC205⁺CD40⁺86⁺) in draining lymph nodes, inducing high levels of IFN-g production (CD8 T cells: precursor frequencies 1/5000 to 1/1000) and high Ab titers in mice. Conjugation of Ag to these nano-beads induced responses that were significantly higher (2- to 10-fold) than those elicited by other bead sizes, and higher than a range of currently used adjuvants (alum, QuilA, monophosphoryl lipid A). Responses were comparable to CFA/IFA immunization for Abs and ex vivo peptide-pulsed dendritic cell immunization for CD8 T cells. A single dose of Ag-conjugated beads protected mice from tumors in 2 different model challenges and caused rapid clearance of established tumors in mice. Thus, a range of Ags conjugated to nano-beads was effective as immunogens in both therapeutic and prophylactic scenarios^ref.
• human proteins :
• IL-2: the systemic administration of IL-2 can act as a potent adjuvant for T cell-directed vaccine strategies. However, not only is the administration of IL-2 potentially toxic, but recent evidence suggests that it may also paradoxically limit the duration and magnitude of the cytotoxic T cell respons, induce susceptibility to AICD and inhibit the survival of memory T cells.
• IL-15 is capable of augmenting the primary CD8⁺ T cell response to vaccination, with neither induced susceptibility to AICD nor inhibited survival of memory T cells
• human tumor-derived heat shock proteins (HSP) peptide complexes can trigger CD8⁺ T cells via cross-presentation
• peptide G : a peptide from human MHC II b chain second domain, aa 135-149)
• CEL-1000 (derG, DGQEEKAGVVSTGLIGGG) is an improved form of peptide G known to enhance immune responses of other immunogenic peptides. It has demonstrated protective activity in 2 infectious disease challenge models (HSV and malaria) and an allogenic tumor vaccine model. Results for CEL-1000 and G peptide conjugates with HIV and malaria peptide were compared with results for KLH conjugates of the same HIV peptide from the p17 molecule (87-116) referred to as HGP-30. Studies demonstrated that comparable titers were seen on day 28, 42, 63, and 77 with either G or KLH-HGP-30 peptide conjugates. In another study, CEL-1000 conjugates (CEL-1000-HGP-30) demonstrated a 4-10-fold higher titer antibody response than seen with several other peptide conjugates of the same HGP-30 peptide. Improved adjuvant activity of CEL-1000 in peptide conjugates was also demonstrated by a shift in the antibody isotypes toward a T_h1 response (IgG_2a). The IgG_2a/IgG₁ ratio for G-HGP-30 HIV or KLH-HGP-30 HIV conjugates were lower than for the CEL-1000-HGP-30 HIV conjugate. A similar favoring of the IgG_2a/IgG₁ ratio was seen for a malaria peptide conjugate (CEL-1000-SF/GF) compared to the un-conjugated peptide (SF-GF). CEL-1000 also showed adjuvant activity in an allogenic tumor vaccine model. As expected for an adjuvant, CEL-1000 or G does not induce detectable self-directed or cross reactive antibodies. CEL-1000 is currently being investigated for use as an adjuvant with conventional vaccines. It is expected that IgG_2a antibodies would be preferably generated by CEL-1000 adjuvancy and could enhance in vivo clearance of antigens or pathogens.
• antigens conjugated to 2 or 3 copies of C3d (Immudaptin^® enhanced vaccines; source : Adprotech) induce high titer, long-lasting Ab production (animated demonstration). C3 covalently attaches to Ags following activation, where the C3d cleavage fragment can function as a molecular adjuvant to augment humoral immune responses. C3d is proposed to exert its adjuvant-like activities by targeting Ags to the C3d receptor (CD21/35) expressed by B cells and follicular dendritic cells (FDCs), but in CD21/35^-/- streptavidin (SA)- and gp120-specific Ab responses were significantly augmented when these Ags were complexed with C3d in comparison to Ag alone. In fact, primary and secondary Ab responses and Ab-forming cell responses of CD21/35^-/- mice approached those of wild-type mice immunized with SA-C3dg and gp120-C3d. Thus, C3d can function as a molecular adjuvant in the absence of CD21/35 expression^ref. The use of C3d as a molecular adjuvant is quite effective in enhancing the efficacy of DNA vaccines expressing
• hemagglutinin (HA) from influenzaviruses A and B administered intranasally : it induces nasal IgA and serum IgG antibodies (Abs) in BALB/c mice^ref. Plasmids were generated that encoded a transmembrane HA (tmHA), a secreted form of HA (sHA), or a sHA fused to 3 tandem copies of the murine homologue of the C3d (sHA-3C3d). Immunizations with sHA-3C3d DNA accelerated both the avidity maturation of antibody to HA and the appearance of hemagglutinin-inhibition activity. These accelerated antibody responses correlated to a more rapid appearance of protective immunity and to complete protection from live virus challenge by a single vaccination at a dose 10 times lower than the protective dose for non-C3d forms of HA^ref. Cross-protection between different subtypes of influenza A virus has been attributed to heterosubtypic immunity (HSI). Although HSI can occur in the absence of anti-HA or anti-NA antibodies, HSI seems to be mediated, in part, by cross-reactive antibodies. HA-mC3d₃ elicited heterosubtypic immunity more efficiently than non-fused forms of HA and protected mice from lethal challenge of influenza with different subtypes. Plasmid encoding for various forms of HA were constructed from 2 influenza strains, A/Puerto Rico/8/34 (H₁N₁) or A/Aichi/2/68-x31 (H₃N₂). Vaccinated mice were analyzed for enhancement of anti-HA titers, affinity maturation of antibody, hemagglutinin-inhibition activity, and altered cytokine profiles. The HA-mC3d₃-DNA vaccinated mice were protected from heterologous influenza challenge, even though sera from these mice had no viral-neutralizing activity against heterologous virus^ref.
• envelope (Env) protein of HIV-1 in all mouse strains tested. However, the immunoglobulin subclass elicited in inbred mice vaccinated with sgp120-mC3d-DNA was predominately IgG₁, but was a mixture of IgG₁ and IgG_2a in vaccinated outbred mice. Also, splenocytes from all sgp120-mC3d-DNA vaccinated inbred mouse strains secreted primarily IL-4 indicating a T_h2 response. In contrast, mice vaccinated with sgp120-mC3d-DNA had splenocytes that secreted both IL-4 and INF-g indicating a mixed T_h response. In addition, the avidity maturation of the anti-Env antisera was enhanced in outbred mice with DNA expressing sgp120-mC3d. Overall, C3d conjugated DNA vaccines elicited enhanced immune responses in outbred mice compared to inbred mice^ref. As oligomeric or trimeric (gp140) forms of Env that more closely mimic the native proteins on the virion are often more effective immunogens than monomeric (gp120) envelopes, several forms of Env constructed from the HIV-1 isolate YU-2 (HIV-1(YU-2)) were tested for their immunogenic potential: a trimeric form of uncleaved (-) Env stabilized with a synthetic trimer motif isolated from the fibritin (FT) protein of the T4 bacteriophage, sgp140(YU-2)(-/FT), was compared to sgp140(YU-2)(-) without a synthetic trimerization domain, as well as to monomeric gp120(YU-2). DNA plasmids were constructed to express Env alone or fused to various copies of murine C3d (mC3d). BALB/c mice were vaccinated (day 1 and week 4) with DNA expressing a codon-optimized envelope gene insert, alone or fused to mC3d. Mice were subsequently boosted (week 8) with the DNA or recombinant Env protein. All mice had high anti-Env antibody titers regardless of the use of mC3d. Sera from mice vaccinated with DNA expressing non-C3d-fused trimers elicited neutralizing antibodies against homologous HIV-1(YU-2) virus infection in vitro. In contrast, sera from mice inoculated with DNA expressing Env-C3d protein trimers elicited antibody that neutralized both homologous HIV-1(YU-2) and heterologous HIV-1(ADA), albeit at low titers. Therefore, DNA vaccines expressing trimeric envelopes coupled to mC3d, expressed in vivo from codon-optimized sequences, elicit low titers of neutralizing antibodies against primary isolates of HIV-1^ref. DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, 3 copies of the murine C3d (mC3d₃) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d₃. In addition, both sCD4-gp120 and sCD4-gp120-mC3d₃ bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. BALB/c mice vaccinated with DNA vaccines expressing either gp120-mC3d₃ or sCD4-gp120-mC3d₃ elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d₃-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d₃ had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d^ref. DNA vaccines were constructed to express the gp120 subunit of Env from the isolate HIV-1(R2) using both wild-type and codon-optimized gene sequences. 3 copies of the murine C3d were added to the carboxyl terminus to enhance the immunogenicity of the expressed fusion protein. Mice (BALB/c) vaccinated with DNA plasmid expressing the gp120(R2) using codon-optimized Env sequences elicited high-titer anti-Env antibodies regardless of conjugation to C3d. In contrast, only mice vaccinated with DNA using wild-type gp120(R2) sequences fused to mC3d₃, had detectable anti-Env antibodies. Interestingly, mice vaccinated with DNA expressing gp120(R2) from codon-optimized sequences elicited antibodies that neutralized both homologous and heterologous HIV-1 isolates. To determine if the unique sequence found in the crown of the V3 loop of the Env(R2) was responsible for the elicitation of the cross-clade neutralizing antibodies, the codons encoding for the Pro-Met (amino acids 313-314) were introduced into the sequences encoding the gp120(ADA) (R5) or gp120(89.6) (R5X4). Mice vaccinated with gp120(ADA)-mC3d₃-DNA with the Pro-Met mutation had antibodies that neutralized HIV-1 infection, but not the gp120(89.6)-mC3d₃-DNA. Therefore, the use of the unique sequences in the Env(R2) introduced into an R5 tropic envelope, in conjunction with C3d fusion, was effective at broadening the number of viruses that could be neutralized. However, the introduction of this same sequence into an R5X4-tropic envelope was ineffective in eliciting improved cross-clade neutralizing antibodies^ref. DNA vaccines that express either the wild type or the codon optimized gp120 gene coding for the envelope (Env) glycoprotein of HIV-1 from the primary isolate JR-FL strain were compared to the same forms fused to 3 tandem copies of the murine C3d genes. Either codon optimization or C3d fusion alone was effective at generating early appearance, higher binding and neutralizing antibody responses. CMI responses against HIV Env could also be enhanced by C3d fusion. However, for both humoral and CMI responses, there were no synergistic effects when the combination of codon optimization and C3d fusion was employed^ref. Fusion of Env and C3d in a DNA vaccine enhances the titers of antibody to Env. Plasmids were generated that expressed a secreted form of Env (sgp120) from 3 isolates of HIV and these same forms fused to 3 tandem copies of the murine homologue of C3d (sgp120-3C3d). Analyses of titers and avidity maturation of the raised antibody indicated that immunizations with each of the sgp120-3C3d-expressing DNAs accelerated both the onset and the avidity maturation of antibody to Env^ref. Murine and human homologues of C3d were used in a DNA vaccine to enhance the titers of antibody to Env. Initially, plasmids expressing a secreted form of Env (sgp120) fused to 1, 2, or 3 copies of the murine homologue of C3d (mC3d) were constructed. Mice were inoculated with 4 vaccinations of DNA or 2 DNA vaccinations, followed by 2 boosts of affinity-purified gp120 protein. Analyses of titers demonstrated that multiple copies of mC3d coupled to sgp120 induced long-lasting, high-titer anti-Env antibody. Priming mice with sgp120-mC3d-DNA, followed by inoculation of purified gp120 protein, elicited the strongest antibody titers; however, the avidity maturation of the antibody was accelerated in the mice inoculated with sgp120-mC3d₃-DNA. In addition, DNAs expressing sgp120 fused to 3 copies of the human homologue of C3d (hC3d₃) efficiently enhanced the anti-Env antibody in rabbits. Lastly, antisera from both mice and rabbits vaccinated with DNA expressing sgp120-C3d₃ elicited higher titers of neutralizing antibody than did nonfused forms of Env. These results indicate that C3d, conjugated to sgp120, enhances the antibody responses to Env compared to non-C3d fused forms of Env, and this approach may be one way to overcome the poor ability of DNA vaccines to generate antibodies to Env^ref.
• measles virus hemagglutinin (H) protein : plasmids were generated that expressed a secreted (s) form of H and the same form fused to 3 tandem copies of the murine homologue of C3d (sH-3C3d). Analysis of titers of the antibody raised in vaccinated mice indicated that immunizations with the DNA expressing sH-3C3d had higher titers of anti-H antibodies compared to serum from mice vaccinated with DNA expressing sH only. In addition, sH-3C3d elicited higher neutralizing antibody titers that inhibited MV induced plaque formation^ref.
• Plasmodium yoelii antigen MSP1₁₉
• hCG has been used as an anti-fertility vaccine and as a target for cancer immunotherapy. The immune response induced by pcDNA3-hCGb-C3d₃ has been enhanced 243-fold compared with pcDNA3-hCGb following DNA immunization in BALB/c mice. In the present study, a new functionally active DNA vaccine of hCGb-C3d₃ chimera based on pCMV4 vector has been described. The expression efficiency of pCMV4 and pcDNA3 eukaryotic vectors for hCGb and hCGb-C3d₃ fusion protein and the immune response of mice immunized with pcDNA3-hCGbeta, pCMV4-hCGb, pcDNA3-hCGb-C3d₃ and pCMV4-hCGb-C3d₃, were compared respectively, at 25, 50 and 100 pmol dose, and further analyzed the levels of T_h1 and T_h2 cytokines produced by spleen lymphocytes of the immunized mice upon hCG restimulation in vitro. It was found that pCMV4 vector achieved 1.3-1.5-fold higher protein expression and raised 1.1-1.2 (primary) and 1.2-1.3 (booster) logs higher titer of anti-hCGb IgG than pcDNA3. Mice vaccinated with 50 pmol of hCGb-C3d₃-DNAs elicited the highest titer of hCGbeta-specific antibody among the serial doses and the immune response induced by pCMV4-hCGb-C3d₃ were, respectively, 1.3, 1.3 and 1.2 logs higher than that of pcDNA3-hCGbeta-C3d₃ and 2.2, 2.9 and 2.4 logs higher than that of pCMV4-hCGb at week 2 following the booster immunization. Moreover, the production of IL-4 and IL-10 increased in mice vaccinated with hCGb-C3d₃-DNAs and the ratio of IL-4/IFN-g showed a T_h2 bias of immune response in the mice immunized with hCGb-C3d₃-DNAs. These findings indicated that gene fusion of C3d₃ to hCGb, as a means of harnessing the adjuvant potential of the innate immune system, may improve the antigen-specific T_h2 humoral immune response of the hCGb DNA vaccine and the pCMV4 vector is a more ideal eukaryotic vector for DNA vaccine than pcDNA3^ref
• gene gun drives T_h2 polarization (?)
• Pseudomonas aeuruginosa exotoxin A truncated form (PE38)^ref : synthetic peptides derived from the G-H loop of the foot and mouth disease virus (FMDV) capsid protein VP1 are relatively poor at recapitulating the native conformation present in the virus, and thus are often poor immunogens. We hypothesized that a candidate mucosal vaccine against FMDV could be developed using the non-toxic Pseudomonas aeruginosa exotoxin A (ntPE) to deliver the G-H loop in its native conformation. An added benefit of this approach is the potential for ntPE to serve as an effective carrier/adjuvant molecule for delivery of the fusion protein across the epithelial barrier by virtue of its capacity to bind to CD91. A chimeric protein (ntPE-GH) was generated by inserting the coding sequence of the G-H loop into an expression plasmid encoding ntPE, in place of the native Ib loop. Recombinant ntPE-GH and wild-type ntPE were each expressed in Escherichia coli, purified over a nickel resin, then administered intranasally to the pigs, with or without the mucosal adjuvant cholera toxin (CT). Both the ntPE and ntPE-GH induced mucosal and systemic immune responses against ntPE; moreover, ntPE-GH administered without CT induced anti-GH loop serum IgG antibodies. In conclusion, these data demonstrate that ntPE can be used as a mucosal carrier/adjuvant to induce an immune response against the VP1 G-H loop of FMDV^ref
• possible cross-talk between mannose receptor (MR) and TLRs could be a mechanism that may be exploited by antibody vaccines targeting DCs : the MR expressed on monocyte-derived dendritic cells (DCs) using a fully human MR-specific antibody, B11, as a vehicle to deliver whole protein tumor antigens such as the human chorionic gonadotropin hormone (hCGb). Since MRs play a role in bridging innate immunity with adaptive immunity we have explored several TLR-specific ligands that may be compatible with MR targeting and be applicable as adjuvants in the clinic. MDDCs from peripheral blood of normal donors as well as cancer patients were treated in vitro with an antibody-antigen fusion product targeting mannose receptors and co-cultured with autologous T cells. Effector lymphocytes generated from repeated in vitro stimulations were tested for cytolytic and T_h cell functions. Antigen-specific helper and CTL from both healthy donors and cancer patients were effectively primed with B11-hCGb-treated autologous DCs when a combination of one or several TLR ligands is used. Specifically, concomitant signaling of DC TLR3 with dsRNA (poly I:C) and DC TLR 7/8 with resiquimod (R-848), respectively, elicited efficient antigen presentation-mediated by MR-targeting^ref
• DCs that phagocytosed a particulate form of antigens present peptides derived from the antigens on MHC class I molecules. Second, DCs that incorporated antigens via certain endocytic receptors such as Fc receptors efficiently present peptides on MHC class I molecules. These 2 strategies were combined and antigen-containing IgG-conjugated liposomes (IgG-liposomes) were prepared. The feasibility of IgG-liposomes as antigen delivery carriers in cancer immunotherapy with DCs was investigated. Immunization of mice with DCs that endocytosed ovalbumin (OVA)-containing IgG-liposomes, but not OVA-containing bare liposomes or soluble OVA, completely prevented the growth of OVA-expressing lymphoma cells. These results suggest that IgG-liposomes represent an efficient antigen delivery carrier for DCs in cancer immunotherapy^ref.
• SOCS “silencing” enhances the adjuvant effect of TLR ligands : SOCS family members are also activated by TLR ligation, and serve as an internal control to diminish the intensity and duration of inflammation. In the left-hand panel, R837 mimics ssRNA as a ligand for TLR-7, and initiates signaling through the MyD88 pathway, eventually resulting in the release of NF-kB and in the upregulation of SOCS1 and many genes involved in inflammation. PolyI:C is a synthetic mimic of dsRNA and triggers TLR-3-associated JAK/STAT signaling through TRIF, activation of IRF-3, and increased production of type 1 interferon. LPS can activate both pathways. SOCS1 specifically interferes with JAK2 and may also inhibit a step between MyD88 and NF-kB release into the nucleus, although this is controversial. This balanced internal feedback mechanism results in a controlled inflammatory process with adjuvant and antiviral effects. When SOCS1 production is blocked by siRNA (right-hand panel), the control of the inflammatory process is temporarily lost, leading to a greater adjuvant and antiviral response that appears to improve vaccine-induced immune responses^{ref1, ref2}.

• transcutaneous immunization (TCI) with proteins or peptides in combination with adjuvants efficiently induces specific cellular and humoral immune responses. However, depending on the kind of skin pretreatment, induction of cellular immune responses was restricted to generation of either specific CTLs or T_h cells. Ovalbumin protein or an ovalbumin-derived fusion peptide containing a CTL and Th epitope together with a combination of cholera toxin (CT) and CpG oligodeoxynucleotide (CpG) was applied onto cold wax-depilated and hydrated bare skin of C57BL/6 mice. TCI with the ovalbumin fusion peptide induced more robust CTL and T_h responses than that with ovalbumin protein. The fusion peptide in combination with the nontoxic CT derivative CTA1-D2D1 and CpG induced an antigen-specific CTL response, albeit less efficiently than in combination with complete CT. Further, the potency of HPV-16 E7 oncoprotein-derived peptides containing single (CTL) or multiple (CTL + T_h + B cell) epitopes to induce effective CTL responses was compared. Strong E7-specific CTL responses were detected only after TCI with the E7 multiepitope peptide. This peptide was also shown to protect mice against tumor growth after challenge with HPV-16 E7⁺ tumor cells. TCI with E7 protein and CT/CpG led to formation of an E7-specific humoral immune response^ref

• vaccines that are composed of all types of antigens, other nucleic acids, use mainly the exogenous pathway for the delivery of antigen to APCs. This, in turn, favours the stimulation of CD4⁺ T lymphocytes and the production of antibody
• continuous antigenic stimulation system (CASS) : encapsulated cells (e.g. C2C12 myoblasts) secreting the antigen induce T_h1 polarization^ref
• 2 classes of adjuvants effectively deliver antigens to the cytoplasm:
• microparticles, such as poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres and virus-like particles : pcDNA-SG encoding T and B cell epitopes of foot-and-mouth disease virus (FMDV) was encapsulated into PLGA microparticles. PLGA microparticles could protect themselves from nuclease degradation in vitro. PLGA-pcDNA-SG microparticles could be uptaken by cells and expressed His-tagged SG immunogen in vitro and in vivo. A prolonged expression and presentation of SG immunogen were observed by confocal laser scanning microscopy in the lymphocytes from the mice incubated with PLGA-pcDNA-SG microparticles, compared with the mice immunized with naked pcDNA-SG. PLGA-pcDNA-SG microparticles displayed a significant stronger immunogenicity than naked DNA vaccines with a higher titer of virus-specific antibody, elevated IFN-g production and enhanced lymphocyte proliferation. PLGA-DNA microparticle could elicit augmented humoral and cellular responses with reduced amounts and times of immunization^ref.
• ISCOMs

Tolerance inducing mechanisms reduce the levels of therapeutic antibodies in the vaccinated subject, and anti-self antibody titers are frequently > 50-fold lower than the anti-non-self response to the carrier. In order to overcome this limitation in efficacy we have explored various methods to enhance the immunogenicity of the vaccine antigen. Vaccination with a molecule containing two IgE Cvare3 domains and thereby a low level of repetitiveness markedly increased the efficacy. The anti-IgE antibody titers in the animals treated with the dimeric vaccine antigen were 4.5, 5 and 8 times higher than in the animals treated with the monomer, in three independent experiments. In addition, this increase in efficacy was not masked by the use of potent adjuvants. The effect persisted even in the presence of Freunds or Montanide ISA 51, 2 mineral oil based adjuvants. This in contrast to most TLR agonists, which appear to enhance the immune response only when administrated together with weak adjuvants. This clearly shows that the introduction of a moderately repetitive structure is enough to substantially increase the efficacy of a therapeutic vaccine^ref

C57BL/6 mice were fed a 5% or 20% casein diet for 30 d. The mice were immunized with an ovalubumin (OVA)-expression plasmid by the gene gun-based method 3 times at 10-d intervals. Body weight and serum albumin concentration in protein-deficient mice were significantly lower than those in mice fed the 20% casein diet (p<0.01, p<0.05). The percentage of OVA-specific CD8⁺ T cells was significantly decreased in the 5% casein group compared to that in the 20% casein group (p<0.05). Furthermore, CD4⁺ T cells from mice fed the low-protein diet showed lower IL-2 production than did those from the 20% group. In contrast to the T-cell function, protein deficiency did not affect OVA-specific Ab responses (p>0.05). Protein deficiency impairs the induction of Ag-specific T-cell but not B-cell response in DNA-immunized mice. Our observation indicates that, in addition to development of an effective of DNA vaccine, the management of nutritional state is important for the prevention of infectious disease by DNA vaccination^ref.

UV-inactivated, replication-defective Sendai virus particles [hemagglutinating virus of Japan envelope (HVJ-E)] injected into murine colon carcinoma (CT26) tumors growing in syngeneic BALB/c mice eradicated 60% to 80% of the tumors and obviously inhibited the growth of the remainder. Induced adaptive antitumor immune responses were dominant in the tumor eradication process because the effect was abrogated in severe combined immunodeficient mice. Murine and human dendritic cells underwent dose-dependent maturation by HVJ-E in vitro. Profiles of cytokines secreted by dendritic cells after HVJ-E stimulation showed that the amount of IL-6 released was comparable to that elicited by live HVJ. Real-time reverse transcription-PCR and immunohistochemistry revealed that HVJ-E induced a remarkable infiltration of dendritic cells and CD4⁺ and CD8⁺ T cells into tumors. In addition, CT26-specific CTLs were induced with the evidence of enhanced CD8⁺ T-cell activation in a CD4⁺CD25- T cell-dependent manner. On the other hand, conditioned medium from dendritic cells stimulated by HVJ-E rescued CD4⁺CD25^- effector T-cell proliferation from Foxp3⁺CD4⁺CD25⁺ regulatory T cell (T_reg)-mediated suppression and IL-6 was presumably dominant for this phenomenon. Such rescue was also confirmed in mice treated with HVJ-E in vivo. Moreover, antitumor effect of HVJ-E was significantly reduced by an in vivo blockade of IL-6 signaling. HVJ-E alone can eradicate tumors and the mechanism through which it induces antitumor immune responses. Because it can enhance antitumor immunity and simultaneously remove T_reg-mediated suppression, HVJ-E shows promise as a novel therapeutic for cancer immunotherapy^ref.

Web resources :

• Information resources for adjuvants and antibody production : comparisons and alternative technologies
• Compendium of adjuvants by NIAID

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