This chapter contains a summary of the development of a very effective adjuvant that contains a muramyl dipeptide (MDP) analogue (threonyl-MDP, temurtide) in an oil-inwater emulsion vehicle. The oil-in-water emulsion system contains squalane, Pluronic® LI21, and polysorbate 80 in an isotonic, pH 7.4, phosphate-buffered saline solution. This adjuvant elicits both cell-mediated and humoral immune responses. While threonyl-MDP serves to increase antibody production and cell-mediated responses, the emulsion vehicle enhances immunogenicity by facilitating presentation of antigens to responding lymphocytes. Because threonyl-MDP does not exhibit toxicity usually associated with alanyl-MDP (pyrogenicity, uveitis, adjuvant-induced arthritis), no safety concerns are anticipated at therapeutic doses. In several animal species, this vehicle proved safe and efficacious, having been used successfully with a variety of antigens.

Guinea pigs were immunized with glycoprotein gD-2t in SAF-m or saline, then challenged with herpes simplex virus, type 2 (HSV-2). Animals given gD-2t in SAF-m had higher anti-gD-2t antibodies, fewer and less severe vaginal lesions, and decreased ganglionic latency compared to animals given gD-2t in saline. Leucocytes from animals vaccinated with gD-2t in SAF-m had greater proliferative responses to gD-2t in vitro than cells from control animals. MHC II-restricted, gD-2t-specific cytotoxic T cells were induced in guinea pigs vaccinated with gD-2t in SAF-m. Thus, immunization of guinea pigs with gD-2t in SAF-m markedly reduced the incidence and severity of primary, HSV-2 by eliciting both humoral and cell-mediated responses.

Adjuvants can be used with recombinant antigens to elicit cell-mediated immunity and antibodies of protective isotypes (IgG2a in the mouse and IgG1 in primates). Adjuvants should not produce reactions at injection sites, be pyrogenic or induce anterior uveitis or arthritis. Among 130 analogs of muramyl dipeptides tested, N-acetylmuramyl-L-threonyl-D-isoglutamine showed the greatest separation of potency as an adjuvant from potency in the production of side-effects. A stable emulsion of squalane and the Pluronic polymer L-121 provides a versatile vehicle for targeting of antigens to antigen-presenting cells. The combination of this emulsion with the threonyl analog of MDP is termed Syntex Adjuvant Formulation. This formulation increases the efficacy of influenza, hepatitis B virus, herpes simplex virus, lentivirus and tumor vaccines in experimental animals.

Humoral and cellular immune responses of mice and guinea-pigs to hepatitis B virus surface antigen when alum-precipitated or administered with Syntex Adjuvant Formulation (SAF) were compared. Two doses of HBsAg in SAF were sufficient to elicit antibody responses, and using SAF the dose of antigen could be reduced to one-tenth of that required to elicit antibody responses by alum-adjuvanted HBsAg. The use of SAF increased and made more consistent the antibody responses in young mice and in strains of mice with inherited low responses to HBsAg. Cellular responses to HBsAg were more consistently observed when SAF was used than when alum was used. SAF increased the formation of IgG2a antibodies in mice except in the B10.M strain; antibodies of this isotype activate complement and act synergistically with antibody-dependent effector cells more efficiently than antibodies of other isotypes. If SAF proves acceptable for human use it could improve vaccines against hepatitis B virus.

Mice and guinea pigs were immunized with the haemagglutinin (HA) of influenza B-USSR/100 virus, either in Syntex Adjuvant Formulation-1 (SAF-1) or in saline. Antibody titres were determined by ELISA, haemagglutination inhibition and virus neutralization. Animals immunized with HA in SAF-1 had significantly higher antibody titres than did animals immunized with HA in saline. Both 3-week-old and 13 1/2-month-old mice had greater and more uniform antibody responses to HA in SAF-1 than to HA in saline.

We have developed an adjuvant formulation (SAF) consisting of a synthetic muramyl dipeptide analogue (N-acetylmuramyl-L-threonyl-D-isoglutamine) in a squalane-Pluronic polymer emulsion. Used with a variety of antigens SAF elicits cell-mediated immunity and antibodies of protective isotypes (IgG2a in the mouse). SAF augments responses to influenza virus haemagglutinin and hepatitis B virus surface antigen. Vaccines using SAF have protected guinea pigs against genital herpes simplex virus infections and subhuman primates against Epstein-Barr virus and simian immunodeficiency virus infections. Properties of SAF are compared with those of other adjuvants, including lipopolysaccharide analogs, ISCOMs and liposomes.

An efficacious vaccine adjuvant which elicits both cell-mediated immunity (CMI) and humoral immune response was developed using [thr1-Muramyldipeptide (MDP) in an oil-in-water emulsion vehicle containing poloxamer 401, polysorbate 80, and squalane. Processing optimization was performed to increase the physical stability of this adjuvant emulsion which, when prepared by conventional mixing methods, demonstrated good bioactivity but poor physical stability. Various manufacturing methods were compared with a microfluidization process, which produced the most stable and elegant emulsion vehicle. The microfluidized emulsion also elicited equivalent biological response in the animal model tested.

(1989). Development of a New Therapeutic Approach to B Cell Malignancy. The Induction of Immunity by the Host Against Cell Surface Receptor on the Tumor. International Reviews of Immunology: Vol. 4, No. 4, pp. 251-270.

Muramyl peptides are the monomeric components of bacterial cell wall peptidoglycans. Many muramyl peptides, such as muramyl dipeptide (MDP), (N-acetylmuramyl-l-alanyl-d-isoglutamine), are immune response modifiers, pyrogenic and somnogenic. The purposes of this study were to measure the somnogenic effects of MDP in conjunction with a biochemical measure of the host defense response, plasma Cu, and to determine if plasma Cu levels, like sleep, are regulated by a central nervous system process. MDP administered into a lateral cerebral ventricle induced a dose-dependent rise in plasma copper at 28 h postinfusion. This was usually associated with dose-dependent fevers, increases in SWS and reductions in rapid eye movement (REM) sleep during the first 6 h after infusion. Intravenous (i.v.) administration of the same amount of MDP did not affect any of these variables. We conclude that the syndrome induced by centrally administered MDP includes activation of the host defense response with respect to a rise in plasma copper in addition to fever and enhanced sleep.

Adjuvant formulations which elicit both humoral and cell-mediated immunity will be required for vaccines based on peptides, viral and bacterial subunits and genetically engineered antigens. This report describes an adjuvant formulation which increases both cell-mediated and humoral immunity and is free of significant side effects encountered with other adjuvants or vehicles. The components include the threonyl analogue of muramyl dipeptide, Tween 80, Pluronic LI21 and squalane. This formulation was found to be effective with several antigens, in several species, including rodents, cats and monkeys. These results suggest that the formulation will be useful for both human and veterinary vaccines.

Adjuvants are required to elicit protective immune responses with bacterial toxoids, inactivated viruses and subunit antigens produced by recombinant DNA technology. Some adjuvants, such as muramyl dipeptide (MDP) analog formulations, preferentially induce the formation of antibodies of isotypes that interact with complement and antibody-dependent effector cells, and do not elicit reaginic antibodies. Aluminum salts and mineral oil emulsions increase antibody formation but not cell-mediated immunity (CMI), whereas MDP formulations also elicit CMI. Adjuvants such as MDP and lipopolysaccharide (LPS) stimulate the production by accessory cells of IL-1 that increases the circulation of lymphocytes through draining lymph nodes and act as a growth factor for lymphocytes. Vehicles such as mineral oil emulsions, liposomes and Pluronic polymer formulations provide large surface areas on which antigens can be retained in a two-dimensional matrix, from which they can readily be transferred to antigen-presenting cells. The development of an adjuvant formulation able to elicit the formation of protective antibodies and CMI without unacceptable side effects is described.

MDP)was observed. However, we foundseveral adjuvant-active analogs whichwere completely innocuous inguinea pigs. 2-(2-Acetamido-2-deoxy-D-glucos-3-O-yl)-D-hexanoyl-L-alanyl-D-isoglutamine (3'-n-propyl- MDP)was onesuchcompound. Tounderstand thedifferences between thetoxic andnontoxic compounds, macrophage stimulation invitro was studied. Bothcompounds stimulated guinea pigperitoneal macro- phages toproduce lymphocyte-activating factor invitro. However, thesupernatant from3'-n-propyl-MDP- stimulated macrophages hadno observable effects on guinea pigs, whereasthedialyzed supernatant from (Abu')-MDP-stimulated cells readily induced theguinea pigdistress syndrome. Sephadex G-150chroma- tography showedthatbothanalogs stimulated production ofa major(60,000) andminor(11,000) peakof lymphocyte-activating factor activity. Thefactor causing theguinea pigdistress syndrome was foundonly inthelow-molecular-weight