Alan Coulter

Adjuvants—a classification and review of their modes of action

Since early this century, various substances have been added to vaccines and certain formulations have been devised in an attempt to render vaccines more effective. Despite a plethora of options, only aluminium salts have gained acceptance as human vaccine adjuvants and even veterinary vaccines are largely dependent upon the use of aluminium salts. Currently, many new vaccines are under development and there is a desire to simplify vaccination schedules both by increasing the number of components per vaccine and decreasing the number of doses required for a vaccine course. New, more effective adjuvants will be required to achieve this.

Intranasal vaccination with ISCOMATRIX adjuvanted influenza vaccine.

Mucosal delivery of inactivated vaccines that are able to elicit protective immune responses against respiratory diseases has been a long time goal of vaccinologists. Such vaccines would enable a more appropriate means of vaccination against respiratory diseases than those currently delivered by a parenteral route. The intranasal delivery of inactivated influenza vaccine plus the ISCOMATRIX (IMX) adjuvant, simply mixed together, was able to induce serum haemagglutination inhibition (HAI) titres in mice far superior to those obtained with unadjuvanted vaccine delivered subcutaneously. Furthermore, the IMX adjuvanted vaccine delivered intranasally induced mucosal IgA responses in the lung, nasal passages and large intestine, together with high levels of serum IgA. Intranasal delivery of IMX adjuvanted influenza vaccine in sheep gave antibody responses in both serum and nasal secretions that surpassed the levels obtained with unadjuvanted vaccine administered subcutaneously. These observations suggest that it may be possible to induce effective immunity to influenza in humans by intranasal vaccination with an IMX adjuvanted inactivated vaccine.

Simplified preparation of rabbit fab fragments

Papain attached to solid-phase CH-Sepharose 4B was used to digest rabbit IgG. Protein A-Sepharose CL-4B was used to remove undigested IgG and Fc fragments. Pure Fab fragments free of IgG, Fc fragments and papain were readily obtained by this procedure with a yield of about 75%. Polyacrylamide gel electrophoresis of the Fab in the presence of sodium dodecyl sulphate gave a single band under both reducing and non-reducing conditions. The molecular weight of the Fab determined by sedimentation equilibrium was 49,200. Unlike the IgG, the Fab obtained did not form precipitin lines when used in immunoelectrophoresis.

Local immune responses to influenza antigen are synergistically enhanced by the adjuvant ISCOMATRIX

The peripheral (draining) lymph node, as the primary site of immune induction, determines the course of systemic responses to an injected antigen. Lymphatic duct cannulation procedures in sheep were used to investigate local immunoreactivity to human influenza virus antigen (Flu ag) admixed with the adjuvant ISCOMATRIX (IMX). Compared to Flu ag or IMX alone, the co-administration of Flu ag and IMX (Flu ag+IMX) synergistically enhanced a number of immunological responses (lymphocyte and blast migration from the node, antigen-specific antibody levels and IL6 output in efferent lymph, and antigen-induced proliferation in cultured efferent lymph cells). Together, these results demonstrate that IMX is an immune modulator, and that lymphatic duct cannulation procedures may be used to evaluate antigen/adjuvant combinations for vaccine development.

Studies on experimental adjuvanted influenza vaccines: comparison of immune stimulating complexes (Iscoms™) and oil-in-water vaccines

Detergent-disrupted influenza virus vaccines, formulated as Iscoms, or oil-in-water (o/w) emulsions, were administered parenterally to mice and evaluated for immunogenicity and protective efficacy. Both formulations enhanced both primary and secondary serum antibody responses. The magnitude of these responses with o/w emulsions was further enhanced by the addition of the non-ionic block copolymer L121 in the emulsion. Four weeks after primary immunization, mice were challenged by exposure to an aerosol containing infectious virus. Resistance to challenge in terms of survival rate and weight change correlated well with serum antibody titre for all formulations. Two major differences were observed between the adjuvant formulations. Iscom vaccines, formulated with Quil-A or the less toxic Quillaia saponin preparation Iscoprep 703, induced specific cytotoxic T-lymphocyte responses, whereas the o/w-based vaccines did not. In addition, dose-site reactivity studies in sheep showed that Iscom vaccines were less reactive than o/w-based vaccines, the degree of reactivity of the latter increasing sharply with increasing L121 concentration. On the basis of these studies, Iscoms were chosen for development as a potential adjuvant for human influenza vaccines.

Induction of lymphocyte recruitment in the absence of a detectable immune response.

Lymphocyte recruitment from blood into the lymph node is thought to be initiated by the presence of antigen. In this study, we have used lymphatic cannulation in sheep to demonstrate that the adjuvant ISCOMATRIX can induce dramatic lymph node activation in the absence of antigen. Consistent patterns of node shutdown (decreased output) and cell recruitment (increased output) with minimal blast cell responses were observed indicating that an antigen-specific immune response is not required. Production of IL-6, IL-8 and IFN-gamma, and the transient presence of red blood cells and neutrophils in the efferent lymph were associated with changes in efferent lymph cell trafficking. These early events may facilitate the screening of low frequency antigen-specific cells for binding to antigen and the subsequent amplification of the immune response.

Immunopotentiation of humoral and cellular responses to inactivated influenza vaccines by two different adjuvants with potential for human use

Two quite different adjuvants, currently under development for use in humans, have been examined for their effects on the magnitude and type of immunity elicited in response to inactivated influenza vaccine. Immunostimulating complexes (ISCOM adjuvant) contain the saponin ISCOPREP 703, and SPT is an oil-in-water emulsion of squalane, non-ionic block copolymer (L121) and Tween 80. Influenza virus vaccines formulated in either adjuvant were far superior to the non-adjuvanted aqueous vaccine in eliciting antibody and T-cell responses in mice, particularly at lower doses of antigen. In addition, the vaccines containing adjuvant were superior in eliciting protective immunity. One of the shortcomings of the unadjuvanted inactivated influenza vaccine was its inability to elicit a primary proliferative T-cell response. However, after one dose of either adjuvanted vaccine, strong proliferative responses were achieved. We also show that subcutaneous vaccination with inactivated vaccines is capable of modulating the isotype profile of antibody secreting cells generated in the lungs of mice in response to intranasal challenge with live virus. In this system, the isotype of antibody elicited after challenge of mice that had received ISCOM vaccine more closely mimicked that of animals vaccinated with live virus.

Assay of snake venoms in tissue fluids

Abstract Snake venom components were measured by solid-phase radioimmunoassay. Circulating venom was detected in cases of human and experimental envenomation. The assay could detect less than 10−8 g of venom. The technique was also used for estimating cross-reaction of antivenenes and for indirectly assaying antiveness.

A Study of the Major Australian Snake Venoms in the Monkey (Macaca Fascicularis): I. The Movement of Injected Venom, Methods Which Retard this Movement, and the Response to Antivenoms

Summary Conscious monkeys were comfortably restrained for several hours whilst the movement and effects of injected snake venom was studied. Routine clinico-pathological studies were carried out on blood samples obtained at regular intervals and a solid phase radioimmunoassay was used to assay plasma levels of snake venoms and an individual neurotoxin. When snake venom was injected subcutaneously into the lower limb of a monkey and no first aid was applied, venom could be detected in the plasma of the animals within 15 min of the injection. Plasma venom and neurotoxin levels peaked about 60 min after the injection but signs of neurotoxicity were usually not seen until 120 min after injection. Post mortem studies showed that very little Tiger snake venom remained at the injection site, but high concentrations were found in the regional lymph nodes. Tiger snake venom components including the neurotoxin, notexin, were excreted in both the bile and urine of monkeys. The high levels of venom and neurotoxin found in the urine of monkeys parallel those found in some human snake bite victims. Recently developed first aid measures were applied to the envenomed limb of a number of monkeys. These measures consisted of the application of a firm crepe bandage to the length of the injected limb which was then immobilized with a splint. The first aid measures were found to delay the movement of all the major Australian land snake venoms and also the venom of the beaked sea snake (Enhydrina schistosa). A number of envenomed monkeys infused with antivenom immediately neurotoxic signs developed, barely survived even when given 10 times the amount of antivenom required to neutralize in vitro the dose of venom they had received. Surviving monkeys had persistent ptosis and lethargy for up to 5 d after envenoming. Failure to respond to antivenom was more likely if no effective first aid measure had been employed.

Prospects for the Development of New Vaccine Adjuvants

This review focuses on the impact of various new adjuvant formulations on the efficacy of existing and new human vaccines.Despite major advances in our understanding of immunology and vaccine adjuvants, existing and even new prophylactic vaccines seem likely to maintain their dependence upon aluminium salts for the foreseeable future. Additional immunomodulators may be included in these formulations to improve efficacy.A number of immunotherapeutic cancer vaccines appear likely to be registered soon and these will be dependent for efficacy upon new adjuvant formulations. The most useful to date have been the saponins e.g. QS-21, detox-B and Mycobacterial cells (either live as BCG, or killed). Vaccines to treat chronic infections will doubtless benefit from these developments.Adjuvant formulations and technologies exist to permit development of mucosal delivery, needle-free parenteral delivery and single dose vaccines. However, each of these will require intensive development, which will doubtless arrive when demanded by a specific application.Possibilities exist to improve responses in the elderly and to overcome the inhibitory effects of maternal antibodies in neonates. However, considerable work is required to establish the practicality and general utility of new approaches.