Shari Lofthouse

Immunological aspects of controlled antigen delivery

Recent advances in controlled delivery systems for protein pharmaceuticals such as microspheres, liposomes, pumps and implants, have provided a new avenue for delivery of vaccine antigens. There has, however, been considerable confusion over the way in which continuous antigen delivery affects the outcome of an immune response. To date, there has been little systematic study of the influence of varying antigen exposure times and release profiles on the phenotype of the immune response, or indeed the balance between immunity and tolerance as most studies have concentrated on optimising responses to a particular antigen of interest in a single model system. As these delivery systems would find particular advantages in management of livestock species, where the use of a single administration vaccine would significantly enhance management practices, it is important to understand the relationship between controlled antigen delivery and immunity. This paper describes how existing controlled antigen delivery studies have contributed to our understanding of the development of the immune response and demonstrates how continuous antigen delivery is useful, and possibly advantageous in the generation of immunity, the maturation of the immune response and the extension of the effector response.

Continuous antigen delivery from controlled release implants induces significant and anamnestic immune responses.

Two continuous delivery injectable silicone implants were tested to determine if they were capable of delivering vaccines in a single shot. The Type A implant delivers antigen in vitro over a 1-month-period and the Type B over several months. Vaccination studies in sheep were designed to compare the responses induced by the Type A and B implants, Alzet mini-osmotic pumps and conventional antigen delivery. A model antigen, avidin, was used along with IL-1beta or alum as adjuvants. Sheep were immunised with various formulations and the titre and isotype of the antigen specific antibodies monitored. The Type B implant induced antibody (Ab) titres of greater magnitude and duration than soluble vaccines or the Type A implant with adjuvant, but only if IL-1beta was included in the formulation. Both implants induced antibodies of IgG1 and IgG2 isotype. A memory response to soluble antigen challenge was induced by the Type B+IL-1beta implant, which was predominantly of an IgG1 isotype.

Recombinant cytokines as immunological adjuvants

This paper describes the bacterial expression and purification of bioactive recombinant ovine interleukin‐2 (rovIL‐2), interleukin‐lα (rovlL‐lα) and tumour necrosis factor α. These purified proteins had specific activities in appropriate bioassays of 1 × 107, 1 times; 107 and 1 × 105 U/mg, respectively. Recombinant ovIL‐lα was assessed as an immunological adjuvant for the sheep response to the model protein avidin. When delivered either intradermally or intramuscularly in conjunction with avidin in aluminium hydroxide the rovIL‐1α significantly enhanced the secondary humoral response. Doses of 1, 10 or 100 μg per sheep enhanced the humoral response to a similar extent. Recombinant ovIL‐1β had similar adjuvant activity in that it was demonstrated to significantly enhance the sheep humoral response to an experimental H. contortus antigen. This increase in specific antibody, however, did not correlate with enhanced protection against infection with third stage H. contortus larvae. In addition incorporation of rovIL‐1 P into the formulation was shown not to alter the isotype profile of H. contortus antigen specific antibody.

Humoral and cellular responses induced by intradermally administered cytokine and conventional adjuvants

Serum antibody responses to the model protein antigen avidin were monitored in sheep following intradermal injection of avidin formulated with a range of commercially available and experimental adjuvants, including muramyl dipeptide (MDP), aluminium hydroxide gel (alum), recombinant ovine interleukin1 beta (rovIL-1 beta), rovIL-1 beta + alum, Quil A + alum or Emulsigen Plus. The highest antibody responses were recorded for animals immunised with avidin in rovIL-1 beta + alum, Quil A + alum or Emulsigen Plus, with moderate responses resulting from use of rovIL-1 beta or alum alone as adjuvants. Lower antibody responses to avidin were recorded when avidin was administered alone or with MDP. Delayed-type hypersensitivity (DTH) responses to avidin indicated that the most pronounced cellular response occurred in animals immunised with rovIL-1 beta + alum. Local cellular changes induced after primary and secondary intradermal injections indicated that distinct patterns of cellular recruitment were induced by the different adjuvants. Avidin with MDP resulted in an elevation of CD4+ T cells in the upper dermis while Emulsigen Plus induced an infiltration of large numbers of neutrophils throughout the dermis and reticular layers. CD4+, CD8+ and gamma delta + T cells increased in number and were found evenly distributed throughout these regions. Alum-based adjuvants resulted in the development of distinct cellular accumulations comprising primarily CD4+ T cells and CD45R + B cells arranged in distinct foci in the reticular layer. These cells were strongly class II positive as were the majority of macrophage like cells surrounding the foci. Staining for factor VIII related antigen indicated the presence of endothelial venules in the T and B cell foci and surrounding tissues.

Induction of T1 (cytotoxic lymphocyte) and/or T2 (antibody) responses to a mucin-1 tumour antigen.

Effective vaccination-based control of intracellular pathogens or parasites and various tumours is dependent upon induction of cytotoxic lymphocytes and other mechanisms of cellular immunity. Such responses are usually described as being antagonistic to an antibody-based immune response. This paper elaborates on previous studies that have demonstrated that conjugation of a fusion protein (FP, incorporating copies of the variable number of tandem repeat sequence of human mucin-1 (MUC1)) to oxidized mannan results in a significant shift from a type-2 response towards a type-1 response. This response induces complete protection upon challenge of immunized mice with MUC1 expressing tumour cells. This report details experiments in which the balance between type-1 and type-2 anti-MUC1 responses is manipulated by altering the dose of mannan-FP (M-FP) delivered. It is also shown that type-1 and type-2 responses may be induced simultaneously by administration of both forms of the antigen (FP/M-FP). Further, when a type-2 response is induced after FP immunization, a type-1 response can also be established by subsequent immunization with M-FP without adversely affecting the initial response. The converse also applies when M-FP is used for the initial immunizations, followed by FP administration. Delivery of interleukin-1 beta as a cytokine adjuvant with M-FP immunizations also enhanced antibody responses to levels fourfold that induced by M-FP alone without adversely affecting the cytotoxic activity induced by M-FP immunization. Contrary to the type-1/type-2 paradigm, cellular and antibody responses to MUC1 were not antagonistic. These results have important implications for the development of vaccination strategies against pathogens for which both the cellular and humoral compartments of the immune response contribute to protection.

Production and in vivo use of recombinant ovine IL-1β as an immunological adjuvant

To determine the potential of ovine interleukin 1 (IL-1) as a vaccine adjuvant in sheep, we have expressed and purified recombinant ovine IL-1 beta (rovIL-1 beta) from bacterial cultures using a modified form of the ovine IL-1 beta cDNA. Adjuvant trials using the model protein avidin demonstrated that rovIL-1 beta when administered in association with a compound providing a slow-release mechanism, resulted in significant enhancement of specific serum antibody levels in both mice and sheep. In a dose-response experiment in sheep, intradermal immunization with avidin plus either 10 or 100 micrograms of rovIL-1 beta in aluminium hydroxide resulted in antibody levels four- to eightfold higher than immunizations without rovIL-1 beta. The addition of rovIL-1 beta also resulted in a more severe DTH response to avidin indicating that rovIL-1 beta is able to enhance both humoral and cell-mediated responses to avidin. The highest antibody titres were observed when sheep received rovIL-1 beta in both the primary and secondary immunizations although the addition of rovIL-1 beta in only one of the immunizations still resulted in a significant increase in antibody levels. Additional experiments showed that rovIL-1 beta and avidin must be administered in a site drained by the same lymph node for the adjuvant effect of rovIL-1 beta to be observed.

Alternative routes of mucosal immunization in large animals

Mucosal immunization regimes that employ the oral route of delivery are often compromised by antigen degradation in the stomach. Moreover, tolerance or immunological unresponsiveness to orally delivered vaccine antigens is also a major problem associated with this route of immunization. Immunization by alternative routes including intrarectal (i.r.) and intranasal (i.n.) is becoming increasingly recognized in large animals for generating protective antibody responses at mucosal surfaces. These approaches are particularly useful in ruminant species which have four stomachs that can potentially interfere with antigen presentation to mucosal inductive sites of the gut. Modifications to enhance existing mucosal immunization regimes have also been explored through the use of alternative antigen delivery systems and mucosal adjuvants. The combination of alternative immunization routes and the use of appropriate antigen delivery systems appear to be a rational approach for providing protective immunity at mucosal surfaces. There has been a considerable amount of research conducted on evaluating the efficacy of emerging antigen delivery systems and novel adjuvants for improved immunity to mucosal immunization but very little of this work has been specific to the mucosal compartment of large animals. The aim of this review is therefore to assess the feasibility and practicality of using large animals (particularly sheep, cattle and pigs) for inducing and detecting specific immune responses to alternative mucosal routes of immunization.

Oxidised mannan antigen conjugates preferentially stimulate T1 type immune responses.

It is desirable to be able to produce either T1 or T2 responses and we have found that, in mice, mannose--coupled antigens stimulated T2 type responses antibodies and CTLs, whereas if oxidized, mannose--coupled antigens stimulated T1 responses little antibody and a potent CTL response. In addition, the cytokine profiles support the T1rT2 differentiation with these immunizations, in that oxidized mannan antigen gives IFNg, IL-2 and IL-12 production, whereas in the absence of oxidization, IL-4 and not the other cytokines is produced. A number of antigens have been examined--particularly Mucin 1 and the delivery method using mannose may be applicable to the other antigens.

Parameters related to the application of recombinant ovine interleukin-1β as an adjuvant

This paper describes aspects of the safety and efficacy of recombinant ovine interleukin-1 beta (rovIL-1 beta) as an immunological adjuvant. A dose-response relationship was established using the intramuscular route, and significant adjuvant activity was observed following delivery of 10 or 100 micrograms of the cytokine delivered either in PBS or in combination with alum. Similar doses of rovIL-1 beta also showed adjuvant activity when delivered via the subcutaneous route. In experiments in both mice and sheep, rovIL-1 beta-mediated adjuvant activity was neutralised by a monoclonal antibody (mAb), 3.41, confirming that the adjuvant effect was due to the biological activity of the cytokine. Serum clearance rates and physiological responses to intravenous, intramuscular or subcutaneous administration of rovIL-1 beta in sheep were also determined. RovIL-1 beta was shown to have a serum half-life of 2 min. Transient body temperature increases of 2 degrees C following intravenous or subcutaneous delivery, or 1 degrees C following intramuscular delivery, were observed. White blood cell counts also fluctuated post-injection, which was shown to be due to changes in the number of circulating neutrophils. The action of the neutralising mAb on serum clearance, body temperatures and white cell counts was also determined. Co-injection of rovIL-1 beta with the mAb 3.41 prevented rapid clearance of the cytokine from the serum, and was associated with an extension in elevated body temperature. The mAb appeared to have no significant influence on the white blood cell profile induced following injection with rovIL-1 beta.

In vitro testing of a pulsatile delivery system and its in vivo application for immunisation against tetanus toxoid

Abstract Problems such as patient compliance and overdosing call for the development of pulsatile drug delivery devices. The in vitro and in vivo testing of a pulsatile delivery device for tetanus toxoid are described. In vitro release studies indicate that the delivery of the entrained active is dependent upon the driving mechanism, a fluid-activated swelling agent. The delivery profile is influenced by the physicochemical properties of the biomaterials used to construct the implant, such as polyethylene porosity, excipients and the design properties of the device. Solid formulations of tetanus toxoid antigen were intermittently released into the subcutaneous tissues in sheep using the delivery device. The in vivo release of the lyophilised and compressed powder of tetanus toxoid containing excipient materials (including lactose, cellulose and magnesium stearate) was monitored indirectly by measuring levels of antibodies against tetanus toxoid in sera of mice and sheep. The antibody titres generated by our devices were no different to the titres generated by subcutaneous injection of an alum-adjuvanted liquid formulation of tetanus toxoid. The pulsatile delivery devices described should facilitate the delivery of most vaccine antigens and pharmaceuticals.