Ke-Fei Hu

Immunostimulating complexes (ISCOMs) for nasal vaccination

The immunostimulating complex (ISCOM) is documented as a strong adjuvant and delivery system for parenteral immunization. Its effectiveness for mucosal immunization has also been proven with various incorporated antigens. Lövgren et al. were the first to demonstrate the capacity of influenza virus ISCOMs to induce mucosal immune response and protection after one comparatively low nasal dose. Further studies show that similar to Cholera toxin (CT) and Escherichia coli heat-labile toxin (LT), ISCOMs break immunological tolerance and exert strong mucosal adjuvant activity, resulting in secretory IgA and systemic immune responses. Striking is the capacity of ISCOMs to induce CTL response also after nasal administration. In contrast to CT, ISCOMs initiate mucosal as well as systemic immune responses in an IL-12 dependent manner but independently of IL-4. The recombinant B subunit of cholera toxin (rCTB) was incorporated in the same ISCOM particle to explore symbiotic effects. The IgA response to rCTB in lungs was increased 100-fold when rCTB was administered nasally in ISCOMs and more than 10-fold in the remote mucosa of the genital tract. An enhanced IgA response to a passenger antigen OVA was recorded in the remote genital tract. After i.n. administration of the envelope proteins of respiratory syncytial virus in ISCOMs, high serum antibodies were induced, almost at the same levels as those following parenteral immunization and potent IgA responses were also evoked both at the local respiratory mucosa, and in the cases tested at the distant mucosae of the genital and intestinal tracts. Similar results have also been recorded with ISCOMs containing envelope proteins from Herpes simplex virus, Influenza virus and Mycoplasma mycoides. The mucosal targeting property of envelope proteins of RSV was utilized in an HIV-gp120 RSV ISCOM formulation. After nasal administration an enhanced mucosal IgA response to gp120 was observed in the female reproductive tract. In general, antigens derived from envelope viruses or cell membranes incorporated into ISCOMs retain their biological activity and conformation, encompassing the mucosal targeting and virus neutralizing properties.

Iscom and iscom-matrix enhance by intranasal route the IgA responses to OVA and rCTB in local and remote mucosal secretions.

Iscoms, with rCTB incorporated via the GM1 receptor, enhanced in mice the mucosal immunogenicity of rCTB as antigen after intranasal (i.n.) administration both by inducing IgA response in the remote intestinal tract mucosa and by a 100-fold increase of the specific IgA locally in the lungs. Iscom-matrix as a separate entity mixed with rCTB enhanced the rCTB-IgA response similarly. While OVA in iscoms induced high mucosal IgA responses, iscom-matrix co-administered with OVA induced low or no mucosal IgA response to OVA. A synergism between iscoms and rCTB could only be seen as an adjuvant targeting effect enhancing the IgA response to OVA in the remote genital tract mucosa. In serum, the immunomodulatory effect of iscoms after i.n. administration was seen as an enhanced serum IgG2a response.

ISCOM: A Delivery System for Neonates and for Mucosal Administration

This chapter describes Immune stimulating complex (ISCOM). ISCOM is a delivery system for antigen and adjuvant together in the same particle. The unique component of ISCOM is a mixture of Quillaja saponins extracted from the bark of the tree Quillaja saponaria Molina. The ISCOM is created to make antigens optimally immunogenic by (1) presenting the antigens in a physically immunogenic form—that is, several copies in a submicroscopic particle to resemble that of an infectious agent, (2) optimizing the targeting of antigen and adjuvant after both mucosal and parenteral modes of administration to lymphatic organs and cells by enclosure of adjuvant and antigen in a stable, uniform particle, and (3) optimizing the immunomodulatory capacity both for neonate and adult immune system by presenting the adjuvant and antigen components in the same particle, reducing the amount of antigen and adjuvant required for efficiently enhancing the immune response.

Bovine respiratory syncytial virus ISCOMs—Immunity, protection and safety in young conventional calves

Abstract Bovine respiratory syncytial virus (BRSV) is a major cause of bronchiolitis and pneumonia in cattle and causes yearly outbreaks with high morbidity in Europe. Commercial vaccines against this virus needs improvement of efficacy, especially in calves with BRSV-specific maternally derived antibodies (MDA). We previously reported that an experimental BRSV-ISCOM vaccine, but not a commercial vaccine, induced strong clinical and virological protection in calves with MDA, immunized at 7–15 weeks of age. The aim of the present study was to characterize the immune responses, as well as to investigate the efficacy and safety in younger animals, representing the target population for vaccination. Four groups of five 3–8 week old calves with variable levels of BRSV-specific MDA were immunized s.c. twice at a 3 weeks interval with (i) BRSV immunostimulating complexes (BRSV-ISCOMs), (ii) BRSV-protein, (iii) adjuvant, or (iv) PBS. All calves were challenged with virulent BRSV by aerosol 2 weeks later and euthanized on day 6 after infection. The cellular and humoral responses were monitored as well as the clinical signs, the viral excretion and the pathology following challenge. Despite presence of MDA at the time of the immunization, only a minimum of clinical signs were observed in the BRSV-ISCOM group after challenge. In contrast, in all control groups, clinical signs of disease were observed in most of the animals (respiratory rates up to 76min−1 and rectal temperatures up to 41°C). The clinical protection was associated to a highly significant reduction of virus replication in the upper and lower respiratory tract of calves, rapid systemic and local antibody responses and T helper cell responses dominated by IFNγ production. Animals that did not shed virus detectable by PCR or cell culture following challenge possessed particularly high levels of pulmonary IgA. The protective immunological responses to BRSV proteins and the ability to overcome the inhibiting effect of MDA were dependent on ISCOM borne antigen presentation.

Characterization of an Experimental Vaccine for Bovine Respiratory Syncytial Virus

ABSTRACT Bovine respiratory syncytial virus (BRSV) and human respiratory syncytial virus (HRSV) are major causes of respiratory disease in calves and children, respectively, and are priorities for vaccine development. We previously demonstrated that an experimental vaccine, BRSV-immunostimulating complex (ISCOM), is effective in calves with maternal antibodies. The present study focuses on the antigenic characterization of this vaccine for the design of new-generation subunit vaccines. The results of our study confirmed the presence of membrane glycoprotein (G), fusion glycoprotein (F), and nucleoprotein (N) proteins in the ISCOMs, and this knowledge was extended by the identification of matrix (M), M2-1, phosphoprotein (P), small hydrophobic protein (SH) and of cellular membrane proteins, such as the integrins αVβ1, αVβ3, and α3β1. The quantity of the major protein F was 4- to 5-fold greater than that of N (∼77 μg versus ∼17 μg/calf dose), whereas G, M, M2-1, P, and SH were likely present in smaller amounts. The polymerase (L), M2-2, nonstructural 1 (NS1), and NS2 proteins were not detected, suggesting that they are not essential for protection. Sera from the BRSV-ISCOM-immunized calves contained high titers of IgG antibody specific for F, G, N, and SH. Antibody responses against M and P were not detected; however, this does not exclude their role in protective T-cell responses. The absence of immunopathological effects of the cellular proteins, such as integrins, needs to be further confirmed, and their possible contribution to adjuvant functions requires elucidation. This work suggests that a combination of several surface and internal proteins should be included in subunit RSV vaccines and identifies absent proteins as potential candidates for differentiating infected from vaccinated animals.

Using Distilled Water for the Extraction of Mucosal Antibodies and the Subsequent Application in RSV Neutralization Test

Virus neutralization (VN) is an important functional test for evaluating RSV vaccines, also encompassing in mucosal secretion of the respiratory tract considering the infection route. In our previous study, an immunoglobin extraction method described by Bergquist et al. was adopted for RSV ELISA, but it was not suitable for virus neutralization test due to the cell toxicity of the 2% saponin solution used for the antibody extraction. In order to overcome this problem, several solvents including distilled water were tested in the present study for the capacity to extract immunogloblins. Antibodies in the extracts were evaluated and compared by ELISA. Distilled water was as efficient as the 2% saponin solution for extraction of total IgA, RSV specific IgA and IgG. More importantly, the organ extracts obtained subsequently could be used for virus neutralization test without causing adverse effect on the cell culture. Therefore, distilled water was finally chosen as the solvent for immunoglobulin extraction from mucosal organs when both ELISA and virus neutralization test are required.