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Immunoconjugates composed of avidin linked to biotinylated antibodies specific for different surface determinants on cells of the immune system were evaluated for their ability to induce adjuvant-independent anti-avidin IgG responses in mice. Previously, we demonstrated that allele-specific murine anti-class II MHC-avidin immunoconjugates were immunogenic in mice bearing the appropriate haplotype. Herein we report the immunotargeting potential of heterologous anti-class II MHC antibodies specific for framework determinants, and extend the range of effective targets to include certain non-MHC structures present on APC (e.g., 33D1 on dendritic cells, 14.8 on B cells, and CD45--the leukocyte common antigen). However, antibodies with other specificities (e.g., leukocyte integrins and some macrophage markers) were not effective targeting vehicles. Surprisingly, immunoconjugates specific for CD3 and CD4 were immunogenic. The isotype distribution of the anti-avidin antibody response induced in mice by immunotargeting to class II MHC or 33D1 was similar to that induced by immunization with Ag emulsified in complete Freund's adjuvant. Most of the antibody induced was IgG1 (65-75%), but a significant proportion was IgG2a (20-30%). We also demonstrate that immunotargeting is able to prime for long-term immunologic memory in mice.

Alum, the only adjuvant currently licensed for use in humans, fails to adsorb influenza virus haemagglutinin (BHA) and is a poor adjuvant for this antigen. A specific monoclonal anti-BHA antibody adsorbed to alum promoted adhesion of the antigen to the adjuvant. The 'alum-anti-BHA-BHA' complex was found to be 1500-fold more immunogenic in mice and 5-fold more immunogenic in rabbits than a mixture of alum and BHA lacking the anti-BHA antibody. The biotin-binding protein, avidin, also mediated adsorption of biotinylated BHA to alum, and enhanced its immunogenicity to a comparable extent. These results indicate that the adjuvant activity of alum can be markedly enhanced by promoting a physical association between the antigen and the adjuvant.

By conjugating the bromelain fragment of the influenza virus haemagglutinin (BHA) to a mouse monoclonal antibody specific for a species-crossreactive, framework determinant on class II major histocompatibility complex molecules, the adjuvant-independent immunotargeting approach could be compared with more conventional immunizations involving Freund's complete adjuvant (FCA) and alum, in different species. Both rabbits and ferrets made significant virus-blocking IgG responses to the immunotargeted BHA in the absence of adjuvant, and no adverse reactions were observed with the conjugate immunizations. As measured by enzyme-linked immunosorbent assay, these responses were equal to those observed with alum, and were approximately one-quarter of the level seen with FCA. In terms of influenza virus haemagglutination inhibition, immunotargeting appeared to be more effective than alum, but less effective than FCA. Collectively, the results indicate that immunotargeting may provide a safe and effective alternative to the use of adjuvants in the development of defined subunit vaccines.

A previous study from this laboratory (D. P. Snider, D. Skea, and B. J. Underdown, Infect. Immun. 56:2838-2842, 1988) indicated that immunodeficient mice expressing the xid gene develop prolonged infections with Giardia muris, unlike immunocompetent mice, which eliminate the intestinal protozoan parasite in 8 to 10 weeks. In this study, CBA/N (xid) and CBA/Ca mice were infected with G. muris cysts and at various times following this primary infection were cured by treatment with metronidazole. In contrast to the marked differences in the ability of xid and normal mice to eliminate a primary infection, mice of both strains were resistant to a secondary challenge of G. muris cysts. These data imply that the mechanism(s) responsible for elimination of a primary infection is not identical to those required to resist a secondary challenge infection. Splenocytes from immunocompetent CBA/Ca mice (but not immunodeficient CBA/N mice) could transfer the ability to eliminate a primary G. muris infection to irradiated mice of either strain. In contrast, splenocytes from previously infected CBA/Ca mice could not transfer resistance to a challenge infection, further supporting the hypothesis that there are differences between mechanisms required to eliminate a primary infection and those necessary to resist a second challenge infection.

Using avidin as a model protein antigen, and biotinylated monoclonal antibodies as a convenient means of forming stable complexes with avidin, we have investigated the adjuvant-independent immunization of three mouse strains, C57BL/6, C3H and (C57BL/6 x C3H)F1, with immunoconjugates targeted to different class II MHC and non-MHC sites. The results confirm the effectiveness of anti-I-Ak and anti-I-Ab immunoconjugates with respect to priming for secondary IgG responses in (H-2b x H-2k)F1 mice, while indicating a lack of response in strains which are homozygous for the targeted allele. In terms of non-MHC targets in the monocyte-macrophage lineage, neither anti-MAC-1 nor anti-MAC-2 immunoconjugates were effective in any of the three strains. However, the 33D1 anti-dendritic cell antibody gave significant responses in all three strains, with the F1 response being more than 10-fold greater than the anti-class II immunoconjugates in either strain. These findings indicate that immunotargeting a protein antigen to a non-MHC determinant on dendritic cells in vivo can be an effective means of inducing an adjuvant-independent serological response, and that this approach can have significant advantages over anti-class II MHC immunotargeting.

The interactions of IgA with the jackfruit lectin, jacalin, were investigated with regard to the specificity of jacalin for species and subclasses of IgA. It was found that jacalin selectively bound to human IgA1, but not to human IgA2, mouse IgA or rat IgA. Binding studies with human IgA1 fragments produced by different IgA1 proteases revealed that jacalin bound to galactose-terminal oligosaccharides in the hinge region of human IgA1. Affinity chromatography employing jacalin-Sepharose provided a means to separate the subclasses of IgA in human whey.

Grain dust extract has been found to activate complement. We investigated the possibility that this activity was due to tannin by comparing the characteristics of grain dust extract with those of tannic acid. The properties that were studied were the interaction with albumin and immunoglobulin G, the stability to heat, and the adsorption to polyvinylpolypyrrolidone and hide powder. Grain dust extract was found to show similarities with tannic acid which were consistent with the presence of tannin accounting for most of its complement fixing activity.