In recent years there has been considerable progress towards the development of expression systems for the display of heterologous polypeptides and, to a lesser extent, oligosaccharides on the surface of bacteria or yeast. The availability of protein display vectors has in turn provided the impetus for a range of exciting technologies. Polypeptide libraries can be displayed in bacteria and screened by cell sorting techniques, thus simplifying the isolation of proteins with high affinity for ligands. Expression of antigens on the surface of nonvirulent microorganisms is an attractive approach to the development of high-efficacy recombinant live vaccines. Finally, cells displaying protein receptors or antibodies are of use for analytical applications and bioseparations.

A live oral recombinant Salmonella vaccine strain expressing pneumococcal surface protein A (PspA) was developed. The strain was attenuated with Δcya Δcrp mutations. Stable expression of PspA was achieved by the use of the balanced-lethal vector-host system, which employs an asd deletion in the host chromosome to impose an obligate requirement for diaminopimelic acid. The chromosomal Δasd mutation was complemented by a plasmid vector possessing the asd + gene. A portion of the pspA gene from Streptococcus pneumoniae Rx1 was cloned onto a multicopy Asd+vector. After oral immunization, the recombinantSalmonella-PspA vaccine strain colonized the Peyer’s patches, spleens, and livers of BALB/cByJ and CBA/N mice and stimulated humoral and mucosal antibody responses. Oral immunization of outbred New Zealand White rabbits with the recombinant Salmonellastrain induced significant anti-PspA immunoglobulin G titers in serum and vaginal secretions. Polyclonal sera from orally immunized mice detected PspA on the S. pneumoniae cell surface as revealed by immunofluorescence. Oral immunization of BALB/cJ mice with the PspA-producing Salmonella strain elicited antibody to PspA and resistance to challenge by the mouse-virulent human clinical isolate S. pneumoniae WU2. Immune sera from orally immunized mice conferred passive protection against otherwise lethal intraperitoneal or intravascular challenge with strain WU2.

The effect on immunogenicity of coupling the immunostimulatory nonapeptide sequence (residues 163-171) from human interleukin 1f3 (IL-1if) to a small immunogen was examined. A 21-amino acid sequence spanning positions 12-32 on the large protein of hepatitis B surface antigen was chosen as a model. Three peptides were synthesized corresponding to the IL-13-derived sequence ], the hepatitis B surface antigen-derived sequence [peptide S1-(12-32)] and a composite peptide that included both these sequences separated by a spacer of two glycine residues [peptide S1-(12-32)-IL-(163-171)]. In an in vitro thymocyte proliferation assay, both peptides S1-(12-32)-IL-(163-171) and showed comparable activity, whereas peptide S1-(12-32) was inactive. Groups of five to seven mice each from C3H/CH, BALB/c, SJL/J, and C57BL/6 strains were immunized with equimolar amounts of either peptide S1-(12-32), peptide S1-(12-32)-IL-(163-171), or a mixture of peptides S1-(12-32) and IL-(163-171), and sera were screened for anti-S1-(12-32) antibodies. In all strains, peptide S1-(12-32)-IL-(163-171) elicited an increased primary and secondary anti-S1-(12-32) antibody response compared to the other two groups. Further, peptide S1-(12-32)- also induced an increased number of responders to primary immunization, though the number of responders was quantitative in all groups following secondary immunization. At least part of the enhanced immunogenicity of the S1-(12-32) sequence in peptide S1-(12-32)-IL-(163-171) appears to be due to augmented T-helper cell activity. These results suggest that coupling of the immunostimulatory IL-113derived sequence in tandem with an immunogen may confer inbuilt adjuvanticity.The cytokine interleukin 1 (IL-1) has been credited with a wide array of biological properties, which include amplification of both cellular and humoral immune responses to infectious or foreign agents (reviewed in refs. 1 and 2). Although precise details at the molecular level remain obscure, it is now widely believed that IL-1 plays an important role in T-and B-cell activation (1, 2). The immunoregulatory effects of IL-1 on T cells include enhanced proliferation in response to mitogen or antigen (3)(4)(5) and enhanced production of autocrine growth factors (3, 6). IL-1 has also been shown to act directly on B cells. It augments primary antibody responses both in vivo and in vitro (7-10) and can prime human B cells for subsequent activation (11). A B-cell growth and differentiation activity for IL-1 has also been described (12). Recently Uhl et al. (13) have identified IL-1 receptors on human monocytes, suggesting that initiation of the immune response by IL-1 can also be at the level of accessory cells.Though it has been suggested that IL-1 could serve as an adjuvant in conjunction with weakly immunogenic vaccines (14), its potent inflammatory activity (1, 2) restricts this application. Antoni et al. (15) have synthesized several short peptide fragments from hydrophilic sequences on human and murine IL-1 to determine the minima...

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Inbred BALB/c mice were either immunized with Triton X-100-extracted antigens of blood-stage Plasmodium yoelii or infected with P. yoelii and cured in three successive schedules. Whereas the immunized BALB/c became only partially protected from subsequent challenge infection with blood-stage P. yoelii, the convalescent mice acquired total immunity. When total P. yoelii antigen extract was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with anti-P. yoelii serum, five major protein bands of 150, 84, 40, 19, and 16 kDa were recognized by the sera of fully protected convalescent mice but not by the sera of partially protected mice. The utility of comparing reactivities of sera from fully protected and partially protected malaria hosts and the possibility that antigens uniquely recognized by the convalescent mouse sera may contribute to immunity against P. yoelii infection are discussed. Although previously reported to be an effective adjuvant for immunization against P. yoelii infection in (BALB/c x C57BL)F1 hybrid mice, saponin did not promote protection any better than did Freund adjuvant in BALB/c mice immunized with detergent-extracted P. yoelii antigen. Most of the P. yoelii proteins (14 to 250 kDa) found in Triton X-100 extracts ofP. yoelii-parasitized erythrocytes isoelectrofocused as a single peak in the pH region 4.4 to 4.6, suggesting a rationale for previous findings that the most anti-P. yoelii protective and T-helper activities are induced by antigens isoelectrically focused in a fraction of similar pH. Most antigens of species of Plasmodium, the malaria parasite, not only are variable between different species and stages of the parasite but also are often strain specific (16, 19, 25). These antigens contain highly immunogenic repetitive and nonrepetitive sequences, most of which induce no protective immune responses. Thus, the infected host is exposed and immunologically reacts to vast numbers of diverse immunogenic determinants, of which only a few can induce cellular and antibody responses that contribute to protective immunity (1, 20).