Complex Serology and Immune Response of Mice to Variant High-Molecular-Weight O Polysaccharides Isolated from Pseudomonas aeruginosa Serogroup O2 Strains

The O antigen of the Pseudomonas aeruginosa lipopolysaccharide is the optimal target for protective antibodies, but the unusual and complex nature of their sugar substituents has made it difficult to define the range of these structures needed in an effective vaccine. Most clinical isolates of P. aeruginosa can be classified into 10 O-antigen serogroups, but slight chemical differences among O polysaccharides within a serogroup give rise to subtype epitopes. These epitopes could impact the reactivity of O-antigen-specific antibodies, as well as the susceptibility of a target strain to protective, opsonic antibodies. To define parameters of serogroup and subtype-epitope immunogenicity, antigenicity, and surface expression on P. aeruginosa cells, we prepared high-molecular-weight O-polysaccharide vaccines from strains of P. aeruginosa serogroup O2, for which eight structurally variant O antigens expressing six defined subtype epitopes (O2a to O2f) have been identified. A complex pattern of immune responses to these antigens was observed following vaccination of mice. The high-molecular-weight O polysaccharides were generally more immunogenic at low doses (1 and 10 μg) than at a high dose (50 μg) and usually elicited antibodies that opsonized the homologous strain for phagocytic killing. Some of the individual polysaccharides elicited cross-opsonic antibodies to a variable number of strains that express all of the defined serogroup O2 subtype epitopes. Combination into one vaccine of two antigens that individually elicited cross-reactive opsonic antibodies to most members of the O2 serogroup inhibited, instead of enhanced, the production of antibodies broadly reactive with most serogroup O2 subtype strains. Thus, immune responses to P. aeruginosa O antigens may be restricted to a limited range of epitopes on structurally complex O antigens, and combining multiple related antigens into a single vaccine formulation may inhibit the production of those antibodies best able to protect against most P. aeruginosa strains within a given O-antigen serogroup.It has been established through animal and human experimentation that the lipopolysaccharide (LPS) O antigen of Pseudomonas aeruginosa is a target for protective antibodies (3, 36, 38). The studies of Knirel and colleagues (17, 19) on the chemical composition and structure of the major O-side-chain polysaccharides have provided important insights into the immunochemical properties of these antigens, but our understanding of their antigenic and immunogenic properties is incomplete. This point is highlighted by the inability to date to develop effective, LPS-specific immunotherapies for human P. aeruginosa infection (7).Results obtained with animals by using immunogens and antibodies specific to the O polysaccharides have indicated that slight chemical differences among bacterial strains with otherwise closely related O-side-chain structures can produce a complex pattern of reactions between antibodies and related antigens (13). With standard serologic methods using whole-cell agglutinations, strains of P. aeruginosa can be classified as members of one serogroup (serotype); members of each serogroup share a group-specific antigen. Further subdivision into subtypes, which correlate with structural variants determined by Knirel and colleagues (17), can be accomplished with appropriate antisera (22).To develop safe and effective O-antigen-specific P. aeruginosa vaccines, we have utilized the high-molecular-mass (>100,000-Da) fraction of O polysaccharides. These antigens are safe and immunogenic in humans and animals (13, 27, 37) and elicit protective antibodies to the strains from which they are isolated. However, in recent studies of animals immunized with a heptavalent high-molecular-weight O-polysaccharide vaccine whose individual components were isolated from single strains representative of the major serogroups causing P. aeruginosa infection, opsonic antibody responses to the group-specific antigens were not commonly elicited (13). Thus, in spite of chemical and serologic relatedness among subtype strains within a P. aeruginosa serogroup, single antigens isolated from one subtype strain do not always elicit opsonic antibodies to all of the strains within the serogroup (13). Previous results showed that a particular O antigen from a given serogroup may elicit group-specific immunity, while an O antigen from another serogroup may elicit only immunity specific to the subtype epitopes expressed on that particular O antigen.To explore this situation further and gain additional insight into the serologic diversity among P. aeruginosa LPS O antigens, we prepared high-molecular-weight O-polysaccharide immunogens from five strains of P. aeruginosa serogroup O2 that, together, express all six of the identified subtype antigens (Table ​(Table1).1). These polysaccharides were used to immunize mice, and the resultant sera were assessed by enzyme-linked immunosorbent assay (ELISA) and for opsonic killing activity. The results showed a complex interaction among the strains with regard to high-molecular-weight O-polysaccharide immunogenicity, antigenicity, serogroup and subtype epitope density, and susceptibility to opsonic killing. These findings indicate that the current serogroup classifications of P. aeruginosa are probably inadequate to define the full range of LPS antigens needed to elicit comprehensive immunity to a wide range of clinical isolates.

TABLE 1

Strains used for immunogen production, their serologic classification by subtype epitope, and chemical structures of the associated O antigens Open in a separate window^aBoldface type indicates a feature of a structure that distinguishes it from a related structure of the same serogroup. Abbreviations: FucNAc, 2-acetamido-2,6-dideoxygalactose (N-acetylfucosamine); Man(NAc)₂A, 2,3-diacetamido-2,3-dideoxymannuronic acid; Man(2NAc3N)A, 2-acetamido-3-acetamidino-2,3-dideoxymannuronic acid; Gul(NAc)₂A, 2,3-diacetamido-2,3-dideoxyguluronic acid. ^bThe lower structure is also part of the O antigen of strain 170007; there is about a 2:1 ratio of the upper and lower structures.