Uniformed Services University of the Health Sciences
Department of Pathology
Institute for Vaccine Research E-mail: email@example.com
4301 Jones Bridge Road, C1094
Bethesda, Maryland 20814-4799
Phone: (301) 295-3490
fax: (301) 295-1640
Most studies comparing the regulation of antibody responses to proteins and polysaccharides (PS) utilize isolated, soluble antigens as immunogens (1). Thus, the current dogma that anti-protein and anti-PS responses are T cell-dependent (TD) and T cell-independent (TI) respectively, is potentially divorced from the physiologic context of the intact microbe that co-expresses these antigens. Indeed, covalent linkage of protein and PS antigens to create a soluble conjugate vaccine appears to convert the PS into a classic TD antigen, including the ability to generate PS-specific memory (2). Capsular PS expressed by Gram-positive extracellular bacteria are covalently linked to the underlying cell wall peptidoglycan to which a number of proteins are also bound through covalent attachments (3, 4). This raises the question of whether PS antigens expressed by such bacteria also behave as classic TD antigens, similar to the conjugate vaccine.
In this regard, studies from our laboratory have indicated that PS in the context of an intact gram-positive extracellular bacterium, Streptococcus pneumoniae (Pn), is in fact, not a classical TD antigen. Rather, it combines features of both an isolated PS antigen (TI) and a PS-protein conjugate vaccine (TD) (5). Thus, whereas the IgM anti-PS response to intact Pn is TI, both the IgG anti-protein and anti-PS responses are dependent upon CD4+ T cell help, B7- and CD40- dependent costimulation, and comprise IgG of all 4 isotypes. However, in contrast to the anti-protein response, the IgG anti-PS response to intact Pn exhibits attenuated primary kinetics, fails to generate immunologic memory, and is dependent upon a shorter period of T cell help and B7-dependent costimulation. Further, the IgG anti-PS response to Pn is ICOS-independent and extrafollicular (6) and more apoptosis-prone (7), in contrast to the IgG anti-protein response. The mechanism underlying these differences, as well as the essential difference between PS expressed by an intact bacterium versus a conjugate vaccine is a matter of ongoing interest.
Covalent linkage of pneumococcal PS type 14 (PPS14) to the immunogenic cell wall protein, pneumococcal surface protein A (PspA) (8), radically changes the nature of the in vivo IgG anti-PPS14 response relative to both purified capsular PPS14 as well as intact Pn. Thus, the IgG anti-PPS14 response to soluble PPS14-PspA conjugate is largely similar to the IgG anti-PspA responses to both conjugate and intact Pn14, including prolonged primary kinetics of induction and the generation of a CD4+ T cell-dependent, PPS14-specific IgG memory response (7, 9). Collectively, these data indicate that PPS14 in association with intact Pn14 is immunologically distinct from a soluble covalent conjugate of PPS14 and an immunogenic pneumococcal protein, despite the CD4+ T cell-dependence of the IgG responses to both immunogens
The studies described above will be extended to include a number of additional Gram-positive and Gram-negative extracellular bacteria including 1) Streptococcus agalactiae (group B Streptococcus [GBS]), 2) Staphylococcus aureus, 3) Neisseria meningitidis, type A and C, 4) Hemophilus influenzae, and 5) Salmonella typhi. All bacteria will be U.V.-inactivated and initially injected through the i.p. route. Mice will be boosted in a similar manner to elicit a secondary response. Purified capsular =polysaccharides expressed by these bacteria have been obtained for use in ELISA assays to measure serum polysaccharide-specific IgM and IgG isotype titers. A purified natural or recombinant protein from each of these bacteria has also been obtained for use in ELISA assays, to measure serum protein-specific Ig responses. All polysaccharides have also been obtained in the form of a soluble polysaccharide-protein conjugate vaccine for comparative immunization studies with intact bacteria.
The overall goal of these studies is to determine whether different bacteria differ in their ability to elicit memory for polysaccharide-specific IgG responses (e.g. boosted secondary responses) and/or whether these IgG responses are dependent on CD4+ T cells, and if so, the mechanism underlying the T cell help. Potential differences in the kinetics of induction of the primary and secondary Ig responses, the IgG isotype profile, and induction of long-term plasma cells in the bone marrow and prolonged serum IgG responses will also be assessed. Finally, we will determine the relative contribution of marginal zone B cells for a given anti-polysaccharide (or anti-protein) response to intact bacteria or conjugate vaccine, and whether the responses are predominantly extrafollicular or proceed through a germinal center reaction. In light of the fundamentally different overall structure of Gram-positive versus Gram-negative bacteria (see Figure below), we hypothesize that these two classes of bacteria may each generate their own unique humoral immune responses. If so, the mechanism underlying these differences will be investigated.