| Abstract: | Moraxella catarrhalis is a causative agent of otitis media in children and lower respiratory tract infections in adults suffering from chronic obstructive pulmonary disease (COPD). This strict human pathogen continues to be a significant cause of disease in this broad spectrum of patients because there is no available vaccine. Although numerous putative vaccine antigens have been described, little is known about the human immune response to M. catarrhalis infection in vivo. Human serum antibodies are directed at a number of surface proteins, and lipooligosaccharides (LOS) and detoxified LOS may be an effective immunogen in mice. In this study, we used a specific LOS-based enzyme-linked immunosorbent assay (ELISA), containing the three major M. catarrhalis serotypes together with a complete series of truncated LOS mutants, to detect the development of new antibodies to specific regions of the oligosaccharide molecule. We compared serum samples from COPD patients who had recently cleared an M. catarrhalis infection to serum samples collected prior to their infection. Variability in the antibody response to LOS was observed, as some patients developed serotype-specific antibodies, others developed antibodies to the LOS of each serotype, others developed broadly cross-reactive antibodies, and some did not develop new antibodies. These newly developed human antibodies are directed at both side chains and core structures in the LOS molecule. This LOS-based ELISA can be used to dissect the human antibody response to both internal and external carbohydrate epitopes, thus providing a better understanding of the humoral immune response to M. catarrhalis LOS epitopes developed during natural infection.Chronic obstructive pulmonary disease (COPD) is currently the fourth leading cause of death in the United States and Europe. The morbidity and mortality associated with exacerbations of COPD together with the associated health care-related costs of $32 billion reported in the United States in 2002 demonstrate that there is a need for a better understanding of the etiology and pathogenesis of these events (6, 36, 43). Bacteria have been isolated in large numbers from the lower respiratory tract during exacerbations, and up to 50% of COPD exacerbations are due to a bacterial agent, primarily nontypeable Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae (31). M. catarrhalis accounts for up to 10% of these infections in adults, and this strictly human pathogen is currently among the three most prominent causes of otitis media in children (13, 15, 28). Some of the primary reasons why M. catarrhalis continues to cause disease can be attributed to the fact that greater than 90% of the clinical isolates express beta-lactamase, there is a high frequency of recurrent disease observed for children that have recovered from infection, and there is a lack of a vaccine (13, 27, 42, 47). Thus, the identification of potential drug targets and vaccine antigens is clearly a priority.One of the major problems hindering the identification of putative vaccine antigens involves the fact that M. catarrhalis is a strictly human pathogen, and the human immune response to this bacterium remains poorly understood. Previous studies investigated the production of new antibodies against different bacterial pathogens in patients suffering from COPD and lower respiratory tract infections. These patients exhibited increased antibody responses to bacterial outer membrane proteins (OMP) and surface-exposed lipooligosaccharides (LOS) after clearing the bacterial strain following an exacerbation (4, 29, 38, 51). Human serum immunoglobulin G (IgG), sputum IgA, or salivary IgA antibodies against M. catarrhalis surface proteins such as UspA1, UspA2, Hag, TbpB, CopB, OMP CD, OMP E, and OMP G1b have been developed (1, 3, 25, 28, 29). In addition, new antibodies to LOS have also been detected in some COPD patients (3, 28, 29).The LOS structure of M. catarrhalis has been well studied. There are three major serotypes, serotypes A, B, and C, previously defined by polyclonal antisera and structural analyses (8-10, 23, 46). The LOS glycosyltransferase (lgt) genes that encode the enzymes required to transfer carbohydrate residues to the M. catarrhalis LOS molecule were previously identified and characterized (11, 33, 41, 49). In addition, the lgt present in a given strain of M. catarrhalis can be used to identify the specific LOS serotype of that isolate using our previously described multiplex PCR method (12). Serotypes A and B are the predominant glycoforms expressed by most clinical isolates analyzed to date (12, 46). In recently reported animal studies, other researchers suggested that detoxified M. catarrhalis LOS has potential as a vaccine antigen in a mouse pulmonary clearance model (16, 19, 52, 53). While these data are both valuable and interesting, it is sometimes difficult to link observations of animals to those of humans (34).Currently, we have constructed a panel of defined LOS mutants that are defective in the expression of each specific glycosyltransferase gene identified in all three major M. catarrhalis LOS serotypes. These truncations are a comprehensive set of mutations with various oligosaccharide (OS) chain lengths representing most, if not all, possible LOS epitopes (11, 33, 41, 49). Purified LOS samples from these mutants were used in enzyme-linked immunosorbent assays (ELISAs) to assess the development of new human antibodies to all LOS epitopes developed following an M. catarrhalis infection. ELISAs were previously employed to determine levels of antibody to Neisseria meningitidis lipopolysaccharide (LPS), including inner core mutations, in patients following disease (35). Thus, this LOS-based ELISA system with the full set of mutations has the potential to determine the regions of the LOS molecule that elicit new antibodies in both children and adults following infection, providing a unique opportunity to analyze the human immune response to these major surface glycolipids in the native host. |
