Mutant lipooligosaccharide-based conjugate vaccine demonstrates a broad-spectrum effectiveness against Moraxella catarrhalis

Abstract

There is no licensed vaccine available against Moraxella catarrhalis, an exclusive human pathogen responsible for otitis media in children and respiratory infections in adults. We previously developed conjugate vaccine candidates based on lipooligosaccharides (LOSs) of M. catarrhalis serotypes A, B, and C, each of which was shown to cover a portion of the clinical strains. To generate conserved LOS antigens and eliminate a potential autoimmune response to a similar epitope between M. catarrhalis LOS moiety Galα1–4Galβ1–4Glc and human P^k antigen, two LOS mutants from strain O35E were constructed. Mutant O35Elgt5 or O35EgalE revealed a deletion of one or two terminal galactose residues of wild type O35E LOS. Each LOS molecule was purified, characterized, detoxified, and coupled to tetanus toxoid (TT) to form conjugates, namely dLOS-TT. Three subcutaneous immunizations using dLOS-TT from O35Elgt5 or O35EgalE elicited significant increases (a 729- or 1263-fold above the preimmune serum levels) of serum immunoglobulin (Ig)G against O35E LOS in rabbits with an adjuvant or without an adjuvant (an 140- or 140-fold above the preimmune serum levels). Rabbit antisera demonstrated elevated complement-mediated bactericidal activities against the wild type strain O35E. The rabbit sera elicited by O35Elgt5 dLOS-TT were further examined and showed cross bactericidal activity against all additional 19 M. catarrhalis strains and clinical isolates studied. Moreover, the rabbit sera displayed cross-reactivity not only among three serotype strains but also clinical isolates in a whole-cell enzyme-linked immunosorbent assay (ELISA), which was further confirmed under transmission electron microscopy. In conclusion, O35Elgt5 dLOS-TT may act as a vaccine against most M. catarrhalis strains and therefore can be used for further in vivo efficacy studies.

Introduction

Moraxella (Branhamella) catarrhalis is a Gram-negative aerobic diplococcus, a frequent pathogen of otitis media in children or respiratory tract infections in adults [1]. It causes 15–20% of acute otitis media episodes with at least 50% of recurrences in children below 2 years [1], [2], [3]. M. catarrhalis also induces an estimated 2–4 million exacerbations of chronic obstructive pulmonary disease in adults annually, which is the fourth highest cause of mortality in the United States [4]. Otitis media accounts for more than 13 million antibiotic prescriptions and approximately $6 billion in health care costs in the United States annually [5]. Despite the availability of antibiotics for treatment, frequent use of antibiotics might result in antibiotic resistance since greater than 90% of the clinical isolates express a drug-resistance enzyme, beta-lactamase [6], [7]. Thus, vaccine development is a key approach to preventing primary and recurrent M. catarrhalis infections, which would have an enormous human and economic impact [3], [6], [8].

Vaccine development against M. catarrhalis has focused primarily on surface antigens including outer membrane proteins (OMPs) and lipooligosaccharides (LOSs) [8], [9]. OMP antigens such as ubiquitous surface protein A (UspA) [10], catarrhalis outer membrane protein B (CopB) [11], and CD [12] elicit bactericidal antibodies. Furthermore, immunization with UspA [10], CopB [13], or recombinant CD [14] has been reported to enhance pulmonary clearance of M. catarrhalis in a mouse model. These functional activities may predict a potential efficacy of a candidate vaccine in humans since there is no appropriate animal model available. M. catarrhalis LOS is another major surface antigen, which is not only an important virulence factor in the bacterial pathogenesis but also a potential vaccine candidate due to its relatively conserved structure [15], [16]. There are three major LOS serotypes: A, B, and C. The ‘A’ serotype accounts for 61.3%, the ‘B’ serotype for 28.8% and the ‘C’ serotype for 5.3% of the 302 tested strains [17]. Serotypes A and C share a common N-acetyl-d-glucosamine (GlcNAc) residue in their LOS branches and induce cross-reactive antibodies [18], [19], [20]. We previously developed conjugate vaccine candidates derived from LOSs of M. catarrhalis serotypes A, B and C [21], [22], [23]. Our data indicate that these conjugates are immunogenic and the antibodies toward them have bactericidal activity. Mice immunized with a conjugate vaccine derived from the serotype A LOS clear both a homologous and a heterologous bacterial challenges in a mouse model of pulmonary clearance [24]. However, each of these conjugates is implicated to cover only a portion of pathogenic strains of M. catarrhalis [21], [22], [23], [24]. In this study, we aim to develop conjugate vaccines with broad coverage, which may act as substitutes for any of the conjugates from M. catarrhalis serotype A, B, and C.

Like other non-enteric Gram-negative bacteria, M. catarrhalis produces LOSs containing an oligosaccharide (OS) linked to lipid A without an O-specific polysaccharide. Structural studies indicate that the LOSs from all three serotypes are branched with a common inner core, and the lipid A portion is similar to that of other Gram-negative bacteria (Fig. 1A) [18], [19], [25], [26]. The LOS glycosyltransferase (lgt)5 gene and the UDP-glucose-4-epimerase (galE) gene are responsible for the catalysis of glycosidic linkage in the LOS biosynthesis of M. catarrhalis strains (Fig. 1B) [27], [28]. Because O35E is a major clinical isolate of serotype A that makes up 61.3% of M. catarrhalis strains, we chose O35E as a parental strain to generate mutants, from which conjugate vaccines were derived. To create conserved LOS antigens, two O35E LOS mutants, O35Elgt5 with the lgt5 gene knockout and O35EgalE with the galE gene knockout, were constructed. The O35Elgt5 or O35EgalE had the deletion of one or two terminal galactose (Gal) residues, respectively, in the LOS oligosaccharide (OS) branch, Galα1-4Galβ1-4Glc (Fig. 1B) [27], [28], [29]. In addition, there is a similarity between the LOS epitope Galα1-4Galβ1-4Glc and human P^k antigen [30], [31], [32]. Our conjugate vaccines derived from the mutant LOSs will eliminate a possible cross-reaction with the human antigen. In this study, we extracted LOSs from both mutants O35Elgt5 and O35EgalE, and coupled the LOSs to a common carrier, tetanus toxoid (TT) to form carbohydrate-based conjugates. The composition, antigenicity, immunogenicity, efficacy, and coverage of the conjugates were then examined.