Abstract express drug resistance enzyme, ?-lactamase (1–3). Currently,


Moraxella catarrhalis and nontypeable Haemophilus influenzae are important bacterial causes of otitis media
in children and respiratory diseases in adults. Lipooligosaccharide from M. catarrhalis and outer membrane
protein 26 from NTHi are major
surface antigens identified as potential vaccine components against these
organisms. Our lab previously constructed double knockout LOS mutant
2951lgt1/4? from serotype A 2951 strain and serotype B 3292 strain LOS and
identified OMP26 as a viable vaccine candidate. In this study, we assessed the
biological role of LOS from 2951lgt1/4? mutant, which showed significantly
reduced toxicity by the Limulus
amebocyte lystate assay, reduced adherence to mammalian epithelial cells,
reduced resistance to normal human serum and more sensitive towards a series of
hydrophobic agents. LOS from the double knockout mutant 2951lgt1/4? was
isolated, detoxified and conjugated to OMP26 to form dLOS-OMP26 vaccine candidate. Three subcutaneous immunisations of
the conjugate formulated with Ribi adjuvant elicited >500-fold
increase in anti-LOS and >600-fold
increase in anti-OMP29 IgG levels in mice. Analysis of IgG isotypes present in
the mice serum revealed that predominantly a T-helper 1 type response was
induced. Importantly, mouse antisera showed complement mediated bactericidal
activity against 5 of 7 testable stains, accounting for 100% inhibition of
serotype A and B LOS. Results about opsonisation
experiment. These data suggest that the LOS and OMP based conjugate can
be used as vaccine components and require further investigation in animal

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Moraxella catarrhalis; Non-typeable Haemophilus
influenzae; lipooligosaccharide mutant; Outer membrane protein; conjugate
vaccine; conserved antigens


1.0 Introduction

Moraxella catarrhalis and nontypeable Haemophilus influenzae (NTHi)
are Gram-negative microorganisms responsible for otitis media in children and respiratory
tract infections in adults (1). Both bacteria share a similar ecological
niche and are strictly human pathogens (1). M.
catarrhalis a few decades ago was thought to be a harmless commensal, which
now is a major respiratory tract pathogen. Both bacteria have shown to express
drug resistance enzyme, ?-lactamase (1–3). Currently, there is no licensed vaccine
against prevention of infection by both pathogens. Studies on vaccine
development have primarily been focused on surface antigens such as
lipooligosaccharides (LOSs) and outer membrane proteins (OMPs) of M. catarrhalis and NTHi (1,4).

NTHi lacks a polysaccharide capsule and has
been shown to demonstrate enormous genetic diversity, making it difficult to
differentiate NTHi strains due to no
genetic makers (4). Research has identified anti-OMPs antibodies
in human serum, therefore most studies are focused on antigenic and immunogenic
properties of this molecules in NTHi
infection (4). OMPs are low-molecular-weight proteins, with
major focus being on P2, P4, P6 and minor OMPs including OMP26, protein D and
transferrin binding proteins 1 and 2 (4,5). Some of this OMPs have been found to elicit
immune responses in animals, however not all are protective against infection
from heterogenic stains of NTHi.
Current laboratory previously identified a novel protein OMP26, a 26 kDa
protein from NTHi-289, to show
significantly enhanced pulmonary clearance in animal model (5). OMP26 is highly conserved across NTHi strains with very minimal differences
among the amino acid sequence. Previous
studies using OMP26 as a vaccine candidate for NTHi, has shown that OMP26 elicits a strong immune response, specific
antibody IgG and IgA, via activation of either complement mediated killing or
opsonophagocytosis (5–7,3). This activation is a result of TH1 type
immune response being activated. OMP26 has also been shown to elevate
bactericidal activity and increase levels of pulmonary clearance in both
homologues and heterologous strains, thus indicating its reliability (5,3).       

M. catarrhalis OMP antigens such as UspA
(8,9), CopB (10), and CD (11) are protentional candidates to be
incorporated into a vaccine candidate. This OMPs have shown to elicit
bactericidal activity and promote pulmonary clearance in animal model (8–11). The LOS of M. catarrhalis is another major antigen that is an important
virulence factor implicated in the pathogenesis of this bacteria (12). M.
catarrhalis produce’s LOS; consisting of an oligosaccharide (OS) linked to
lipid A without an O-antigen, on the membrane surface (13). Structural studies conducted on LOS of M. catarrhalis indicate that it’s in a
branched form, and that lipid A is like other Gram-negative bacteria and have
common inner core (14) . There are three M. catarrhalis LOS serotypes A, B and C, which differ in the
structure of the core OS of LOS but are distinct in the terminal sugars
(Fig.1). Serotype A accounts for 61.3% of strains and shares an N-acetyl-D-glucosamine
residue with serotype C (5.3%), while serotype B accounts for 28.8% of the
strains (13,15). Serotypes B and C OS chains are of variable
length, and serotype C terminates the OS chain with Gal-?-(1-4)-Gal-?-(1-4)-Glc,
otherwise known as the PK epitope (16).

vaccines derived from LOS of M.
catarrhalis serotypes A, B and C have shown immunogenic response, however
they are only effective against some pathogenic strains of M. catarrhalis (17–19). An immunogenicity study by Yu and Gu using
LOS from serotype B strain 26397 conjugated to tetanus toxoid forming
detoxified LOS, showed that when this LOS conjugate is introduced to rabbit and
mice, a 180-230 fold increase in serum anti-LOS IgG are observed for mice and
more than 2000 fold increase for rabbits (19). Additionally, elevated complement-mediated
bactericidal activity was shown in the antiserum. They proposed that this
serotype B LOS based conjugate is a vaccine candidate. A prior study by the
same authors, using serotype A OS, found similar results to that described
above (17).  Ren
et al. developed OS conjugate vaccine candidates from M. catarrhalis serotypes A, B and C, following Yu et al. studies
very closely; they found immunogenic responses from all three conjugates and
antibodies with bactericidal activity (15). Similarly, like other studies serotype A
derived conjugates generated protection against both homologues and
heterologous strains (pulmonary clearance). The implication of these conjugates
is that they only cover certain M.
catarrhalis strains. In this study we constructed a double knockout mutant
2951lgt1/4? from LOS of 2951
(serotype A) and 3292 (serotype B) strains, therefore creating a conserved LOS
mutant covering serotype A and B LOS (14). M.
catarrhalis wild-type serotype A OS (strain 2951) consists of 9 residues
including Kdo (Fig.1). A glycosyltransferase double mutant lgt 1/4? (Fig.1), was transformed using the lgt13292::KAN (from serotype B strain 3292) with 2951
strain. From the mutation introduced into the genes encoding for Lgt 1 and Lgt 4 a highly truncated OS mutant, when compared to
OS of wild-type, was formed. The mutation arises on the basis that alleles from
serotype B strain preventing Lgt 4 from
being expressed when introduced in the 2951 serotype A strain, therefore the OS
only consisting of a central ?-D-glucose residue linked to three ?-D-Glucose

this study, we extracted LOSs from M.
catarrhalis 2951 and 3292 wild-type and mutant 2951lgt1/4? and coupled to carrier protein, OMP26 (expressed from a
recombinant OMP26 gene in E. coli)
forming carbohydrate-based conjugates. Although M. catarrhalis OMPs look promising as vaccine candidates, it would
be intriguing to investigate conjugation of OMP26 (from NTHi) to M. catarrhalis LOS
and observe the composition, immunogenicity, antigenicity and efficiency of this
conjugates as a potential vaccine candidate for both against M.
catarrhalis and NTHi infections.