Overcoming Waning Immunity in Pertussis Vaccines: Workshop of the National Institute of Allergy and Infectious Diseases.

Despite high vaccine coverage in many parts of the world, pertussis is resurging in a number of areas in which acellular vaccines are the primary vaccine administered to infants and young children. This is attributed in part to the suboptimal and short-lived immunity elicited by acellular pertussis vaccines and to their inability to prevent nasal colonization and transmission of the etiologic agent Bordetella pertussis In response to this escalating public health concern, the National Institute of Allergy and Infectious Diseases held the workshop "Overcoming Waning Immunity in Pertussis Vaccines" in September 2019 to identify issues and possible solutions for the defects in immunity stimulated by acellular pertussis vaccines. Discussions covered aspects of the current problem, gaps in knowledge and possible paths forward. This review summarizes presentations and discussions of some of the key points that were raised by the workshop.

Moraxella catarrhalis lipooligosaccharide selectively upregulates ICAM-1 expression on human monocytes and stimulates adjacent naïve monocytes to produce TNF-alpha through cellular cross-talk.

To elucidate the role of Moraxella catarrhalis lipooligosaccharide (LOS) in otitis media with effusion (OME), the effects of LOS on adhesion antigens of human monocytes were investigated. M. catarrhalis LOS selectively enhanced intercellular adhesion molecule 1 (ICAM-1 or CD54) expression on human monocytes by significantly increasing both the surface expression intensity and the percentage of ICAM-1(+) cells. ICAM-1 upregulation on human monocytes by the LOS required surface CD14, TLR4, NF-kappaB p65 and c-Jun N-terminal kinase (JNK) activity. Our study also revealed that the LOS-induced surface ICAM-1 expression was partially mediated through a TNF-alpha dependent autocrine mechanism and could be further augmented by lipopolysaccharide-binding protein in serum. In addition, M. catarrhalis LOS also stimulated human monocytes to produce pro-inflammatory cytokines in both TLR4- and CD14-dependent pathways. Our results also indicated that enhanced surface ICAM-1 expression on monocytes may hinder their adherence to the lung epithelial monolayer. Furthermore, the LOS-activated human monocytes secreted a significantly high level of IL-8, and could stimulate adjacent naïve monocytes to produce TNF-alpha which was partially mediated via membrane ICAM-1 and IL-8/IL-8RA. These results suggest that M. catarrhalis LOS could induce excessive middle ear inflammation through a cellular cross-talk mechanism during OME.

Galactose residues on the lipooligosaccharide of Moraxella catarrhalis 26404 form the epitope recognized by the bactericidal antiserum from conjugate vaccination.

Lipooligosaccharide (LOS) from Moraxella catarrhalis has the potential to elicit bactericidal antibodies against the pathogen. We generated LOS-based conjugate vaccines that elicited bactericidal antibodies in animal models. However, epitopes on the LOS recognized by the functional anti-LOS antibodies remain unidentified. In this study, a mutant strain, D4, which lost the recognition by a bactericidal anti-LOS rabbit serum in Western blotting was generated from a serotype C strain 26404 by random transposon mutagenesis. Sequence analysis revealed there was an insertion of a kanamycin resistance gene in the lgt2 gene of D4, which encodes beta(1-4)-galactosyltransferase. An isogenic lgt2 mutant, 26404lgt2, was constructed. Structural analysis indicated that the mutant strain produced a truncated LOS lacking terminal galactoses from 4- and 6-linked oligosaccharide chains of strain 26404. Further studies showed that the antiserum lost the recognition of both mutant cells and LOSs in Western blotting, an enzyme-linked immunosorbent assay (ELISA), or a flow cytometry assay. The antiserum also lost the ability to kill the mutant strain in a bactericidal assay, whereas it showed a bactericidal titer of 1:80 to strain 26404. In an inhibition ELISA, d-(+)-galactose or 26404lgt2 LOS showed no inhibition. However, the 26404 LOS and a serotype A O35E LOS with terminal galactoses on its 6-linked oligosaccharide chain showed >90% inhibition, while a serotype B 26397 LOS showed >60% inhibition. These studies suggest that the terminal alpha-Gal-(1-->4)-beta-Gal on the 6-linked oligosaccharide chain of 26404 LOS plays a critical role in forming the epitope recognized by the bactericidal antiserum induced by immunization with our conjugate vaccine.

Role of different moieties from the lipooligosaccharide molecule in biological activities of the Moraxella catarrhalis outer membrane.

Lipooligosaccharide (LOS), a major component of the outer membrane of Moraxella catarrhalis, consists of two major moieties: a lipid A and a core oligosaccharide (OS). The core OS can be dissected into a linker and three OS chains. To gain an insight into the biological activities of the LOS molecules of M. catarrhalis, we used a random transposon mutagenesis approach with an LOS specific monoclonal antibody to construct a serotype A O35Elgt3 LOS mutant. MALDI-TOF-MS of de-O-acylated LOS from the mutant and glycosyl composition, linkage, and NMR analysis of its OS indicated that the LOS contained a truncated core OS and consisted of a Glc-Kdo(2) (linker)-lipid A structure. Phenotypic analysis revealed that the mutant was similar to the wild-type strain in its growth rate, toxicity and susceptibility to hydrophobic reagents. However, the mutant was sensitive to bactericidal activity of normal human serum and had a reduced adherence to human epithelial cells. These data, combined with our previous data obtained from mutants which contained only lipid A or lacked LOS, suggest that the complete OS chain moiety of the LOS is important for serum resistance and adherence to epithelial cells, whereas the linker moiety is critical for maintenance of the outer membrane integrity and stability to preserve normal cell growth. Both the lipid A and linker moieties contribute to the LOS toxicity.

Waning Immunity and Microbial Vaccines-Workshop of the National Institute of Allergy and Infectious Diseases.

Since the middle of the 20th century, vaccines have made a significant public health impact by controlling infectious diseases globally. Although long-term protection has been achieved with some vaccines, immunity wanes over time with others, resulting in outbreaks or epidemics of infectious diseases. Long-term protection against infectious agents that have a complex life cycle and antigenic variation remains a key challenge. Novel strategies to characterize the short- and long-term immune responses to vaccines and to induce immune responses that mimic natural infection have recently emerged. New technologies and approaches in vaccinology, such as adjuvants, delivery systems, and antigen formulations, have the potential to elicit more durable protection and fewer adverse reactions; together with in vitro systems, these technologies have the capacity to model and accelerate vaccine development. The National Institute of Allergy and Infectious Diseases (NIAID) held a workshop on 19 September 2016 that focused on waning immunity to selected vaccines (for Bordetella pertussis, Salmonella enterica serovar Typhi, Neisseria meningitidis, influenza, mumps, and malaria), with an emphasis on identifying knowledge gaps, future research needs, and how this information can inform development of more effective vaccines for infectious diseases.

Kinetics of mouse antibody and lymphocyte responses during intranasal vaccination with a lipooligosaccharide-based conjugate vaccine.

We investigated the kinetics of humoral immunity and its related cellular immune responses to intranasal (IN) immunization with a detoxified lipooligosaccharide (dLOS)-tetanus toxoid (TT) conjugate against nontypeable Haemophilus influenzae (NTHi) in mice. IN vaccination with dLOS-TT elicited high titers of LOS-specific IgA in nasal washes and IgG in sera during a course of 4 inoculations while high titers of TT-specific IgA and IgG were found in sera. A significant increase of LOS-specific IgA antibody forming cells (AFCs) was observed in nasopharyngeal-associated lymphoid tissue (NALT) and nasal passages. However, TT induced broad responses with higher numbers of IgA and IgG AFCs found in NALT and nasal passages, less but significant IgA AFCs in cervical lymphoid nodes (CLN), spleen, and lungs. Phenotypic analysis revealed a significant rise of total B220+ B-lymphocytes in NALT and CLN, particularly a rise in IgA+/IgM+ cells in the NALT after the immunization. The latter result was complied with a significant rise of IL-4 but not IFN-gamma positive CD4+ T-lymphocytes in NALT. Analysis of IgG antibody subclasses showed that an IgG1 response to both LOS and TT epitopes dominated in serum when compared to IgG2a. These kinetic antibody patterns and cellular responses may provide useful information regarding to effective mucosal vaccines against NTHi infections.

Intranasal immunization with a lipooligosaccharide-based conjugate vaccine from nontypeable Haemophilus influenzae enhances bacterial clearance in mouse nasopharynx.

Nontypeable Haemophilus influenzae (NTHi) is a major cause of otitis media in children. We investigated whether intranasal immunization with a detoxified lipooligosaccharide-tetanus toxoid (dLOS-TT) conjugate vaccine would generate protective immunity against NTHi in a mouse model of nasopharyngeal clearance. The results demonstrated that intranasal immunization with dLOS-TT plus adjuvant cholera toxin (CT) significantly induced LOS-specific IgA antibodies in mouse external secretions, especially in nasal wash (90-fold), bronchoalveolar lavage fluid (25-fold), saliva (13-fold) and fecal extract (three-fold). LOS-specific IgA antibody-forming cells were also found in mucosal and lymphoid tissues with their highest numbers in the nasal passage (528 per 10(6) cells). In addition, the intranasal immunization elicited a significant rise in LOS-specific IgG (32-fold) and IgA (13-fold) in serum. For the immunized mice which had been challenged through the nose with 10(7) live NTHi strain 9274 cells, the vaccine group showed a significant reduction (74-77%) of NTHi, compared to that of control groups with CT alone or dLOS plus CT (P<0.05). Negative correlations were found between bacterial counts and the levels of nasal wash IgA or IgG, saliva IgA and serum IgG. The clearance of five heterologous strains was investigated and revealed a significant clearance of strains 3198, 5657 and 7502 but not of strains 1479 and 2019. These data suggest that intranasal immunization with dLOS-TT vaccine elicits both mucosal and systemic immunity against NTHi and enhances bacterial clearance from nasopharynx in mice. Such a vaccine and vaccination regime may be applicable to humans with an appropriate formulation.

Exploration of Moraxella catarrhalis outer membrane proteins, CD and UspA, as new carriers for lipooligosaccharide-based conjugates.

Moraxella catarrhalis outer membrane proteins, CD and ubiquitous surface protein A (UspA), were used as carriers for M. catarrhalis detoxified lipooligosaccharide (dLOS)-based conjugates. Our study was designed to investigate the feasibility of CD and UspA as protein carriers for dLOS-based conjugates and their possible synergic effects on protection from both anti-LOS and anti-CD or anti-UspA antibody responses. Female Balb/c mice were immunized subcutaneously three times with dLOS-CD or dLOS-UspA conjugate in Ribi adjuvant. Antisera elicited by the conjugates showed high titers of specific anti-LOS antibodies with complement-dependent bactericidal activity towards M. catarrhalis strain 25238. In a mouse aerosol challenge model, mice immunized with both conjugates showed a significant enhancement of the clearance of strain 25238 from lungs as compared with the control mice. Although both conjugates elicited reduced (relative to unconjugated CD or UspA) but significant levels of anti-CD or UspA antibodies, they did not show synergetic effects with anti-LOS antibodies on the bactericidal activity or the pulmonary bacterial clearance. Nevertheless, CD and UspA are safe and effective new carriers for dLOS-based or other potential carbohydrate-based conjugate vaccines to help thymus-independent carbohydrate antigens for production of anti-carbohydrate antibodies against target pathogens.

Immortalization of rat eustachian tube epithelial cells by adenovirus 12-simian virus 40 hybrid virus.

The eustachian tube epithelial cells play an important role in the initial pathogenesis of otitis media. In order to study the role of the eustachian tube epithelial cells in the pathogenesis of otitis media, we have established a rat eustachian tube epithelial cell line. The cell line was derived by infecting primary cultures of eustachian tube epithelial cells with the adenovirus 12-simian virus 40 (Adl2-SV40) hybrid virus. The immortalized cells have retained the morphological characteristics of the parental cells and show positive staining with anti-cytokeratin antibodies (a marker for epithelial cells), but not with anti-vimentin antibodies (a fibroblast marker). The cells have been in continuous culture for more than 10 months and have undergone 38 passages. Western blotting and cell staining have confirmed the expression of the SV40 T antigen and p53. Chromosomal analysis indicates that the cell line is aneuploid and derived from male rat epithelial cells. Together, our results suggest that the cell line originated from eustachian tube epithelial cells from a male rat and was successfully immortalized by the Ad12-SV40 virus.

Towards New Broader Spectrum Pneumococcal Vaccines: The Future of Pneumococcal Disease Prevention.

Seven-valent pneumococcal conjugate vaccine (PCV7) introduction and routine pediatric use has substantially reduced the burden of Streptococcus pneumoniae disease worldwide. However, a significant amount of disease burden, due to serotypes not contained in PCV7, still exists globally. A newly recognized serotype, 6C, was until recently, identified and reported as serotype 6A. This review summarizes the serotype epidemiology of pneumococcal disease pre- and post-introduction of PCV7, available post-marketing surveillance data following the introduction of higher valency pneumococcal vaccines (PCV10, PCV13) and future prospects for the development of new pneumococcal vaccines.

Late acyltransferase genes lpxX and lpxL jointly contribute to the biological activities of Moraxella catarrhalis.

Lipo-oligosaccharide (LOS) is a major surface component and virulence factor of the human respiratory pathogen Moraxella catarrhalis. Two late acyltransferase genes, lpxX and lpxL, have been identified involved in the incorporation of acyloxyacyl-linked secondary acyl chains into lipid A during M. catarrhalis LOS biosynthesis. In this study, a double mutant with a deletion of both the lpxX and lpxL genes in M. catarrhalis strain O35E was constructed and named O35ElpxXL. Structural analysis of lipid A showed that the O35ElpxXL mutant lacked two decanoic acids (10 : 0) and one dodecanoic (lauric) acid (12 : 0). In comparison with the O35E parental strain and the single mutants O35ElpxX and O35ElpxL, the double mutant O35ElpxXL displayed prominently decreased endotoxin content, reduced resistance to normal human serum and accelerated bacterial clearance at 0, 3 and 6 h after an aerosol challenge in a mouse model of bacterial pulmonary clearance. These results indicate that these two genes encoding late acyltransferases responsible for lipid A biosynthesis jointly contribute to the biological activities and pathogenicity of M. catarrhalis. The double mutant O35ElpxXL with dramatically reduced toxicity is proposed as a potential vaccine candidate against M. catarrhalis infections for further investigation.

Role of c-Jun N-terminal protein kinase 1/2 (JNK1/2) in macrophage-mediated MMP-9 production in response to Moraxella catarrhalis lipooligosaccharide (LOS).

Moraxella catarrhalis is a gram negative bacterium and a leading causative agent of otitis media (OM) in children. Recent reports have provided strong evidence for the presence of high levels of matrix metalloproteinase (MMPs) in effusion fluids from children suffering with OM, however, the precise mechanisms by which MMPs are generated are currently unknown. We hypothesized that MMPs are secreted from macrophages in the presence of M. catarrhalis lipooligosaccharide (LOS). In this report, we demonstrate that in vitro stimulation of murine macrophage RAW 264.7 cells with LOS leads to secretion of MMP-9 as determined by ELISA and zymogram assays. We have also shown that inhibition of ERK1/2 and p38 kinase completely blocked LOS induced MMP-9 production. In contrast, inhibition of JNK1/2 by the specific inhibitor SP600125 actually increased the level of expression and production of MMP-9 at both mRNA and protein levels, respectively by almost five fold. This latter result was confirmed by knocking down JNK1/2 using siRNA. Similar results have been observed in murine bone marrow derived macrophages in vitro. In contrast to and in parallel with the LOS-induced increased levels of MMP-9 in the presence of SP600125, we found a corresponding dose-dependent inhibition of TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) secretion. Results of subsequent in vitro studies provided evidence that when JNK1/2 was inhibited prior to stimulation with LOS, it significantly increased both the extent of macrophage cell migration and invasion compared to control cells or cells treated with LOS alone. The results of these studies contribute to an increased understanding of the underlying pathophysiology of OM with effusion in children.

Moraxella catarrhalis activates murine macrophages through multiple toll like receptors and has reduced clearance in lungs from TLR4 mutant mice.

Moraxella catarrhalis is a gram negative bacterium and a leading causative agent of otitis media (OM) in children. Several recent reports have provided strong evidence for an association between toll like receptors and OM. It has been found that both Streptococcus pneumoniae and nontypeable Haemophilus influenzae activate host protective immune responses through toll like receptors (TLRs), however, the precise mechanism by which Moraxella catarrhalis initiates the host immune response is currently unknown. In this report, using murine macrophages generated from a series of knock-out mice, we have demonstrated that M. catarrhalis lipooligosaccharide (LOS) and either heat killed or live bacteria are recognized by one or more TLRs. LOS activates the host immune response through a membrane bound CD14-TLR4 complex, while both heat killed and live M.cat require recognition by multiple toll like receptors such as TLR2, TLR4 and TLR9 without the requirement of CD14. We have also shown that M.cat stimuli are capable of triggering the host innate immune response by both MyD88- and TRIF- dependent signaling pathways. We further showed that M.cat induced activation of mitogen activated protein kinase (MAPK) is essential in order to achieve optimal secretion of pro-inflammatory cytokine TNF-α. We finally showed that TLR4 mutant C3H/HeJ mice produce significantly lower levels of pro-inflammatory cytokines TNF-α and IL-6 in vivo, An increased bacterial loads at 12 and 24 hours (P<0.001) in their lungs upon challenge with live M.cat in an aerosol chamber compared to wild-type (WT) control mice. These data suggest that TLRs are crucial for an effective innate immune response induced by M.cat. The results of these studies contribute to an increased understanding of molecular mechanism and possible novel treatment strategies for diseases caused by M.cat by specifically targeting TLRs and their signaling pathways.

Characterization of extended co-culture of non-typeable Haemophilus influenzae with primary human respiratory tissues.

Non-typeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that comprise part of the normal flora of the human upper airway, but are also responsible for a number of mucosal infections such as otitis media and bronchitis. These infections often recur and can become chronic. To characterize the effect of long-term co-culture of NTHi with human tissues, we infected primary respiratory epithelial cells grown at the air-liquid interface with three NTHi strains over a range of 1-10 days. Scanning and transmission electron microscopy of tissues confirmed that intact NTHi were persisting paracellularly, while organisms observed in intracellular vacuoles appeared degraded. Furthermore, the apical surface and tight junctions of the infected tissues were undisturbed, with high transepithelial electrical resistances, while the basal cell layer displayed more junctional disorganization and wider intercellular spaces than the uninfected control tissues. Although the tissues elaborated the cytokine profile reported for NTHi-caused otitis media in vivo, there was little change in the dynamics of cytokine secretion over the time points tested. Finally, we report that NTHi strains released outer membrane vesicles (OMVs) during extended co-culture with the tissues, and show that these OMVs directly interact with host cell membranes.

Intranasal immunization of the combined lipooligosaccharide conjugates protects mice from the challenges with three serotypes of Moraxella catarrhalis.

BACKGROUND: There are no licensed vaccines available against Moraxella catarrhalis, a significant human respiratory pathogen. Lipooligosaccharide (LOS) based conjugate vaccines derived from individual serotype M. catarrhalis only showed partial protection coverage. A vaccine combining LOS conjugates of two or three serotypes might provide a broader protection. METHODS: Mice were immunized intranasally with the combined conjugates consisting of LOS from serotype A and B or serotype A, B, and C followed by challenge with different M. catarrhalis strains of three serotypes. Mouse lungs, nasal washes, and sera were collected after each challenge for bacterial counts, histological evaluation, cytokine profiles, antibody level and binding activity determinations. RESULTS: Intranasal administration of the combined LOS conjugates not only enhanced pulmonary bacterial clearance of all three serotypes of M. catarrhalis strains in vaccinated mice, but also elevated serotype-specific anti-LOS immunoglobulin (Ig)A and IgG titers in nasal wash and serum respectively. Mice vaccinated with the combined LOS conjugates also showed increased interferon (IFN)-γ, interleukin (IL)-12, and IL-4 in the lungs after challenges. Compared to the control group, mice immunized with the combined LOS conjugates also showed reduced lung inflammation after M. catarrhalis infections. The hyperimmune sera induced by the combined conjugates exhibited a broad cross-reactivity toward all three serotypes of M. catarrhalis under transmission electron microscopy. CONCLUSIONS: The combined vaccine of serotype A and B LOS conjugates provides protection against most M. catarrhalis strains by eliciting humoral and cellular immune responses.

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

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 Galα1-4Galß1-4Glc 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.

Protection against nontypeable Haemophilus influenzae challenges by mucosal vaccination with a detoxified lipooligosaccharide conjugate in two chinchilla models.

Otitis media (OM) can occur following outset of upper respiratory tract infections. Inhibition of bacterial colonization in nasopharynx (NP) by mucosal vaccination may prevent OM by reducing bacterial invasion of the middle ears (MEs). In this study, 80 chinchillas were intranasally (i.n.) immunized with a detoxified lipooligosaccharide (dLOS)-tetanus toxoid conjugate vaccine of nontypeable Haemophilus influenzae (NTHi) mixed with cholera toxin (CT) or CT alone. All vaccinated animals responded with elevated levels of mucosal and serum anti-LOS antibodies. Two weeks after the last immunization, 40 chinchillas were challenged i.n. with NTHi to evaluate NP colonization and ME infection while the rest of the animals were challenged transbullarly (T.B.) to examine the development of OM. Compared to the control group, the vaccination inhibited not only bacterial colonization in NP and transmission to MEs in the i.n. challenge group but also bacterial colonization in NP and transmission to unchallenged ears in the T.B. challenge group. Though no difference was found in the challenged ears of either group right after the T.B. challenge, an early clearance of NTHi from NP and unchallenged ears as well as less severity of OM in the unchallenged ears were observed in vaccinated animals. Current results along with our previous data indicate that mucosal vaccination is capable of inhibiting NTHi NP colonization and preventing OM occurrence in chinchillas; the i.n. challenge model is preferable for testing the mucosal vaccines while the T.B. challenge model is superior for testing the systemic vaccines.

Identification of two late acyltransferase genes responsible for lipid A biosynthesis in Moraxella catarrhalis.

Lipid A is a biological component of the lipo-oligosaccharide of a human pathogen, Moraxella catarrhalis. No other acyltransferases except for UDP-GlcNAc acyltransferase, responsible for lipid A biosynthesis in M. catarrhalis, have been identified. By bioinformatics, two late acyltransferase genes, lpxX and lpxL, responsible for lipid A biosynthesis were identified, and knockout mutants of each gene in M. catarrhalis strain O35E were constructed and named O35ElpxX and O35ElpxL. Structural analysis of lipid A from the parental strain and derived mutants showed that O35ElpxX lacked two decanoic acids (C10:0), whereas O35ElpxL lacked one dodecanoic (lauric) acid (C12:0), suggesting that lpxX encoded decanoyl transferase and lpxL encoded dodecanoyl transferase. Phenotypic analysis revealed that both mutants were similar to the parental strain in their toxicity in vitro. However, O35ElpxX was sensitive to the bactericidal activity of normal human serum and hydrophobic reagents. It had a reduced growth rate in broth and an accelerated bacterial clearance at 3 h (P < 0.01) or 6 h (P < 0.05) after an aerosol challenge in a murine model of bacterial pulmonary clearance. O35ElpxL presented similar patterns to those of the parental strain, except that it was slightly sensitive to the hydrophobic reagents. These results indicate that these two genes, particularly lpxX, encoding late acyltransferases responsible for incorporation of the acyloxyacyl-linked secondary acyl chains into lipid A, are important for the biological activities of M. catarrhalis.