WO2006085910A2 - Moraxella catarrhalis exempte d'endotoxine - Google Patents

Moraxella catarrhalis exempte d'endotoxine Download PDF

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WO2006085910A2
WO2006085910A2 PCT/US2005/019479 US2005019479W WO2006085910A2 WO 2006085910 A2 WO2006085910 A2 WO 2006085910A2 US 2005019479 W US2005019479 W US 2005019479W WO 2006085910 A2 WO2006085910 A2 WO 2006085910A2
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los
bacterium
vaccine
moraxella catarrhalis
mutant
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WO2006085910A3 (fr
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Xin-Xing Gu
Daxin Peng
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The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/104—Pseudomonadales, e.g. Pseudomonas
    • A61K39/1045—Moraxella
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01129—Acyl-[acyl-carrier-protein]-UDP-N-acetylglucosamine O-acyltransferase (2.3.1.129)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated

Definitions

  • the invention relates to a Moraxella catarrhalis bacterium comprising a mutation such that it is viable, and lacks endotoxic lipooligosaccharide (LOS), and related vaccines and methods of immunization.
  • LOS endotoxic lipooligosaccharide
  • Moraxella catarrhalis a Gram-negative diplococcus
  • Streptococcus pneumoniae and nontypeable Haemophilus influenzae as the causative agent of otitis media in children, and a cause of lower respiratory tract infections such as bronchitis and pneumonia in the elderly (Catlin, B.W. 1990 Clin Microbio Rev
  • M. Catarrhalis causes a variety of severe infections including septicemia and meningitis.
  • Clinical and epidemiological studies revealed high carriage rates in young children and suggested that a high rate of colonization is associated with an increased risk of the development of M. catarrhalis-mediated diseases (Murphy, T.F. 1996
  • M. catarrhalis LOS Unlike the lipopolysaccharide (LPS) of enteric bacteria, M. catarrhalis LOS consists of only an oligosaccharide core and lipid A (Edebrink P. et al. 1994 Carbohydr Res 257:269-284). In contrast to most of the LOS or LPS molecules, the inner core oligosaccharide of M.
  • LPS lipopolysaccharide
  • catarrhalis LOS is attached to 3-deoxy-D ⁇ manno-octulosonic acid (Kdo) with a glucosyl residue instead of heptosyl residue (Holme, T. et al. 1999 Eur J Biochem 265:524-529; Raetz, C.R., and Whitfield, C. 2002 Annu Rev Biochem 71:635- 700).
  • the lipid A portion of the LOS is similar to that of other bacteria (Holme, T. et al. 1999 Eur J Biochem 265:524-529; Masoud, H. et al. 1994 Eur J Biochem 220:209-216).
  • Zaleski et al. identified a galE gene encoding UDP-glucose-4-epimerase in M. catarrhalis and showed inactivation of the gene resulting in a truncated LOS structure lacking two terminal galactosyl residues (Zaleski, A. et al. 2000 Infect Immun 68:5261- 5268).
  • Luke et al. identified a kdsA gene encoding Kdo-8-phosphate synthase and found a kdsA deficient mutant consisting only of lipid A on its LOS molecule (Luke, N.R. et al. 2003 Infect Immun 71:6426-6434). However, the toxic lipid A moiety of the above mutants remains intact.
  • the UDP-GIcNAc acyltransferase (ipxA) is selective for catalyzing the transfer of a ⁇ -hydroxymyristoyl moiety from R-3- hydroxymyristoyl acyl carrier protein to the 3 position of the glucosamine ring of UDP- GIcNAc (Anderson, M.S. and Raetz CR. 1987 J Biol Chem 262:5159-5169). Inactivation of the IpxA gene may block the initial step of the lipid A biosynthesis pathway, resulting in an LOS-def ⁇ cient structure bacterium. Previous attempts to construct knockout IpxA mutants in E.
  • coli or other Gram-negative bacteria failed because a minimal structure of Kdo 2 -lipid A is required for bacterial viability (Raetz, C.R., and Whitfield, C. 2002 Annu Rev Biochem 71:635-700; Gronow S. and Brade H. 2001 J Endotoxin Res 7:3-23; Schnaitman, CA and Klena, L-D. 1993 Microbiol Rev 57:655-682).
  • An LOS-deficient mutant of Neisseria meningitidis was reported when its IpxA gene was inactivated (Steeghs, L. et al. 1998 Nature 392:449-450).
  • the invention relates to a Moraxella catarrhalis bacterium comprising a mutation such that it is viable, and lacks endotoxic lipooligosaccharide (LOS), and related vaccines and methods of immunization.
  • LOS endotoxic lipooligosaccharide
  • Fig. 1 Structure and biosynthesis of Kdo 2 -lipid A in E. coli K-12.
  • the symbols indicate the relevant structural genes encoding each of the enzymes.
  • Fig. 2 Genetic organization of M. catarrhalis LOS biosynthesis gene cluster containing ipxD-fabZ-lpxA. Large arrows represent the direction of transcription, and the location of deletion replaced by the kanamycin-resistant gene (kanK) is between two
  • Hznd ⁇ i cleavage sites The sites of primers used are indicated as small arrows (ipxAl, ipxAI & lpxA3, Table 3).
  • Fig. 3 LOS patterns of SDS-PAGE followed by silver staining (A, B) or Western blot (C) of M. catarrhalis wild type strain O35E (1) and mutant O35ElpxA (2).
  • Panel A represents extracts from proteinase K treated whole cells lysates from 1 ml of each bacterial suspension and panels B and C represent 1.6 ⁇ g of each OMV.
  • a rabbit anti-LOS antibody was used at 1:50 dilution for panel C.
  • Molecular markers (Markl2, Invitrogen) were indicated on the left.
  • TIC Total ion current
  • GC-MS gas-chromatography mass spectrometric
  • the components of the O35ElpxA were identified as indicated and show the fatty acids C16:0, C18:l and C18:0 which are constituents of phospholipids (A).
  • the components of the 035E showed the sugar constituents (Kdo, Gal, GIc, GIcNAc) and specific lipid A fatty acids (3-OHC12:0) (B). Inositol was added as internal standard.
  • Fig. 5 Bactericidal activity of a normal human serum against M. catarrhalis wild type strain O35E (black bar) and mutant O35ElpxA (gray bar). "-" stands for 25% of heat- inactive normal human serum. The data represent the averages of three independent assays.
  • Fig. 6. Time courses of bacterial recovery in mouse lungs (A) and nasal washes (B) after an aerosol challenge with M. catarrhalis wild type strain O35E ( ⁇ ) and mutant O35ElpxA (D). Each time point represents a geometric mean of six mice.
  • the IpxA gene sequence of Moraxella catarrhalis strain 035E SEQ ID NO:1
  • Lipooligosaccharide is a major surface component of Moraxella catarrhalis and possible virulence factor in the pathogenesis of human infections caused by this organism.
  • the presence of LOS on the bacterium is an obstacle to develop vaccines derived from whole cells or outer membrane of the bacterium.
  • An IpxA gene encoding UDP-N-acetylglucosamine acyltransferase responsible for the first step of lipid A biosynthesis was identified by the construction of an isogenic M. catarrhalis IpxA mutant in strain O35E. The resulting mutant is viable despite complete loss of LOS.
  • the mutant strain showed significantly reduced adherence to epithelial cells and virulence in a mouse aerosol challenge model.
  • the nontoxic mutant When used as a vaccine, the nontoxic mutant elicited high levels of antibodies with bactericidal activity and provided protection against a challenge with the wild type strain. These data indicate that the null LOS mutant is attenuated and shall be envisioned as a vaccine against M. catarrhalis.
  • the mutation will arise early in the biosynthesis pathway such that no stage leading past the ipxB stage is reached as these products already closely resemble lipid A structure.
  • the IpxA, IpxB and ipxD genes are clustered. Steeghs et al 1997 Gene 190:263-270 provides references disclosing such details for E. coli, etc. Knowledge of the sequences of these microorganisms is thus available to the person skilled in the art and homologous sequences in other organisms can be ascertained.
  • Both IpxA and IpxD contain a characteristic hexapeptide repeat structure.
  • the IpxB gene is generally transcribed with IpxA and as such can also be readily found.
  • the cluster also comprises the fabZ gene which can also be used to ascertain the location of the gene cluster involved in lipid A biosyntheis (Steeghs et al 1997 Gene 190:263-270).
  • fabZ gene can also be used to ascertain the location of the gene cluster involved in lipid A biosyntheis (Steeghs et al 1997 Gene 190:263-270).
  • mutate one or more genes associated with lipid A biosynthesis can be either such that enzyme is produced in an inactive form or such that the gene is mutated such that it is not expressed, i.e., thereby forming a knockout mutant.
  • the manners in which this objective can be achieved are numerous and will be readily available to a person skilled in the art of genetic engineering.
  • the subject invention comprises the mutants described above.
  • it comprises new vaccines made from such organisms or component parts thereof.
  • Such new vaccines are free of lipid A.
  • Such vaccines can in fact be completely free of either active or inactive lipid A.
  • the vaccines are free of LOS.
  • the Limulus test can be applied as described herein to ascertain that a preparation is in fact free of LOS.
  • a vaccine against a Moraxella catarrhalis bacterium said vaccine being substantially free of LOS, wherein substantially free can be ascertained by the Limulus test, said vaccine comprising a microorganism according to the invention as disclosed above as active component falls within the scope of the invention.
  • This is termed a whole cell vaccine.
  • a vaccine against a Moraxella catarrhalis bacterium comprising one or more components of the aforementioned microorganism which is also substantially free of LOS as defined above is covered by the invention.
  • an outer membrane protein (OMP) or outer membrane vesicle (OMV) comprising vaccine substantially free of LOS as defined above falls within the scope the invention.
  • a method of producing a vaccine against Moraxella catarrhalis employing a microorganism according to the invention and/or parts derived therefrom as active component in a manner known per se for producing whole cell or acellular vaccines is covered by the invention as are the products of said method.
  • the vaccines according to the invention will preferably be further characterized by the presence of an adjuvant to enhance the immunogenic activity thereof.
  • a number of adjuvants commonly used in vaccines are known. Any of these can be suitably applied. Any dosage form and additional components commonly used for vaccines is suitable for the subject invention. Mutants with Modifications on Lipid A Portion
  • the inactivation of early stage enzymes associated with LOS biosynthesis may result in not only the null LOS mutant (ipxA) but also mutants with modifications on lipid A portion, e.g., partial synthesis of fatty acid chains such as lack of ester-linked fatty acids or replacement of C 12:0 or C10:0 with shorter or longer fatty acid chains by the expression of homologous genes from other bacterial species, etc.
  • mutants with truncated LOS in fatty acid portion (or reduced toxicity in lipid A), the truncated LOS itself, or outer membrane components from these mutants are envisioned as serving as the basis of a vaccine against M. catarrhalis.
  • the attenuated micro-organism of the invention may further comprise at least one heterologous nucleic acid sequence inserted into its genome. This is useful for reproducing or replicating heterologous nucleic acid molecules and/or for expression of heterologous nucleic acid molecules, either in vivo or in vitro.
  • the heterologous nucleic acid sequence advantageously codes for an immunogen, antigen or epitope from a pathogenic viral, parasitic or bacterial agent which is different from those naturally expressed by the attenuated micro-organism.
  • This heterologous sequence may encode an immunogen, antigen or epitope from another strain of the microorganism or bacteria, e.g., another Moraxella catarrhalis strain.
  • An immunogen or antigen is a protein or polypeptide able to induce an immune response against the pathogenic agent or a secreted antigen of the pathogenic agent, and contains one or more epitopes; an epitope is a peptide or polypeptide which is able to induce an immune response against the pathogenic agent or a secreted antigen of the pathogenic agent.
  • Heterologous nucleic acid sequences which are suitable for this use in such a vector will be apparent to the skilled person and include for example those for the control of plague caused by Yersinia pestis and other Yersinia species such as Y pseudotuberculosis and Y enterocolitica, of gonorrhea caused by Neisseria gonorrhoea, of syphilis caused by Treponema pallidum, and of venereal diseases as well as eye infections caused by Chlamydia trachomatis; and those coming from species of Streptococcus from both group A and group B, such as those species that cause sore throat or heart diseases, Neisseria meningitidis, Mycoplasma pneumoniae and other Mycoplasma species, Hemophilus influenza, Bordetella Pertussis, Mycobacterium tuberculosis, Mycobacterium leprae, Bordetella species, Escherichia coli, Streptococcus e
  • RNA viruses for example from the classes Papovavirus, Adenovirus, Herpesvirus, Poxvirus, Parvovirus, Reovirus, Picornavirus, Myxovirus, Paramyxovirus, or Retrovirus; and those coming from pathogenic fungi, protozoa and parasites.
  • the heterologous sequence is advantageously inserted so as to be expressed by the micro-organism in the host when administered in order to develop an immune response against both the attenuated micro-organism and said expressed immunogen.
  • the heterologous sequence is advantageously inserted with or operably linked to or downstream from the regulatory elements allowing its expression, such as a promoter. Nucleotide sequences useful for the addressing and the secretion of the protein may also be added. Accordingly, leader or signal sequences may be included in expressed products to facilitate transport through the cell wall and/or secretion.
  • heterologous sequence is inserted within the selected nucleotide sequence or the selected gene used for the attenuation.
  • the codon usage can be adapted to the bacterial vector used.
  • the attenuated mutants of the invention may also comprise a nucleic acid sequence encoding a therapeutic protein, an allergen, a growth factor or a cytokine or an immunomodulator or immunostimulator.
  • Attenuated micro-organisms are used to produce live attenuated immunogenic compositions or live attenuated vaccine compositions.
  • the attenuated microorganism is a Moraxella catarrhalis, mutated according to the invention.
  • the microorganism may act as a recombinant vector to immunize and/or vaccinate animals or humans against infections caused by other agents than Moraxella.
  • the immunogenic compositions or the vaccine compositions comprise the attenuated mutant and a pharmaceutically or veterinarily acceptable carrier, excipient, diluent or vehicle, and optionally a stabilizer and/or an adjuvant.
  • the attenuated mutant can be a vector that additionally expresses nucleic acid molecules heterologous to the vector, such as a heterologous epitope, antigen, immunogen, and/or growth factor, cytokine, immunoregulator or immunostimulator.
  • immunogenic composition covers herein any composition able, once it has been injected to animals or to a human to elicit an immune response against the targeted pathogen.
  • vaccine composition covers herein any composition able, once it has been injected to animals or to a human to induce a protective immune response against the targeted pathogen.
  • the pharmaceutically or vetermarily acceptable vehicle may be water or saline, but it may, for example, also comprise bacteria culture medium.
  • the live attenuated bacteria according to the invention may be freeze-dried advantageously with a stabilizer. Freeze-drying can be done according to well known standard freeze-drying procedures.
  • the pharmaceutically or veterinarily acceptable stabilizers may be carbohydrates (e.g., sorbitol, mannitol, lactose, sucrose, glucose, dextran, trehalose), sodium glutamate, proteins such as peptone, albumin, lactalbumin or casein, protein containing agents such as skimmed milk, and buffers (e.g., phosphate buffer, alkaline metal phosphate buffer).
  • An adjuvant may be used.
  • adjuvants are oil-in-water, water-in-oil-in- water emulsions based on mineral oil and/or vegetable oil and non ionic surfactants such as block copolymers, Tween®, Span®.
  • suitable adjuvants are for example vitamin E, saponins, and Carbopol®, aluminium hydroxide or aluminium phosphate ("Vaccine Design, The subunit and adjuvant approach", Pharmaceutical Biotechnology, vol. 6, Edited by Michael F. Powell and Mark J. Newman, 1995, Plenum Press New York).
  • the live attenuated bacteria may be stored at -70 0 C in a medium containing glycerol.
  • the immunogenic composition or vaccine can be combined with one or more immunogens, antigens or epitopes selected from other pathogenic micro-organisms or viruses in an inactivated or live form.
  • the present invention relates to methods to immunize against or to prevent bacterial infection or protect against bacterial infection in animals or humans.
  • a live attenuated immunogenic composition or vaccine of the invention is administered.
  • embodiments of the invention may be employed with other vaccines or immunogenic compositions that are not of the invention, e.g., in prime-boost processes, such as where a vaccine or immunogenic composition of the invention is administered first and a different vaccine or immunogenic composition is administered thereafter, or vice versa.
  • the administration may be notably made by intramuscular (IM), intradermal (ID) or subcutaneous (SC) injection or via intranasal, intratracheal or oral administration.
  • IM intramuscular
  • ID intradermal
  • SC subcutaneous
  • the immunogenic composition or the vaccine according to the invention is advantageously administered by syringe, needleless apparatus, spray, drinking water, eye-drop.
  • Advantageous administrations for the live attenuated immunogenic composition or vaccine are via the oral, ocular, tracheal, intradermal, subcutaneous (SC) or intramuscular (EVl) routes.
  • the quantity of live attenuated microorganisms can be determined and optimized by the skilled person, without undue experimentation from this disclosure and the knowledge in the art. Generally an animal (including a human) may be administered approximately
  • the putative IpxA gene in M. catarrhalis strain 035E was identified by BLAST searches of the M. catarrhalis genome.
  • the strain 035E IpxA gene homologue was amplified using primers ipxAl and IpxAl, and cloned into pCR2.1. Nucleotide sequence analysis showed that the DNA fragment contained a single open reading frame of 774 bp with a predicted gene product of 257 amino acids.
  • the deduced polypeptide sequence showed 53% or 45% identity compared with a known IpxA amino acid sequence of E. coli or N. meningitidis.
  • IpxA gene Upstream sequence analysis of IpxA gene revealed the presence of ipxD and fabZ; two genes linked to IpxA (Fig. 2). The same gene order is seen in the chromosomal DNA of E. coli and of N. meningitides (Raetz, C.R., and Whitfield, C. 2002 Annu Rev Biochem 71:635-700; Steeghs, L. et al. 1997 Gene 190:263-270). Construction of a knockout IpxA mutant
  • IpxA mutant was constructed by allelic exchange with a substitution of a kanamycin-resistant cassette within the IpxA coding region, where the IpxA gene was disrupted (Fig. 2).
  • the disrupted IpxA gene was amplified from kanamycin-resistant colonies using primers ipxAi and IpxAl. Nucleotide sequence analysis of PCR products confirmed that the kanamycin-resistant cassette had been inserted into IpxA of O35E chromosomal DNA at the predicted position. Thus, a mutant O35ElpxA was generated. Morphology and growth rate of the IpxA mutant
  • the O35ElpxA formed large, thin, flat, and transparent colonies on the chocolate agar plates when compared with the wild type strain.
  • the growth rate of the mutant was similar to that of the wild type in BHI broth. Such growth rate was slightly slower than the wild type strain in logarithmic phase. However, the wet weight of mutant cells was greater than that of wild type at stationary phase (over night growth) by 30-50%. Determination of LOS in the IpxA mutant
  • LOS was detected in O35E but not in O35ElpxA (Fig. 3A). Since the truncated LOS, possibly present in O35ElpxA, may be more hydrophobic and soluble in the organic solvents during extraction, OMV with whole structure of outer membranes was also analyzed. An LOS band was detected in OMV of 035E by silver staining while the mutant O35E//H.4 did not produce a detectable LOS band in OMV sample (Fig. 3B). The LOS band was confirmed by Western blot analysis, in which a specific anti-LOS antibody detected LOS in the O35E but not in the O35ElpxA (Fig. 3C).
  • the IpxA mutant was tested for LOS-associated biological activity in an LAL assay.
  • the mutant O35E/pzA was extremely susceptible to most hydrophobic antibiotics and reagents, and also to a hydrophilic glycopeptide, vancomycin (Table 1).
  • hi a bactericidal assay with normal human serum strain O35E survived at the highest concentration of 25% normal human serum.
  • Polymyxin B (300 iu) 11.5 ⁇ 0.5 16.0 ⁇ 0.0
  • Vancomycin (5 ⁇ g) ⁇ 6.0 b 12.2 ⁇ 0.5
  • Triton X-100 (5% [wt/vol]) 15.5 ⁇ 0.5 40.0 ⁇ 0.9
  • human epithelial cells Chang, HeLa, and A549 cell lines were used.
  • the adherence rates of the O35ElpxA to Chang and HeLa epithelia were 5.5 + 0.6 and 7.3 + 0.6, respectively, while those of wild type were 54.9 ⁇ 4.9 and 64.6 ⁇ 4.7, respectively, a 9 to 10-fold reduction in the mutant.
  • the adherence rate of O35ElpxA was 6.9 ⁇ 2.4 while that of the wild type was 19.3 ⁇ 2.0, near a 3-fold reduction.
  • Table 2 shows that both the O35ElpxA and wild type whole cells elicited similar levels of serum anti-whole cell IgG antibodies. The antibody levels were significantly enhanced by 5 to 7-fold when an adjuvant was used.
  • the bactericidal titer elicited by immunization of mice with O35ElpxA was comparable to that of the wild type (1:640 vs. 1:640 or 1:320 vs 1:160).
  • active immunization of mice with the mutant strain O35ElpxA resulted in a significant bacterial reduction (77%) in the lungs after an aerosol challenge with the wild type strain when compared to that of the controls. This level of bacterial reduction was similar to that of the wild type strain (62% reduction).
  • PBS 1 ⁇ 1 5 ND ND a Mice were immunized subcutaneously with 0.2 ml of 10 CFU of killed strain 035E or
  • O35ElpxA with or without Ribi adjuvant for three times at 10 -day intervals and blood were collected two weeks after the last injection.
  • LOS biosynthesis in Gram-negative bacteria is usually lethal to the organism (Galloway, S.M. and Raetz, CR. 1990 J Biol Chem 265:6394-6402; Kelly, T.M. et al. 1993 J Biol Chem 268:19866-19874).
  • a meningococcal ipxA mutant has been shown to be viable without LOS (Steeghs, L. et al. 1998 Nature 392:449-450), while further effort to construct IpxA knockout mutants in N. gonorrhoeae and H. influenzae have failed (van der Ley, P. and Steeghs, L. 2003 J Endotoxin Res 9:124-128).
  • M. catarrhalis bacterium does not require LOS for viability, LOS deficiency results in alterations in colony morphology, permeability of OM, and serum resistance.
  • O35 ⁇ lpxA colonies are large, flat and transparent, almost like those from an OMP CD mutant (Holm, M. M. et al. 2004 Infect Immun 72:1906-1913), and the altered opacity of the IpxA mutant is consistent with H. influenzae (Weiser, J. N. and Pan, N. 1998 MoI Microbiol 30:767-775) and N. meningitidis (Albiger, B. et al.
  • the mutant O35E IpxA was extremely susceptible to most hydrophobic reagents. This may be caused by the lack of a continuous LOS layer in the outer membrane leaflet and the resultant compensatory presence of glycerophospholipid in this leaflet, creating glycerophospholipid bilayers or patches in the outer membrane that allow for diffusion of hydrophobic solutes.
  • the mutant was also susceptible to a hydrophilic glycopeptide which is normally excluded by the intact enterobacterial outer membrane indicating that the outer membrane of M. catarrhalis IpxA mutant is probably fragile and transiently ruptured (Vaara, M.
  • M. catarrhalis is a respiratory tract mucosal pathogen, it is feasible to evaluate its virulence by observation of interaction with host epithelial cells in vitro and in an aerosol challenge mouse model. Attachment of microbes to host epithelial cells represents the first step in the pathogenesis of microbial infection, with the target specificity being defined by precise adhesin-receptor interactions (St. Geme, J. W. 1997 Adv Pediatr 44:43-72). In our in vitro study, attachment to human epithelia by the IpxA mutant showed near a 10-fold reduction to Chang (conjunctival), or HeLa (cervix) cells but near a 3 -fold reduction to A549 (lung) cells.
  • the mutant had reduced adherence to different types of epithelial cells, m the mouse challenge model, the IpxA mutant showed reduced rates of attachment to mouse respiratory tracts, especially in lungs immediately after a bacterial challenge. However, following the challenge the mutant was rapidly cleared from the nasopharynx, but presented a similar clearance rate in lungs when compared with that of the parental strain. These data indicate that the O35ElpxA mutant is deficient in its interaction with the lungs right after the bacterial challenge and is more easily cleared from the nasopharynx post challenge. Both early and late stages are critical for bacterial adherence proceeding towards microbial infections (St. Geme, J.W. 1997 Adv Pediatr 44:43-72).
  • Loss of LOS structure in the O35ElpxA resulted in reduced bacterial attachment in vitro and/or enhanced bacterial clearance in vivo, indicating that LOS is an important virulence factor in the pathogenesis of M. catarrhalis infections.
  • the LOS-deficient mutant O35ElpxA is nontoxic with reduced virulence features, and is envisioned as being used as the basis of a vaccine. Recent studies involving vaccine antigens have shown that multiple bacterial components might be preferred and given the ability of the bacteria to vary surface components in response to immunologic pressures (McMichael, J.C. 2000 Microbe Infect 2:561-568; Karalus, R., and Campagnari, A.
  • M. catarrhalis LOS is not essential for bacterial survival, although it is an important virulence factor.
  • a completely LOS-deficient M. catarrhalis strain with normal growth rate is attenuated and highly immunogenic, and can be used as the basis of a vaccine.
  • Such a nontoxic mutant is also envisioned as being useful as a vaccine vehicle for carrying and expressing other pathogenic components or epitopes as vaccine components for further utilities.
  • EXAMPLE 1 Strains, plasmids, and growth conditions Bacterial strains, plasmids and primers are described in Table 3. M. catarrhalis strains were cultured on chocolate agar plates (Remel, Lenexa, KS), or BHI (Difco, Detroit, MI) agar plates at 37°C in 5% CO 2 . Mutant strains were selected on BHI agar supplemented with kanamycin at 20 ⁇ g/ml. Growth rates of wild type and mutant strains were measured from an overnight culture inoculated in 10 ml of BHI broth (adjusted OD 600 0.05) and incubated at 37 0 C with 250 rpm.
  • DNA restriction endonucleases T4 DNA ligase, E. coli DNA polymerase I Klenow fragment and Taq DNA polymerase were purchased from Fermentas (Hanover, MD).
  • Preparation of plasmid and purification of PCR products or DNA fragments were performed using kits manufactured by Qiagen (Santa Clarita, CA).
  • Bacterial chromosomal DNA was isolated using a genomic DNA purification kit (Promega, Madison, WI).
  • DNA nucleotide sequences were obtained via 3070x1 DNA analyzer (Applied Biosystems, Foster city, CA) and analyzed with DNASTAR software (DNASTAR, Inc, Madison, WI). Cloning of an IpxA homologue and construction of a knockout IpxA mutant
  • PCR primers for cloning of the IpxA homologue from strain 035E were designed based on an assumptive M. catarrhalis IpxA sequence predicted by BLAST searches with other known IpxA homologues at GenBank of National Center for Biotechnology Information.
  • a PCR product was amplified from chromosomal DNA of strain O35E using primers ipxAX and lpxA2 (Table 3), and cloned into pCR2.1 using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA) to obtain pCRL.
  • the construct (containing an insertion of kanamycin-resistant cassette within a 241-bp deletion of IpxA coding region) was amplified by PCR using primers IpxAX and lpxA2.
  • a 20- ⁇ l portion of this cell suspension was mixed with 1.0 ⁇ g of the PCR product in 2 ⁇ l of water and transferred into a micro-electroporation chamber and electroporated using a field strength of 2.2 kV over a 0.1 -cm distance for electroporation (Micropulser, BIO-RAD, Hercules, CA).
  • the resulting cell suspension was mixed with 1 ml of BHI broth, shaken at 250 rpm at 37 0 C for 6 h, and plated on BHI agar containing kanamycin. After 24 h incubation, the kanamycin-resistant colonies were selected for PCR analysis of chromosomal DNA using primers lpxA2 and lpxA3 (Fig. 2).
  • the inactivated IpxA mutant was confirmed by sequence analysis and designated as O35ElpxA.
  • the nucleotide sequence of the IpxA gene was deposited at GenBank under accession number AY648946.
  • the chromogenic LAL assay for endotoxin activity was performed using the QCL- 1000 kit (Bio-Whittaker Inc, Walkersville, MD). Overnight cultures from chocolate agars were suspended in BHI broth to OD 62O of 0.1 and serial dilutions of these stocks were used as samples. Susceptibility
  • the sensitivity of strains to a panel of hydrophobic agents or hydrophilic glycopeptide was performed using standard disk-diffusion assays (West, N. et al. 2000 Infect Immun 68:4673-4680). Bacteria were cultured in BHI broth to an OD 600 of 0.2 and 100 ⁇ l portions of the bacteria were spred onto chocolate agar plates. Antibiotic disks or sterile blank paper disks (6 mm, Becton Dickinson, Cockeysville, MD) saturated with various agents were plated on the lawn in triplicate, and the plates were incubated at 37°C for 18 hr. Sensitivity was assessed by measuring the diameter of the zone of growth inhibition in two axes and the mean value was calculated.
  • Bactericidal assay with normal human serum A complement-sufficient normal human serum was prepared and pooled from 8 healthy adult donors. A 200- ⁇ l scale bactericidal assay was performed in a 96-well plate (Luke, N.R. et al. 2003 Infect Immun 71:6426-6434). Normal human serum was diluted to 0.5, 2.5, 5.0, 12.5, and 25% in pH 7.4 Dulbecco's PBS buffer (containing magnesium and calcium with 0.05% gelatin). Bacteria (10 ⁇ l of 10 6 CFU) were inoculated into 190- ⁇ l reaction wells containing the diluted normal human serum, 25% of heat-inactivated normal human serum or the PBS buffer alone and incubated at 37°C for 30 min. Samples were diluted serially (1:10) and plated onto chocolate agar plates. The resulting colonies were counted after 24 h of incubation. Adherence assay
  • Chang conjunctival; CCL20.2), HeLa (cervix; CCL-2), and A549 (lung; CCL-185) human epithelial lines were cultured in Eagle's Minimal Essential medium (ATCC, Manassas, VA) supplemented with 10% heat-inactivated fetal bovine serum at 37°C in 5% CO 2 .
  • a quantitative adherence assay was performed (Aebi, C. et al. 1998 Infect Immun 66:3113-3119). Briefly, 1 ml of 2x10 5 cells was seeded into each well of 24-well tissue culture plates and incubated for 24 h.
  • mice Female BALB/c mice (6-8 weeks of age), from Taconic Farms Inc. (Germantown, NY), were housed in an animal facility in accordance with National Institutes of Health guidelines under animal study protocol 1158-04.
  • a bacterial aerosol challenge was carried out in mice using 10-ml of 1.65xlO 9 or 1.8OxIO 9 CFU/ml of wild type strain O35E or mutant O35ElpxA respectively (Hu, W.G. et al. 2000 Vaccine 18:799-804).
  • the numbers of bacteria present in the lungs and nasal washes were measured at various time points post challenge.
  • the minimum detecting numbers of viable bacteria were 100 CFU per lung (10 ml) and 4 CFU per nasal washing (0.4 ml). Clearance of M. catarrhalis was expressed as the percentage of bacterial CFU detectable at each time point compared with the number deposited at time zero.
  • Antibody response and challenge study was carried out in mice using 10-ml of 1.65xlO 9 or 1.8OxIO 9
  • mice in each group received three subcutaneous injections with 0.2 ml of Ix 10 8 CFU whole cells of wild type or mutant strains or PBS with or without Ribi-700 adjuvant (Corixa Corporation, Hamilton, MT) at 10-day intervals. Blood samples were collected at 2 weeks after the last injection. Serum antibodies were assayed by whole cell ELISA, where ELISA plates were coated with 50 ⁇ l of O35ElpxA (4x10 7 CFU/ml), and dried at 5O 0 C overnight. Other steps were performed as described (Hu, W. G. et al. 2001 Infect Immun 69:1358-1363).
  • bactericidal activity against wild type strain O35E pooled mouse sera from each group were used after inactivation at 56°C for 30 min. A bactericidal assay was performed (Gu, X.X. et al. 1998 Infect Immun 66:1891-1897), and titers were the last dilution of the sera causing at least 50% killing as compared with that of a control.
  • mice immunized with whole cells of wild type or mutant strains plus Ribi adjuvant were challenged by an aerosol of 10 ml of wild type strain 035E (4 xlO 9 CFU/ml). The number of CFU present in the lungs was measured at 6 h post challenge (Hu, W.G. et al. 2000 Vaccine 18:799-804).
  • the number of viable bacteria were expressed as geometric mean (GM) CFUs of n independent observations + SD.
  • the antibody titers were expressed as reciprocal GM of n independent observations ⁇ SD.
  • the significance was analyzed using two-tailed independent Student's t test.
  • the bacterial clearance rate was analyzed by Chi-square test.
  • Ribi - 10 a Mice were immunized subcutaneously with 0.2 ml of purified 035E or O35ElpxA outer 5 membrane protein with or without Ribi adjuvant for three times at 10-day intervals, and blood samples collected two weeks after the last injection.

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Abstract

L'invention concerne une bactérie de Moraxella catarrhalis comprenant une mutation, de sorte qu'elle est viable, et ne contient pas de lipooligosaccharide endotoxique (LOS). L'invention concerne également des vaccins associés et des méthodes d'immunisation correspondantes.
PCT/US2005/019479 2004-06-04 2005-06-03 Moraxella catarrhalis exempte d'endotoxine WO2006085910A2 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998053851A1 (fr) * 1997-05-28 1998-12-03 University Of Iowa Research Foundation Mutants laft de bacteries pathogenes gram negatif
WO1999010497A1 (fr) * 1997-08-21 1999-03-04 De Staat Der Nederlanden, Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur Nouveaux mutants de bacteries des muqueuses gram negatives et leur application dans des vaccins
WO1999036086A1 (fr) * 1998-01-13 1999-07-22 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Vaccin a base de lipooligosaccharide destine a la prevention d'infections dues a moraxella (branhamella) catarrhalis chez les mammaliens

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998053851A1 (fr) * 1997-05-28 1998-12-03 University Of Iowa Research Foundation Mutants laft de bacteries pathogenes gram negatif
WO1999010497A1 (fr) * 1997-08-21 1999-03-04 De Staat Der Nederlanden, Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur Nouveaux mutants de bacteries des muqueuses gram negatives et leur application dans des vaccins
WO1999036086A1 (fr) * 1998-01-13 1999-07-22 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Vaccin a base de lipooligosaccharide destine a la prevention d'infections dues a moraxella (branhamella) catarrhalis chez les mammaliens

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GU X X ET AL: "Synthesis and characterization of lipooligosaccharide-based conjugates as vaccine candidates for Moraxella (Branhamella) catarrhalis" INFECTION AND IMMUNITY, AMERICAN SOCIETY FOR MICROBIOLOGY. WASHINGTON, US, vol. 66, no. 5, May 1998 (1998-05), pages 1891-1897, XP002102901 ISSN: 0019-9567 *
LEY VAN DER P ET AL: "MODIFICATION OF LIPID A BIOSYNTHESIS IN NEISSERIA MENINGITIDIS IPXLMUTANTS: INFLUENCE ON LIPOPOLYSACCHARIDE STRUCTURE, TOXICITY AND ADJUVANT ACTIVITY" INFECTION AND IMMUNITY, AMERICAN SOCIETY OF MICROBIOLOGY, WASHINGTON, DC, US, vol. 69, no. 10, October 2001 (2001-10), pages 5981-5990, XP001055221 ISSN: 0019-9567 *
LUKE NICOLE R ET AL: "Identification of a 3-deoxy-D-manno-octulosonic acid biosynthetic operon in Moraxella catarrhalis and analysis of a KdsA-deficient isogenic mutant." INFECTION AND IMMUNITY, vol. 71, no. 11, November 2003 (2003-11), pages 6426-6434, XP002392434 ISSN: 0019-9567 *
PENG DAXIN ET AL: "Moraxella catarrhalis bacterium without endotoxin, a potential vaccine candidate" INFECTION AND IMMUNITY, vol. 73, no. 11, November 2005 (2005-11), pages 7569-7577, XP002392435 ISSN: 0019-9567 *
STEEGHS L ET AL: "Isolation and characterization of the Neisseria meningitidis lpxD-fabZ-lpxA gene cluster involved in lipid A biosynthesis" GENE: AN INTERNATIONAL JOURNAL ON GENES AND GENOMES, ELSEVIER, AMSTERDAM, NL, vol. 190, no. 2, 1997, pages 263-270, XP004116090 ISSN: 0378-1119 *
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