C群奈瑟氏脑膜炎球菌荚膜多糖-TT结合疫苗的制备及其在小鼠体内免疫原性的研究

将C群脑膜炎球菌荚膜多糖以ADH作为间隔剂与TT结合形成GCMP-TT结合疫苗,然后用此结合疫苗免疫NIH小鼠,结果显示使用GCMP免疫小鼠后仅能产生较低水平抗GCMP的IgG抗体,而用GCMP-TT免疫后小鼠血清中产生了较GCMP免疫显著增高抗GCMP的IgG抗体,并且GCMP-TT组第二次和第三次免疫后与初次免疫相比,IgG抗体水平均有显著升高(P<0.01),表明GCMP-TT结合疫苗具有免疫记忆和再次免疫加强应答效应。补体介导的血清抗体体外杀菌试验结果证明,GCMP-TT结合疫苗组免疫小鼠诱导的抗体IgG比GCMP组具有增强的体外杀菌活性。     

The emerging pathogen Moraxella catarrhalis interacts with complement inhibitor C4b binding protein through ubiquitous surface proteins A1 and A2

Moraxella catarrhalis ubiquitous surface protein A2 (UspA2) mediates resistance to the bactericidal activity of normal human serum. In this study, an interaction between the complement fluid phase regulator of the classical pathway, C4b binding protein (C4BP), and M. catarrhalis mutants lacking UspA1 and/or UspA2 was analyzed by flow cytometry and a RIA. Two clinical isolates of M. catarrhalis expressed UspA2 at a higher density than UspA1. The UspA1 mutants showed a decreased C4BP binding (37.6% reduction), whereas the UspA2-deficient Moraxella mutants displayed a strongly reduced (94.6%) C4BP binding compared with the wild type. In addition, experiments with recombinantly expressed UspA1(50-770) and UspA2(30-539) showed that C4BP (range, 1-1000 nM) bound to the two proteins in a dose-dependent manner. The equilibrium constants (K(D)) for the UspA1(50-770) and UspA2(30-539) interactions with a single subunit of C4BP were 13 microM and 1.1 microM, respectively. The main isoform of C4BP contains seven identical alpha-chains and one beta-chain linked together with disulfide bridges, and the alpha-chains contain eight complement control protein (CCP) modules. The UspA1 and A2 bound to the alpha-chain of C4BP, and experiments with C4BP lacking CCP2, CCP5, or CCP7 showed that these three CCPs were important for the Usp binding. Importantly, C4BP bound to the surface of M. catarrhalis retained its cofactor activity as determined by analysis of C4b degradation. Taken together, M. catarrhalis interferes with the classical complement activation pathway by binding C4BP to UspA1 and UspA2.     

Method for reducing endotoxin in Moraxella catarrhalis UspA2 protein preparations

The UspA2 protein from the bacterium Moraxella catarrhalis is a potential vaccine candidate for preventing human diseases caused by this organism. Before a vaccine can be administered parentally, the level of endotoxin must be reduced as much as possible. However, in this case the endotoxin was very tightly complexed with the UspA2 protein and could not be dissociated with Triton X-100. It was found that it dissociated from the protein with the zwitterionic detergents Zwittergent 3-12 and Zwittergent 3-14. The endotoxin could then be separated from the protein by either ion-exchange or gel filtration chromatography. Using the limulus amoebocyte lysate assay for quantitation, the endotoxin was reduced approximately 20 000-fold. The removal of residual endotoxin from UspA2 preparations had no detrimental effect on the immunological properties of the protein. Mouse antisera raised against UspA2 prior to, and following endotoxin reduction exhibited comparable antibody and bactericidal titers against the tested strains. Further, mice immunized with both preparations, followed by pulmonary challenge with either a homologous or a heterologous isolate, exhibited comparable levels of clearance.     

卡他莫拉菌UspA1蛋白多克隆抗体的制备及鉴定

为制备抗卡他莫拉菌(Moraxella catarrhalis,Mc)表面蛋白UspA1胞外结构域的多克隆抗体(PcAb),对UspA1蛋白进行生物信息学分析,获取胞外结构域中抗原表位最为丰富的肽段,找到其对应的基因序列并引入大肠杆菌偏好性密码子,对其优化后化学合成全基因序列。将该基因序列按常规方法克隆入表达载体p ET-28a(+)后表达重组UspA1-His融合蛋白并纯化。以该纯化抗原免疫新西兰大白兔,经4次免疫后,用Protein A亲和层析柱从抗血清中纯化出抗UspA1-His融合蛋白PcAbIgG。经免疫荧光法、酶联免疫吸附法及Western blotting鉴定,抗UspA1-His融合蛋白PcAb能特异性识别UspA1蛋白的表面暴露区。该多抗的制备为下一步建立卡他莫拉菌快速检测技术奠定了基础。     

Outer membrane protein UspA1 and lipooligosaccharide are involved in invasion of human epithelial cells by Moraxella catarrhalis

Invasion of non-professional phagocytes is a strategy employed by several mucosal pathogens, but has not been investigated in detail for Moraxella catarrhalis, a major cause of human respiratory tract infections. We investigated the role of outer membrane protein (OMP) UspA1 and lipooligosaccharide (LOS) in M. catarrhalis invasion into epithelial cells. An isogenic mutant of strain O35E, which lacked expression of the UspA1 adhesin, demonstrated not only severely impaired adherence (86%) to but also reduced invasion (77%) into Chang conjunctival cells in comparison with the wild-type strain. The isogenic, LOS-deficient mutant strain O35E.lpxA was attenuated in adherence (93%) and its capacity to invade was severely reduced (95%), but not abolished. Inhibition assays using sucrose and cytochalasin D, respectively, demonstrated that clathrin and actin polymerization contribute to internalization of M. catarrhalis by Chang cells. Furthermore, inhibition of UspA1-mediated binding to cell-associated fibronectin and alpha5beta1 integrin decreased invasion of M. catarrhalis strain O35E (72% and 41%, respectively). These data indicate that OMP UspA1 and LOS profoundly affect the capacity of M. catarrhalis to invade epithelial cells.     

The major outer membrane protein, CD, extracted from Moraxella (Branhamella) catarrhalis is a potential vaccine antigen that induces bactericidal antibodies

The major outer membrane protein of Moraxella (Branhamella) catarrhalis, CD, was detergent-extracted from the bacterial cell wall and purified to homogeneity in high yields by a simple process. The purified protein appeared to exhibit immunogenic properties similar to those of native CD exposed on the surface of the bacterium. Antibodies to CD raised in mice specifically bound to intact B. catarrhalis, as determined by flow cytometry analysis. The IgG subclass distributions of anti-CD antibodies in sera from mice immunized with purified CD or with B. catarrhalis were also similar. CD was found to be antigenically conserved among a panel of B. catarrhalis isolates, as demonstrated by the consistent reactivities of mouse anti-CD antisera with a common 60 kDa protein on immunoblots. Furthermore, convalescent sera collected from patients with otitis media due to B. catarrhalis infection were found to be reactive with the CD protein by immunoblotting. Finally, the purified protein induced antibodies in guinea pigs and mice that exhibited in vitro bactericidal activity against the pathogen. Therefore, the native CD outer membrane protein represents a potentially useful antigen for inclusion in a vaccine against B. catarrhalis.     

The UspA1 protein and a second type of UspA2 protein mediate adherence of Moraxella catarrhalis to human epithelial cells in vitro

The UspA1 and UspA2 proteins of Moraxella catarrhalis are structurally related, are exposed on the bacterial cell surface, and migrate as very high-molecular-weight complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Previous analysis of uspA1 and uspA2 mutants of M. catarrhalis strain 035E indicated that UspA1 was involved in adherence of this organism to Chang conjunctival epithelial cells in vitro and that expression of UspA2 was essential for resistance of this strain to killing by normal human serum (C. Aebi, E. R. Lafontaine, L. D. Cope, J. L. Latimer, S. R. Lumbley, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 66:3113-3119, 1998). In the present study, isogenic uspA1, uspA2, and uspA1 uspA2 mutations were constructed in three additional M. catarrhalis strains: 012E, TTA37, and 046E. The uspA1 mutant of strain 012E had a decreased ability to attach to Chang cells. However, inactivation of the uspA1 gene in both strain TTA37 and strain 046E did not cause a significant decrease in attachment ability. Inactivation of the uspA2 gene of strain TTA37 did result in a loss of attachment ability. Nucleotide sequence analysis revealed that the predicted protein encoded by the uspA2 genes of both strains TTA37 and 046E had a N-terminal half that resembled the N-terminal half of UspA1 proteins, whereas the C-terminal half of this protein was nearly identical to those of previously characterized UspA2 proteins. The gene encoding this "hybrid" protein was designated uspA2H. PCR-based analysis revealed that approximately 20% of M. catarrhalis strains apparently possess a uspA2H gene instead of a uspA2 gene. The M. catarrhalis uspA1, uspA2, and uspA2H genes were cloned and expressed in Haemophilus influenzae cells, which were used to prove that both the UspA1 and UspA2H proteins can function as adhesins in vitro.     

Ionic binding of C3 to the human pathogen Moraxella catarrhalis is a unique mechanism for combating innate immunity

Moraxella catarrhalis ubiquitous surface proteins A1 and A2 (UspA1/A2) interfere with the classical pathway of the complement system by binding C4b-binding protein. In this study we demonstrate that M. catarrhalis UspA1 and A2 noncovalently and in a dose-dependent manner bind both the third component of complement (C3) from EDTA-treated serum and methylamine-treated C3. In contrast, related Moraxella subspecies (n = 13) or other human pathogenic bacteria (n = 13) do not bind C3 or methylamine-treated C3. Experiments with recombinant proteins and M. catarrhalis mutants devoid of UspA1/A2 revealed that UspA1/A2 exert their actions by absorbing and neutralizing C3 from serum and restrain complement activation. UspA2 was responsible for most of the effect, and the Moraxella mutant lacking UspA2 was more sensitive to the lytic effect of human serum compared with the wild type. Interestingly, among the large number of bacteria analyzed, only M. catarrhalis has this unique ability to interfere with the innate immune system of complement by binding C3.     

Progress toward the development of a vaccine to prevent Moraxella (Branhamella) catarrhalis infections

Moraxella catarrhalis is a major cause of otitis media and respiratory disease. Vaccine development is at the antigen identification stage. This review examines the more promising antigens, including the 200K protein, the hemagglutinins, the lactoferrin-binding proteins, the UspA proteins, the CopB protein, the transferrin-binding proteins, the CD protein, the E protein and lipooligosaccharide conjugates. Clinical testing of some of these antigens should begin soon.     

Investigating the potential of conserved inner core oligosaccharide regions of Moraxella catarrhalis lipopolysaccharide as vaccine antigens: accessibility and functional activity of monoclonal antibodies and glycoconjugate derived sera

We investigated the conservation and antibody accessibility of inner core epitopes of Moraxella catarrhalis lipopolysaccharide (LPS) in order to assess their potential as vaccine candidates. Two LPS mutants, a single mutant designated lgt2 and a double mutant termed lgt2/lgt4, elaborating truncated inner core structures were generated in order to preclude expression of host-like outer core structures and to create an inner core structure that was shared by all three serotypes A, B and C of M. catarrhalis. Murine monoclonal antibodies (mAbs), designated MC2-1 and MC2-10 were obtained by immunising mice with the lgt2 mutant of M. catarrhalis serotype A strain. We showed that mAb MC2-1 can bind to the core LPS of wild-type (wt) serotype A, B and C organisms and concluded that mAb MC2-1 defines an immunogenic inner core epitope of M. catarrhalis LPS. We were unsuccessful in obtaining mAbs to the lgt2/lgt4 mutant. MAb MC2-10 only recognised the lgt2 mutant and the wt serotype A strain, and exhibited a strong requirement for the terminal N-acetyl-glucosamine residue of the lgt2 mutant core oligosaccharide, suggesting that this residue was immunodominant. Subsequently, we showed that both mAbs MC2-1 and MC2-10 could facilitate bactericidal killing of the lgt2 mutant, however neither mAb could facilitate bactericidal killing of the wt serotype A strain. We then confirmed and extended the candidacy of the inner core LPS by demonstrating that it is possible to elicit functional antibodies against M. catarrhalis wt strains following immunisation of rabbits with glycoconjugates elaborating the conserved inner core LPS antigen. The present study describes three conjugation strategies that either uses amidases produced by Dictyostelium discoideum, targeting the amino functionality created by the amidase activity as the attachment point on the LPS molecule, or a strong base treatment to remove all fatty acids from the LPS, thus creating amino functionalities in the lipid A region to conjugate via maleimide-thiol linker strategies targeting the carboxyl residues of the carrier protein and the free amino functionalities of the derived lipid A region of the carbohydrate resulted in a high loading of carbohydrates per carrier protein from these carbohydrate preparations. Immunisation derived antisera from rabbits recognised fully extended M. catarrhalis LPS and whole cells. Moreover, bactericidal activity was demonstrated to both the immunising carbohydrate antigen and importantly to wt cells, thus further supporting the consideration of inner core LPS as a potential vaccine antigen to combat disease caused by M. catarrhalis.     

Plasmid pWW115, a cloning vector for use with Moraxella catarrhalis

The plasmid shuttle vector pWW102B is able to replicate in only a modest number of Moraxella catarrhalis strains. Plasmid pWW115, a spontaneous deletion mutant of pWW102B, was shown to lack both the pACYC184-derived origin of replication and the associated chloramphenicol-resistance gene but was able to replicate in every M. catarrhalis strain tested in this study, including one strain that had been previously refractory to all types of genetic manipulations. To test the utility of this plasmid, a M. catarrhalis gene encoding the UspA2 serum-resistance factor was cloned into pWW115 and the resultant recombinant plasmid was shown to confer serum-resistance on a serum-sensitive M. catarrhalis uspA2 mutant.     

Vaccination equally enables both genetically susceptible and resistant mice to control infection with group A streptococci

There is substantial evidence that host genetic factors are important in determining susceptibility to infection with group A streptococci (GAS). Several studies have revealed that, similarly to humans, a genetic component may be important in determining susceptibility to GAS infection in mice. Thus, C3H/HeN mice are much more susceptible to streptococcal infection than BALB/c mice. We have determined here whether vaccination makes genetically susceptible mice as capable as genetically resistant mice to control GAS infection. Resistant BALB/c and susceptible C3H/HeN mice were immunized either systemically with heat-killed GAS or through the mucosal route with an M protein-based subunit vaccine, and challenged with live bacteria. Vaccination elicited in both mouse strains similar levels of bactericidal anti-GAS IgG antibodies and also antigen-specific mucosal IgA. Vaccination provided mice of both strains with an increased and equal capacity to express immunity against GAS as indicated by the reduced level of bacteria in the organs and the ability of vaccinated mice to survive infection. Protection in vaccinated mice was dependent on the presence of T cell-dependent bactericidal antibodies as shown by the ability of serum elicited in immunocompetent mice but not of serum elicited in T cell-deficient nu/nu mice to passively transfer anti-GAS immunity. In conclusion, the results presented here demonstrated that the presence of anti-GAS specific, T cell-dependent bactericidal antibodies elicited after vaccination overcomes the innate genetic susceptibility of C3H/HeN mice and makes both resistant and susceptible mice equally capable of controlling GAS infection.     

T-cell-mediated clearance of mouse hepatitis virus strain JHM from the central nervous system

Clearance of the neurotropic JHM strain of mouse hepatitis virus from the central nervous system was examined by the transfer of spleen cells from immunized donors. A T cell with the surface phenotype of Thy1.2+ CD4+ CD8- asialo-GM1+ Mac-1- was found to be necessary for viral clearance. The surface phenotype and adherence to nylon wool suggest that these cells are activated helper-inducer T cells. Adoptive transfer to congenic histocompatibility strains demonstrated the necessity for compatibility at the D locus of the major histocompatibility complex. The expression of the CD4 surface marker and the requirement for major histocompatibility complex class I were further studied by the transfer of cells to recipients treated with anti-CD4 or anti-CD8 monoclonal antibodies. Treatment of recipients with either the anti-CD8 or the anti-CD4 antibodies inhibited virus clearance from the central nervous system. This suggests that the CD4+ cell acts as a helper and that virus is cleared from the central nervous system. This suggests that the CD4+ cell acts as a helper and that virus is cleared from the central nervous system by CD8+ cells that recognize viral antigen in the context of the H-2Db gene product.     

Antigenic Determinants of Salmonella for Production of “Clearance” Factor in Mouse Typhoid

The “clearance” factor produced in the peritoneal cavity of mice immunized with killed vaccines prepared from Salmonella typhimurium or S. enteritidis was identified as the specific antibodies elicited by the O side chain of the cell wall polysaccharides in the organisms used as immunogens. After immunization of mice with vaccines prepared from virulent Salmonella strains, complement-dependent antibacterial antibodies in the serum and “clearance” factors in the peritoneal cavity were found to appear coincidentally, to last for more than one year, and to have the same specificity against the virulent bacterial strains. The relationship between the complement-dependent antibacterial antibodies and “clearance” factor, and the mechanisms of bactericidal action of these antibacterial agents in experimental typhoid were discussed.     

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.     

Vaccination with the T cell antigen Mtb 8.4 protects against challenge with Mycobacterium tuberculosis

The development of an effective vaccine against Mycobacterium tuberculosis is a research area of intense interest. Mounting evidence suggests that protective immunity to M. tuberculosis relies on both MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells. By purifying polypeptides present in the culture filtrate of M. tuberculosis and evaluating these molecules for their ability to stimulate PBMC from purified protein derivative-positive healthy individuals, we previously identified a low-m.w. immunoreactive T cell Ag, Mtb 8.4, which elicited strong Th1 T cell responses in healthy purified protein derivative-positive human PBMC and in mice immunized with recombinant Mtb 8.4. Herein we report that Mtb 8.4-specific T cells can be detected in mice immunized with the current live attenuated vaccine, Mycobacterium bovis-bacillus Calmette-Guérin as well as in mice infected i.v. with M. tuberculosis. More importantly, immunization of mice with either plasmid DNA encoding Mtb 8.4 or Mtb 8.4 recombinant protein formulated with IFA elicited strong CD4(+) T cell and CD8(+) CTL responses and induced protection on challenge with virulent M. tuberculosis. Thus, these results suggest that Mtb 8.4 is a potential candidate for inclusion in a subunit vaccine against TB.