Epitopes of serogroup B Neisseria meningitidis analysed in vitro and directly from cerebrospinal fluid

Two type B15 P1.16 strains of Neisseria meningitidis were examined by immunogold electron microscopy for accessibility of two outer membrane protein (OMPs) to monoclonal antibodies. Both strains exhibited cell-to-cell variation of one epitope of the Class 3 OMP (P3.15) and one of the Class 1 OMPs (P1.16) when grown in vitro. One strain, a nasopharyngeal isolate revealed this variation to be growth-phase independent and double labelling of both epitopes showed independent variation. CSF containing N. meningitidis was stored in liquid nitrogen without laboratory processing at the time of isolation of the second strain. Direct analysis of the organisms showed no cell-to-cell variation and immunoglobulin G on the surface. However, while there were similar labelling densities of the Class 1 epitope in vivo compared with either strain grown in vitro, there was a lower labelling density of the Class 3 epitope in vivo that was not caused by freeze-thawing. This reduction may be due to decreased expression of this epitope in vivo which casts doubts on the use of the Class 2/3 OMP as a vaccine candidate.     

FetA Antibodies Induced by an Outer Membrane Vesicle Vaccine Derived from a Serogroup B Meningococcal Isolate with Constitutive FetA Expression

Invasive meningococcal disease causes over 3500 cases each year in Europe, with particularly high incidence among young children. Among serogroup B meningococci, which cause most of the cases, high diversity in the outer membrane proteins (OMPs) is observed in endemic situations; however, comprehensive molecular epidemiological data are available for the diversity and distribution of the OMPs PorA and FetA and these can be used to rationally design a vaccine with high coverage of the case isolates. The aim of this study was to determine whether outer membrane vesicles (OMVs) derived from an isolate with constitutive FetA expression (MenPF-1 vaccine) could be used to induce antibodies against both the PorA and FetA antigens. The immunogenicity of various dose levels and number of doses was evaluated in mice and rabbits, and IgG antibody responses tested against OMVs and recombinant PorA and FetA proteins. A panel of four isogenic mutants was generated and used to evaluate the relative ability of the vaccine to induce serum bactericidal activity (SBA) against FetA and PorA. Sera from mice were tested in SBA against the four target strains. Results demonstrated that the MenPF-1 OMVs were immunogenic against PorA and FetA in both animal models. Furthermore, the murine antibodies induced were bactericidal against isogenic mutant strains, suggesting that antibodies to both PorA and FetA were functional. The data presented indicate that the MenPF-1 vaccine is a suitable formulation for presenting PorA and FetA OMPs in order to induce bactericidal antibodies, and that proceeding to a Phase I clinical trial with this vaccine candidate is justified.     

The Development of an Experimental Multiple Serogroups Vaccine for Neisseria meningitidis

A native outer membrane vesicles (NOMV) vaccine was developed from three antigenically diverse strains of Neisseria meningitidis that express the L1,8, L2, and L3,7 lipooligosaccharide (LOS) immunotypes, and whose synX, and lpxL1 genes were deleted.. Immunogenicity studies in mice showed that the vaccine induced bactericidal antibody against serogroups B, C, W, Y and X N. meningitidis strains. However, this experimental NOMV vaccine was not effective against serogroup A N. meningitidis strains. N. meningitidis capsular polysaccharide (PS) from serogroups A, C, W and Y were effective at inducing bactericidal antibody when conjugated to either tetanus toxoid or the fHbp1-fHbp2 fusion protein fHbp(1+2). The combination of the NOMV vaccine and the N. meningitidis serogroup A capsular polysaccharide (MAPS) protein conjugate was capable of inducing bactericidal antibodies against a limited number of N. meningitidis strains from serogroups A, B, C, W, Y and X tested in this study.     

Antibody studies in mice of outer membrane antigens for use in an improved meningococcal B and C vaccine

Abstract Since 1988, N. meningitidis , B:4:P1.15, ET-5 complex, has been responsible for an epidemic of meningococcal disease in Greater São Paulo, Brazil. Despite current trials to develop an effective vaccine against group B meningococci, children less than 2 years old have not been protected. It has been suggested that iron-regulated proteins (IRPs) should be considered as potential antigens for meningococcal vaccines. The vaccines under study consisted of outer-membrane vesicles depleted of lipooligosaccharide from three serogroup B strains and one serogroup C strain, IRPs, meningococcal group C polysaccharide and aluminum hydroxide. Four different protein and C polysaccharide concentrations were studied. The ELISA and bactericidal results showed a higher antibody response when 2 injections of 2.0 μg doses were administered. Despite higher IgG reactivity against antigen preparations containing IRPs seen in ELISA, the bactericidal activity was not increased if the target strain was grown in iron-restricted medium. The influence of addition of alkaline-detoxified lipooligosaccharide (dLOS) on immunogenicity of the vaccine was also investigated, and the dLOS provided for a more functionally specific antibody response.     

Conformation-dependent antibody response to Pseudomonas aeruginosa outer membrane proteins induced by immunization in humans

Outer membrane proteins (OMPs) of pathogenic bacteria have been used as protective antigens in developing bacterial vaccines. In the present study, we compared the antibody responses to a Pseudomonas aeruginosa OMP vaccine elicited in humans and rabbits by immunization. Immunization with the vaccine induced high titers of serum IgG antibody both in rabbits and humans but reactivities of the induced antibodies with the OMPs were different. The rabbit immune sera recognized most of the OMPs in the vaccine both in immunoblot and immunoprecipitation analyses. In contrast, a great variation in band pattern and intensity was observed among the human immune sera in immunoblot analysis, but not in immunoprecipitation analysis. Denaturation of the OMPs did not affect the binding activity of the rabbit immune sera as determined by ELISA, but substantially reduced those of the human immune sera and anti-OMP IgG purified from a pooled normal human plasma. These data suggest that antibody response to P. aeruginosa OMPs elicited by immunization in humans is mainly directed against discontinuous or conformation-dependent epitopes, which should be taken into account in developing vaccines, especially for OMP-derived synthetic peptides.     

Characterization of OmpK, GAPDH and their fusion OmpK-GAPDH derived from Vibrio harveyi outer membrane proteins: their immunoprotective ability against vibriosis in large yellow croaker (Pseudosciaena crocea)

AIM: To investigate the immunoprotection of three recombinant proteins derived from the Vibrio harveyi outer membrane proteins (OMPs) OmpK, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and their fusion OmpK-GAPDH as vaccine candidates from vibriosis of large yellow croaker (Pseudosciaena crocea). METHODS: The ompK gene, of which the leader sequence was omitted, was fused with the gapdh gene. Three recombinant proteins r-OmpK, r-GAPDH and r-OmpK-GAPDH were expressed and purified. Western blots were carried out to detect the specificity of the antibodies raised against the recombinant proteins; Fish were immunized with recombinant proteins and challenged by native V. harveyi. The immunoresponse to the recombinant proteins were determined by ELISA and phagocytic activity assay. CONCLUSIONS: The fusion protein r-OmpK-GAPDH can afford greater protection against the wild V. harveyi than r-OmpK or r-GAPDH alone or their mixture in humoral and cellular immunity, indicating that OmpK and GAPDH could produce a synergistic immunoprotection against vibriosis of large yellow croaker (Pseudosciaena crocea) when fused into OmpK-GAPDH with a linker. SIGNIFICANCE AND IMPACT OF THE STUDY: It has been realized that a multi-component OMP antigen can induce a higher frequency of immune effectors than a single OMP. The results presented here bring forth a good suggestion for the subunit vaccine design based on the OMPs of gram-negative pathogens.     

Bactericidal antibody response against P6, protein D, and OMP26 of nontypeable Haemophilus influenzae after acute otitis media in otitis-prone children

The bactericidal antibody response to three nontypeable Haemophilus influenzae (NTHi) outer membrane proteins (D, P6, and OMP26) was studied in 24 otitis-prone children (aged 7-28?months) after an acute otitis media (AOM) caused by NTHi. The study was carried out to understand the contribution of antigen-specific bactericidal antibody responses in the class of children who are most vulnerable to recurrent otitis media infections. Levels of protein D (P?=?0.005) and P6 (P?=?0.026) but not OMP26 antibodies were higher in bactericidal sera compared with nonbactericidal sera. For five (24%) and 16 (76%) of 21 bactericidal sera tested, removal of anti-protein D and P6 antibody, respectively, resulted in a two- to fourfold drop in bactericidal antibody. Antibodies to OMP26 did not make any contribution to the overall bactericidal activity in any serum samples. Eleven of 21 sera (52%) had bactericidal activity against a heterologous NTHi (86-028 NP) strain but the titers were significantly lower (P?相似文献   

Involvement of Neisseria meningitidis Lipoprotein GNA2091 in the Assembly of a Subset of Outer Membrane Proteins

GNA2091 of Neisseria meningitidis is a lipoprotein of unknown function that is included in the novel 4CMenB vaccine. Here, we investigated the biological function and the subcellular localization of the protein. We demonstrate that GNA2091 functions in the assembly of outer membrane proteins (OMPs) because its absence resulted in the accumulation of misassembled OMPs. Cell fractionation and protease accessibility experiments showed that the protein is localized at the periplasmic side of the outer membrane. Pulldown experiments revealed that it is not stably associated with the β-barrel assembly machinery, the previously identified complex for OMP assembly. Thus, GNA2091 constitutes a novel outer membrane-based lipoprotein required for OMP assembly. Furthermore, its location at the inner side of the outer membrane indicates that protective immunity elicited by this antigen cannot be due to bactericidal or opsonic activity of antibodies.     

Genetic and immunologic characterization of a novel serotype 4, 15 strain of Neisseria meningitidis

The porin proteins of Neisseria meningitidis are important components of outer membrane protein (OMP) vaccines. The class 3 porin gene, porB, of a novel serogroup B, serotype 4, 15 isolate from Chile (Ch501) was found to be VR1-4, VR2-15, VR3-15 and VR4-15 by porB variable region (VR) typing. Rabbit immunization studies using outer membrane vesicles revealed immunodominance of individual PorB (class 3) VR epitopes. The predominant anti-Ch501 PorB response was directed to the VR1 epitope. Anti-PorB VR1 mediated killing was suggested by the bactericidal activity of Ch501 anti-sera against a type 4 strain not expressing PorA or class 5 OMPs. Studies that examine the molecular epidemiology of individual porB VRs, and the immune responses to PorB epitopes, may contribute to the development of broadly protective group B meningococcal vaccines.     

Role of antibodies against outer-membrane proteins in murine resistance to infection with encapsulated Klebsiella pneumoniae

In the assessment of immunity to the encapsulated virulent strain of Klebsiella pneumoniae and its avirulent mutant defective for capsular polysaccharide (CPS), killed bacterial vaccine of both strains could protect mice equally against challenge with 100 x LD50 of encapsulated wild strain. Antisera to each strain conferred the same level of protection on naive mice upon transfer; the protective anti-mutant serum was highly capable of opsonizing the encapsulated bacteria. In addition to the common antigenic components shared by both strains, the wild strain had antigen(s) unrelated to the mutant since the protective capacity of the anti-wild serum was not affected by preabsorption with the mutant strain; the protection conferred by the anti-mutant serum was mediated by antibodies against non-capsular antigens since the antiserum did not contain antibodies against purified CPS detectable by ELISA. As possible candidates among the non-capsular antigens, outer-membrane proteins (OMPs) extracted from the mutant strain were examined for their immunogenicity. Immunoblotting of the protein-containing fraction and ELISA using LPS-free OMP suggested that a number of proteins were involved in the immune response evoked by K. pneumoniae. Furthermore, mice immunized with OMP or anti-OMP serum could overcome a lethal challenge with the wild strain. These results indicated that OMPs of K. pneumoniae are implicated as the protective antigens and may pave the way for the development of non-capsular, proteinaceous vaccines.     

Evaluation of the immunological activity and safety of group B meningococcal vaccine prepared from a natural complex of specific polysaccharide and outer membrane proteins

Immunological activity and safety of group B meningococcal vaccine prepared from a natural complex of specific polysaccharide and outer membrane proteins were under study. The immunological safety of the vaccine was evaluated by the absence of antibodies to denaturated and native DNA (d-DNA and n-DNA). As shown with the use of the enzyme immunoassay (EIA), the administration of the vaccine did not induce antibody formation to d-DNA and n-DNA during the observation period. The titer of bactericidal antibodies in the immune bacteriolysis assay (IBA) to the vaccine strain B:2b:P1.2 after immunization increased four-fold and greater in 80% of the vaccinated persons. The significant increase of bactericidal antibodies to heterologous strains B:2a:P1.2 and B:15:P1.7 was registered in 20-30% of the vaccinees, respectively. A month after the repeated vaccination an increase in specific IgG antibodies to the complex antigen was found to occur according to EIA results. The use of RIB made it possible to evaluate the preventive activity of group B meningococcal vaccine as a whole and to suppose that the vaccine induced mainly type-specific response.     

Acellular and whole cell pertussis vaccines protect against the lethal effects of intracerebral challenge by two different T-cell dependent humoral routes

Athymic (nu/nu) and euthymic (+/nu) BALB/c mice were immunized with a whole cell pertussis vaccine or with an acellular vaccine which contained detoxified pertussis toxin (PT) and filamentous hemagglutinin (FHA). Only the euthymic mice were protected against intracerebral challenge with virulent Bordetella pertussis which implies involvement of T-cells. As a cell transfer from mice immunized with whole cell or acellular vaccine prior to the challenge did not protect naive euthymic recipients, cellular immunity seems to be non-protective as an effector mechanism. Mice could be protected passively against a challenge by administration of immune sera. Therefore, T-cell dependent humoral immune responses to B. pertussis appear to be crucial for protection. The humoral response was further studied with athymic and euthymic mice. In euthymic mice the whole cell vaccine induced antibodies to FHA, pililipopolysaccharides (LPS) and an outer membrane protein (OMP) preparation, whereas the acellular vaccine induced antibodies to PT, FHA and OMP. Both IgM and IgG could be detected. From the nude mice only those immunized with the whole cell vaccine showed an antibody response which consisted of low titres of IgM directed to LPS. Sera from both +/nu and nu/nu mice immunized with the whole cell vaccine were bactericidal in vitro. These data demonstrate that in the mouse model protection to intracerebral challenge with B. pertussis is T-cell dependent as is the humoral response to PT, FHA, OMP and pili. The T-independent B-cell activation by the whole cell preparation is due to the presence of LPS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human immune response to epitopes on the meningococcal outer membrane class 5 protein following natural infection

Abstract Two monoclonal antibodies (mAbs) were produced against a serogroup B Neisseria meningitidis strain. These mAbs recognized two epitopes in the class 5 outer membrane proteins (OMP), designated P5.7 and P5.Bm, and were able to kill the homologous strain through complement activation. Both epitopes were surface exposed and 68% of group B meningococcal clinical isolates had one or both epitopes present in their class 5 OMP. Antibodies to one or both epitopes were demonstrated in 17 patients with meningococcal meningitis using an ELISA inhibition assay. Of the 17 paired sera, 41% and 29% of the acute-phase sera had antibodies to the P5.7 and P5.Bm epitopes, respectively. Immunoglobulin G to P5.Bm were found in all 17 convalescent-phase sera while specific antibodies against P5.7 were only found in 6 of these sera. These results demonstrate the potential importance of the P5.Bm and P5.7 epitopes on the class 5 OMP as candidates for vaccine composition.     

Epitope specificity of murine and human bactericidal antibodies against PorA P1.7,16 induced with experimental meningococcal group B vaccines

Synthetic peptides derived from the predicted loops 1 and 4 of meningococcal PorA, sero-subtype P1.7,16, were used to study the epitope specificity of murine and human PorA P1.7,16 bactericidal antibodies. The predicted loops 1 and 4 are surface exposed and carry in their apices the sero-subtype epitopes P1.7 (loop 1) or P1.16 (loop 4), respectively. Peptides were synthesized as mono- and multimeric peptides. Murine monoclonal and polyclonal antibodies were induced with meningococcal whole cell preparations. Polyclonal antibodies were evoked in volunteers after one immunization with 50 μg or 100 μg protein of a hexavalent meningococcal PorA vesicle vaccine. The induction of PorA antibodies was determined in ELISA using purified PorA P1.7,16. The epitope specificity of anti-PorA antibodies for both murine and human antibodies could be demonstrated by direct peptide ELISA using overlapping multimeric peptides almost spanning the entire loops 1 or 4 of the protein. The capacity of peptides to inhibit the bactericidal activity of murine and human antibodies was investigated using meningococcal strain H44/76 (B:15:P1.7,16) as a target strain. Bactericidal activities could be inhibited with both monomeric and multimeric peptides derived from epitopes P1.7 and P1.16.     

The reactogenicity and antigenic activity of a meningococcal group B vaccine made from a natural complex of the specific polysaccharide and outer membrane proteins

Group B meningococcal vaccine consisting of the natural complex of specific polysaccharide and outer membrane protein (OMP) has been shown to be moderately reactogenic, safe with respect to the effect of undermining tolerance to human brain tissue antigens and to produce no allergization of humans. The vaccine under study possesses antigenic activity: (a) immunization with this vaccine ensures the fourfold rise of the level of antibodies to the group-specific polysaccharide of group B meningococcus in about 80% of persons with the initially low level of antibodies, this percentage being retained during the whole period of observation, i. e. 85 days; (b) the vaccine enhances the level of antibodies to meningococcal OMP, determined in the enzyme immunoassay and the passive hemagglutination test; (c) these data are indicative of the expediency of immunizing the risk groups of persons with the initially low level of antibodies.     

Bactericidal antibody response to Neisseria meningitidis serogroup B in patients with bacterial meningitis: effect of immunization with an outer membrane protein vaccine

We evaluated the bactericidal antibody response to Neisseria meningitidis serogroup B in convalescent patients (n=65) from bacterial meningitis. Patients infected with B meningococci were stratified according to their vaccination status (Cuban BC vaccine) into group 1 (immunized) (n=12) and group 2 (non-immunized) (n=15). The results suggested that antibody titers > or =2 (log(2)) indicate a specific immune response to N. meningitidis. In group 1, 64% of patients had a significant antibody titer (> or =2) in their acute sera against a B:4:P1.15 strain, compared to only 21% of group 2 patients. All patients from group 1 without bactericidal antibodies in their acute sera had a significant increase (at least 2-fold increase in log(2) titers) in antibody titers in their convalescent sera, in contrast, to only 27% of patients from group 2 (P=0.06). Using mutant strains lacking OMP1 or OMP5, it was shown that OMP1 was an important antigen recognized by immunized patients but not by non-immunized patients.     

Characterization of intermediate phenotypes induced by chemically undefined laboratory media in virulent Bordetella bronchiseptica strains

The expression of many virulence factors of Bordetella bronchiseptica is regulated by the bvgAS locus and reduced in response to environmental signals called modulators. Virulent strains can alternate between virulent (Bvg(+)), intermediate (Bvg(i)), and modulated (Bvg(+)mod) phenotypes. Potential vaccine antigens can be expressed by Bvg1 strains grown only in the absence of modulators. In the present study we evaluated filamentous hemagglutinin (FHA) and outer membrane protein (OMP) expression in Bvg(+) B. bronchiseptica strains grown in chemically undefined media: nutrient agar (NA), tryptic soy agar (TSA), tryptose phosphate broth (TPB), and brain-heart infusion (BHI). Our results suggest that TSA and TPB usually induce semimodulation, since Bvg(+) strains cultured in these media retained the expression of FHA and virulence-associated OMPs in the 30 kDa region, but failed to express other virulence markers such as OMPs in the regions of 90 and 200 kDa, though they expressed flagellin (avirulence marker). On the other hand, NA and BHI usually induce modulation. Thus the assayed chemically undefined media should not be used in vaccine production. Semimodulation induced by TSA and TPB can be accurately detected by SDS-PAGE Sarkosyl-insoluble OMP-enriched profiles. The reduction or absence of OMPs in the regions of 90 and 200 kDa is the most sensitive marker, and in some cases the presence of flagellin in intermediate profiles is another trait of the Bvg(i) phenotypes. Therefore these markers could be useful for selecting media for vaccine production. We also characterized the phenotype of Bvg(+) strains grown in Stainer-Scholte broth, an expensive medium, with and without glutathione, and we have detected no differences; this is the first attempt to reduce the cost of a Bordetella growth medium for veterinary vaccine production.     

Mutant Native Outer Membrane Vesicles Combined with a Serogroup A Polysaccharide Conjugate Vaccine for Prevention of Meningococcal Epidemics in Africa

Background

The meningococcal serogroup A (MenA) polysaccharide conjugate vaccine used in Sub-Saharan Africa does not prevent disease caused by MenW or MenX strains, which also cause epidemics in the region. We investigated the vaccine-potential of native outer membrane vesicles with over-expressed factor H-binding protein (NOMV-fHbp), which targeted antigens in African meningococcal strains, and was combined with a MenA polysaccharide conjugate vaccine.

Methodology/Principal Findings

The NOMV-fHbp vaccine was prepared from a mutant African MenW strain with PorA P1.5,2, attenuated endotoxin (ΔLpxL1), deleted capsular genes, and over-expressed fHbp in variant group 1. The NOMV-fHbp was adsorbed with Al(OH)₃ and used to reconstitute a lyophilized MenA conjugate vaccine, which normally is reconstituted with liquid MenC, Y and W conjugates in a meningococcal quadrivalent conjugate vaccine (MCV4-CRM, Novartis). Mice immunized with the NOMV-fHbp vaccine alone developed serum bactericidal (human complement) activity against 13 of 15 African MenA strains tested; 10 of 10 African MenX strains, 7 of 7 African MenW strains, and 6 of 6 genetically diverse MenB strains with fHbp variant group 1 (including 1 strain from The Gambia). The combination NOMV-fHbp/MenA conjugate vaccine elicited high serum bactericidal titers against the two MenA strains tested that were resistant to bactericidal antibodies elicited by the NOMV-fHbp alone; the combination elicited higher titers against the MenA and MenW strains than those elicited by a control MCV4-CRM vaccine (P<0.05); and high titers against MenX and MenB strains. For most strains, the titers elicited by a control NOMV-fHbp knock out vaccine were <1∶10 except when the strain PorA matched the vaccine (titers >1∶000).

Conclusion/Significance

The NOMV-fHbp/MenA conjugate vaccine provided similar or higher coverage against MenA and MenW strains than a quadrivalent meningococcal conjugate vaccine, and extended protection against MenX strains responsible for epidemics in Africa, and MenB strains with fHbp in variant group 1.     

Differentiation of outer membrane proteins from Salmonellaenterica serotypes using Fourier transform infrared spectroscopy and chemometrics

AIMS: To differentiate between outer membrane proteins (OMPs) from six Salmonellaenterica serotypes using a Fourier transform infrared (FTIR) spectroscopy method and chemometrics. METHODS AND RESULTS: The OMPs from Salmonella serotypes (Typhimurium, Enteritidis, Thomasville, Hadar, Seftenberg and Brandenburg) were isolated using a sarcosyl extraction method. OMP profiles on SDS-PAGE exhibited two or three bands between 48 and 54 kDa. Spectra of 10 microl of OMP preparations (5 mg ml(-1)) dried on a gold reflective slide were collected using 128 scans at 4 cm(-1) resolution and units of log (1/R) and analyzed using canonical variate analysis (CVA) and linear discriminant analysis (LDA). The CVA of Salmonella OMP spectra in the 1800-1500 cm(-1) region separated the serotypes and LDA provided a 100% correct classification. CONCLUSIONS: The use of a FTIR method combined with chemometrics provided better differentiation of Salmonella OMPs than the OMP pattern analysis by SDS-PAGE. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate that spectra of OMP extracts from Salmonella serotypes can be used for 100% correct classification of the serotypes studied.     

鸭疫里默氏杆菌外膜蛋白生物学特性研究

血清2型鸭疫里默氏杆菌强毒菌株体外传200代获得了无毒力无免疫原性菌株,采用超声波裂解和超速离心法提取二株菌的外膜蛋白, 以比较分析鸭疫里默氏杆菌外膜蛋白的生物学特性。电镜观察细菌超微结构显示传代菌株外膜膜密度降低, 外膜泡的数量明显减少, 细胞质不均匀、内有空泡产生;免疫印迹结果表明二株菌的外膜蛋白免疫原性多肽存在明显区别;原代菌株的外膜蛋白仅与2型RA抗体出现特异性凝集, 而传代菌株的外膜蛋白与 1、2、10与11型RA抗体均出现凝集;二株菌的外膜蛋白均可诱导雏鸭产生抗体, 但原代菌株外膜蛋白诱导雏鸭产生抗体滴度显著高于200代次菌株;原代菌株外膜蛋白免疫鸭对同源RA菌株的攻击可产生100%的免疫保护, 而传代菌株外膜蛋白免疫鸭对同源RA菌株的攻击不产生免疫保护。序列分析显示两者的外膜蛋白A同源性达到99.9%。结果表明强毒菌株的外膜蛋白为良好的亚单位疫苗候选, 体外连续传代对RA外膜蛋白生物学特性影响显著。