Serological Characterization of Haemophilus Pleuropneumoniae (Actinobacillus Pleuropneumoniae) Strains and Proposal of a New Serotype: Serotype 9

Ten strains of H. pleuropneumoniae isolated from 10 herd outbreaks of pleuropneumonia were studied by means of the slide agglutination test, the indirect haemaggluitiniation (IHA) test and by gel diffusion. The strains were antigenically homogeneous and serologically distinct from serotypes 1 through 8. It is therefore proposed to refer these strains to a new serotype: serotype 9, with strain CVJ 13261 as the type strain. In addition to the serotype-specific capsular antigens, capsular antigen of serotype 1 (strain 4074) could be demonstrated in the 10 strains by means of gel diffusion analyses. In cross protection studies it was shown that the antigenic determinants shared by serotypes 9 and 1 were unable to yield a sufficient protection against disease. Thus, parenteral immunization with a killed 6-h culture of serotype 9 did not afford an acceptable protection against challenge with serotype 1 since only 3 of the 5 vaccinates were protected. The reverse experiment showed that parenteral immunization with serotype 1 only protected 1 out of 4 vaccinates.     

Serological Characterization of Actinobacillus pleuropneumoniae Biotype 1 Strains Antigenically Related to both Serotypes 2 and 7

Nine Danish Actinobacillus pleuropneumoniae biotype 1 isolates were shown by latex agglutination and indirect haemagglutination to possess capsular polysaccharide epitopes identical to those of serotype 2 strain 1536 (reference strain of serotype 2) and strain 4226 (Danish serotype 2 strain). Immunodiffusion confirmed the antigenic relationship with serotype 2 and further demonstrated an antigenic relationship with strain WF83 (reference strain of serotype 7). SDS-PAGE with LPS from strains 1536, 4226, WF83 and strain 7317 (representative of the 9 isolates examined) showed that strains WF83 and 7317 had an identical smooth ladder pattern whereas LPS from strains 1536 and 4226 showed a distinctly different pattern. The antigenic similarities of the LPS of strains WF83 and 7317 were confirmed by immunoblots using rabbit or pig antisera prepared against the 3 strains. No antigenic similarities in the LPS of strains 1536 and 7317 were revealed. Since an antigenic determinant specific for the 9 isolates could not be demonstrated with the methods used, the strains are proposed to be designated K2:O7.     

Role of L3T4+ and Lyt-2+ T cell subsets in protective immune responses of mice against infection with a low or high virulent strain of Toxoplasma gondii

In order to elucidate the role of T cell subsets in protective immunity against infection with high virulent and low virulent strains of Toxoplasma gondii, monoclonal antibodies specific for T cell subsets were injected into mice before immunization or challenge infection. Treatment of mice with monoclonal antibody to either L3T4+ or Lyt-2+ T cells before they were immunized with Toxoplasma cell homogenate prepared from high virulent RH strain tachyzoites markedly reduced survival after mice were challenged with low virulent bradyzoites of the Beverley strain. Thus, induction of protective immunity against bradyzoites of the Beverley strain requires the presence of both L3T4+ and Lyt-2+ T cells. In contrast, mice injected with living bradyzoites of the low virulent Beverley strain after immunization with Toxoplasma cell homogenate acquired protective immunity against high virulent tachyzoites of the RH strain. Lyt-2+ T cells alone appear to be final effector cells for protection against the challenge with high virulent RH strain tachyzoites, since treatment of the bradyzoite-immune mice with anti-Lyt-2 antibody, but not anti-L3T4 antibody, before challenge significantly increased mortality.     

Influences of ORF1 on the Virulence and Immunogenicity of Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae is a Gram-negative pathogen that causes porcine pleuropneumonia. The pathogenicity of A. pleuropneumoniae is strongly correlated with the production of active repeat-in-toxin (RTX) proteins such as ApxIVA. We evaluated the contribution of a potential ApxIVA activator, ORF1, to the virulence and immunogenicity of A. pleuropneumoniae in pigs. The orf1 gene in A. pleuropneumoniae SLW03 (serovar 1, ΔapxICΔapxIIC) was deleted, producing strain SLW05 (ΔapxICΔapxIICΔorf1). The virulence of strains SLW03 and SLW05 was compared in pigs. Clinical signs and pulmonary lesions induced by strain SLW05 were slighter than that of strain SLW03 (P < 0.05). The immunogenicity and protective efficacy of strains SLW03 and SLW05 were similar. All pigs immunized with strain SLW03 or SLW05 developed high antibody titers against ApxIA, ApxIIA, and ApxIVA before challenge. Two weeks after a second immunization, pigs were challenged intratracheally with either a fully virulent A. pleuropneumoniae serovar 1 or serovar 3 strain. Vaccination with strains SLW03 or SLW05 provided significantly greater protection compared to the negative control (P < 0.01). Immunized pigs displayed significantly fewer clinical signs and lower lung lesion scores than non-immunized pigs. These results suggested that ORF1 plays an important role in the development of ApxIVA toxicity. Furthermore, strain SLW05 is a highly attenuated strain able to induce protective immunity against A. pleuropneumoniae infection.     

Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum

The fish pathogen Vibrio anguillarum causes a lethal infection in rainbow trout (Salmo gairdneri). Three different avirulent mutants, constructed by transposon insertion mutagenesis (VAN20 and VAN70) or as antibiotic-resistant mutants (VAN1000), were isolated by screening 200 individual isolated mutants for avirulence. When used as live vaccines, all three avirulent mutants were able to induce protective immunity against the homologous as well as a heterologous strain of V. anguillarum. When VAN1000 was used, protective immunity could be recorded 1 week after bath vaccination with 10(7) bacteria per ml of water for 30 min. A single-dose immunization was effective for at least 12 weeks. Western immunoblotting showed that strains of V. anguillarum have antigenic determinants in common with Aeromonas strains. Therefore, we tested and confirmed that VAN1000 also was able to induce protective immunity against challenge with Aeromonas salmonicida.     

Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum.

The fish pathogen Vibrio anguillarum causes a lethal infection in rainbow trout (Salmo gairdneri). Three different avirulent mutants, constructed by transposon insertion mutagenesis (VAN20 and VAN70) or as antibiotic-resistant mutants (VAN1000), were isolated by screening 200 individual isolated mutants for avirulence. When used as live vaccines, all three avirulent mutants were able to induce protective immunity against the homologous as well as a heterologous strain of V. anguillarum. When VAN1000 was used, protective immunity could be recorded 1 week after bath vaccination with 10(7) bacteria per ml of water for 30 min. A single-dose immunization was effective for at least 12 weeks. Western immunoblotting showed that strains of V. anguillarum have antigenic determinants in common with Aeromonas strains. Therefore, we tested and confirmed that VAN1000 also was able to induce protective immunity against challenge with Aeromonas salmonicida.     

Structural studies of the O-chains of the lipopolysaccharides produced by strains of Actinobacillus (Haemophilus) pleuropneumoniae serotype 5.

The water-phase lipopolysaccharides isolated by phenol-water extraction from the cells of four strains of Actinobacillus pleuropneumoniae serotype 5 (K17, L20, B78-3760, and 81-750) were shown on structural analysis to have O-chain components with the same basic polysaccharide structure of a linear unbranched homopolymer of 1,6-linked beta-D-galactopyranosyl residues, although the linear length of the O-chain varied among different strains. While those of strains B78-3760 and 81-750 were partially O-acetylated, the O-chains of strains K17 and L20 were unsubstituted.     

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.     

Identification of VP7 epitopes associated with protection against human rotavirus illness or shedding in volunteers.

Sera from 17 of 18 adult volunteers challenged with a virulent serotype 1 rotavirus strain (D) were examined for prechallenge antibody levels against several well-defined rotavirus VP7 and VP4 neutralization epitopes by a competitive epitope-blocking immunoassay (EBA) in order to determine whether correlates of resistance to diarrheal illness could be identified. The presence of prechallenge serum antibody at a titer of greater than or equal to 1:20 that blocked the binding of a serotype 1 VP7-specific monoclonal antibody (designated 2C9) that maps to amino acid residue 94 in antigenic site A on the serotype 1 VP7 was significantly associated with resistance to illness or shedding (P less than 0.001) or illness and shedding (P less than 0.01) following challenge with the serotype 1 virus. In addition, an EBA antibody titer of greater than or equal to 1:20 in prechallenge serum against a serotype 3 VP7-specific epitope (defined by monoclonal antibody 954/159) that maps to amino acid 94 on the serotype 3 VP7 was also significantly associated with resistance to illness or shedding (P = 0.02), with a trend for protection against illness and shedding. A trend was also noted between the presence of EBA antibody against a cross-reactive VP4 epitope common to many human rotavirus strains, including the challenge virus, or a rhesus monkey rotavirus strain-specific VP4 antigenic site, and resistance to illness or shedding. These data confirm that the presence of serum antibody correlates with resistance to rotavirus illness or shedding but, in addition, demonstrate the association of antibody to a specific epitope with resistance to illness or shedding. These data also suggest that antigenic site A on the rotavirus VP7, composed of amino acids 87 to 96, may be involved in the formation of a major protective epitope. Further study of the role of this epitope in the development of homotypic and heterotypic immunity to rotaviruses following natural or vaccine-induced infection may be important in the development of strategies for control of rotavirus diarrheal disease.     

Envelope determinants of equine infectious anemia virus vaccine protection and the effects of sequence variation on immune recognition

A highly effective attenuated equine infectious anemia virus (EIAV) vaccine (EIAV(D9)) capable of protecting 100% of horses from disease induced by a homologous Env challenge strain (EIAV(PV)) was recently tested in ponies to determine the level of protection against divergent Env challenge strains (J. K. Craigo, B. S. Zhang, S. Barnes, T. L. Tagmyer, S. J. Cook, C. J. Issel, and R. C. Montelaro, Proc. Natl. Acad. Sci. USA 104:15105-15110, 2007). An inverse correlation between challenge strain Env variation and vaccine protection from disease was observed. Given the striking differences in protective immunity, we hypothesized that analysis of the humoral and cellular immune responses to the Env protein could reveal potential determinants of vaccine protection. Neutralization activity against the homologous Env or challenge strain-specific Env in immune sera from the vaccinated ponies did not correlate with protection from disease. Cellular analysis with Env peptide pools did not reveal an association with vaccine protection from disease. However, when individual vaccine-specific Env peptides were utilized, eight cytotoxic-T-lymphocyte (CTL) peptides were found to associate closely with vaccine protection. One of these peptides also yielded the only lymphoproliferative response associated with protective immunity. The identified peptides spanned both variable and conserved regions of gp90. Amino acid divergence within the principal neutralization domain and the identified peptides profoundly affected immune recognition, as illustrated by the inability to detect cross-reactive neutralizing antibodies and the observation that certain peptide-specific CTL responses were altered. In addition to identifying potential Env determinants of EIAV vaccine efficacy and demonstrating the profound effects of defined Env variation on immune recognition, these data also illustrate the sensitivity offered by individual peptides compared to peptide pools in measuring cellular immune responses in lentiviral vaccine trials.     

Construction and characterization of a live, attenuated apxIICA inactivation mutant of Actinobacillus pleuropneumoniae lacking a drug resistance marker

The apxIIC gene of Actinobacillus pleuropneumoniae serotype 7 was inactivated by homologous recombination using a sucrose counter-selectable marker system, resulting in a mutant strain that had no antibiotic resistance marker and expressed an inactivated ApxII toxin. The safety and immunogenicity of the mutant were evaluated in mice. The mutant strain caused no adverse effects in mice at doses up to 2 x 10(9) CFU via the intraperitoneal route while the parental strain induced total mortality at a dose of 2 x 10(7) CFU. Mice vaccinated intraperitoneally with the mutant strain had 100% and 70% protection against homologous (serotype 7) or heterologous (serotype 1, 3) challenge with A. pleuropneumoniae, respectively. The A. pleuropneumoniae mutant strain HB04C- and the counterselection method used in the study show promise in developing effective live vaccines for porcine pleuropneumonia and for other infections diseases of the respiratory system.     

The relationship of the antigenic characteristics of the tick-borne encephalitis virus to the level of the protective activity of inactivated cultured vaccines]

As shown in this study, the immunization of animals with killed vaccines against tick-borne encephalitis (TBE) leads to the formation of specific immunity, depending on the antigenic structure of the vaccine strain and the test strains used for challenge. Vaccines obtained on the basis of the TBE virus strain of the Eastern antigenic variant induced the development of a wider spectrum of specific protective activity than vaccines obtained on the basis of the TBE virus strain of the Western antigenic variant.     

Growth of Actinobacillus pleuropneumoniae is promoted by exogenous hydroxamate and catechol siderophores.

Siderophores bind ferric ions and are involved in receptor-specific iron transport into bacteria. Six types of siderophores were tested against strains representing the 12 different serotypes of Actinobacillus pleuropneumoniae. Ferrichrome and bis-catechol-based siderophores showed strong growth-promoting activities for A. pleuropneumoniae in a disk diffusion assay. Most strains of A. pleuropneumoniae tested were able to use ferrichrome (21 of 22 or 95%), ferrichrome A (20 of 22 or 90%), and lysine-based bis-catechol (20 of 22 or 90%), while growth of 36% (8 of 22) was promoted by a synthetic hydroxamate, N5-acetyl-N5-hydroxy-L-ornithine tripeptide. A. pleuropneumoniae serotype 1 (strain FMV 87-682) and serotype 5 (strain 2245) exhibited a distinct yellow halo around colonies on Chrome Azurol S agar plates, suggesting that both strains can produce an iron chelator (siderophore) in response to iron stress. The siderophore was found to be neither a phenolate nor a hydroxamate by the chemical tests of Arnow and Csaky, respectively. This is the first report demonstrating the production of an iron chelator and the use of exogenous siderophores by A. pleuropneumoniae. A spermidine-based bis-catechol siderophore conjugated to a carbacephalosporin was shown to inhibit growth of A. pleuropneumoniae. A siderophore-antibiotic-resistant strain was isolated and shown to have lost the ability to use ferrichrome, synthetic hydroxamate, or catechol-based siderophores when grown under conditions of iron restriction. This observation indicated that a common iron uptake pathway, or a common intermediate, for hydroxamate- and catechol-based siderophores may exist in A. pleuropneumoniae.     

Protection against lethal subcutaneous challenge of virulent Y. pestis strain 141 using an F1-V subunit vaccine

In this study, we designed and engineered a two-component recombinant fusion protein antigen as a vaccine candidate against the possible biological threat of Yersinia pestis. The recombinant F1-V pro-tein was formulated with Alhydrogel. A four-time injection with a dosage of 10, 20 and 50 μg/mouse in about two months was adopted for vaccination. Serum antibodies and subclass of T helper cells were measured and analyzed. After the final vaccination, the mice were challenged by 141 strain with 25― 600 LD50. In conclusion, the recombinant vaccine was capable of inducing protective immunity against subcutaneous challenge. The level of serum IgG was supposed to be a main factor that affected the final protection of challenge. 20 μg recombinant protein could induce an endpoint titre of serum IgG as high as 51200, which was enough to afford 100% protection against 400 LD50 virulent 141 challenge. The antibody isotype analysis showed that the vaccine induced predominantly an IgG1 rather than IgG2a response. Flow cytometric analysis revealed that Alhydrogel significantly helped induce a stronger humoral immunity instead of CTL cellular response. These findings suggested that the plague F1-V subunit vaccine is promising for the next plague vaccine.     

Characterization of monoclonal antibodies that recognize common epitopes located on O antigen of lipopolysaccharide of serotypes 1, 9 and 11 of Actinobacillus pleuropneumoniae

Abstract Seven murine monoclonal antibodies (mAbs) against serotype 1 of Actinobacillus (Haemophilus) pleuropneumoniae (reference strain Shope 4074) were produced and characterized. All hybridomas secreting mAbs were reactive with whole-cell antigens from reference strains of serotypes 1, 9 and 11, except for mAb 5D6 that failed to recognize serotype 9. They did not react with other taxonomically related Gram-negative organisms tested. The predominant isotype was immunoglobulin (Ig) M, although IgG_2a, IgG_2b, and IgG₃ were also obtained. The epitopes identified by these mAbs were resistant to proteinase K treatment and boiling in the presence of sodium dodecyl sulfate and reducing conditions; however, they were sensitive to sodium periodate treatment. Enhanced chemiluminescence-immunodetection assay showed that mAbs could be divided in two groups according to the patterns of immunoreaction observed. Group I (mAbs 3E10, 4B7, 9H5 and 11C3) recognized a ladder-like banding profile consistent with the O antigen of lipopolysaccharide (LPS) from smooth strains. Group II (mAbs 3B10 and 9H1) recognized a long smear of high molecular weight which ranged from 60 to 200 kDa. The mAbs were tested against 96 field isolates belonging to serotypes 1, 5, 9, 11 and 12, which had previously been classified by a combination of serological techniques based on polyclonal rabbit sera (counterimmunoelectrophoresis, immunodiffusion and coagglutination). The panel of mAbs identified all isolates of serotypes 9 and 11, but only 66% of those belonging to serotype 1. This may suggest the existence of antigenic heterogeneity among isolates of A. pleuropneumoniae serotype 1. These mAbs reacted with epitopes common to serotypes 1, 9 and 11 of Actinobacillus pleuropneumoniae which were located on the O antigen of LPS.     

IL-15 prolongs the duration of CD8+ T cell-mediated immunity in mice infected with a vaccine strain of Toxoplasma gondii.

Immunization of mice with a vaccine (ts-4) strain of Toxoplasma gondii is known to induce complete protection against subsequent lethal infection. Ts-4-mediated protection has been reported to be primarily dependent on IFN-gamma-producing CD8+ T cells. However, duration of CD8+ T cell-mediated immunity in the ts-4-vaccinated animals is not known. In the present study, the kinetics of the CD8+ T cell response in mice immunized with the ts-4 strain of T. gondii was evaluated. Optimal CD8+ T cell immunity persisted at least 6 mo after vaccination, and mice at this time point continued to overcome lethal challenge with a more virulent strain. However, at 9 mo postimmunization, CD8+ T cell immunity was severely diminished and the mice succumbed to Toxoplasma challenge. Pretreatment of animals, vaccinated 9 mo earlier, with rIL-15 prevented the mortality induced by Toxoplasma challenge. The protective effect of IL-15 treatment was due to a rise in the frequency of Ag-specific CD8+ T cells. CD8+ T cells from IL-15-administered animals showed increased proliferation and IFN-gamma production in response to antigenic restimulation. These findings suggest that rIL-15 can reverse the decline in the long-term CD8+ T cell immune response in mice immunized with vaccine strain of T. gondii.     

Enhanced CD8+ T cell immune responses and protection elicited against Plasmodium berghei malaria by prime boost immunization regimens using a novel attenuated fowlpox virus

Sterile immunity can be provided against the pre-erythrocytic stages of malaria by IFN-gamma-secreting CD8(+) T cells that recognize parasite-infected hepatocytes. In this study, we have investigated the use of attenuated fowlpox virus (FPV) strains as recombinant vaccine vectors for eliciting CD8(+) T cells against Plasmodium berghei. The gene encoding the P. berghei circumsporozoite (PbCS) protein was inserted into an FPV vaccine strain licensed for use in chickens, Webster's FPV, and the novel FPV vaccine strain FP9 by homologous recombination. The novel FP9 strain proved more potent as a vaccine for eliciting CD8(+) T cell responses against the PbCS Ag. Sequential immunization with rFP9 and recombinant modified vaccinia virus Anakara (MVA) encoding the PbCS protein, administered by clinically acceptable routes, elicited potent CD8(+) T cell responses against the PbCS protein. This immunization regimen elicited substantial protection against a stringent liver-stage challenge with P. berghei and was more immunogenic and protective than DNA/MVA prime/boost immunization. However, further improvement was not achieved by sequential (triple) immunization with a DNA vaccine, FP9, and MVA.     

Protein heterogeneity ofFrancisella tularensis: Detection of proteins with antigenic determinants

The autolyzates of three different strains ofFrancisella tularensis 15L, 130 and SCHU were tested for their immunogenic potential and protein heterogeneity. The autolyzates induce the production of specific antibodies, the delayed type of hypersensitivity, and some degree of protection against European virulent strain 130. This material (as antigen) was especially suitable for ELISA. When the autolyzates were subjected to SDS gradient PAGE, a variety of polypeptides were distinguished. The composition of polypeptides from all three strains on SDS-PAGE was almost identical. After the detection of antigenic determinants by Western blotting more than twenty bands appeared. The visualization with polyclonal antisera against live laboratory strain 15L and against autolyzates 15L, 130 and SCHU revealed differences in the composition of the antigenic determinants of these strains.     

Class Ia MHC-deficient BALB/c mice generate CD8+ T cell-mediated protective immunity against Listeria monocytogenes infection

CD8(+) T cells are required for protective immunity against intracellular pathogens such as Listeria monocytogenes. In this study, we used class Ia MHC-deficient mice, which have a severe reduction in circulating CD8(+) T cells, to determine the protective capacity of class Ib MHC-restricted T cells during L. monocytogenes infection. The K(b-/-)D(b-/-) mutation was backcrossed onto a C.B10 (BALB/c congenic at H-2 locus with C57BL/10) background, because BALB/c mice are more susceptible to Listeria infection than other commonly studied mouse strains such as C57BL/6. C.B10 K(b-/-)D(b-/-) mice immunized with a sublethal dose of L. monocytogenes were fully protected against a subsequent lethal infection. Adoptive transfer of Listeria-immune splenocyte subsets into naive K(b-/-)D(b-/-) mice indicated that CD8(+) T cells were the major component of this protective immune response. A CD8(+) T cell line isolated from the spleen of a Listeria-infected class Ia MHC-deficient mouse was shown to specifically recognize Listeria-infected cells in vitro, as determined by IFN-gamma secretion and cytotoxicity assays. Adoptive transfer of this T cell line alone resulted in significant protection against L. monocytogenes challenge. These results suggest that even a limited number of class Ib MHC-restricted T cells are sufficient to generate the rapid recall response required for protection against secondary infection with L. monocytogenes.     

T cells as mediators of protective immunity against liver stages of Plasmodium

T cells from different subsets play a major role in protective immunity against pre-erythrocytic stages of malaria parasites. Exposure of humans and animals to malaria sporozoites induces (alphabeta CD8(+) and CD4(+) T cells specific for antigens expressed in pre-erythrocytic stages of Plasmodium. These T cells inhibit parasite development in the liver, and immunization with subunit vaccines expressing the respective antigenic moieties confers protection against sporozoite challenge. gammadelta and natural killer T cells can also play a role in protective immunity. Recent studies with mice transgenic for the alphabeta T-cell receptor have revealed the existence of complex mechanisms regulating the induction and development of these responses.