Biosynthetic capacity for type-specific antigen synthesis determines the virulence of serotype III strains of group B streptococci

The level of type-specific antigen (that covalently associated with the cell wall peptidoglycan and that released extracellularly) synthesized by virulent and avirulent strains of type III group B streptococci was quantitated and compared. Additionally, the effect of the physiological age of the cells and the influence of the exogenous phosphate ion concentration on the level of antigen synthesis by these organisms were also examined. Approximately 4% of the total antigen synthesized by the organism is noncovalently bound to the cell surface, and the difference in level of the noncovalently associated type-specific antigen between virulent and avirulent strains was negligible. In contrast, when the cell-associated covalently bound type antigens were evaluated, virulent strains were demonstrated to have two- to threefold higher levels than those of avirulent strains during the exponential and stationary phases of growth under various growth conditions. Furthermore, virulent strains that had high levels of cell-associated type antigen also secreted more extracellular type antigen than did avirulent strains. Thus, the data were consistent with the hypothesis that an overall production of type-specific antigen correlated with virulence in mice. However, the cell-associated type-specific antigen probably represented a better indicator for virulence potential since the addition of purified extracellular type-specific antigen to a mutant strain that lacks cell surface type antigen did not alter the 50% lethality value of the organism. To account for variation in the level of type-specific antigen produced by these strains, the kinetics of both the group- and type-specific antigens synthesis was investigated at the cell membrane level by utilizing an intact protoplast system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anthrax Spores Make an Essential Contribution to Vaccine Efficacy

Anthrax is caused by Bacillus anthracis, a gram-positive spore-forming bacterium. Septicemia and toxemia rapidly lead to death in infected mammal hosts. Currently used acellular vaccines against anthrax consist of protective antigen (PA), one of the anthrax toxin components. However, in experimental animals such vaccines are less protective than live attenuated strains. Here we demonstrate that the addition of formaldehyde-inactivated spores (FIS) of B. anthracis to PA elicits total protection against challenge with virulent B. anthracis strains in mice and guinea pigs. The toxin-neutralizing activities of sera from mice immunized with PA alone or PA plus FIS were similar, suggesting that the protection conferred by PA plus FIS was not only a consequence of the humoral response to PA. A PA-deficient challenge strain was constructed, and its virulence was due solely to its multiplication. Immunization with FIS alone was sufficient to protect mice partially, and guinea pigs totally, against infection with this strain. This suggests that spore antigens contribute to protection. Guinea pigs and mice had very different susceptibilities to infection with the nontoxigenic strain, highlighting the importance of verifying the pertinence of animal models for evaluating anthrax vaccines.     

Serological relationship between a pathogenic strain of Marek's disease virus, its attenuated derivative and herpes virus of turkeys.

Precipitating antigens present in extracts of chick embryo cells infected with the HPRS-16 attenuated strain of Marek's disease virus (att-MDV) were separated by gel filtration on Sephadex G200 and some of their properties determined. The two main antigens detected with convalescent MD serum, referred to as 'B' and 'C' antigens, had mobilities of 0-55 and 0-25 respectively relative to phenol red on electrophoresis in 7-5% acrylamide gel. The B antigen was relatively stable and of low mol. wt. in comparison with the C antigen. B and C antigens were in some instances also detected in culture medium of infected cells, but were distinguishable from the A antigen, a major glycoprotein antigen released into the culture medium of cells infected with HPRS-16. The results of immunodiffusion studies suggest that B antigen is common to MDV and strains of herpes virus of turkeys(HVT) and that at least 2 antigens (including C) are MDV specific. The A antigen was also common to MDV and HVT strains. It was noted however that the capacity of HPRS-16/att to synthesize A antigen was considerably reduced in comparison with HPRS-16 and HVT strains, and in some preparations the A antigen could not be detected. Evidence was also obtained for the presence of HVT-specific antigens associated mainly with the cell fraction.     

Antibody responses to toxoplasma antigens in mice infected with strains of different virulence.

Swiss Webster mice were infected with either the relatively virulent C56 strain or the relatively avirulent C37 strain of Toxoplasma gondii, and the sequence of their antibody response to surface antigens of the parasite was studied. An immunoglobulin M (IgM) agglutinating antibody was the first serologically detectable antibody and was first detected on day 2 in mice infected with the C37 strain and on day 5 in mice infected with the C56 strain. IgG antibodies were first detected on day 8 for both strains. The major component of the IgG antibodies was IgG2:IgG3 antibodies had lower titers, and no IgG1 antibodies were detected. The IgG antibodies were active in direct parasite agglutination and in the complement-dependent cytotoxicity assay of Sabin and Feldman (Science 108:660-663, 1948). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, sera from mice infected with T. gondii detected all major radioiodinated surface proteins of toxoplasma tachyzoites. The earliest time point at which these antigens were detected differed for the two strains. Serum from mice infected with C56 strain immunoprecipitated all cell surface antigens by day 10 of infection, whereas serum from mice infected with the C37 strain did not do so until day 15 of infection.     

Monoclonal antibodies identify new Toxoplasma gondii soluble antigens

In order to characterize Toxoplasma gondii antigens, we have produced a panel of monoclonal antibodies specific for the parasite. A total of 22 hybridomas were derived from the spleen cells of mice immunized either with a 100,000 g supernatant of a sonicate from the RH strain (called F3), or chronically infected with the Wiktor or the 76K strain. Except for one hybridoma producing an IgM, all the hybridomas derived from mice immunized with F3 produced IgG1 antibodies while those obtained from chronically infected mice produced antibodies belonging to the IgG2b, IgG2a and IgM subclasses. Western-blot analysis showed that the panel of monoclonal antibodies defines at least 7 distinct antigens or antigen families. An antigen of apparent Mw 25 kD present exclusively in the 100,000 g supernatant of the T. gondii sonicate was recognized by the majority of monoclonal antibodies derived from mice immunized with the F3 fraction. Two other antigens of apparent Mw 27 kD and 29 kD present in the soluble and insoluble fractions of the sonicate were also identified. Monoclonal antibodies against the previously described 21 kD and 31 kD surface antigens and belonging to the IgG2a but also to the IgG1 subclasses were able to mediate lysis of the parasite in the presence of human non immune serum. The 22 monoclonal antibodies did not identify antigenic differences between the two independently isolated RH and Wiktor strains.     

Infection of Hela cells with a virulent and an attenuated strain of pseudorabies virus studied by electron microscopy.

Hollow-core particles, forming crystals in nuclei, prevailed in HeLa cells infected with an attenuated strain of pseudorabies virus (PRV). After infection with a virulent PRV strain, the cells contained mainly fully infectious dense-core particles. These findings might explain the lower susceptibility of HeLa and some other human cells to infection with attenuated strains of PRV as compared to virulent strains.     

A DNA prime-live vaccine boost strategy in mice can augment IFN-gamma responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis

The Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine has variable efficacy for both human and bovine tuberculosis. There is a need for improved vaccines or vaccine strategies for control of these diseases. A recently developed prime-boost strategy was investigated for vaccination against M. bovis infection in mice. BALB/c and C57BL/6 mice were primed with a DNA vaccine, expressing two mycobacterial antigens, ESAT-6 and antigen 85 A and boosted with attenuated M. bovis strains, BCG or WAg520, a newly attenuated strain, prior to aerosol challenge. Before challenge, the antigen-specific production of interferon-gamma (IFN-gamma) was evaluated by ELISPOT and antibody responses were measured. The prime-boost stimulated an increase in the numbers of IFN-gamma producing cells compared with DNA or live vaccination alone, but this varied according to the attenuated vaccine strain, time of challenge and the strain of mouse used. Animals vaccinated with DNA alone generated the strongest antibody response to mycobacterial antigens, which was predominantly IgG1. BCG and WAg520 alone generally gave a 1-2 log10 reduction in bacterial load in lungs or spleen, compared to non-vaccinated or plasmid DNA only control groups. The prime-boost regimen was not more effective than BCG or WAg520 alone. These observations demonstrate the comparable efficacy of BCG and WAg520 in a mouse model of bovine tuberculosis. However, priming with the DNA vaccine and boosting with an attenuated M. bovis vaccine enhanced IFN-gamma immune responses compared to vaccinating with an attenuated M. bovis vaccine alone, but did not increase protection against a virulent M. bovis infection.     

Marek's disease virus serotype-1 antigens A and B and their unglycosylated precursors detected by western blot analysis of infected cells

The antigenic profile of cell cultures infected with Marek's disease virus (MDV) was determined by the immunoblotting method using convalescent immune serum obtained from chickens that survived infection with MDV strain GA5. The MDV antigen profile in infected cell lysates could be accurately determined since this method has advantages over the immunoprecipitation method used in other studies. We studied six very virulent MDV isolates and the prototype of serotype 1 MDV, the GA5 strain. Immunoblots of NaDodSO₄-polyacrylamide gel electrophoresis (PAGE) performed under reducing conditions revealed a main viral antigen (B) of 120–130 kD, which was present in all cell lysates infected with MDV isolates. Analysis of infected cell proteins by NaDodSO₄-PAGE performed under nonreducing conditions, revealed a 205 kD major MDV antigen, which, under reducing conditions, was identical to the 130 kD major antigen. The unglycosylated precursors of the major MDV antigens were elucidated. Two polypeptides of 43 and 45 kD were found to be the unglycosylated precursors of MDV antigen A (the glycosylated form of which appears in 4 distinct bands). The unglycosylated precursors of the MDV major antigen B were found to be three polypeptides of 80, 110, and 125 kD.     

Characterization of the Structural-Protein-Coding Region of SAT 2 Type Foot-and-Mouth Disease Virus

The NSP4 protein of rotavirus is a nonstructural glycoprotein and has a crucial function in virus morphogenesis during infection of host cells. It was recently reported that NSP4 may also function as a viral enterotoxin in the induction of rotavirus diarrhea by causing Ca⁺⁺ influx in the cytoplasm of the infected cells. We sequenced and analyzed two (Wa and M strains) pairs of NSP4 genes of virulent (v) and attenuated (a) (after 30 to 40 passages in cell culture) human group A rotaviruses and a pair of NSP4 genes of virulent and attenuated porcine group C rotavirus (Cowden strain). These strains were previously identified as virulent (induce diarrhea) or attenuated (no diarrhea) in a gnotobiotic pig model of rotavirus infection [Bohl et al. (4), Saif et al. (13), Ward et al. (17)]. The NSP4 genes of the Wa, M and Cowden strains were amplified with RT-PCR using a proof reading polymerase (Tli) and the RT-PCR product was sequenced directly. Analysis of the NSP4 deduced amino acid sequences showed that only 3 (Wa) and 2 (M and Cowden) amino acids differed between the virulent and attenuated strains. For the Wa strain, the changes from the virulent to attenuated strain were in amino acids 13 (V to A), 16 (L to S) and 34 (P to L); in the M strain, the difference was in amino acids 53 (T to I) and 104 (K to E), and in the Cowden strains, amino acids 50 (L to F) and 97 (D to N) differed between virulent and attenuated strains. To our knowledge, this is the first sequence comparison between NSP4 of a virulent and attenuated pair of group C rotaviruses. The potential impact of these few amino acid changes on the pathogenesis of the NSP4 protein for piglets is unclear, relative to previous findings in mice (1), but requires further study using purified recombinant NSP4 proteins or peptides.     

Enhancement of resistance of mice Toxoplasma gondii by 2 polysaccharides beta 1-3, beta 1-6 (PSAT and Scleroglucan)

The protective effect of PSAT and Scleroglucan (beta 1-3, beta 1-6 polysaccharides), has been studied in mice infected with different strains of Toxoplasma gondii (RH, attenuated RH or Prugniaud strains). Intramuscular or intravenous administration of PSAT before infection of mice by the attenuated RH strain, resulted in a significant enhancement of survival: 100% of survival in the treated group, compared to 33% in controls and 50% in mice pretreated with Scleroglucan (10 mg/kg, intravenously). In contrast, there was no difference in the survival rate between the group treated with PSAT (1 mg/kg, IV) and the control in mice infected with the virulent RH strain: all mice died between day 7 and day 8. In mice infected with 15 cysts of the Prugniaud strain a decrease in the chemiluminescence (CL) of peritoneal macrophages and of the number of cysts in the brain of mice were observed in mice treated either with PSAT (1 mg/kg, IM) or scleroglucan (10 mg/kg, IM).     

Marek's disease in turkeys: the induction of lesions and the establishment of lymphoid cell lines

The inoculation of turkeys with large doses of a virulent strain of Marek's disease virus (GA strain), but not of two other virulent strains (HPRS-16 and JM), was found to induce a disease resembling Marek's disease of the chicken. The most prominent lesions were lymphocytic leukaemia and lymphoid and reticular hyperplasia in the spleen and the liver. These developed after a prolonged latent period and the early histological changes (lymphoid cell destruction and reticuloendothelial cell hyperplasia) reported in chickens were not observed. Twelve cell lines were established from suspensions of spleen cells or of buffy coat cells from infected turkeys. These cells expressed both Marek's disease tumour-associated surface antigen and T-cell antigens. The cells carried the Marek's disease virus genome and when inoculated into chickens induced typical Marek's disease lymphomas. Nine of the cell lines were infected with an avian leukosis virus, but three lines were free of such infection. All cell lines had normal turkey karyotypes.     

Precipitating antigens associated with Marek's disease viruses and a herpesvirus of turkeys

Precipitating antigens associated with a number of Marek's disease virus strains and with a turkey herpesvirus have been analyzed. The 'A' antigen has been defined as the major soluble antigen in feather follicles of infected chickens, which is identical with the major antigen usually present in supernatants of chicken kidney cell cultures infected with strains of Marek's disease virus. 'BC' antigens are 2 or more antigens which are usually not noted in skin extracts but present in cultured cells infected with Marek's disease virus or turkey herpesvirus, in addition to the 'A' antigen. Some of the virus strains examined were positive and others negative for 'A' antigen, but all contained the 'BC' antigens. Results of agar-gel precipitin tests suggested a serological classification of the group of avian herpesviruses formed by Marek's disease viruses and turkey herpesvirus into 3 types. Pathogenic strains of Marek's disease virus and their attenuated A- variants, represented by the HPRS-16 strain (HPRS-16, JM, GA, VC, Oldenburg). Apathogenic Marek's disease virus, represented by the HPRS-24 strain. Turkey herpesvirus and its A- variants, represented by the FC126 strain. A serological subdivision corresponding to the different grades of pathogenicity of virus strains of the first type was not possible. Differences between antigens associated with the 3 types of virus were apparent from the antigen and antibody titres against homologous and heterologous reagents. Precipitin bands produced by homologous antigen and antibody were stronger than those produced by heterologous reagents. Differences between 'A' antigens of the 3 virus types were characterized by spur patterns of precipitin bands indicating a partial identity. At least 3 'BC' precipitin bands were noted; at least one was group-specific and one appeared to be type-specific.     

Reactivity of synthetic SAG1 (p30) peptide sequences with RH, S273 and Beverley strain-induced anti-Toxoplasma gondii antibodies.

OBJECTIVES: We compared the reactivity of IgG1 and IgG2a antibodies in mouse sera after infection with virulent RH and low-virulent S273 and Beverley strains of Toxoplasma gondii against RH SAG1 recombinant p30 (rp30) and synthetic SAG1 peptides. METHODS: Infected mouse serum samples were collected 9 days after infection, and the level of total IgG, IgG1 and IgG2a against the RH SAG1 rp30 protein and twenty peptides of the RH SAG1 protein were assessed. The glycosylphosphatidylinositol (GPI) modification site, the hydrophilic-hydrophobic structure, the transmembrane region and the secondary structure of the SAG1 sequence of virulent and low-virulent strains were analyzed using software. RESULTS: The virulent strain-infected mice produced a higher level of IgG1 but a lower IgG2a against the rp30 antigen, while the low-virulent strain-infected mice produced a higher level of IgG2a than the virulent strain. The difference in the secondary structure of SAG1 protein between the virulent and low-virulent strain was largely confined to amino acid positions 291-336, showing mutations and GPI anchor site. CONCLUSION:The difference in the reactivity of IgG against the rp30 antigen and synthetic peptides between virulent and low-virulent strains points to the importance of the primary and secondary structure assumed by antigens in the activation of Th cells and, subsequently, in the induction of IgG and its subclasses.     

Monoclonal antibodies to a virulent strain of transmissible gastroenteritis virus: comparison of reactivity with virulent and attenuated virus

Summary Twelve hybridomas secreting monoclonal antibodies (MAbs) against Miller virulent strain of transmissible gastroenteritis virus (TGEV) were generated and characterized. In a cell culture immunofluorescence (CCIF) assay, three MAbs directed against peplomer protein (E 2) had perinuclear fluorescence and four unclassified MAbs showed cell membrane fluorescence. Six of these seven MAbs neutralized both attenuated and virulent TGEV, and the seventh (an unclassified MAb) neutralized only the latter virus. Two MAbs able to bind the cell membrane of infected cells had low neutralizing antibody titers (8 to 72) but were able to distinguish between virulent and attenuated TGEV (9- to 72-fold differences in neutralizing titers). Two E 2-specific MAbs had higher neutralizing antibody titers (782 to 34,117) and showed 4- to 13-fold differences in titers against the attenuated and virulent TGEV strains. Five MAbs which were specific for nucleocapsid (N) protein had cytoplasmic, particulate fluorescence in CCIF, and did not neutralize TGEV. Comparison of CCIF antibody titers of MAbs to the virulent and attenuated strains of TGEV indicated that differences existed in titers of most E 2 and all N-specific MAbs, with titers consistently higher against virulent TGEV (homologous strain).Hyperimmune antisera prepared in gnotobiotic pigs against the attenuated, virulent and a recent isolate of TGEV immunoprecipitated the 3 major structural proteins of both the attenuated and virulent TGEV strains. Relative mol. wt. differences in the E 1 and E 2 proteins between the two virus strains were revealed using either the hyperimmune pig sera or MAbs. In addition to the 48 K N protein, a 44 K protein was coimmunoprecipitated by the hyperimmune sera and MAbs, but mainly from lysates of attenuated TGEV.     

Virulence ranking of some Mycobacterium tuberculosis and Mycobacterium bovis strains according to their ability to multiply in the lungs, induce lung pathology, and cause mortality in mice.

Three virulent strains of Mycobacterium tuberculosis (H37Rv, Erdman, and NYH-27) and two virulent strains of M. bovis (Ravenel and Branch) were compared in terms of their growth rates in the livers and the lungs of mice, their ability to cause lung pathology, and the time taken for them to cause death. In immunocompetent mice, all strains caused an infection that progressed for 20 days or more and then underwent resolution in the liver but not in the lungs. In the lungs, infection persisted and induced progressive pathology. According to host survival time, Ravenel was the most virulent strain, followed, in decreasing order of virulence, by Branch, H37Rv, Erdman, and NYH-27. The much longer survival times of mice infected with M. tuberculosis strains allowed time for lung histopathology to change from a histiocytic alveolitis to a chronic fibroblastic fibrosis that eventually obliterated most of the lung architecture. By contrast, in mice infected with M. bovis strains, the alveolitis that developed during early infection was rapid and expansive enough to cause death before chronic lung pathology became evident. In mice depleted of CD4+ T cells, increased growth of all virulent strains induced necrotic exudative lung lesions that rapidly filled most of the alveolar sacs with inflammatory cells. These mice died much earlier than infected control mice did. Attenuated strains had longer population doubling times in vivo and failed to cause progressive disease or pathology in the lungs or livers of immunocompetent mice.     

Virulence of Mycobacterium tuberculosis affects interleukin-8, monocyte chemoattractant protein-1 and interleukin-10 production by human mononuclear phagocytes.

Microbial virulence and cytokine-mediated immune responses to Mycobacterium tuberculosis infection are important determinants of the pathogenesis of human tuberculosis. To determine the interrelationship between mycobacterial virulence and cytokine induction, human monocytes and monocyte-derived macrophages were infected with attenuated (H37Ra) and virulent (H37Rv and CH306) strains of M. tuberculosis and the amount of proinflammatory [interleukin (IL)-8 and monocyte chemoattractant protein (MCP)- 1] and inhibitory (IL- 10) cytokines was measured in the culture supernatants by enzyme-linked immunosorbent assay (ELISA). Infection with live bacilli induced de novo synthesis of IL-8, MCP-1 and IL-10, since cytokine release was abolished when cells were preincubated with the protein synthesis inhibitor cycloheximide. A differential production of antiinflammatory and inhibitory cytokines was observed. The amount of IL-8 and MCP-1 release was inversely related to strain virulence, the attenuated H37Ra strain being more prone than virulent strains to induce secretion of chemokines. In contrast, virulent strains induced greater amounts of the inhibitory cytokine IL-10. Efficient upregulation of IL-10 synthesis, but not of chemokines, required infection of cells with live bacilli, since heat killing of organisms or challenge with soluble mycobacterial products completely abrogated the effect. Moreover, cells infected with virulent strains produced IL-10 even at a very low bacillus-to-cell ratio and secreted IL-10 continuously during the 96 h that followed infection. The results suggest that the degree of virulence affects host cell responses to M. tuberculosis infection. Continued production of IL-10 may be one of the means by which M. tuberculosis downregulates acute local inflammatory reactions, favoring the development of tuberculosis.     

Cytokine production in lethal and non-lethal murine malaria

Levels of IFN-gamma, IL-2 and IL-4 were measured in vitro during the course of non-lethal Plasmodium chabaudi adami and lethal P. chabaudi strain 1309 infections in BALB/cByJ mice. Spleen cells from mice infected with the non-lethal Plasmodium had a higher initial response to P. chabaudi antigens than mice infected with P. chabaudi strain 1309, as determined by measuring all three lymphokines. We conclude that both Th1 and Th2 subsets of T helper lymphocytes are activated during P. chabaudi adami infection but that these responses are suppressed in mice infected with the more virulent P. chabaudi strain 1309.     

Host and bacterial factors control the Mycobacterium avium-induced chronic peritoneal granulocytosis in mice.

Persistent peritoneal granulocytosis and elevated macrophage counts have been found in nine mouse strains from 8 to 90 days after infection with Mycobacterium avium. Peritoneal granulocytosis was higher in M. avium-resistant BALB/c. Bcgr (C.D2) mice, compared with congenic M. avium-susceptible BALB/c (Bcgs) animals. Although maximal granulocytosis values were not related to virulence of the inocula, the kinetics of the granulocytic response varied with the virulence of M. avium. Following infections by avirulent (rough) strains of M. avium, the peritoneal granulocytosis progressively declined in BALB/c and C3H/He mice. A similar decline in granulocyte number was observed in resistant C3H/He mice infected with virulent M. avium (smooth transparent strain). In both instances the decline in the peritoneal granulocytosis was associated with a progressive elimination of the inoculum. In the susceptible BALB/c mice, virulent M. avium strains induced progressive infection accompanied with a rapid decline in granulocyte number, whereas the infection with attenuated M. avium, which caused a chronic infection, induced persistent granulocytosis. The ability to recruit granulocytes following the intraperitoneal inoculation of a phlogistic substance (casein hydrolysate) was decreased in infected susceptible but not in infected resistant mice at 90 days of infection with virulent M. avium.     

Experimental ocular toxoplasmosis in genetically susceptible and resistant mice

Genetic factors determining the pathogenesis and course of ocular toxoplasmosis are poorly understood. In this study, we explored the development of experimental ocular pathogenesis in genetically dissimilar mice infected with either the RH strain, the PLK strain, or the immunodominant surface antigen 1 (SAG1 [P30])-deficient mutant of the RH strain of Toxoplasma gondii. At 11 days postinfection, ocular infection of C57BL/6 mice with all of the strains of parasites resulted in severe inflammatory lesions and high numbers of parasites in eye tissue; less severe ocular lesions at earlier histopathology and prolonged survival were observed in this mouse strain infected with either the major surface antigen 1-deficient SAG1(-/-) strain or the less virulent PLK strain compared with RH infection. In contrast, both BALB/c and CBA/J mice had less severe lesions and low numbers of parasites in their eye tissue, and infection developed into the chronic stage in these mice. There were significantly higher serum levels of gamma interferon and tumor necrosis factor alpha in C57BL/6 mice than in BALB/c and CBA/J mice following ocular infection. These observations confirm earlier reports on systemic immunity to these parasites that the route of Toxoplasma infection markedly influences survival of mice. Our data indicate that genetic factors of the host as well as the parasite strain are critical in determining susceptibility to experimental ocular toxoplasmosis in murine models.