Virulence-associated Antigens Research Articles

Identification of two anti-Candida antibodies associated with the survival of patients with candidemia.

Candidemia (bloodstream invasion by Candida species) is a major fungal disease in humans. Despite the recent progress in diagnosis and treatment, therapeutic options are limited and under threat of antimicrobial resistance. The disease mortality remains high (around 40%). In contrast with deep-seated invasive candidiasis, particularly that occurring in patients with hematologic malignancies and organ transplants, patients with candidemia are often not immunocompromised and therefore able to mount memory anticandidal immune responses, perhaps primed by Candida commensalism. We investigated antibody immunity in candidemia patients and report here on the ability of these patients to produce antibodies that react with Candida antigens. In particular, the patients with high titers of IgG reactive with two immunodominant, virulence-associated antigens (Als3 and MP65) had a higher 30-day survival. If confirmed by controlled, prospective clinical studies, our data could inform the development of antibody therapy to better treat a severe fungal infection such as candidiasis.

Intranasal multivalent adenoviral-vectored vaccine protects against replicating and dormant M.tb in conventional and humanized mice

Viral-vectored vaccines are highly amenable for respiratory mucosal delivery as a means of inducing much-needed mucosal immunity at the point of pathogen entry. Unfortunately, current monovalent viral-vectored tuberculosis (TB) vaccine candidates have failed to demonstrate satisfactory clinical protective efficacy. As such, there is a need to develop next-generation viral-vectored TB vaccine strategies which incorporate both vaccine antigen design and delivery route. In this study, we have developed a trivalent chimpanzee adenoviral-vectored vaccine to provide protective immunity against pulmonary TB through targeting antigens linked to the three different growth phases (acute/chronic/dormancy) of Mycobacterium tuberculosis (M.tb) by expressing an acute replication-associated antigen, Ag85A, a chronically expressed virulence-associated antigen, TB10.4, and a dormancy/resuscitation-associated antigen, RpfB. Single-dose respiratory mucosal immunization with our trivalent vaccine induced robust, sustained tissue-resident multifunctional CD4+ and CD8+ T-cell responses within the lung tissues and airways, which were further quantitatively and qualitatively improved following boosting of subcutaneously BCG-primed hosts. Prophylactic and therapeutic immunization with this multivalent trivalent vaccine in conventional BALB/c mice provided significant protection against not only actively replicating M.tb bacilli but also dormant, non-replicating persisters. Importantly, when used as a booster, it also provided marked protection in the highly susceptible C3HeB/FeJ mice, and a single respiratory mucosal inoculation was capable of significant protection in a humanized mouse model. Our findings indicate the great potential of this next-generation TB vaccine strategy and support its further clinical development for both prophylactic and therapeutic applications.

Rhodococcus equi infection in Marwari male foal: A case report

A 2-months-old Marwari male foal was anorectic, febrile, and showed difficulty in breathing. Haematology revealed anemia and severe neutrophilic leukocytosis with left shift. Symptomatic treatment was given, however the animal died within 48 hours. On necropsy, multifocal, raised, well-demarcated, off-white to grey, firm, abscesses of 10 to 15 cm diameter effaced the normal lung parenchyma. Colon wall showed multifocal to coalescing, raised, well-demarcated, off-white to grey, firm, transmural, variable-sized abscesses. On histopathological examination, the lung showed pyogranulomatous bronchopneumonia. Frequently cytoplasm of macrophages was filled with numerous fine basophilic bacteria. Colon showed necro-pyogranulomatous inflammation with intracytoplasmic bacilli. On Taylor’s Gram staining of tissue, the cytoplasm of macrophages was filled with numerous blue Gram-positive bacilli. Sample collected at the time of necropsy were subjected to microbiological and molecular investigation. Based on colony morphology, bacterial morphology, gram reactivity, and biochemical tests, the isolates were presumptively diagnosed as Rhodococcus equi (R.equi). Further, the isolates were confirmed as R. equi by species-specific primers. Moreover, all the isolates were also found positive for the presence of virulence-associated antigens (VapA), indicative of the pathogenic nature of the isolates. This case report described R. equi induced bronchopneumonia, and colitis in foal

Analysis of Humoral Immune Responses to Surface and Virulence-Associated Chlamydia abortus Proteins in Ovine and Human Abortions by Use of a Newly Developed Line Immunoassay.

The obligate intracellular bacterium Chlamydia abortus is the causative agent of enzootic abortion of ewes and poses a significant zoonotic risk for pregnant women. Using proteomic analysis and gene expression library screening in a previous project, we identified potential virulence factors and candidates for serodiagnosis, of which nine were scrutinized here with a strip immunoassay. We have shown that aborting sheep exhibited a strong antibody response to surface (MOMP, MIP, Pmp13G) and virulence-associated (CPAF, TARP, SINC) antigens. While the latter disappeared within 18 weeks following abortion in a majority of the animals, antibodies to surface proteins persisted beyond the duration of the study. In contrast, nonaborting experimentally infected sheep developed mainly antibodies to surface antigens (MOMP, MIP, Pmp13G), all of which did not persist. We were also able to detect antibodies to these surface antigens in C abortus-infected women who had undergone septic abortion, whereas a group of shepherds and veterinarians with occupational exposure to C abortus-infected sheep revealed only sporadic immune responses to the antigens selected. The most specific antigen for the serodiagnosis of human C abortus infections was Pmp13G, which showed no cross-reactivity with other chlamydiae infecting humans. We suggest that Pmp13G-based serodiagnosis accomplished by the detection of antibodies to virulence-associated antigens such as CPAF, TARP, and SINC may improve the laboratory diagnosis of human and animal C abortus infections.

Wild-typeLeishmania donovanipromastigotes block maturation, increase integrin expression and inhibit detachment of human monocyte-derived dendritic cells – the influence of phosphoglycans

The protective immune response against the parasite, including the role of dendritic cells (DC) in the course of infection, plays a fundamental role. This study shows that wild-type (WT) Leishmania promastigotes and specifically the phosphoglycans family of virulence-associated antigens inhibit human monocyte-derived dendritic cells (MoDC) maturation and detachment to distinct surfaces. Immature phagocytosis of Leishmania donovani promastigotes by immature MoDC results in the increased expression of CD11b and CD51, and inhibition of cell detachment to distinct surfaces, which was dependent on the presence of phosphoglycans. These findings demonstrate that phosphoglycans of WT L. donovani might also inhibit human DC migration to lymphoid organs.

Progress in the development of mastitis vaccines

Mastitis is a combination of clinical signs caused by the infection of the mammary gland with one or more different micro-organisms. These pathogens have been the subject of vaccine experimentation for almost a century. However, vaccine development has been difficult because the immune response to a natural infection does not protect efficiently against a subsequent infection. Recent advances in both molecular biology and the understanding of bovine immunology have lead to important progress in the generation of more efficient vaccines. In the past two years, reports have been published which described prototype vaccines against Streptococcus uberis, Streptococcus agalactiae, Escherichia coli and Staphylococcus aureus. These vaccines are based either on the use of a bacterial extract which contains antigens from the most common bacterial serotypes, or on purified antigens which may or may not be conjugated to carriers in order to increase their immunological efficiency. However, these vaccines are not yet in common use because of problems associated with their lack of widespread efficiency, their production costs and, in some cases, their stability. In addition, chronic forms of mastitis have been associated with intracellular forms of bacteria that are protected from some immune mechanisms and are often not a reachable target for these vaccines. A more recent strategy is to use DNA expression vector plasmids as vaccines which express virulence-associated antigens in vivo. These plasmids, once introduced into the animal, lead to the activation of both humoral and cellular immune responses to the antigen. The type of plasmid, the route of injection and the inclusion of genetic adjuvants can all be adjusted so as to achieve the type of immune response desired. S. aureus mastitis has been particularly difficult to control and in some areas of the world it causes over 50% of the reported cases of mastitis. S. aureus expresses and secretes a large number of proteins that are essential for the virulence of the bacteria and these have been the focus of recent interest for vaccine production. In the last few years, in order to identify the antigens that can be used to induce a protective immune response by a DNA vaccine in cattle; we have produced a series of plasmids and recombinant proteins that represent some of the major virulence factors of this bacterium. The most promising antigen candidates currently being tested are the bacterial adhesion molecules, the extra-cellular processing enzymes and the extra-cellular quorum sensing molecules. Results demonstrate that high titres of antibodies can be obtained by DNA vaccination, and that these antibodies can have an effect on the clearance of the bacteria. It is expected that the successful composition of an S. aureus vaccine will include a combination of DNA-expressed antigens with a booster injection of either recombinant protein or virally vectored antigen.

Isolation and characterisation of Mycobacterium avium and Rhodococcus equi from granulomatous lesions of swine lymph nodes in Slovenia.

Granulomatous lesions in bovine and especially swine lymph nodes are still frequently observed during routine veterinary meat inspections even though Mycobacterium bovis infections are no longer detected in domestic animals in Slovenia. Different lymph nodes of pigs (n = 260) were investigated using classical bacteriological and molecular methods. Mycobacterium avium alone was isolated in 47.3% of pigs and in mixed infection with Rhodococcus equi in 3.9% of pigs. R. equi alone was isolated in 27.3% and in mixed infection with mycobacteria other than M. avium in 1.5% of pigs. A total of 133 M. avium isolates were typed using the IS1245, IS901 and FR300 PCR. Almost two thirds (60.9%) of isolates belonged to M. avium hominissuis (IS901-, IS1245+ genotype), 33.8% of isolates belonged to M. avium avium (IS901+, IS1245+ genotype) and 5.3% of isolates remained non-typed. Fifty out of 85 R. equi isolates were tested for the virulence-associated antigens (VapA and VapB). Nearly two thirds (60.0%) were positive for VapB while all the other isolates were VapA- and VapB-negative.

Infection: Animal and Human Health

With the advent of AIDS, Rhodococcus equi infection has become more common in humans. Identification of virulence-associated antigens and virulence

Virulence of Rhodococcus equi isolated from cats and dogs.

Nine cat isolates and nine dog isolates of Rhodococcus equi from clinical material were investigated for the presence of the virulence-associated antigens (VapA and VapB) and virulence plasmids. Five of the cat isolates and one dog isolate were VapA positive and contained an 85-kb type I or an 87-kb type I plasmid. The remaining 12 isolates were avirulent R. equi strains and contained no virulence plasmids.

Identification of virulence-associated antigens and plasmids in Rhodococcus equi from patients with acquired immune deficiency syndrome and prevalence of virulent R. equi in soil collected from domestic animal farms in Chiang Mai, Thailand.

The prevalence of virulent Rhodococcus equi in soil collected from 17 domestic animal farms (from 12 cattle, 1 pig, and 4 horse farms) and in 6 clinical specimens from patients with acquired immune deficiency syndrome (AIDS) in Chiang Mai, Thailand, was investigated. The isolates were tested for the presence of 15-17-kDa antigens (VapA) and a 20-kDa antigen (VapB) by immunoblotting and for the presence of virulence plasmid DNA. Rhodococcus equi was isolated from most soil samples (68 of 80) obtained from the 17 farms, with 2.0 x 10(2) to 6.0 x 10(5) colony-forming units per gram of soil. We detected VapA in none of the 537 isolates from the soil samples. In one isolate from a pig farm, both VapB and virulence plasmid DNA were detected. Of the 6 clinical isolates from patients with AIDS, however, 4 isolates contained both VapB and virulence plasmid DNA. The remaining 2 isolates were avirulent.

Pathogenicity of Rhodococcus equi expressing a virulence-associated 20 kDa protein (VapB) in foals

Rhodococcus equi strains of intermediate virulence (IMV) for mice possess a 20 kDa protein designated Virulence Associated Protein B (VapB) and a virulence plasmid of 79–100 kb, and can be recovered from the submaxillary lymph nodes of pigs. The pathogenicity of such R. equi strains for foals is unknown. In this study, two foals, 42 and 43 days of age, were infected intratracheally with 10 6 and 10 9 cells of R. equi IMV strain A5, respectively. The foal infected with 10 9 cells of strain A5 became clinically ill, with the onset of illness (pyrexia and depression) occurring 21 days after inoculation. R. equi was isolated from the feces and tracheal washings of the foal from 14 to 28 days after inoculation. The foal infected with 10 6 cells of A5 showed no clinical signs, and no R. equi was isolated from any of the samples of feces or tracheal washings during the 28 days of observation. Two foals of 45 and 50 days of age were infected with 10 5 or 10 6 of virulent R. equi ATCC 33701 having 15–17 kDa surface proteins designated VapA. Both exhibited severe clinical signs (pyrexia, depression and anorexia) at 12 and 13 days after inoculation. Histopathological examination revealed that strain A5 caused focal granulomatous pneumonia in the foals. R. equi IMV strain A5 was isolated from lung lesions of both foals and from the contents of the intestinal tracts of the foal infected with 10 9 bacteria. These results suggest that IMV R. equi having VapB is less virulent than virulent R. equi having VapA in foals. This finding supports our previous results on the pathogenicities of R. equi strains having these virulence-associated antigens assessed by mouse pathogenicity tests.

Detection of the 20-kDa virulence-associated antigen of Rhodococcus equi in malakoplakia-like lesion in pleural tissue obtained from an AIDS patient

A malakoplakia-like lesion was detected in a pleural biopsy from an AIDS patient presenting clinical and radiologic features of pneumonia. Cultures of bronchoalveolar lavage and pleural fluid evidenced Rhodococcus equi as the causative agent of pleuro-pulmonary infection. Immunochemical characterization of the R. equi isolate showed the presence of a strain similar to the ATCC 33704 reference strain presenting the capsular antigen of serotype 4, and the intermediate virulence-associated antigen of 20-kDa. Histopathology of the patient's pleural biopsy showed plaques of macrophages interspersed with lymphocytes, and intracytoplasmic cocci and bacilli in macrophages, which were variably acid-fast positive. Immunohistochemistry of cocci, bacilli and their degradation products resulted strongly positive when stained with a mouse monoclonal antibody (MAb) produced against the 20-kDa antigen. This finding could have important implications for the pathogenicity of R. equi for human beings, since we do not know yet all the factors involved in the formation of malakoplakia. Indeed, the results obtained in the present study, taken together with the results obtained for pigs inoculated with R. equi strains of intermediate virulence (Madarame et al. 1998), raise the possibility that most strains presenting the 20-kDa antigen may be capable of inducing malakoplakia. If this hypothesis is confirmed by immunohistochemical analysis of human pulmonary malakoplakia cases due to R. equi, the detection of this antigen may be extremely helpful in the diagnosis and treatment of such patients. This is the first report of R. equi infection in human beings that suggests a relationship between pleural malakoplakia and the virulence-associated antigen of 20-kDa.

Search for an antibody profile of Rhodococcus equi infection in AIDS patients despite the diversity of isolates and patient immune dysfunction

Diversity of virulence-associated antigens of Rhodococcus equi was detected among thirteen strains isolated from AIDS patients on two continents. One out of four Brazilian isolates presented the virulence-associated antigen of 15- to 17-kDa, and the other three isolates had the 20-kDa virulence-associated antigen. In contrast, only three out of nine Italian isolates were positive for virulence-associated antigens – two for the 15- to 17-kDa antigen and one for the 20-kDa antigen. In four other Italian strains, one or more other low-molecular-weight antigens were identified. Because of R. equi variability and host immune dysfunction, no characteristic antibody profile was detected among patients, although the presence of specific antibodies in serum samples suggested prognostic value: good patient outcome and recovery from pneumonia were correlated with R. equi antibody detection, whereas the lack or disappearence of specific antibodies, mainly those to low-molecular-weight antigens, was correlated with disease progression and patient death. These results confirmed the nonobligatory presence of the well-known virulence-associated antigens for the pathogenicity of R. equi in humans, and also the diversity of R. equi strains isolated from AIDS patients, which may be related to the geographic origin of the isolates or may be a consequence of the route of R. equi transmission in different countries. Some mechanisms underlying the results obtained are discussed, suggesting immune complex formation during the progress of the disease.

Live virulent Rhodococcus equi, rather than killed or avirulent, elicits protective immunity to R. equi infection in mice

Mice inoculated intravenously with a sublethal dose of live virulent Rhodococcus equi ATCC 33701 that contained an 85-kb virulence plasmid were immune to a lethal intravenous challenge of ATCC 33701. This immunity depended upon the dose of immunization and developed rapidly: mice primed with 10 5 live ATCC 33701 eliminated the challenged bacteria more rapidly than mice primed with doses ranging from 10 2 to 10 4 bacteria, and mice given 10 5 live ATCC 33701 intravenously withstood the lethal challenge as early as 5 days after the initial inoculation. However, this protective immunity did not develop in mice immunized with doses of heat-killed ATCC 33701 ranging from 10 6 to 10 8, or in mice immunized with doses of live ATCC 33701P −, a plasmid-cured derivative (avirulent), in doses ranging from 10 5 to 10 7. These mice had positive antibody titers against R. equi at the challenge (14 days after priming). Adoptive transfer of resistance to virulent R. equi was obtained with spleen cells from mice immunized with live ATCC 33701, but not monoclonal antibody to 15- to 17-kDa virulence-associated antigens. These results revealed that live ATCC 33701P −, a plasmid-cured derivative of virulent R. equi, could not elicit protective immunity, and are consistent with previous observations that protective immunity was induced by live virulent, but not killed organisms.

Pathogenicity of Rhodococcus equi strains possessing virulence-associated 15- to 17-kDa and 20-kDa antigens: Experimental and natural cases in pigs

The pathogenic role of Rhodococcus equi in pigs remains controversial. Small numbers of pigs were inoculated intravenously (i.v.), or intramuscularly (i.m.) around the mouth, with a virulent, an intermediately virulent, or an avirulent strain of R. equi and killed 14 days later. None showed clinical signs other than transient fever and weight loss. The virulent and intermediately virulent strains were recovered in culture from various organs and lymph nodes of pigs inoculated i.v., but only from the mandibular lymph nodes of pigs inoculated i.m. The avirulent strain was not recovered from any site. None of the pigs developed macroscopically visible lesions, but they showed reactive hyperplasia of the mandibular lymph nodes. The latter contained scattered phagocytic cells, which were labelled immunohistochemically for virulence-associated antigens (15- to 17-kDa antigens or 20-kDa antigen). Intermediately virulent and virulent strains of R. equi were isolated from mandibular lymph nodes of 5.5% of apparently healthy abattoir pigs (n = 1615). Virulence-associated antigens were detected in phagocytic cells of culture-positive nodes, but the latter showed no lesions other than reactive lymphoid hyperplasia. The results would seem to question the pathogenic role of R. equi in pigs, and it is speculated that the organism survives in the lymph nodes without causing pathognomonic lesions.

Comparison of tracheal aspiration with other tests for diagnosis of Rhodococcus equi pneumonia in foals

The diagnostic value of tracheal aspiration was evaluated through comparison with other diagnostic methods using an experimental model of Rhodococcus equi (R. equi) pneumonia in foals. Pneumonia was induced by spraying of the virulent R. equi strain ATCC 33701 into the trachea of foals. All foals developed fever from 11 to 16 days after bacterial inoculation. One foal was euthanized on day 26 due to its poor prognosis, and other foals euthanized on day 43. During the experiment, some tests for diagnosis of Rhodococcus equi pneumonia such as tracheal aspiration, radiography, serodiagnosis and fecal culture were carried out. R. equi was continually isolated from tracheal aspirates collected via a silicone catheter inserted transnasally on day 8 to day 32 after bacterial inoculation. On the other hand, radiography, serodiagnosis and fecal culture were demonstrated to be valuable diagnostic methods, but to be limited compared with tracheal aspiration. Indirect fluorescent antibody technique (IFA) using a monoclonal antibody against the 15- to 17-kDa virulence-associated antigens (VapA) of R. equi and PCR targeting the structural gene of VapA detected bacteria in tracheal aspirates less sensitively than the isolation technique although they were more rapid. Therefore, we conclude that a combination of tracheal aspiration and bacterial isolation was the most valuable method for routine diagnosis of R. equi pneumonia in foals.

Identification of intermediately virulent Rhodococcus equi isolates from pigs.

We recently reported the existence of Rhodococcus equi isolates with at least three virulence levels, isolated from AIDS patients: virulent R. equi having 15- to 17-kDa antigens that kills mice with 10(6) cells, intermediately virulent R. equi having a 20-kDa antigen that kills mice with 10(7) cells, and avirulent R. equi that does not kill mice with 10(8) cells or more (S. Takai, Y. Imai, N. Fukunaga, Y. Uchida, K. Kamisawa, Y. Sasaki, S. Tsubaki, and T. Sekizaki, J. Infect. Dis. 172:1306-1311, 1995). Virulent R. equi having the 15- to 17-kDa antigens has been isolated frequently from horses and their environment, but the source of intermediately virulent R. equi having the 20-kDa antigen is poorly understood. There are many reports of the isolation of R. equi from the lymph nodes of pigs with and without lesions resembling those of tuberculosis. Therefore, we analyzed antigens of R. equi isolates from the submaxillary lymph nodes of pigs by immunoblotting with monoclonal antibodies against these virulence-associated antigens. Immunoblots of whole-cell antigen preparations of R. equi pig isolates revealed the presence of the 20-kDa antigen in almost all the pig isolates studied, and these isolates were intermediately virulent for mice. We also demonstrated that the expression of the 20-kDa antigen and its pathogenicity in mice were associated strongly with the presence of five large, distinct plasmids of 70 to 95 kb; two of the five plasmids from pig isolates were the same sizes as those from human isolates. These results suggest that R. equi having the 20-kDa antigen exists in the submaxillary lymph nodes of pigs and that the source of infection in some human cases might be associated with pigs and their environment.

Animal models and Lyme disease

Animal models and Lyme disease

Identification of virulence-associated antigens and plasmids in Rhodococcus equi from patients with AIDS.

Rhodococcus equi is an emerging opportunistic pathogen of human immunodeficiency virus-infected patients. However, little is known about the characteristics of R. equi isolates from humans. This study characterized the plasmid content, expression of a virulence-associated antigen, and mouse virulence of 19 R. equi isolates from patients with and without AIDS. EcoRI digestion patterns and Southern, Western, and virulence analyses of these isolates with cryptic plasmids allowed definition of a new category of R. equi. Isolates from patients with AIDS tended either to be virulent and have 15- to 17-kDa antigens and an 85-kb plasmid (10(6) bacteria needed for lethality) or have intermediate virulence (10(7) bacteria needed for lethality) and one of four distinct large plasmids that share DNA homology and express a 20-kDa antigen. Most of the non-AIDS isolates were avirulent (> 10(8) bacteria needed for lethality) and did not express any of these antigens.

Rhodococcus equi Virulence-Associated Antigens and Specific Antibody Response in AIDS Patients Infected with R. equi

OBJECTIVES: To analyze the expression of the 15- to 17-kDa plasmid-encoded antigens from Rhodococcus equi isolates of 7 AIDS patients and determine the immunologic response to these proteins in the patients' sera. METHODS: The expression of the virulence proteins in R. equi isolates and the specific antibody response were investigated by immunoblotting. Plasmid DNA was analyzed by agarose gel electrophoresis. RESULTS: The only patient infected with a strain carrying the virulence 85-kb plasmid and expressing the 15- to 17-kDa antigens developed a fatal pneumonia and did not produce specific antibodies to the virulence proteins. Of the 6 patients infected with R. equi strains lacking both proteins and plasmid, only 1 subject had a pulmonary disease with poor clinical outcome and exhibited a negligible humoral immune response to R. equi antigenic components, whereas the other patients who produced a remarkable antibody response developed either an asymptomatic infection (1 case) or pneumonia (4 cases) which completely cleared up. CONCLUSIONS: Our findings suggest that R. equi disease can be induced without the expression of the 15- to 17-kDa virulence-associated plasmid-encoded antigens in HIV-infected patients with very low CD4+ cell counts. Nevertheless, both the synthesis of the virulence proteins and a defective humoral immune response to R. equi may contribute to the severity of rhodococcal disease.