Bartonella henselae Recombinant Pap31 for the Diagnosis of Canine and Human Bartonelloses.

Bartonella spp. comprise a genus of Gram-negative alphaproteobacteria that are slow growing, fastidious, and facultative intracellular pathogens with zoonotic potential. Immunofluorescent antibody assays (IFAs), Western blot (WB), and enzyme-linked immunosorbent assays (ELISAs), the frequently used modalities for the serological diagnosis of canine and human Bartonelloses, generate numerous false negative results. Therefore, the development of a reliable serodiagnostic assay for Bartonelloses is of clinical and epidemiological importance. Pap31, a heme binding surface protein of B. henselae, is associated with bacterial adhesion and related to bacterial colonization. To our knowledge, B. henselae Pap31 and its fragments (N-terminal (NTD), middle (MD), and C-terminal (CTD) domains) have not been investigated for the serodiagnosis of canine and human Bartonelloses. In this study, we evaluate the diagnostic utility of B. henselae recombinant whole Pap31 (rPap31) and Pap31 fragments by ELISA using sera from 70 dogs (36 Bartonella spp. IFA-positive (naturally infected), and 34 Bartonella spp. IFA- and PCR-negative (control dogs)) and 36 humans (18 Bartonella spp. IFA-positive (naturally infected) and 18 controls)). In the dogs, the area under the curve (AUC) score of recombinant whole Pap31 was 0.714 with a sensitivity of 42% and specificity of 94% at an OD cutoff value of 0.8955. Among the evaluated recombinant Pap31 proteins for the diagnosis of canine Bartonelloses, rPap31-NTD yielded the highest AUC score of 0.792 (95% CI 0.688-0.895) with a sensitivity of 44% and specificity of 100% at a cutoff value of 1.198. In concordance with this finding, rPap31-NTD also had the highest AUC score of 0.747 (95% CI 0.581-0.913) among the Pap31 recombinant proteins for the diagnosis of human Bartonelloses, with 39% sensitivity and 94% specificity at a cutoff value of 1.366. Recombinant whole Pap31 (rPap31) resulted in 72% sensitivity and 61% specificity at a cutoff value of 0.215 for human Bartonelloses. Due to either low sensitivity or questionable specificity, our findings indicate that recombinant Pap31 and the selected fragments may not be appropriate diagnostic targets in detecting anti-Bartonella antibodies in Bartonella-infected dogs and humans. The findings from this study can be used to further assess the antigenicity and immunogenicity of B. henselae Pap31 as a diagnostic target.

Purification and characterization of a recombinant Yersinia pestis V-F1 "Reversed" fusion protein for use as a new subunit vaccine against plague.

We previously developed a unique recombinant protein vaccine against plague composed of a fusion between the Fraction 1 capsular antigen (F1) and the V antigen. To determine if overall expression, solubility, and recovery of the F1-V fusion protein could be enhanced, we modified the original fusion. Standard recombinant DNA techniques were used to reverse the gene order such that the V antigen coding sequence was fused at its C-terminus to the N-terminus of F1. The F1 secretion signal sequence (F1S) was subsequently fused to the N-terminus of V. This new fusion protein, designated F1S-V-F1, was then co-expressed with the Y. pestis Caf1M periplasmic chaperone protein in BL21-Star Escherichia coli. Recombinant strains expressing F1-V, F1S-F1-V, or F1S-V-F1 were compared by cell fractionation, SDS-PAGE, Western blotting, and suspension immunolabelling. F1S-V-F1 exhibited enhanced solubility and secretion when co-expressed with Caf1M resulting in a recombinant protein that is processed in a similar manner to the native F1 protein. Purification of F1S-V-F1 was accomplished by anion-exchange and hydrophobic interaction chromatography. The purification method produced greater than 1mg of purified soluble protein per liter of induced culture. F1S-V-F1 polymerization characteristics were comparable to the native F1. The purified F1S-V-F1 protein appeared equivalent to F1-V in its ability to be recognized by neutralizing antibodies.

Multiple asparagine deamidation of Bacillus anthracis protective antigen causes charge isoforms whose complexity correlates with reduced biological activity.

Protective antigen is essential for the pathology of Bacillus anthracis and is the proposed immunogen for an improved human anthrax vaccine. Known since discovery to comprise differentially charged isoforms, the cause of heterogeneity has eluded specific structural definition until now. Recombinant protective antigen (rPA) contains similar isoforms that appear early in fermentation and are mostly removed through purification. By liquid chromatography-tandem mass spectrometry sequencing of the entire protein and inspection of spectral data for amino acid modifications, pharmaceutical rPA contained measurable deamidation at seven of its 68 asparagine residues. A direct association between isoform complexity and percent deamidation was observed such that each decreased with purity and increased with protein aging. Position N537 consistently showed the highest level of modification, although its predicted rate of deamidation ranked 10th by theoretical calculation, and other asparagines of higher predicted rates were observed to be unmodified. rPA with more isoforms and greater deamidation displayed lower activities for furin cleavage, heptamerization, and holotoxin formation. Lethal factor-mediated macrophage toxicity correlated inversely with deamidation at residues N466 and N408. The described method measures deamidation without employing theoretical isotopic distributions, comparison between differentially treated samples or computational predictions of reactivity rates, and is broadly applicable to the characterization of other deamidated proteins.

Design and testing for a nontagged F1-V fusion protein as vaccine antigen against bubonic and pneumonic plague.

A two-component recombinant fusion protein antigen was re-engineered and tested as a medical counter measure against the possible biological threat of aerosolized Yersinia pestis. The active component of the proposed subunit vaccine combines the F1 capsular protein and V virulence antigen of Y. pestis and improves upon the design of an earlier histidine-tagged fusion protein. In the current study, different production strains were screened for suitable expression and a purification process was optimized to isolate an F1-V fusion protein absent extraneous coding sequences. Soluble F1-V protein was isolated to 99% purity by sequential liquid chromatography including capture and refolding of urea-denatured protein via anion exchange, followed by hydrophobic interaction, concentration, and then transfer into buffered saline for direct use after frozen storage. Protein identity and primary structure were verified by mass spectrometry and Edman sequencing, confirming a purified product of 477 amino acids and removal of the N-terminal methionine. Purity, quality, and higher-order structure were compared between lots using RP-HPLC, intrinsic fluorescence, CD spectroscopy, and multi-angle light scattering spectroscopy, all of which indicated a consistent and properly folded product. As formulated with aluminum hydroxide adjuvant and administered in a single subcutaneous dose, this new F1-V protein also protected mice from wild-type and non-encapsulated Y. pestis challenge strains, modeling prophylaxis against pneumonic and bubonic plague. These findings confirm that the fusion protein architecture provides superior protection over the former licensed product, establish a foundation from which to create a robust production process, and set forth assays for the development of F1-V as the active pharmaceutical ingredient of the next plague vaccine.

Recombinant F1-V fusion protein protects black-footed ferrets (Mustela nigripes) against virulent Yersinia pestis infection.

Black-footed ferrets (Mustela nigripes) are highly susceptible to sylvatic plague, caused by the bacterium Yersinia pestis, and this disease has severely hampered efforts to restore ferrets to their historic range. A study was conducted to assess the efficacy of vaccination of black-footed ferrets against plague using a recombinant protein vaccine, designated F1-V, developed by personnel at the U.S. Army Medical Research Institute of Infectious Diseases. Seven postreproductive black-footed ferrets were immunized with the vaccine, followed by two booster immunizations on days 23 and 154; three control black-footed ferrets received a placebo. After the second immunization, antibody titers to both F1 and V antigen were found to be significantly higher in vaccinates than controls. On challenge with 7,800 colony-forming units of virulent plague by s.c. injection, the three control animals died within 3 days, but six of seven vaccinates survived with no ill effects. The seventh vaccinate died on day 8. These results indicate that black-footed ferrets can be immunized against plague induced by the s.c. route, similar to fleabite injection.

Infection of C57BL/6 mice by Trypanosoma musculi modulates host immune responses during Brucella abortus cocolonization.

Brucellosis, which results in fetal abortions in domestic and wildlife animal populations, is of major concern in the US and throughout much of the world. The disease, caused by Brucella abortus, poses an economic threat to agriculture-based communities. A moderately efficacious live attenuated vaccine (B. abortus strain RB51) exists. However, even with vaccine use, outbreaks occur. Evidence suggests that elk (Cervus canadensis), a wild host reservoir, are the source of recent outbreaks in domestic cattle herds in Wyoming, USA. Brucella abortus establishes a chronic, persistent infection in elk. The molecular mechanisms allowing the establishment of this persistent infective state are currently unknown. A potential mechanism could be that concurrent pathogen burdens contribute to persistence. In Wyoming, elk are chronically infected with Trypanosoma cervi, which may modulate host responses in a similar manner to that documented for other trypanosomes. To identify any synergistic relationship between the two pathogens, we simulated coinfection in the well-established murine brucellosis model using Trypanosoma musculi and B. abortus S19. Groups of C57BL/6 mice (Mus musculus) were infected with either B. abortus strain 19 (S19) or T. musculi or both. Sera were collected weekly; spleens from euthanized mice were tested to determine bacterial load near the end of normal brucellosis infection. Although changes in bacterial load were observed during the later stages of brucellosis in those mice coinfected with T. musculi, the most significant finding was the suppression of gamma interferon early during the infection along with an increase in interleukin-10 secretion compared with mice infected with either pathogen alone. These results suggest that immune modulatory events occur in the mouse during coinfection and that further experiments are warranted to determine if T. cervi impacts Brucella infection in elk.

Vaccination with Brucella abortus recombinant in vivo-induced antigens reduces bacterial load and promotes clearance in a mouse model for infection.

Current vaccines used for the prevention of brucellosis are ineffective in inducing protective immunity in animals that are chronically infected with Brucella abortus, such as elk. Using a gene discovery approach, in vivo-induced antigen technology (IVIAT) on B. abortus, we previously identified ten loci that encode products up-regulated during infection in elk and consequently may play a role in virulence. In our present study, five of the loci (D15, 0187, VirJ, Mdh, AfuA) were selected for further characterization and compared with three additional antigens with virulence potential (Hia, PrpA, MltA). All eight genes were PCR-amplified from B. abortus and cloned into E. coli. The recombinant products were then expressed, purified, adjuvanted, and delivered subcutaneously to BALB/c mice. After primary immunization and two boosts, mice were challenged i.p. with 5 x 104 CFU of B. abortus strain 19. Spleens from challenged animals were harvested and bacterial loads determined by colony count at various time points. While vaccination with four of the eight individual proteins appeared to have some effect on clearance kinetics, mice vaccinated with recombinant Mdh displayed the most significant reduction in bacterial colonization. Furthermore, mice immunized with Mdh maintained higher levels of IFN-γ in spleens compared to other treatment groups. Collectively, our in vivo data gathered from the S19 murine colonization model suggest that vaccination with at least three of the IVIAT antigens conferred an enhanced ability of the host to respond to infection, reinforcing the utility of this methodology for the identification of potential vaccine candidates against brucellosis. Mechanisms for immunity to one protein, Mdh, require further in vitro exploration and evaluation against wild-type B. abortus challenge in mice, as well as other hosts. Additional studies are being undertaken to clarify the role of Mdh and other IVI antigens in B. abortus virulence and induction of protective immunity.

Identification of in vivo-induced conserved sequences from Yersinia pestis during experimental plague infection in the rabbit.

In an effort to identify the novel virulence determinants of Yersinia pestis, we applied the gene "discovery" methodology, in vivo-induced (IVI) antigen technology, to detect genes upregulated during infection in a laboratory rabbit model for bubonic plague. After screening over 70,000 Escherichia coli clones of Y. pestis DNA expression libraries, products from 25 loci were identified as being seroreactive to reductively adsorbed, pooled immune serum. Upon sequence analysis of the predicted IVI gene products, more frequently encountered conserved protein functional categories have emerged, to include type-V autotransporters and components of more complex secretion systems including types III and VI. The recombinant products from eight selected clones were subsequently immunoblotted against pooled immune serum from two naturally infected host species: the prairie dog, and a species refractory to lethal disease, the coyote. Immune prairie dog serum recognized 2-3 of the rabbit-reactive antigens, suggesting at least some overlap in the pathogen's in vivo survival mechanisms between these two hosts. Although the coyote serum failed to recognize most of the IVI antigens, LepA was universally reactive with all three host sera. Collectively, the profiles/patterns of IVI conserved sequences (IVICS) may represent immune "signatures" among different host species, possessing the potential for use as a diagnostic tool for plague. Further, the antigenic nature of IVICS makes them ideal for further evaluation as novel subunit vaccine candidates. The gathering of additional data and analysis of the intact IVI genes and the expressed IVICS products should provide insight into the unique biologic processes of Y. pestis during infection and reveal the genetic patterns of the pathogen's survival strategy in different hosts.

Prophylaxis and therapy of inhalational anthrax by a novel monoclonal antibody to protective antigen that mimics vaccine-induced immunity.

The neutralizing antibody response to the protective antigen (PA) component of anthrax toxin elicited by approved anthrax vaccines is an accepted correlate for vaccine-mediated protection against anthrax. We reasoned that a human anti-PA monoclonal antibody (MAb) selected on the basis of superior toxin neutralization activity might provide potent protection against anthrax. The fully human MAb (also referred to as MDX-1303 or Valortim) was chosen from a large panel of anti-PA human MAbs generated using transgenic mice immunized with recombinant PA solely on the basis of in vitro anthrax toxin neutralization. This MAb was effective in prophylactic and postsymptomatic treatment of rabbits exposed to aerosolized anthrax spores, and a single intramuscular injection of 1 mg/kg of body weight fully protected cynomolgus monkeys challenged with aerosolized anthrax spores. Importantly, MAb 1303 defines a novel neutralizing epitope that requires Fc receptor engagement for maximal activity. F(ab')2 fragments of MAb 1303, which retain equivalent affinity for PA, are 10- to 100-fold less potent in neutralizing anthrax toxin in vitro. Addition of Fc receptor-blocking antibodies also greatly reduced the activity of MAb 1303. Moreover, we found that the neutralizing activity of mouse, rabbit, and human antisera elicited by PA vaccines was effectively abrogated by blocking Fc receptors. Selection of an anti-PA MAb by using a functional assay that is a surrogate for protection has resulted in the identification of a fully human MAb with potent activity in vivo and uncovered a previously unrecognized mechanism of antibody-mediated toxin neutralization that is important for currently used anthrax vaccines.

Yersinia pestis outer membrane type III secretion protein YscC: expression, purification, characterization, and induction of specific antiserum.

The type III secretion system (YscC) protein of Yersinia pestis plays an essential role in the translocation of Yersinia outer proteins (Yops) into eukaryotic target cells through a type III secretion mechanism. To assess the immunogenicity and potential protective efficacy of YscC against lethal plague challenge, we cloned, overexpressed, and purified YscC using two different bacterial expression and purification systems. The resulting expression plasmids for YscC, pETBlue-2-YscC and pTYB11-YscC, were regulated by robust T7 promoters that were induced with isopropyl-beta-D-thiogalactopyranoside. The intein-fusion pTYB11-YscC system and the six-histidine-tagging pETBlue-2-YscC system were both successful for producing and purifying YscC. The intein-mediated purification system produced about 1mg of soluble YscC per liter of bacterial culture while the YscC-His(6)-tag method resulted in 16mg of insoluble YscC per liter of bacterial culture. Protein identity for purified YscC-His(6) was confirmed by ion trap mass spectrometry. Antisera were produced against both YscC and YscC-His(6). The specific immune response generated in YscC-vaccinated mice was relative to the particular purified protein, YscC or YscC-His(6), which was used for vaccination as determined by Western blot analysis and ELISA. Regardless of the purification method, either form of the YscC protein failed to elicit a protective immune response against lethal plague challenge with either F1 capsule forming Y. pestis CO92 or the isogenic F1(-)Y. pestis C12.

Flea-borne transmission model to evaluate vaccine efficacy against naturally acquired bubonic plague.

A flea-to-mouse transmission model was developed for use in testing new candidate vaccines for the ability to protect against flea-borne plague. The model was used to evaluate a recombinant fusion protein vaccine consisting of the Yersinia pestis F1 and V antigens. After one to three challenges with Y. pestis-infected fleas, 14 of 15 unvaccinated control mice developed plague, with an average septicemia level of 9.2 x 10(8) Y. pestis CFU/ml. None of 15 vaccinated mice developed the disease after similar challenges, and serological testing indicated that transmitted bacteria were eliminated by the immune system before extensive replication and systemic infection could occur. The transmission and development of disease in control mice correlated with the number of bites by blocked fleas but not with the total number of fleabites. The model provides a means to directly assess the efficacy of new vaccines to prevent naturally acquired bubonic plague and to study events at the vector-host interface that lead to dissemination and disease.