Quantitative In Vivo Detection of Chlamydia muridarum Associated Inflammation in a Mouse Model Using Optical Imaging.

Chlamydia trachomatis is a bacterial sexually transmitted disease with over 1.3 million cases reported to the CDC in 2010. While Chlamydia infection is easily treated with antibiotics, up to 70% of infections are asymptomatic and go untreated. The current mouse model relies on invasive upper genital tract gross pathology readouts at ~60-80 days postinfection. High throughput optical imaging through the use of biomarkers has been successfully used to quickly evaluate several disease processes. Here we evaluate Neutrophil Elastase 680 (Elastase680) for its ability to measure Chlamydia muridarum associated inflammation in live mice using fluorescence molecular tomography (FMT) and In Vivo Imaging System (IVIS). Optical imaging was able to distinguish with statistical significance between vaccinated and nonvaccinated mice as well as mock-challenged and challenged mice 2 weeks after challenge which was 9 weeks sooner than typical gross pathological assessment. Immunohistochemistry confirmed the presence of neutrophils and correlated well with both in vivo and ex vivo imaging. In this report we demonstrate that Elastase680 can be used as a molecular imaging biomarker for inflammation associated with chlamydial infection in a mouse model and that these biomarkers can significantly decrease the time for pathology evaluation and thus increase the rate of therapeutics discovery.

RSV disease in infants and young children: Can we see a brighter future?

Respiratory syncytial virus (RSV) is a highly contagious seasonal virus and the leading cause of Lower Respiratory Tract Infections (LRTI), including pneumonia and bronchiolitis in children. RSV-related LRTI cause approximately 3 million hospitalizations and 120,000 deaths annually among children <5 years of age. The majority of the burden of RSV occurs in previously healthy infants. Only a monoclonal antibody (mAb) has been approved against RSV infections in a restricted group, leaving an urgent unmet need for a large number of children potentially benefiting from preventive measures. Approaches under development include maternal vaccines to protect newborns, extended half-life monoclonal antibodies to provide rapid long-lasting protection, and pediatric vaccines. RSV has been identified as a major global priority but a solution to tackle this unmet need for all children has yet to be implemented. New technologies represent the avenue for effectively addressing the leading-cause of hospitalization in children <1 years old.

Comparison of rhesus and cynomolgus macaques in a Streptococcus pyogenes infection model for vaccine evaluation.

Animal models predictive of human disease are generally difficult to establish and reproduce. In the case of the Group A Streptococcus (GAS) bacterium, which is predominantly a human pathogen, virulence assessment in animal models is problematic. We compared a monkey colonization and pharyngitis model of infection in two macaque species to determine the optimal model for vaccine candidate evaluation. Rhesus and cynomolgus macaques were intranasally infected with a streptomycin resistant (Str(r)) GAS strain. Monkeys were monitored for body weight and temperature changes, throat swabs and sera were collected, and clinical observations were noted throughout the study. Both species exhibited oropharyngeal colonization by GAS, with rhesus macaques demonstrating a more sustained colonization through day 28 post-challenge. Veterinary observations revealed no significant differences between GAS-infected rhesus and cynomolgus macaques. Mock-infected monkeys did not exhibit clinical symptoms or GAS colonization throughout the study. ELISA results demonstrated that both rhesus and cynomolgus macaques developed anti-streptolysin-O antibody titers, with cynomolgus generating higher titers. Sera from infected monkeys produced opsonophagocytic killing and bound to the bacterium in an immunofluorescence assay. Both rhesus and cynomolgus macaques can be used for colonization studies with this GAS M3 strain, yet only mild clinical signs of pharyngitis and tonsillitis were observed.

A novel automated method for enumeration of Chlamydia trachomatis inclusion forming units.

Chlamydia trachomatis is an obligate intracellular pathogen that primarily infects epithelial cells. Traditional methods for quantification of inclusion forming units (IFUs) rely upon infection of epithelial cell monolayers in vitro. Following incubation for approximately 2 days, inclusion bodies that result from infection of cells are detected by immunofluorescent staining with an antibody conjugated to a fluorescent dye. These inclusion bodies are then manually counted by microscopic examination of multiple, randomly selected fields of view. This requires substantial operator time and is subject to investigator bias. We have developed a novel method in which we utilize an automated microplate ImmunoSpot reader to count C. trachomatis IFUs. Following infection of epithelial cells in a 96-well plate and subsequent incubation, IFUs are fixed and detected with an anti-C. trachomatis LPS monoclonal antibody. Immobilized antibody is detected with a biotinylated secondary antibody and visualized enzymatically with streptavidin-alkaline phosphatase and the colorimetric substrate nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phospate (NBT/BCIP). IFUs are then enumerated with the ImmunoSpot system. This method has been used to quantify IFUs from all cell lines traditionally used for chlamydial propagation, including L929, McCoy, HeLa and HaK cells. IFU numbers obtained are comparable to those determined by traditional microscopic counting. In addition, the method can be applied to rapid determination of serum-neutralizing titers for vaccine studies, and we have also applied this approach to quantify Chlamydia recovered from vaginal swabs collected from infected animals. This method provides for rapid enumeration of IFU counts while minimizing investigator bias and has potential applications for both research and diagnostic use.

Development of a Novel Vaccine Containing Binary Toxin for the Prevention of Clostridium difficile Disease with Enhanced Efficacy against NAP1 Strains.

Clostridium difficile infections (CDI) are a leading cause of nosocomial diarrhea in the developed world. The main virulence factors of the bacterium are the large clostridial toxins (LCTs), TcdA and TcdB, which are largely responsible for the symptoms of the disease. Recent outbreaks of CDI have been associated with the emergence of hypervirulent strains, such as NAP1/BI/027, many strains of which also produce a third toxin, binary toxin (CDTa and CDTb). These hypervirulent strains have been associated with increased morbidity and higher mortality. Here we present pre-clinical data describing a novel tetravalent vaccine composed of attenuated forms of TcdA, TcdB and binary toxin components CDTa and CDTb. We demonstrate, using the Syrian golden hamster model of CDI, that the inclusion of binary toxin components CDTa and CDTb significantly improves the efficacy of the vaccine against challenge with NAP1 strains in comparison to vaccines containing only TcdA and TcdB antigens, while providing comparable efficacy against challenge with the prototypic, non-epidemic strain VPI10463. This combination vaccine elicits high neutralizing antibody titers against TcdA, TcdB and binary toxin in both hamsters and rhesus macaques. Finally we present data that binary toxin alone can act as a virulence factor in animal models. Taken together, these data strongly support the inclusion of binary toxin in a vaccine against CDI to provide enhanced protection from epidemic strains of C. difficile.

Ion-Exchange Chromatography to Analyze Components of a Clostridium difficile Vaccine.

Ion-exchange (IEX) chromatography is one of many separation techniques that can be employed to analyze proteins. The separation mechanism is based on a reversible interaction between charged amino acids of a protein to the charged ligands attached to a column at a given pH. This interaction depends on both the pI and conformation of the protein being analyzed. The proteins are eluted by increasing the salt concentration or pH gradient. Here we describe the use of this technique to characterize the charge variant heterogeneities and to monitor stability of four protein antigen components of a Clostridium difficile vaccine. Furthermore, the IEX technique can be used to monitor reversion to toxicity for formaldehyde-treated Clostridium difficile toxins.

MRI as a Novel In Vivo Approach for Assessing Structural Changes of Chlamydia Pathology in a Mouse Model.

Chlamydia trachomatis is among the most prevalent of sexually transmitted diseases. While Chlamydia infection is a reportable event and screening has increased over time, enhanced surveillance has not resulted in a reduction in the rate of infections, and Chlamydia infections frequently recur. The development of a preventative vaccine for Chlamydia may be the only effective approach for reducing infection and the frequency of pathological outcomes. Current vaccine research efforts involve time consuming and/or invasive approaches for assessment of disease state, and MRI presents a clinically translatable method for assessing infection and related pathology both quickly and non-invasively. Longitudinal T2-weighted MRI was performed over 63 days on both control or Chlamydia muridarum challenged mice, either with or without elementary body (EB) immunization, and gross necropsy was performed on day 65. A scoring system was developed to assess the number of regions affected by Chlamydia pathology and was used to document pathology over time and at necropsy. The scoring system documented increasing incidence of pathology in the unimmunized and challenged mice (significantly greater compared to the control and EB immunized-challenged groups) by 21 days post-challenge. No differences between the unchallenged and EB immunized-challenged mice were observed. MRI scores at Day 63 were consistently higher than gross necropsy scores at Day 65, although two of the three groups of mice showed no significant differences between the two techniques. In this work we describe the application of MRI in mice for the potential evaluation of disease pathology and sequelae caused by C. muridarum infection and this technique's potential for evaluation of vaccines for Chlamydia.

Design and Purification of Subunit Vaccines for Prevention of Clostridium difficile Infection.

Clostridium difficile is a gram-positive bacterium responsible for a large proportion of nosocomial infections in the developed world. C. difficile secretes toxins A and B (TcdA and TcdB) and both toxins act synergistically to induce a spectrum of pathological responses in infected individuals ranging from pseudomembranous colitis to C. difficile-associated diarrhea. Toxins A and B have been actively investigated as components of prophylactic vaccine as well as targets for therapeutic intervention with antibodies. Expression of such toxins by recombinant technology is often difficult and may require special handling and adherence to strict safety regulations during the manufacturing process due to the inherent toxicity of the proteins. Both toxins are large proteins (308 kDa and 270 kDa, respectively) and contain distinct domains mediating cell attachment, cellular translocation, and enzymatic (glucosidase) activity. Here we describe methods to produce fragments of Toxin B for their subsequent evaluation as components of experimental C. difficile vaccines. Methods presented include selection of fragments encompassing distinct functional regions of Toxin B, purification methods to yield high quality proteins, and analytical evaluation techniques. The approach presented focuses on Toxin B but could be applied to the other component, Toxin A, and/or to any difficult to express or toxic protein.

Toxicity assessment of Clostridium difficile toxins in rodent models and protection of vaccination.

Clostridium difficile is the leading cause of hospital-acquired diarrhea, also known as C. difficile associated diarrhea. The two major toxins, toxin A and toxin B are produced by most C. difficile bacteria, but some strains, such as BI/NAP1/027 isolates, produce a third toxin called binary toxin. The precise biological role of binary toxin is not clear but it has been shown to be a cytotoxin for Vero cells. We evaluated the toxicity of these toxins in mice and hamsters and found that binary toxin causes death in both animals similar to toxins A and B. Furthermore, immunization of mice with mutant toxoids of all three toxins provided protection upon challenge with native toxins. These results support the concept that binary toxin contributes to the pathogenicity of C. difficile and provide a method for monitoring the toxicity of binary toxin components in vaccines.

Detecting and preventing reversion to toxicity for a formaldehyde-treated C. difficile toxin B mutant.

The toxicity of Clostridium difficile large clostridial toxin B (TcdB) can be reduced by many orders of magnitude by a combination of targeted point mutations. However, a TcdB mutant with five point mutations (referred to herein as mTcdB) still has residual toxicity that can be detected in cell-based assays and in-vivo mouse toxicity assays. This residual toxicity can be effectively removed by treatment with formaldehyde in solution. Storage of the formaldehyde-treated mTcdB as a liquid can result in reversion over time back to the mTcdB level of toxicity, with the rate of reversion dependent on the storage temperature. We found that for both the "forward" mTcdB detoxification reaction with formaldehyde, and the "reverse" reversion to toxicity reaction, mouse toxicity correlated with several biochemical assays including anion exchange chromatography retention time and appearance on SDS-PAGE. Maintenance of a low concentration of formaldehyde prevents reversion to toxicity in liquid formulations. However, when samples with 0.016% (v/v) formaldehyde were lyophilized and stored at 37 °C, formaldehyde continued to react with and modify the mTcdB in the lyophilized state. Lyophilization alone effectively prevented reversion to toxicity for formaldehyde-treated, formaldehyde-removed mTcdB samples stored at 37 °C for 6 months. Formaldehyde-treated, formaldehyde-removed lyophilized mTcdB showed no evidence of reversion to toxicity, appeared stable by several assays, and was immunogenic in mice, even after storage for 6 months at 37 °C.

An in vitro culture model to study the dynamics of colonic microbiota in Syrian golden hamsters and their susceptibility to infection with Clostridium difficile.

Clostridium difficile infections (CDI) are caused by colonization and growth of toxigenic strains of C. difficile in individuals whose intestinal microbiota has been perturbed, in most cases following antimicrobial therapy. Determination of the protective commensal gut community members could inform the development of treatments for CDI. Here, we utilized the lethal enterocolitis model in Syrian golden hamsters to analyze the microbiota disruption and recovery along a 20-day period following a single dose of clindamycin on day 0, inducing in vivo susceptibility to C. difficile infection. To determine susceptibility in vitro, spores of strain VPI 10463 were cultured with and without soluble hamster fecal filtrates and growth was quantified by quantitative PCR and toxin immunoassay. Fecal microbial population changes over time were tracked by 16S ribosomal RNA gene analysis via V4 sequencing and the PhyloChip assay. C. difficile culture growth and toxin production were inhibited by the presence of fecal extracts from untreated hamsters but not extracts collected 5 days post-administration of clindamycin. In vitro inhibition was re-established by day 15, which correlated with resistance of animals to lethal challenge. A substantial fecal microbiota shift in hamsters treated with antibiotics was observed, marked by significant changes across multiple phyla including Bacteroidetes and Proteobacteria. An incomplete return towards the baseline microbiome occurred by day 15 correlating with the inhibition of C. difficile growth in vitro and in vivo. These data suggest that soluble factors produced by the gut microbiota may be responsible for the suppression of C. difficile growth and toxin production.

Development of a recombinant toxin fragment vaccine for Clostridium difficile infection.

Clostridium difficile infection (CDI) is the major cause of antibiotic-associated diarrhea and pseudomembranous colitis, a disease associated with significant morbidity and mortality. The disease is mostly of nosocomial origin, with elderly patients undergoing anti-microbial therapy being particularly at risk. C. difficile produces two large toxins: Toxin A (TcdA) and Toxin B (TcdB). The two toxins act synergistically to damage and impair the colonic epithelium, and are primarily responsible for the pathogenesis associated with CDI. The feasibility of toxin-based vaccination against C. difficile is being vigorously investigated. A vaccine based on formaldehyde-inactivated Toxin A and Toxin B (toxoids) was reported to be safe and immunogenic in healthy volunteers and is now undergoing evaluation in clinical efficacy trials. In order to eliminate cytotoxic effects, a chemical inactivation step must be included in the manufacturing process of this toxin-based vaccine. In addition, the large-scale production of highly toxic antigens could be a challenging and costly process. Vaccines based on non-toxic fragments of genetically engineered versions of the toxins alleviate most of these limitations. We have evaluated a vaccine assembled from two recombinant fragments of TcdB and explored their potential as components of a novel experimental vaccine against CDI. Golden Syrian hamsters vaccinated with recombinant fragments of TcdB combined with full length TcdA (Toxoid A) developed high titer IgG responses and potent neutralizing antibody titers. We also show here that the recombinant vaccine protected animals against lethal challenge with C. difficile spores, with efficacy equivalent to the toxoid vaccine. The development of a two-segment recombinant vaccine could provide several advantages over toxoid TcdA/TcdB such as improvements in manufacturability.

Development and optimization of a high-throughput assay to measure neutralizing antibodies against Clostridium difficile binary toxin.

Clostridium difficile strains producing binary toxin, in addition to toxin A (TcdA) and toxin B (TcdB), have been associated with more severe disease and increased recurrence of C. difficile infection in recent outbreaks. Binary toxin comprises two subunits (CDTa and CDTb) and catalyzes the ADP-ribosylation of globular actin (G-actin), which leads to the depolymerization of filamentous actin (F-actin) filaments. A robust assay is highly desirable for detecting the cytotoxic effect of the toxin and the presence of neutralizing antibodies in animal and human sera to evaluate vaccine efficacy. We describe here the optimization, using design-of-experiment (DOE) methodology, of a high-throughput assay to measure the toxin potency and neutralizing antibodies (NAb) against binary toxin. Vero cells were chosen from a panel of cells screened for sensitivity and specificity. We have successfully optimized the CDTa-to-CDTb molar ratio, toxin concentration, cell-seeding density, and sera-toxin preincubation time in the NAb assay using DOE methodology. This assay is robust, produces linear results across serial dilutions of hyperimmune serum, and can be used to quantify neutralizing antibodies in sera from hamsters and monkeys immunized with C. difficile binary toxin-containing vaccines. The assay will be useful for C. difficile diagnosis, for epidemiology studies, and for selecting and optimizing vaccine candidates.

Immunogenicity in mice and non-human primates of the Group A Streptococcal J8 peptide vaccine candidate conjugated to CRM197.

Vaccine development for Group A streptococcal (GAS) infection has been extensively focused on the N-terminal hypervariable or the C-terminal conserved regions of the M protein, a major virulence factor of GAS. We evaluated the immunogenicity and functional activity of the conserved C-terminal peptide vaccine candidate, J8, conjugated to CRM197, in two mouse strains: C3H (H2(k)) and Balb/c (H2(d)), and in rhesus macaques. Mice were immunized with J8-CRM197 formulated with Amorphous Aluminum Hydroxyphosphate Sulfate Adjuvant (AAHSA), and non-human primates were immunized with J8-CRM197 formulated with AAHSA, ISCOMATRIX (TM) adjuvant, or AAHSA/ISCOMATRIX adjuvant. J8-CRM197 was immunogenic in mice from both H2(k) and H2(d) backgrounds, and the antibodies generated bound to the surface of four different GAS serotypes and had functional bacterial opsonic activity. Mice immunized with J8-CRM197/AAHSA demonstrated varying degrees of protection from lethal challenge. We also demonstrated that J8-CRM197 is immunogenic in non-human primates. Our data confirm the utility of J8 as a potential GAS vaccine candidate and demonstrate that CRM197 is an acceptable protein carrier for this peptide.

Development and optimization of a novel assay to measure neutralizing antibodies against Clostridium difficile toxins.

Clostridium difficile produces two major virulence toxins, toxin A (TcdA) and toxin B (TcdB). Antitoxin antibodies, especially neutralizing antibodies, have been shown to be associated with a lower incidence of C. difficile infection (CDI) recurrence, and antibody levels are predictive of asymptomatic colonization. The development of an assay to detect the presence of neutralizing antibodies in animal and human sera for the evaluation of vaccine efficacy is highly desired. We have developed such an assay, which allows for the quantification of the effect of toxins on eukaryotic cells in an automated manner. We describe here the optimization of this assay to measure toxin potency as well as neutralizing antibody (NAb) activity against C. difficile toxins using a design-of-experiment (DOE) methodology. Toxin concentration and source, cell seeding density, and serum-toxin preincubation time were optimized in the assay using Vero cells. The assay was shown to be robust and to produce linear results across a range of antibody concentrations. It can be used to quantify neutralizing antibodies in sera of monkeys and hamsters immunized with C. difficile toxoid vaccines. This assay was shown to correlate strongly with traditional assays which rely on labor-intensive methods of determining neutralizing antibody titers by visual microscopic inspection of intoxicated-cell monolayers. This assay has utility for the selection and optimization of C. difficile vaccine candidates.

Pre-clinical evaluation of a 15-valent pneumococcal conjugate vaccine (PCV15-CRM197) in an infant-rhesus monkey immunogenicity model.

The incidence of invasive pneumococcal disease (IPD), caused by the approximately 91 serotypes of Streptococcus pneumoniae (PN), varies geographically and temporally as a result of changing epidemiology and vaccination patterns as well as due to regional measurement differences. Prevnar(®) (Pfizer), the first licensed pneumococcal conjugate vaccine (PCV), comprises polysaccharides (PS) from 7 serotypes conjugated to the mutant diphtheria toxin carrier protein, CRM197. In the United States and elsewhere, this vaccine has been highly efficacious in reducing the incidence of IPD caused by vaccine serotypes, however, the incidence of non-vaccine serotypes (e.g., 19A, 22F, and 33F) has increased, resulting in the need for vaccines with higher valencies. In response, 10- and 13-valent PCVs have recently been licensed. To further increase serotype coverage, we have developed a 15-valent PCV containing PS from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to CRM197 and formulated on aluminum phosphate adjuvant. Vaccine immunogenicity was evaluated in infant rhesus monkeys since they, like human infants, respond poorly to unconjugated PN PS. Infant (2-3 month old) rhesus monkeys were vaccinated three times with PCV-15 or Prevnar(®) at 2 month intervals, and serotype-specific IgG antibodies were measured using a multiarray electrochemiluminescence (ECL) assay. The results indicate that antibody responses to PCV-15 and Prevnar(®) were comparable for the 7 common serotypes and that post-vaccination responses to PCV-15 were >10-fold higher than baseline for the 8 additional serotypes.

A real-time quantitative polymerase chain reaction assay for the detection of Chlamydia in the mouse genital tract model.

Chlamydia trachomatis is a human pathogen that infects genital tracts in women. Disease control may be achieved through development of an efficacious vaccine. A mouse genital tract model serves as a tool for evaluation of vaccine candidates. Currently, assessment of infection in mice is performed by enumeration of inclusion-forming units (IFUs) through microscopic counting of fluorescently stained bacteria. We have developed a highly sensitive real-time quantitative polymerase chain reaction (RT-qPCR) assay for enumeration of Chlamydia from mouse genital tracts to increase assay sensitivity, remove subjectivity, and improve sample throughput. The qPCR assay uses a 16S ribosomal gene sequence that is conserved across Chlamydia species and serovars, resulting in detection of multiple serovars of C. trachomatis, as well as Chlamydia muridarum and Chlamydia pneumoniae. The PCR assay provided results similar to IFU enumeration (94% agreement between the 2 assays) and is highly sensitive and specific with less inherent subjectivity than traditional enumeration methods.

Effect of alternative aluminum adjuvants on the absorption and immunogenicity of HPV16 L1 VLPs in mice.

Aluminum adjuvants are commonly used in prophylactic vaccines to enhance antigen immunogenicity through induction of high-titer antibody responses. Three major forms of aluminum adjuvants with substantially different physical and chemical properties have been described: aluminum phosphate (AlPO(4)), aluminum hydroxide (AlOH) and amorphous aluminum hydroxyphosphate sulfate (AAHS). Here we describe the effect of these different aluminum adjuvants on the formulation and subsequent immunogenicity in mice of virus-like particles (VLPs) consisting of the L1 protein of Human Papillomavirus (HPV) Type 16. Electron microscopy demonstrated that the physical appearance of the phosphate-containing aluminum adjuvants was markedly different from that of aluminum hydroxide. All three aluminum adjuvants were found to display unique surface charge profiles over a range of pH, while AAHS demonstrated the greatest inherent capacity for adsorption of L1 VLPs. These differences were associated with differences in immunogenicity: anti-HPV L1 VLP responses from mice immunized with AAHS-formulated HPV16 vaccine were substantially greater than those produced by mice immunized with the same antigen formulated with aluminum hydroxide. In addition, HPV L1 VLPs formulated on AAHS also induced a substantial interferon-gamma secreting T cell response to L1 peptides indicating the potential for an enhanced memory response to this antigen. These results indicate that the chemical composition of aluminum adjuvants can have a profound influence on the magnitude and quality of the immune response to HPV VLP vaccines.