Loss Of Titer Research Articles

Effect of Extended Transit and Temperature Variations on EID50 Values of Thermostable NDVI-2 Vaccines

Temperature alterations due to poor cold keeping condition have consistently posed a challenge to vaccines dose EID50 titre value. The sensitivity of Newcastle disease vaccine to temperature variations due to poor cold storage condition in rural settings, led to the development of the NDVI-2 thermostable vaccine, which is known to exhibit a thermostability of 3 hours at 56 °C. In this study, three Newcastle disease vaccines (NDVI-2) with EID50 titre per dose values of log10 EID50 7.7, 7.6 and 6.75 respectively from vaccine production facility at the National Veterinary Research Institute (NVRI), Vom Nigeria, were sent for certification at African Union laboratory at Debre Zeit, Ethiopia under cold condition; using ice packs for cold chain maintenance. Tracking of the vaccine in the course of freight to the certifying laboratory revealed changes in the original transit route by the airline and delay by custom at the receiving country port. Thus, a 12 hours transit freight was delayed for over 7 days before arrival at the certifying laboratory. Though, the EID50 titre per dose values of these vaccines were originally well above recommended EID50 5.5 per dose value by log 2.2, log 2.1 and log 1.25 for NDVI-2 respectively. Comparing these vaccines EID50 values obtained from the certifying laboratory in relation to values initially obtained by the Quality Control Division of the producing Institute (NVRI), revealed loss of EID50 titre per dose value by log101.2, and log10 0.8 among two of the certified vaccine batches; with one of the certified vaccines retaining its original EID50 titre per dose value. This result attest that NDVI-2 thermostable vaccine produced at NVRI, Vom Nigeria are robust and of high quality. Thus, the findings of this study have further demonstrated that temperature variations, prolonged vaccine transportation under poor cold chain can affect the EID50 per dose value of a wholesome, field fit vaccine irrespective of the vaccine thermostability.

Influenza hemagglutinin antigenic distance measures capture trends in HAI differences and infection outcomes, but are not suitable predictive tools

Vaccination is the most effective method to combat influenza. Vaccine effectiveness is influenced by the antigenic distance between the vaccine strain and the actual circulating virus. Amino acid sequence based methods of quantifying the antigenic distance were designed to predict influenza vaccine effectiveness in humans. The use of these antigenic distance measures has been proposed as an additive method for seasonal vaccine selection. In this report, several antigenic distance measures were evaluated as predictors of hemagglutination inhibition titer differences and clinical outcomes following influenza vaccination or infection in mice or ferrets. The antigenic distance measures described the increasing trend in the change of HAI titer, lung viral titer and percent weight loss in mice and ferrets. However, the variability of outcome variables produced wide prediction intervals for any given antigenic distance value. The amino acid substitution based antigenic distance measures were no better predictors of viral load and weight loss than HAI titer differences, the current predictive measure of immunological correlate of protection for clinical signs after challenge.

High frequency acoustic nebulization for pulmonary delivery of antibiotic alternatives against Staphylococcus aureus

The increasing prevalence of multidrug resistant bacteria has warranted the search for new antimicrobial agents as existing antibiotics lose their potency. Among these, bacteriophage therapy, as well as the administration of specific bacteriolysis agents, i.e., lytic enzymes, have emerged as attractive alternatives. Nebulizers offer the possibility for delivering these therapeutics directly to the lung, which is particularly advantageous as a non-invasive and direct route to treat bacterial lung infections. Nevertheless, nebulizers can often result in significant degradation of the bacteriophage or protein, both structurally and functionally, due to the large stresses the aerosolization process imposes on these entities. In this work, we assess the capability of a novel low-cost and portable hybrid surface and bulk acoustic wave platform (HYDRA) to nebulize a Myoviridae bacteriophage (phage K) and lytic enzyme (lysostaphin) that specifically targets Staphylococcus aureus. Besides its efficiency in producing phage or protein-laden aerosols within the 1–5 μm respirable range for optimum delivery to the lower respiratory tract where lung infections commonly take place, we observe that the HYDRA platform—owing to the efficiency of driving the aerosolization process at relatively low powers and high frequencies (approximately 10 MHz)—does not result in appreciable denaturation of the phages or proteins, such that the loss of antimicrobial activity following nebulization is minimized. Specifically, a low (0.1 log10 (pfu/ml)) titer loss was obtained with the phages, resulting in a high viable respirable fraction of approximately 90%. Similarly, minimal loss of antimicrobial activity was obtained with lysostaphin upon nebulization wherein its minimum inhibitory concentration (0.5 μg/ml) remained unaltered as compared with the non-nebulized control. These results therefore demonstrate the potential of the HYDRA nebulization platform as a promising strategy for pulmonary administration of alternative antimicrobial agents to antibiotics for the treatment of lung diseases caused by pathogenic bacteria.

Inhalable bacteriophage powders: Glass transition temperature and bioactivity stabilization.

Recent heightened interest in inhaled bacteriophage (phage) therapy for combating antibacterial resistance in pulmonary infections has led to the development of phage powder formulations. Although phages have been successfully bioengineered into inhalable powders with preserved bioactivity, the stabilization mechanism is yet unknown. This paper reports the first study investigating the stabilization mechanism for phages in these powders. Proteins and other biologics are known to be preserved in dry state within a glassy sugar matrix at storage temperatures (T s) at least ~50°C below the glass transition temperature (T g). This is because at (T g − T s) >50°C, molecules are sufficiently immobilized with reduced reactivity. We hypothesized that this glass stabilization mechanism may also be applicable to phages comprising mostly of proteins. In this study, spray dried powders of Pseudomonas phage PEV20 containing lactose and leucine as excipients were stored at 5, 25 or 50°C and 15 or 33% relative humidity (RH), followed by assessment of bioactivity (PEV20 stability) and physical properties. PEV20 was stable with negligible titer loss after storage at 5°C/15% RH for 250 days, while storage at 33% RH caused increased titer losses of 1 log10 and 3 log10 at 5 and 25°C, respectively. The plasticizing effect of water at 33% RH lowered the T g by 30°C, thus narrowing the gap between T s and T g to 19–28°C, which was insufficient for glass stabilization. In contrast, the (T g − T s) values were higher (range, 46–65°C) under the drier condition of 15% RH, resulting in the improved stability which corroborated with the vitrification hypothesis. Furthermore, phage remained stable (≤1 log10) when the (T g − T s) value lay between 26–48°C, but became inactivated as the value fell below 20°C. In conclusion, this study demonstrated that phage can be sufficiently stabilized in spray dried powders by keeping the (T g − T s) value above 46°C, thus supporting the vitrification hypothesis that phages are stabilized by immobilization inside a rigid glassy sugar matrix. These findings provide a guide to better manufacture and storage practices of inhaled phage powder products using for translational medicines.

Reduction of charge variants by CHO cell culture process optimization.

Over the past decade, global interest in the development of therapeutic monoclonal antibodies (mAbs) has risen rapidly. As therapeutic agents, antibodies have shown marked efficacy in combatting a range of cancers and immune diseases with high target specificity and low toxicity (Carla Lucia et al. in PLoS ONE 6:e24071, 2011; Donaghy in MAbs 8:659-671, 2016; Nasiri et al. in J Cell Physiol 9:6441-6457, 2018; Teo et al. in Cancer Immunol Immunother 61:2295-2309, 2012). Recent advances in cell culture technology, such as high-throughput clone screening, have facilitated antibody production at concentrations exceeding 10 g/L (Chen et al. in BMC Immunol 19:35, 2018; Huang et al. in Biotechnol Prog 26:1400-1410, 2010; Lu et al. in Biotechnol Bioeng 110:191-205, 2013; Singh et al. in Biotechnol Bioeng 113:698-716, 2016). As titers have improved, the industry has begun to focus on the adjustment of target antibody quality profiles to improve efficacy. Cell lines, culture media, and culture conditions impact protein quality (Van Beers and Bardor in Biotechnol J 7:1473-1484, 2012). Optimization of critical quality attributes (CQAs), such as charge variants, can be achieved through bioprocess development and is the preferred approach as changes to the cell line or growth media used is considered unfavorable by regulatory bodies (Gawlitzek et al. in Biotechnol Bioeng 103:1164-1175, 2009; Jordan et al. in Cytotechnology 65:31-40, 2013; Pan et al. in Cytotechnology 69:39-56, 2016). In this study, the effect of process control and ion supplementation on charge variants of mAbs produced by Chinese hamster ovary (CHO) cells was investigated. Results of this study demonstrated that the concentration of Zn2+, duration of culturing, and temperature affect charge variants of a given mAb. Under the optimum conditions of 3L bioreactors, the most significant was that Zn2 + and temperature shift could further improve the quality of antibody. The main peak increased by 12%, and the acid peak decreased by 16%. At the same time, there was no significant loss of titer. This study provided supporting evidence for methods to improve charge variants arising during mAb production.

Development of a Lyophilization Process for Campylobacter Bacteriophage Storage and Transport.

Bacteriophages are a sustainable alternative to control pathogenic bacteria in the post-antibiotic era. Despite promising reports, there are still obstacles to phage use, notably titer stability and transport-associated expenses for applications in food and agriculture. In this study, we have developed a lyophilization approach to maintain phage titers, ensure efficacy and reduce transport costs of Campylobacter bacteriophages. Lyophilization methods were adopted with various excipients to enhance stabilization in combination with packaging options for international transport. Lyophilization of Eucampyvirinae CP30A using tryptone formed a cake that limited processing titer reduction to 0.35 ± 0.09 log10 PFU mL−1. Transmission electron microscopy revealed the initial titer reduction was associated with capsid collapse of a subpopulation. Freeze-dried phages were generally stable under refrigerated vacuum conditions and showed no significant titer changes over 3 months incubation at 4 °C (p = 0.29). Reduced stability was observed for lyophilized phages that were incubated either at 30 °C under vacuum or at 4 °C at 70% or 90% relative humidity. Refrigerated international transport and rehydration of the cake resulted in a total phage titer reduction of 0.81 ± 0.44 log10 PFU mL−1. A significantly higher titer loss was observed for phages that were not refrigerated during transport (2.03 ± 0.32 log10 PFU mL−1). We propose that lyophilization offers a convenient method to preserve and transport Campylobacter phages, with minimal titer reduction after the drying process.

Predicting favorable landing pads for targeted integrations in Chinese hamster ovary cell lines by learning stability characteristics from random transgene integrations

Chinese Hamster Ovary (CHO) cell lines are considered to be the preferred platform for the production of biotherapeutics, but issues related to expression instability remain unresolved. In this study, we investigated potential causes for an unstable phenotype by comparing cell lines that express stably to such that undergo loss in titer across 10 passages. Factors related to transgene integrity and copy number as well as the genomic profile around the integration sites were analyzed. Horizon Discovery CHO-K1 (HD-BIOP3) derived production cell lines selected for phenotypes with low, medium or high copy number, each with stable and unstable transgene expression, were sequenced to capture changes at genomic and transcriptomic levels. The exact sites of the random integration events in each cell line were also identified, followed by profiling of the genomic, transcriptomic and epigenetic patterns around them. Based on the information deduced from these random integration events, genomic loci that potentially favor reliable and stable transgene expression were reported for use as targeted transgene integration sites. By comparing stable vs unstable phenotypes across these parameters, we could establish that expression stability may be controlled at three levels: 1) Good choice of integration site, 2) Ensuring integrity of transgene and observing concatemerization pattern after integration, and 3) Checking for potential stress related cellular processes. Genome wide favorable and unfavorable genomic loci for targeted transgene integration can be browsed at https://www.borthlabchoresources.boku.ac.at/

The safety of attenuated and recombinant nasal influenza vaccines in terms of the development of secondary bacterial superinfection

Introduction. Influenza is a severe viral disease. The most common post-influenza complication is pneumonia. Earlier, we developed an experimental mouse model of viralbacterial pneumonia induced by successive infection with influenza virus and St. aureus, in which lethal synergy between pathogens observed in epidemiological observations was detected.Aim. To study the effect of the administration of intranasal vaccines, followed by infection with St. pneumoniae on the development and completion of the disease.Materials and methods. The animals were immunized intranasal with a strain of attenuated cold-adapted live influenza vaccine A/17/California/2009/38 (H1N1)pdm09 (LAIV) and a recombinant vaccine based on virus-like particles HA(Puerto Rico/8/34)- Gag (VLPs). Control groups of animals were infected with virulent strains of influenza virus A/ California/04/2009 (H1N1)pdm09 or A/Puerto Rico /8/34 (H1N1). On the fifth day after intranasal immunization with the vaccine preparations and infection with pathogenic strains, animals were subjected to bacterial infection with a strain of St. pneumoniae. The presence of synergism of vaccine or viral agent with bacterial infection was assessed by survival and weight loss of animals, virus titer and density of bacteria in nasopharyngeal washes and lungs.Results. It was shown that immunization with vaccine preparations did not lead to increased sensitivity of mice to bacterial infection. Elimination of bacteria from the lungs and nasopharynx in groups immunized with vaccine preparations corresponded to the dynamics in the group of animals immunized by PBS.Discussion. The results obtained indicate the safety of intranasal immunization with LAIV A/17/California/2009/38 (H1N1)pdm09 and virus-like particles HA (Puerto Rico/8/34)- Gag (VLPs) in terms of enhancing secondary bacterial superinfection caused by St. pneumoniae.Conclusion. The studied vaccines successfully blocked infections in the lower respiratory tract.

Inclusion of PF68 Surfactant Improves Stability of rAAV Titer when Passed through a Surgical Device Used in Retinal Gene Therapy

Recent advances in recombinant adeno-associated virus (rAAV) gene therapy for choroideremia show gene replacement to be a promising approach. It is, however, well known that contact of vector solution with plastic materials in the surgical device may result in non-specific adsorption with resulting loss of physical titer and/or level of protein expression and activity. Here we assessed the biocompatibility and stability of rAAV2-REP1 (Rab Escort Protein-1) before and following passage through the injection device over a period of time to mimic the clinical scenario. Three identical devices were screened using two concentrations of vector: high (1E+12 DNase-resistant particles [DRP]/mL) and low (1E+11 DRP/mL), to mimic high- and low-dose administrations of vector product. The low dose was prepared using either formulation buffer that contained 0.001% of a non-ionic surfactant (PF68) or balanced salt solution (BSS). We observed significant losses in the genomic titer of samples diluted with BSS for all time points. The addition of 0.001% PF68 did not, however, affect rAAV physical titer, or REP1 protein expression and biological activity. Hence we observed that neither the genomic titer nor the biological activity of a rAAV2-REP1-containing solution was affected following passage through the surgical device when PF68 was present as a surfactant and this was maintained over a period up to 10 h.

Removal of Endotoxin from rAAV Samples Using a Simple Detergent-Based Protocol

Endotoxin is the most common contaminant found in protein samples. Even a small amount of endotoxin can induce strong allergic reaction and death of a host organism. Endotoxin is also often detected in recombinant adeno-associated virus (rAAV) stocks prepared in research laboratories using off-the-shelf reagents; purifying rAAV stocks from endotoxin using commercial reagents sometimes results in significant titer loss. The problem is exacerbated due to the recently expanded diversity of rAAV serotypes and capsid variants, which, due to their variable capsid surface charge, display differential affinity toward endotoxin. In this paper, we describe a simple universal protocol of purifying vector stocks irrespective of AAV serotype. The protocol is based on subjecting endotoxin-contaminated rAAV to mild detergent treatment, followed by repeated buffer-exchange washing and concentrating viral stock by low-speed centrifugation. Multiple assays were employed to test the physical and biological equivalency of the viral stocks before and after purification. The described protocol has been routinely utilized to purify vector stocks contaminated at levels as high as >1,000 endotoxin units (EU)/mL to produce viral vectors with practically undetectable levels of endotoxin (<2.5 EU/mL), with the titer’s recovery in the range of 50%–100%.

Evaluation of storage conditions and efficiency of a novel microencapsulated Salmonella phage cocktail for controlling S. enteritidis and S. typhimurium in-vitro and in fresh foods

S. Enteritidis and S. Typhimurium are typically linked to foodborne outbreaks. Phages have continued to expand in various food applications. In this study, microencapsulation is applied for enhancing the stability and efficacy of phages as bio-control agent. Microencapsulated phage cocktail kept in aluminium laminated foil bag (LF) at 4 °C showed the highest survivability with a titer loss of 0.5 log PFU/g after 12 weeks of storage. Titer loss of phage cocktail lysate >4 log PFU/mL was observed after 12 weeks, at 4 °C. Color change of microencapsulated phage cocktail kept in LF at 4 °C did not show any significant difference during storage, and water activity (free water content) at 0.13 was found in these conditions. In-vitro study, S. Enteritidis and S. Typhimurium were decreased 1.79 and 3.63 log CFU/mL, respectively at 37 °C. Whereas, 0.43 and 0.76 log CFU/mL, respectively were observed at 10 °C. In foods, S. Enteritidis and S. Typhimurium were decreased 0.57 and 1.78 log CFU/cm2, respectively in meat. Whereas, 0.86 and 1.2 log CFU/g, respectively were observed in sprout. Foods with/without microencapsulated phage cocktail showed non-significant differences in liking scores after 2 days of storage. Overall, microencapsulated phage cocktail suggests another alternative for phage-based biocontrol with improved stability and efficacy for food application.

T-bet deficiency impairs CD4 T cell effector trafficking to Influenza A Virus infected lungs but not their anti-viral functions

Abstract CD4 T cell responses against Influenza A Virus (IAV) are predominantly classified as TH1, characterized by strong IFN-γ production. However, the protective role of IFN-γ is unclear, suggesting that full TH1 polarization may be dispensable for effective CD4 immunity. We test this hypothesis by analyzing transgenic CD4 T cells recognizing IAV that are deficient in the transcription factor T-bet (Tbx21−/−), the ‘master regulator’ of TH1 differentiation. 3×106 Wild-type (WT) or Tbx21−/− effector cells, primed under TH1 conditions, were transferred to naïve congenic WT mice then infected with lethal IAV. Tbx21−/− effectors protected mice, promoting similar reductions in viral titer and weight loss recovery as WT cells. However, T-bet deficiency impacted production of several cytokines by the effectors resulting in less IFN-γ and GM-CSF but more IL-2, IL-4 and IL-17. Although the peak magnitude of WT and Tbx21−/− effector responses were similar in secondary lymphoid organs, significantly fewer Tbx21−/− cells were observed in the lungs. We thus titrated effectors to 1×106 cells, to expose any potential differences in protection arising from differential lung homing. Lower effector transfer resulted in higher viral titer and delayed weight loss recovery in mice given Tbx21−/− versus WT cells. Mechanistically, Tbx21−/− cells expressed lower levels of the chemokine receptor CXCR3. CXCR3 blockade in mice given WT effectors reduced their numbers in the lungs and delayed weight loss recovery matching patterns seen in mice given Tbx21−/− effectors. Thus, while prototypical TH1 functions are dispensable for protection against IAV, T-bet-dependent chemokine responses are required to maximize anti-viral CD4 responses at the site of infection.

Serum Analysis of Hydro-pericardium Syndrome (HPS) by Filter-Paper Blood Sampling Technique: As Substitute to Serum Collection Technique

Background: The study aimed to assess the filter paper blood sampling technique for sero- monitoring against Hydro-pericardium syndrome (HPS). In view of the fact, dried blood samples don’t require immediate refrigeration, occupy little space and are easily transported.Methods: Whole blood paired with serum samples were collected from 100 broiler chickens on filter paper strips, dried for 2hrs at 37oC, stored in polythene bag and then eluated in normal saline at 4oC for overnight. Antigen of HPS was isolated, purified and confirmed by agar gel precipitation test (AGPT) with raised hyper-immune serum. Eluates of whole blood dried on filter paper with corresponding serum samples were tested for antibody activity by indirect heam-agglutination (IHA) test. Results: ‘The IHA titers of eluates were similar to titers obtained with serum diluted as 1:10. Normal saline and phosphate buffered saline did not influence the antibody stability, when used as eluating fluid. Whole blood dried on filter paper could be stored sealed in plastic bag at 4oC or ambient temperature for at least one week with no appreciable loss of antibody titers. A strong correlation (r = 0.900) exist between the titers obtained with two methods of blood sampling. Conclusion: This study demonstrated that recovery of antibodies from blood dried on filter paper after eluation produces results comparable to those obtained by recovering antibodies from serum. Based on above findings it is concluded that filter paper blood sampling could serve as a cost effective and convenient tool for HPS sero-monitoring.

Impact of long-term storage of clinical samples collected from 1996 to 2017 on RT-PCR detection of norovirus

Noroviruses are recognized as the leading cause of acute gastroenteritis globally. With improved molecular diagnostics developed over the last two decades, archived clinical specimens are increasingly used to investigate the historic prevalence and molecular epidemiology of human norovirus. Yet the impact of long-term storage on viral integrity in clinical specimens has not been evaluated. In this study, we retested 994 stool specimens collected between 1996 and 2017 that originally tested norovirus-positive to quantify the loss of norovirus RT-PCR positivity with increasing sample storage time at 4 °C. In all, 79% of samples tested positive after retesting, but there was an approximate 3% decline in the positivity ratio and 4% decline in the percentage of samples that could be genotyped with each additional year of sample storage. For samples that were originally quantified by real-time RT-PCR (collected between 2003 and 2017), there was an estimated 1-log loss of viral titer occurring every 7 years of sample storage. Few samples contained PCR inhibitors, assessed using a MS2 extraction control, indicating that loss of RT-PCR signal was due primarily to loss of viral RNA integrity after long-term storage of stool samples at 4 °C. Our results indicate that norovirus positive stool samples can be stored with minimal loss in RT-PCR positivity when stored less than a decade. Longer periods of storage may impair norovirus detection, potentially impacting historic estimates of norovirus prevalence and molecular epidemiology if derived by testing archival clinical specimens.

Storage stability of inhalable phage powders containing lactose at ambient conditions

The aim of this study was to evaluate the storage stability of inhalable phage powders containing lactose and leucine as excipient. As an FDA-approved excipient for inhalation, lactose is preferred over other sugars. PEV phages active against antibiotic-resistant Pseudomonas aeruginosa was spray dried with lactose (55–90%) and leucine (45–10%). Produced powders were heat-sealed in an aluminium pouch at 15% relative humidity (RH) with subsequent storage at 20 °C/60% RH for 12 months. Lactose concentration in the powder positively influenced the phage stability over time. Formulation containing 90% lactose maintained the viability of PEV61 across the study, while ∼1.2 log10 titer reduction was observed in formulations with less lactose. PEV20 was more prone to inactivation (1.7 log10 titer loss at 12-month) when lactose concentration in the particle was below 80%. The fine particle fraction (% wt. particles <5 μm in aerosol) of phage powders was 52–61% and remained the same after 12-month storage. The results demonstrate that spray dried PEV phage powders containing lactose and leucine are biologically and physically stable over long-term storage at ambient temperature. Furthermore, these spray dried phage powders were shown to be non-toxic to lung alveolar macrophage and epithelial cells in vitro.

Long-Term Antimicrobial Activity of Phage–Sugar Glasses is Closely Tied to the Processing Conditions

Bacteriophage-based antimicrobials have proven to be effective in the control of foodborne contaminants at various stages of the food production chain. Incorporating bacteriophages onto food-processing surfaces and food packaging can significantly impact food safety. One of the major roadblocks toward using phage as a natural antimicrobial/preservative in food packaging is phage sensitivity to desiccation. We recently developed a coating (film) composed of pullulan and trehalose that proved to be highly effective in preserving the antimicrobial activity of phage for more than 8 weeks. During this investigation, we realized that the conditions under which the films were formed and stored significantly affected the protective ability of these films toward bacteriophage antimicrobials, leading us to design a more in-depth investigation to identify and evaluate the major factors that affect the efficacy of these antimicrobial films. In this work, we report the outcome of a four-factor, two-level full-factorial randomized design of experiments, in which we examined the effects of four processing/storage factors on the short-term and long-term antimicrobial activity of phage-embedded pullulan/trehalose films. The drying method used to make the films and the storage humidity are the two factors that have the most significant impact on the long-term antimicrobial activity of pullulan/trehalose. The long-term viability of the bacteriophage (as measured by loss of titer) can be increased by over 1000-fold by using dry vacuum to form the films and then storing (for 28 days at room temperature) them in a closed container (tube) with low humidity in the headspace. The presence of divalent ions during the drying process and modification of the surface onto which the films were cast did not have a significant effect on long-term phage viability. Coatings, such as a pullulan/trehalose film, can help realize the promise of phage in the food industry. Insights in the control and stabilization of active ingredients in pullulan/trehalose films can also be applied to stabilization of viral vaccines and diagnostic bioassays.

Circulating AQP4-specific auto-antibodies alone can induce neuromyelitis optica spectrum disorder in the rat

It is well established that the binding of pathogenic aquaporin-4 (AQP4)-specific autoantibodies to astrocytes may initiate a cascade of events culminating in the destruction of these cells and in the formation of large tissue-destructive lesions typical for patients with neuromyelitis optica spectrum disorders (NMOSD). To date, not a single experimental study has shown that the systemic presence of the antibody alone can induce any damage to the central nervous system (CNS), while pathological studies on brains of NMOSD patients suggested that there might be ways for antibody entry and subsequent tissue damage. Here, we systemically applied a highly pathogenic, monoclonal antibody with high affinity to AQP4 over prolonged period of time to rats, and show that AQP4-abs can enter the CNS on their own, via circumventricular organs and meningeal or parenchymal blood vessels, that these antibodies initiate the formation of radically different lesions with AQP4 loss, depending on their mode and site of entry, and that lesion formation is much more efficient in the presence of encephalitogenic T-cell responses. We further demonstrate that the established tissue-destructive lesions trigger the formation of additional lesions by short and far reaching effects on blood vessels and their branches, and that AQP4-abs have profound effects on the AQP4 expression in peripheral tissues which counter-act possible titer loss by antibody absorption outside the CNS. Cumulatively, these data indicate that directly induced pathological changes caused by AQP4-abs inside and outside the CNS are efficient drivers of disease evolution in seropositive organisms.

Jet nebulization of bacteriophages with different tail morphologies – Structural effects

It was previously demonstrated that the loss of infectivity of a myovirus PEV44 after jet nebulization was closely related to a change in bacteriophage (phage) structure. In this follow-up study, we further examined the impact of jet nebulization on tailed phages, which constitute 96% of all known phages, from three different families, Podoviridae (PEV2), Myoviridae (PEV40) andSiphoviridae (D29). Transmission electron microscopy (TEM) identified major changes in phage structures after jet nebulization, correlating with their loss of infectivity. For the podovirus PEV2, jet nebulization had a negligible impact on its activity (0.04 log10 pfu/mL loss) and structural change. On the other hand, the proportion of intact phages in the nebulized samples dropped from 50% to ∼27% for PEV40 and from 15% to ∼2% for D29. Phage deactivation of PEV40 measured by the TEM structural damage (0.52 log10 pfu/mL) was lower than that obtained by plaque assay (1.02 log10 pfu/mL), but within the range of variation (±0.5 log10 pfu/mL). However, TEM quantification considerably underestimated the titer reduction of D29 phage, ∼2 log pfu/mL lower than that obtained in plaque assay (3.25 log10 pfu/mL loss). In conclusion, nebulization-induced titre loss was correlated with morphological damage to phages and in particular, the tail length may be an important consideration for selection of phages in inhaled therapy using jet nebulization.

Phenotypic signatures and genetic determinants of oxacillin tolerance in a laboratory mutant of Staphylococcus aureus.

Addition of β-lactam antibiotics to growing cultures of bacteria inhibit synthesis of the bacterial cell wall peptidoglycan accompanied by killing (loss of viable titer) and lysis (physical disintegration) of the cells. However, it has also been well established that these antibiotics are not effective in killing non-growing or slow-growing bacteria and the mechanism of this “antibiotic tolerance” is not well understood. In this study, we report on the genetic basis and phenotypic properties of an antibiotic tolerant derivative of the methicillin susceptible S. aureus strain 27s. Cultures were exposed to “pulses” of high concentrations of oxacillin followed by outgrowth of the surviving bacteria. This procedure quickly selected for antibiotic tolerant mutants with an increased ability to survive antibiotic treatment without increase in the MIC value for the antibiotic. Such mutants also exhibited longer lag phase, decreased lysis, virtually no change in antibiotic susceptibilities, cross tolerance to D-cycloserine and vancomycin, and increase in biofilm formation in the presence of high concentrations of oxacillin. Whole genome sequencing showed that these altered properties were linked to mutations in the atl and gdpP genes.

Manufacturing and ambient stability of shelf freeze dried bacteriophage powder formulations

The severity of multidrug resistance to antibiotics has urged development of alternative treatment approaches, including bacteriophage therapy. Given the complexity of the bacteriophage structure, formulation and stability are primary concerns. Our present work optimized process and formulations of phage powder manufacturing and investigated the stability of lyophilized bacteriophage powders under ambient storage. The model phage M13 was formulated with trehalose, mannitol, sucrose and PEG6000 and lyophilized in different conditions. Bacteriophage viability was examined by titering and was considered as the assessment of phage stability. Less titer loss of trehalose and sucrose formulations were observed compared to mannitol and PEG groups both immediately after lyophilization and upon long term storage. When evaluating lyophilization conditions, an additional 1 log titer was preserved by reduction of product drying stress. Trehalose was stabilized in the amorphous state whereas mannitol stayed in crystalline state in lyophilized powders. Increased moisture content was demonstrated to have a positive impact on viability of phage after lyophilization and upon storage. Overall, 2% trehalose or sucrose (w/v) can sufficiently stabilize phage during lyophilization process and storage in ambient conditions. There is a positive correlation between residual water and stability of phage. These collective findings highlight the potential of long-term, ambient storage of bacteriophage towards their successful use in diverse healthcare settings.