Discovery of new antigens for serodiagnosis of visceral leishmaniasis in humans and dogs adds valuable tools to our arsenal.

Surveillance and sustained control of visceral leishmaniasis (VL) require reliable serodiagnostic tools. rK39, the gold standard antigen for VL diagnosis, is limited by its documented poor sensitivity in certain endemic regions, such as East Africa, and by the longevity of its antibodies, making it difficult to distinguish active from cured infections. In a recent publication in mBio, Roberts et al. (A. J. Roberts, H.B. Ong, S. Clare, C. Brandt, et al., mBio 15:e00859-24, 2024, https://doi.org/10.1128/mbio.00859-24) identified new immunogenic Leishmania candidates in dogs and humans. In dogs, combined antigens LdBPK_290790.1 + LdBPK_362700.1 (D4 +D46) distinguished symptomatic from asymptomatic infections. For humans, LdBPK_323600.1 (D36) antigen produced short-lived antibodies and performed well in patient cohorts from Bangladesh and Ethiopia, but not Kenya. This study adds promising new candidates to our serodiagnostic toolbox but highlights the need for more antigen discovery studies that may have to be focused on regional performance.

Protein Production at the 100L Scale.

The Baculovirus Expression Vector System (BEVS) has revolutionized the field of recombinant protein expression by enabling efficient and high yield production. The platform offers many advantages including manufacturing speed, flexible design, and scalability. In this chapter, we describe the methods including strategies and considerations to successfully optimize and scale-up using BEVS as a tool for production (Fig. 1). As an illustrative case study, we present an example focused on the production of a viral glycoprotein.

TCID50 Assay: A Simple Method to Determine Baculovirus Titer.

There are many methods that can be used to determine the infectious titer of your baculovirus stock. The TCID50 method is a simple end-point dilution method that determines the amount of baculovirus virus needed to produce a cytopathic effect or kill 50% of inoculated insect cells. Serial dilutions of baculovirus stock are added to Sf9 cells cultivated in 96-well plates and 3-5 days after infection, cells are monitored for cell death or cytopathic effect. The titer can then be calculated by the Reed-Muench method as described in this method.

Deciphering the antiviral nature of endophytic Bacillus spp. against groundnut bud necrosis virus in cowpea and tomato.

Tomato, the important vegetable crop, is severely affected by Orthotospovirus arachinecrosis which impacts heavy economic losses. The application of insecticide to manage viral diseases is not an environmentally safe approach. In view of these issues, we investigated the antiviral efficacy of 21 bacterial endophytes against GBNV in local lesion host (Cowpea-VBN3). Based on the reduction in lesion number and virus titer as estimated through both DAC ELISA and qPCR in cowpea, the bacterial endophytes viz., Bacillus licheniformis Soya1, Bacillus tequilensis NBL6, and Bacillus velezensis VB7 were selected and further tested in tomato. The study revealed the well-defined antiviral efficacy of these endophytes against GBNV. The percentage of disease incidence ranged from 16 to 24% in endophyte-treated tomato plants compared with untreated plants (88%). In addition, symptom severity was reduced, and the application of endophytes also in promotion of the growth compared with untreated control. DAC ELISA revealed that the tomato plants treated with bacterial endophytes challenged with GBNV showed reduction in the virus titer (0.26-0.39 @ OD 405 nm) at different days of interval after inoculation (0, 5, and 10 days) compared with untreated control (3.475 @ OD 405 nm). Additionally, reduction in the viral copy number in bacterial endophyte-treated plants was evident by real-time PCR. Furthermore, tomato plants bacterized with endophytes depicted significant correlation and reduction in viral load and disease incidence as revealed by the principal-component biplot analysis. Thus, the application of bacterial endophytes has a potential role in reducing the disease incidence, severity, and titer value of GBNV, which will be the promising management approach in future to mitigate the virus infection in tomato plants.

Median Tissue Culture Infectious Dose 50 (TCID50) Assay to Determine Infectivity of Cytopathic Viruses.

The emergence of zoonotic viruses like severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 have significantly impacted global health and economy. The discovery of other viruses in wildlife reservoir species present a threat for future emergence in humans and animals. Therefore, assays that are less reliant on virus-specific information, such as neutralization assays, are crucial to rapidly develop diagnostics, understand virus replication and pathogenicity, and assess the efficacy of therapeutics against newly emerging viruses. Here, we describe the discontinuous median tissue culture infectious dose 50 (TCID50) assay to quantitatively determine the titer of any virus that can produce a visible cytopathic effect in infected cells.

'Small volume-big problem': culturing Yarrowia lipolytica in high-throughput micro-formats.

With the current progress in the 'design' and 'build' stages of the 'design-build-test-learn' cycle, many synthetic biology projects become 'test-limited'. Advances in the parallelization of microbes cultivations are of great aid, however, for many species down-scaling leaves a metabolic footprint. Yarrowia lipolytica is one such demanding yeast species, for which scaling-down inevitably leads to perturbations in phenotype development. Strictly aerobic metabolism, propensity for filamentation and adhesion to hydrophobic surfaces, spontaneous flocculation, and high acidification of media are just several characteristics that make the transfer of the micro-scale protocols developed for the other microbial species very challenging in this case. It is well recognized that without additional 'personalized' optimization, either MTP-based or single-cell-based protocols are useless for accurate studies of Y. lipolytica phenotypes. This review summarizes the progress in the scaling-down and parallelization of Y. lipolytica cultures, highlighting the challenges that occur most frequently and strategies for their overcoming. The problem of Y. lipolytica cultures down-scaling is illustrated by calculating the costs of micro-cultivations, and determining the unintentionally introduced, thus uncontrolled, variables. The key research into culturing Y. lipolytica in various MTP formats and micro- and pico-bioreactors is discussed. Own recently developed and carefully pre-optimized high-throughput cultivation protocol is presented, alongside the details from the optimization stage. We hope that this work will serve as a practical guide for those working with Y. lipolytica high-throughput screens.

Anti-A and anti-B titers in A, B and O whole blood donors: Beyond "dangerous O".

BACKGROUND AND OBJECTIVES: Hemolytic transfusion reactions (HTRs) pose significant risks in transfused patients, with anti-A and anti-B antibodies in donor plasma being potential contributing factors. Despite advancements in component preparation, HTRs remain a concern, particularly with apheresis-derived platelets. This study aimed to determine the prevalence of high anti-A and anti-B titers among A, B, and O blood group donors and to explore factors associated with high titers. MATERIALS AND METHODS: A cross-sectional observational study was conducted over 18 months, enrolling 978 participants from a tertiary care teaching hospital in Western India. Anti-A and anti-B titers were determined using the Conventional Tube Technique (CTT). Statistical analysis assessed correlations between high titers and demographic factors. RESULTS: The majority of participants were young males (98.8%). Prevalence of high titers for IgM anti-A was 12.2% and IgG anti-A was 2.5%. For anti-B, IgM titers were 2.3% and IgG titers were 0.2%. The prevalence of dangerous O was found to be 14.1%, while 3.52% and 10.5% of A and B blood group donors were found to have high titers, respectively. Factors associated with high titers included female gender, vegetarian diet, age <30 years, and O blood group. CONCLUSION: The study sheds additional light and provides supplementary information regarding the prevalence and correlation of high anti-A and anti-B titers among O, A and B blood donors. Understanding these factors is crucial for optimizing transfusion safety protocols, including selective screening of platelet units and tailored transfusion strategies based on donor characteristics.

Low-dose immune tolerance induction for severe hemophilia A inhibitor patients: Immunosuppressants are generally not necessary for inhibitor-titer below 200 BU/mL.

Importance: It remained unclear that the efficacy comparison between low-dose immune tolerance induction (LD-ITI) incorporating immunosuppressants (IS) when severe hemophilia A (SHA) patients had inhibitor-titer ≥200 Bethesda Units (BU)/mL (LD-ITI-IS200 regimen) and LD-ITI combining with IS when SHA patients had inhibitor-titer ≥40 BU/mL (LD-ITI-IS40 regimen). Objective: To compare the efficacy of the LD-ITI-IS200 regimen with that of the LD-ITI-IS40 regimen for SHA patients with high-titer inhibitors. Methods: A prospective cohort study on patients receiving LD-ITI-IS200 compared to those receiving LD-ITI-IS40 from January 2021 to December 2023. Both received LD-ITI [FVIII 50 IU/kg every other day]. IS (rituximab + prednisone) was added when peak inhibitor tier ≥200 BU/mL in the LD-ITI-IS200 regimen and ≥40 BU/mL in the LD-ITI-IS40 regimen. Success is defined as a negative inhibitor plus FVIII recovery ≥66% of the expected. Results: We enrolled 30 patients on LD-ITI-IS200 and 64 patients on LD-ITI-IS40, with similar baseline clinical characteristics. A lower IS-use rate was discovered in the LD-ITI-IS200 regimen compared to the LD-ITI-IS40 regimen (30.0% vs. 62.5%). The two regimens (LD-ITI-IS200 vs. LD-ITI-IS40) had similar success rate (70.0% vs. 79.7%), median time to success (9.4 vs. 10.6 months), and annualized bleeding rate during ITI (3.7 vs. 2.8). The cost to success was lower for LD-ITI-IS200 than for LD-ITI-IS40 (2107 vs. 3256 US Dollar/kg). Among patients with peak inhibitor-titer 40-199 BU/mL, 10 non-IS-using (on LD-ITI-IS200 regimen) and 28 IS-using (on LD-ITI-IS40 regimen) had similar success rates (70.0% vs. 78.6%) and time to success (9.0 vs. 8.8 months). Interpretation: In LD-ITI, IS are not necessary for inhibitor titer <200 BU/mL.

Long Prime-Boost Interval and Heightened Anti-GD2 Antibody Response to Carbohydrate Cancer Vaccine.

The carbohydrate ganglioside GD2/GD3 cancer vaccine adjuvanted by ß-glucan stimulates anti-GD2 IgG1 antibodies that strongly correlate with improved progression-free survival (PFS) and overall survival (OS) among patients with high-risk neuroblastoma. Thirty-two patients who relapsed on the vaccine (first enrollment) were re-treated on the same vaccine protocol (re-enrollment). Titers during the first enrollment peaked by week 32 at 751 ± 270 ng/mL, which plateaued despite vaccine boosts at 1.2-4.5 month intervals. After a median wash-out interval of 16.1 months from the last vaccine dose during the first enrollment to the first vaccine dose during re-enrollment, the anti-GD2 IgG1 antibody rose to a peak of 4066 ± 813 ng/mL by week 3 following re-enrollment (p < 0.0001 by the Wilcoxon matched-pairs signed-rank test). Yet, these peaks dropped sharply and continually despite repeated boosts at 1.2-4.5 month intervals, before leveling off by week 20 to the first enrollment peak levels. Despite higher antibody titers, patients experienced no pain or neuropathic side effects, which were typically associated with immunotherapy using monoclonal anti-GD2 antibodies. By the Kaplan-Meier method, PFS was estimated to be 51%, and OS was 81%. The association between IgG1 titer during re-enrollment and ß-glucan receptor dectin-1 SNP rs3901533 was significant (p = 0.01). A longer prime-boost interval could significantly improve antibody responses in patients treated with ganglioside conjugate cancer vaccines.

A case study application of AQbD to the re-development and validation of an affinity chromatography analytical procedure for mAb titer quantitation.

Protein A (ProA) high-performance liquid chromatography (HPLC) is a common analytical procedure for measuring monoclonal antibody (mAb) titers due to its high specificity and efficiency. Accurate and reliable results of this procedure are imperative, as the quantitation of the total mAb present for in-process samples directly impacts downstream purification steps related to the removal of process-related impurities. This study aimed to improve a platform ProA HPLC analytical procedure which was previously developed using traditional approaches and was not always reliable. By retrospectively applying Analytical Quality by Design (AQbD) principles and statistical assessments of performance, a bias in the calibration standard due to protein-adsorption to common sample vial materials was identified. The inclusion of Tween® 20 into the mobile phase used as sample diluent was optimized to ensure procedure performance and improve analytical range. The resulting procedure robustness was evaluated using Design of Experiment (DoE) approaches and performance was verified against Analytical Target Profile (ATP) criteria as recommended by regulatory agencies. The resulting linearity displayed R2 values of 1.00 with intercept biases of 1.2 % (analyst 1) and 0.8 % (analyst 2), accuracy across all levels was reported at 99.2 % recovery, and intermediate precision was reported as 3.0 % RSD. Application of this new platform procedure has since reduced development timelines for new mAb products by 50 % and allowed for accurate titer determination to support >5 early phase product-specific process decisions without requiring extensive analytical procedure development. This work demonstrates the utility and relative ease of adopting AQbD concepts, even for established procedures, and supporting them with a lifecycle approach to managing procedure performance.

Correlation between severe attacks and serum aquaporin-4 antibody titer in neuromyelitis optica spectrum disorder.

Aquaporin 4-immunoglobulin G (AQP4-IgG) specifically targets aquaporin 4 in approximately 80% of Neuromyelitis Optica Spectrum Disorder (NMOSD) cases. NMOSD is presently categorized as anti-AQP4-antibody (Ab) positive or negative based on AQP4-Ab presence. The association between antibody titers and patient prognosis remains unclear. Therefore, the present study explores the correlation between severe attacks and serum AQP4 Ab titers in patients with neuromyelitis optica spectrum disorder. Data were gathered retrospectively from 546 patients with NMOSD between September 1, 2009, and December 1, 2021. Patients were categorized based on their AQP4-Ab titers: AQP4 titer ≥ 1:320 were classified as the high-titer group, AQP4 (+ +), and AQP4 titer of ≤ 1:100 were classified as the low-titer group, AQP4 ( +). Clinical characteristics and prognoses between the two groups were compared. Patients with AQP4 ( +) exhibited few severe optic neuritis (SON) attacks (false discovery rate [FDR] corrected p < 0.001), a reduced percentage experiencing SON attacks, and a lower incidence of visual disability than patients with AQP4 (+ +). Patients with AQP4 (+ +) and AQP4 ( +) NMOSD exhibited significant difference in annual recurrence rate (ARR) (FDR-corrected p < 0.001). The lower AQP4 Ab titer group demonstrated reduced susceptibility to severe relapse with conventional immunosuppressive agents and rituximab (RTX) than the higher titer group. No significant differences in sex, age at onset, coexisting connective tissue diseases, motor disability, or mortality rates were observed between the two groups. Higher AQP4 Ab titers correlated with increased disease severity and visual disability in patients with NMOSD.

Effect of host and strain factors on α-synuclein prion pathogenesis.

Prion diseases are a group of neurodegenerative disorders caused by misfolding of proteins into pathogenic conformations that self-template to spread disease. Although this mechanism is largely associated with the prion protein (PrP) in classical prion diseases, a growing literature indicates that other proteins, including α-synuclein, rely on a similar disease mechanism. Notably, α-synuclein misfolds into distinct conformations, or strains, that cause discrete clinical disorders including multiple system atrophy (MSA) and Parkinson's disease (PD). Because the recognized similarities between PrP and α-synuclein are increasing, this review article draws from research on PrP to identify the host and strain factors that impact disease pathogenesis, predominantly in rodent models, and focuses on key considerations for future research on α-synuclein prions.

Advanced Critical Care Techniques in the Field.

Critical care principles and techniques continue to hold promise for improving patient outcomes in time-dependent diseases encountered by emergency medical services such as cardiac arrest, acute ischemic stroke, and hemorrhagic shock. In this review, the authors discuss several current and evolving advanced critical care modalities, including extracorporeal cardiopulmonary resuscitation, resuscitative endovascular occlusion of the aorta, prehospital thrombolytics for acute ischemic stroke, and low-titer group O whole blood for trauma patients. Two important critical care monitoring technologies-capnography and ultrasound-are also briefly discussed.

Assessment of Key Factors Impacting Variability in AAV Vector Genome Titration by Digital PCR.

Recombinant adeno-associated virus (rAAV) has emerged as a prominent vector for in vivo gene therapy, owing to its distinct advantages. Accurate determination of the rAAV genome titer is crucial for ensuring the safe and effective administration of clinical doses. The evolution of the rAAV genome titer assay from quantitative PCR (qPCR) to digital PCR (dPCR) has enhanced accuracy and precision, yet practical challenges persist. This study systematically investigated the impact of various operational factors on genome titration in a single-factor manner, aiming to address potential sources of variability in the quantitative determination process. Our findings revealed that a pretreatment procedure without genome extraction exhibits superior precision compared with titration with genome extraction. Additionally, notable variations in titration results across different brands of dPCR instruments were documented, with relative standard deviation (RSD) reaching 23.47% for AAV5 and 11.57% for AAV8. Notably, optimal operations about DNase I digestion were identified; we thought treatment time exceeding 30 min was necessary, and there was no need for thermal inactivation after digestion. And we highlighted that thermal capsid disruption before serial dilution substantially affected AAV genome titers, causing a greater than ten-fold decrease. Conversely, this study found that additive components of dilution buffer are not significant contributors to titration variations. Furthermore, we found that repeated freeze-thaw cycles significantly compromised AAV genome titers. In conclusion, a comprehensive dPCR titration protocol, incorporating insights from these impact factors, was proposed and successfully tested across multiple serotypes of AAV. The results demonstrate acceptable variations, with the RSD consistently below 5.00% for all tested AAV samples. This study provides valuable insights to reduce variability and improve the reproducibility of AAV genome titration using dPCR.

Breast milk-mediated exposure to dioxins and antigen in infancy enhances antigen-specific antibody production capacity in adulthood in mice.

The immune system is sensitive to many chemicals. Among dioxin compounds, 2,3,7,8-tetrachlorodizenzo-p-dioxin (TCDD) is the most toxic environmental pollutant. The effects of perinatal maternal exposure to dioxins may persist into childhood. However, there have been no reports to date on the effects of exposure to dioxins during infancy, when the immune organs are developing. Therefore, we investigated the effects of TCDD and antigen exposure during lactation on immune function, especially antibody production capacity, in adult mice. Beginning the day after delivery, lactating mothers were orally administered TCDD or a mixture of TCDD and ovalbumin (OVA) daily for 4 weeks, until the pups were weaned. At 6 weeks of age, progeny mice were orally administered OVA daily for 10 weeks, while non-progeny mice were orally administered OVA or a mixture of TCDD and OVA daily for 10 weeks. Production of serum OVA-specific IgG was examined weekly. The amount of TCDD transferred from the mother to the progeny via breast milk was determined by measuring TCDD in the gastric contents of the progeny. A trend toward increasing IgA titer was observed in TCDD-treated mice, and production of IgE was observed only in progeny whose mothers were treated with TCDD and OVA. The results suggest that exposure to TCDD and OVA in breast milk can affect immune function in newborns.

Combinatorial metabolic engineering of Streptomyces sp. CB03234-S for the enhanced production of anthraquinone-fused enediyne tiancimycins.

BACKGROUND: Anthraquinone-fused enediynes (AFEs) are excellent payloads for antibody-drug conjugates (ADCs). The yields of AFEs in the original bacterial hosts are extremely low. Multiple traditional methods had been adopted to enhance the production of the AFEs. Despite these efforts, the production titers of these compounds are still low, presenting a practical challenge for their development. Tiancimycins (TNMs) are a class of AFEs produced by Streptomyces sp. CB03234. One of their salient features is that they exhibit rapid and complete cell killing ability against various cancer cell lines. RESULTS: In this study, a combinatorial metabolic engineering strategy guided by the CB03234-S genome and transcriptome was employed to improve the titers of TNMs. First, re-sequencing of CB03234-S (Ribosome engineered mutant strains) genome revealed the deletion of a 583-kb DNA fragment, accounting for about 7.5% of its genome. Second, by individual or combined inactivation of seven potential precursor competitive biosynthetic gene clusters (BGCs) in CB03234-S, a double-BGC inactivation mutant, S1009, was identified with an improved TNMs titer of 28.2 ± 0.8 mg/L. Third, overexpression of five essential biosynthetic genes, including two post-modification genes, and three self-resistance auxiliary genes, was also conducted, through which we discovered that mutants carrying the core genes, tnmE or tnmE10, exhibited enhanced TNMs production. The average TNMs yield reached 43.5 ± 2.4 mg/L in a 30-L fermenter, representing an approximately 360% increase over CB03234-S and the highest titer among all AFEs to date. Moreover, the resulting mutant produced TNM-W, a unique TNM derivative with a double bond instead of a common ethylene oxide moiety. Preliminary studies suggested that TNM-W was probably converted from TNM-A by both TnmE and TnmE10. CONCLUSIONS: Based on the genome and transcriptome analyses, we adopted a combined metabolic engineering strategy for precursor enrichment and biosynthetic pathway reorganization to construct a high-yield strain of TNMs based on CB03234-S. Our study establishes a solid basis for the clinical development of AFE-based ADCs.

Relationship of various COVID-19 antibody titer with individual characteristics and prediction of future epidemic trend in Xiamen City, China.

Background: Reinfection of coronavirus disease 2019 (COVID-19) has raised concerns about how reliable immunity from infection and vaccination is. With mass testing for the virus halted, understanding the current prevalence of COVID-19 is crucial. This study investigated 1,191 public health workers at the Xiamen Center for Disease Control, focusing on changes in antibody titers and their relationship with individual characteristics. Methods: The study began by describing the epidemiological characteristics of the study participants. Multilinear regression (MLR) models were employed to explore the associations between individual attributes and antibody titers. Additionally, group-based trajectory models (GBTMs) were utilized to identify trajectories in antibody titer changes. To predict and simulate future epidemic trends and examine the correlation of antibody decay with epidemics, a high-dimensional transmission dynamics model was constructed. Results: Analysis of epidemiological characteristics revealed significant differences in vaccination status between infected and non-infected groups (χ2=376.706, P<0.05). However, the distribution of antibody titers among the infected and vaccinated populations was not significantly different. The MLR model identified age as a common factor affecting titers of immunoglobulin G (IgG), immunoglobulin M (IgM), and neutralizing antibody (NAb), while other factors showed varying impacts. History of pulmonary disease and hospitalization influenced IgG titer, and factors such as gender, smoking, family history of pulmonary diseases, and hospitalization impacted NAb titers. Age was the sole determinant of IgM titers in this study. GBTM analysis indicated a "gradual decline type" trajectory for IgG (95.65%), while IgM and NAb titers remained stable over the study period. The high-dimensional transmission dynamics model predicted and simulated peak epidemic periods in Xiamen City, which correlated with IgG decay. Age-group-specific simulations revealed a higher incidence and infection rate among individuals aged 30-39 years during both the second and third peaks, followed by those aged 40-49, 50-59, 18-29, and 70-79 years. Conclusions: Our study shows that antibody titer could be influenced by age, previous pulmonary diseases as well as smoking. Furthermore, the decline in IgG titers is consistent with epidemic trends. These findings emphasize the need for further exploration of these factors and the development of optimized self-protection countermeasures against reinfection.

The oviposition preference and offspring performance of Aethina tumida (Coleoptera: Nitidulidae).

Given the rapid spread and potential harm caused by the small hive beetle, Aethina tumida (Coleoptera: Nitidulidae) in China, it has become imperative to comprehend the developmental biology of this invasive species. Currently, there is limited knowledge regarding the impact of A. tumida female oviposition site preference on larval growth and development. To examine this, we investigated the ovipositional preference of adult female A. tumida on bee pupae, beebread, banana, and honey through a free choice test. Furthermore, we assessed the impact of these food resources on offspring performance, which included larval development time, survival, wandering larvae weight, emerged adult body mass, reproduction, and juvenile hormone titer. Our results showed that A. tumida females exhibited a strong preference for ovipositing on bee pupae compared to other diets, while showing reluctance toward honey. Moreover, A. tumida larvae that were fed on bee pupae displayed accelerated growth compared to those fed on other diets. Furthermore, A. tumida fed on bee pupae exhibited higher weights for wandering larvae, and emerged adult, increased pupation rates, enhanced fecundity and fertility, as well as a larger number of unilateral ovarioles during the larval stage when compared to those fed on other diets. Overall, the results indicate that the oviposition preferences of A. tumida females are adaptive, as their choices can enhance the fitness of their offspring. This finding aligns broadly with the hypothesis of oviposition preference and larval performance. This study can provide a foundation for the development of attractants aimed at promoting the oviposition of the A. tumida adults.

Cross-validation of chemical and genetic disruption approaches to inform host cellular effects on Wolbachia abundance in Drosophila.

Introduction: Endosymbiotic Wolbachia bacteria are widespread in nature, present in half of all insect species. The success of Wolbachia is supported by a commensal lifestyle. Unlike bacterial pathogens that overreplicate and harm host cells, Wolbachia infections have a relatively innocuous intracellular lifestyle. This raises important questions about how Wolbachia infection is regulated. Little is known about how Wolbachia abundance is controlled at an organismal scale. Methods: This study demonstrates methodology for rigorous identification of cellular processes that affect whole-body Wolbachia abundance, as indicated by absolute counts of the Wolbachia surface protein (wsp) gene. Results: Candidate pathways, associated with well-described infection scenarios, were identified. Wolbachia-infected fruit flies were exposed to small molecule inhibitors known for targeting those same pathways. Sequential tests in D. melanogaster and D. simulans yielded a subset of chemical inhibitors that significantly affected whole-body Wolbachia abundance, including the Wnt pathway disruptor, IWR-1 and the mTOR pathway inhibitor, Rapamycin. The implicated pathways were genetically retested for effects in D. melanogaster, using inducible RNAi expression driven by constitutive as well as chemically-induced somatic GAL4 expression. Genetic disruptions of armadillo, tor, and ATG6 significantly affected whole-body Wolbachia abundance. Discussion: As such, the data corroborate reagent targeting and pathway relevance to whole-body Wolbachia infection. The results also implicate Wnt and mTOR regulation of autophagy as important for regulation of Wolbachia titer.

Neutralization of Rubella Vaccine Virus and Immunodeficiency-Related Vaccine-Derived Rubella Viruses by Intravenous Immunoglobulins.

The association between granulomas and vaccine-derived rubella virus (VDRV) in people with primary immune deficiencies (PID) has raised concerns about the ability of immunoglobulin (IG) preparations to neutralize VDRVs. We investigated the capacity of IG to neutralize rubella vaccine virus and four VDRV strains. As expected, the rubella vaccine virus itself was potently neutralized by IG preparations; however, the VDRV isolates from patients after intra-host evolution, 2-6 times less so. Diagnosis of immune deficiencies before possible live-virus vaccination is thus of critical importance, while IG replacement therapy can be expected to provide protection from rubella virus infection.

The occurrence of granulomas associated with vaccine derived rubella viruses (VDRV) in people with primary immune deficiencies (PID) challenges immunoglobulin (IG) preparations regarding their rubella neutralizing ability. This study confirmed potent rubella virus neutralization capacity of IG preparations and thus suggests protection of IG-treated PID patients against rubella. The study also highlights the importance of early diagnosis and timely given IG to prevent possible systemic spread of VDRV persisting locally in granulomas.