Drop In Titer Research Articles

Antigen Titers in Cryptococcal Meningitis: What Determines How Fast They Fall?

Follow-up of previously healthy patients surviving cryptococcal meningitis found that cryptococcal antigen could be detected for >1 year in serum from 38 of 44 (86%) patients and in cerebrospinal fluid (CSF) from 20 of 31 patients (67%), far beyond the time of culture conversion. The speed of titer decline, measured as the number of days for a 2-fold drop in titer to occur, was slower in serum than in CSF. The speed of decline of antigen titers was much slower in serum and CSF for patients infected with Cryptococcus gattii than Cryptococcus neoformans. The speed of decline in CSF and serum titers was also much slower in patients who had received a ventriculoperitoneal shunt for increased intracranial pressure. The variable and extraordinarily slow rate of clearance in our patients did not appear to reflect differences in disease control but rather differences in species and shunting for increased intracranial pressure.

Obtaining and Characterization of Experimental Diagnostic Brucellosis Rabbit Sera for the Development of a National Standard for Brucellosis Serum

Relevance. The production of brucellosis diagnosticums is currently being established in the medical and veterinary practice of the Republic of Uzbekistan. However, due to the lack of a national standard serum in the country and the high cost of analog standard serums on the market, problems may arise in the production of diagnostic tools. In addition, the lack of national standard serums causes problems in quality control of commercial and local antigens, hinders the development of local drugs – brucellosis diagnostics, which reduces the effectiveness of serological diagnosis of brucellosis in humans and animals. Given the above, it becomes necessary to develop and produce a standard serum with a high level of safety, which will improve the methods of laboratory diagnosis of various forms of brucellosis in patients and animals to identify hidden foci of infection in Uzbekistan. The aim of the study is to obtain and characterize experimental standard diagnostic brucellosis rabbit sera for the development of national and industry standard serum with appropriate titers. Materials and Methods. The experiments were carried out in full compliance with the European Council Directive 2010/63/EU on compliance with ethical principles in working with laboratory animals and the experiments were carried out on the basis of the methodological manual «Methods and rules of work» approved by the Ministry of Health of the Republic of Uzbekistan in 2016 with laboratory animals in experimental microbiological and immunological studies. To obtain positive standard sera against brucellosis pathogens, 12 rabbits weighing from 2.2 kg to 4.6 kg, aged 6 to 12 months, were used. The animals were kept under identical conditions on a standard vivarium diet. For the experiment, animals adapted to the conditions of the study were selected and kept in quarantine for 21 days. The maintenance and conduct of the studies followed international rules for the humane treatment of animals. Results and Discussion. During the evaluation of the heterologic specificity of the obtained sera for 23 collection and hospital strains, a weakly positive response (+) was observed in 16.7% of cases. Rabbit sera positive for heterologic specificity were adsorbed on suspensions of inactivated microorganisms (Citrobacter spp. and Enterobacteriaceae spp.) - no significant drop in titer was observed, and cross-agglutination was eliminated. A significant increase in the total protein level was noted after the first and fourth immunizations. Before the first immunization, the total protein level was 59.57 g/l, and after the fourth immunization, it increased threefold to 157.17 g/l. There was also an increase in albumin and globulin (33.2 g/L and 26.37 g/L, respectively) to 79.37 g/L and 77.43 g/L, respectively. IgM levels remained almost unchanged, increasing 3.5-fold after the second immunization and decreasing to 0.24 mg/ml after the fourth immunization. An increase in the level of IgG after the second and third immunizations was revealed, followed by a decrease after the fourth immunization. Conclusion. The obtained national and industry standard diagnostic polyvalent brucellosis sera, as well as the developed methods of laboratory control of its quality and efficiency testing will make it possible to produce candidate standard serum samples for agglutination reaction test and detection of Brucella strains. The obtained standard serum samples will be further used for control of the produced immunobiological diagnostic preparations and provision of unified requirements to their quality. The goal was achieved – improving the serological diagnosis of brucellosis, laying the foundation for further study and standardization of serum.

Pseudomonas aeruginosa ventricular assist device infections: findings from ineffective phage therapies in five cases.

Left ventricular assist devices (LVAD) are increasingly used for management of heart failure; infection remains a frequent complication. Phage therapy has been successful in a variety of antibiotic refractory infections and is of interest in treating LVAD infections. We performed a retrospective review of four patients that underwent five separate courses of intravenous (IV) phage therapy with concomitant antibiotic for treatment of endovascular Pseudomonas aeruginosa LVAD infection. We assessed phage susceptibility, bacterial strain sequencing, serum neutralization, biofilm activity, and shelf-life of phage preparations. Five treatments of one to four wild-type virulent phage(s) were administered for 14-51 days after informed consent and regulatory approval. There was no successful outcome. Breakthrough bacteremia occurred in four of five treatments. Two patients died from the underlying infection. We noted a variable decline in phage susceptibility following three of five treatments, four of four tested developed serum neutralization, and prophage presence was confirmed in isolates of two tested patients. Two phage preparations showed an initial titer drop. Phage biofilm activity was confirmed in two. Phage susceptibility alone was not predictive of clinical efficacy in P. aeruginosa endovascular LVAD infection. IV phage was associated with serum neutralization in most cases though lack of clinical effect may be multifactorial including presence of multiple bacterial isolates with varying phage susceptibility, presence of prophages, decline in phage titers, and possible lack of biofilm activity. Breakthrough bacteremia occurred frequently (while the organism remained susceptible to administered phage) and is an important safety consideration.

Durability of immune responses to mRNA booster vaccination against COVID-19.

BackgroundMaintaining durable immunity following vaccination represents a major challenge, but whether mRNA booster vaccination improves durability is unknown.MethodsWe measured antibody responses in 55 healthy adults, who received a booster dose of the Pfizer-BioNTech or Moderna vaccine against SARS-CoV-2 and calculated the half-life of the antibody titers. We also measured memory B and T cell responses in a subset of 28 participants. In 13 volunteers who received a second booster vaccine, we measured serum antibody titers and memory B and T cell responses.ResultsThe booster (third immunization) dose at 6 to 10 months increased the half-life of the serum-neutralizing antibody (nAb) titers to 76 days from 56 to 66 days after the primary 2-dose vaccination. A second booster dose (fourth immunization) a year after the primary vaccination further increased the half-life to 88 days. However, despite this modestly improved durability in nAb responses against the ancestral (WA.1) strain, there was a loss of neutralization capacity against the Omicron subvariants BA.2.75.2, BQ.1.1, and XBB.1.5 (48-, 71-, and 66-fold drop in titers, respectively, relative to the WA.1 strain). Although only 45% to 65% of participants demonstrated a detectable nAb titer against the newer variants after the booster (third dose), the response declined to below the detection limit in almost all individuals by 6 months. In contrast, booster vaccination induced antigen-specific memory B and T cells that persisted for at least 6 months.ConclusionThe durability of serum antibody responses improves only marginally following booster immunizations with the Pfizer-BioNTech or Moderna mRNA vaccines.

The role of autoantibodies in myositis associated interstitial lung disease

Myositis-associated interstitial lung disease (M-ILD) is a heterogeneous group of diseases. It can be divided into different subtypes listed below according to different myositis antibodies. (1) ASS-related ILD responds well to glucocorticoid plus immunosuppressant therapy but tends to relapse. (2) Anti-MDA5-related ILD is the worst prognosis of M-ILD with high mortality rate, and most patients die within the first 6 months of the disease course. (3) Non-MDA-5 DM-associated ILD is often mild, and NSIP is the main HRCT pattern. (4) IMNM-associated ILD shows mild clinical symptoms of ILD in majority of the case, and NSIP is also the main HRCT pattern. (5) With MAA-associated ILD, anti-Ku-positive patients are more common complicated with ILD. In addition, the changes of serum levels of myositis antibody are generally positively correlated with disease activity, and titer drop after disease remission. Therefore, myositis antibodies are important immunological biomarkers for clinical typing and prognosis of M-ILD.

Viability evaluation of freeze dried and suspension anthrax spore vaccine formulations stored at different temperatures

Anthrax is endemic in Ethiopia with sporadic outbreaks despite the regular vaccination of domestic livestock. This has raised concerns on the effectiveness of the vaccination strategy which may be associated with breaches in the vaccine cold chain maintenance. This study was aimed at demonstrating the tolerance of anthrax vaccine to cold chain breaches through evaluation of viable spore counts expressed as colony forming units per mL (CFU/mL) of freeze-dried and suspension anthrax vaccines stored at 5 °C, 20 °C and 37 °C for up to 6 months. Both vaccine formulations maintained above the recommended minimum required titre (2 × 106 culturable spores per dose for cattle, buffaloes and horses, and not <1 × 106 for sheep and goats) for up to 6 months at 5 °C storage. In storage at 20 °C, the viability of freeze-dried anthrax vaccine maintained the minimum required titre up to 6 months while up to 90 days in case of the suspension formulation. Both types of vaccine formulations maintained the minimum titre per dose for up to 30 days at 37 °C storage. Generally, both vaccine formulations showed similar trends in titre fall in all of the three storage temperatures (5 °C, 20 °C and 37 °C) as observed in the almost linearly overlapping 95% confidence intervals (CI) up to day 90 at 5 °C and 20 °C storages while up to day 30 at 37 °C storage. However, a significant (P < 0.05) drop in titre was observed after day 90 for storages at 5 °C and 20 °C, and after day 30 for 37 °C storage as observed in the non overlapping 95% CI from the average titres of previous time points. This study showed that if temperature excursion occurs above the recommended temperature range (4–8 °C) during storage or transport, the vaccine should remain effective and can still be used in vaccination programs.

SY6-2. Possible role of statin to prevent preeclampsia

Anthrax is endemic in Ethiopia with sporadic outbreaks despite the regular vaccination of domestic livestock. This has raised concerns on the effectiveness of the vaccination strategy which may be associated with breaches in the vaccine cold chain maintenance. This study was aimed at demonstrating the tolerance of anthrax vaccine to cold chain breaches through evaluation of viable spore counts expressed as colony forming units per mL (CFU/mL) of freeze-dried and suspension anthrax vaccines stored at 5 °C, 20 °C and 37 °C for up to 6 months. Both vaccine formulations maintained above the recommended minimum required titre (2 × 106 culturable spores per dose for cattle, buffaloes and horses, and not <1 × 106 for sheep and goats) for up to 6 months at 5 °C storage. In storage at 20 °C, the viability of freeze-dried anthrax vaccine maintained the minimum required titre up to 6 months while up to 90 days in case of the suspension formulation. Both types of vaccine formulations maintained the minimum titre per dose for up to 30 days at 37 °C storage. Generally, both vaccine formulations showed similar trends in titre fall in all of the three storage temperatures (5 °C, 20 °C and 37 °C) as observed in the almost linearly overlapping 95% confidence intervals (CI) up to day 90 at 5 °C and 20 °C storages while up to day 30 at 37 °C storage. However, a significant (P < 0.05) drop in titre was observed after day 90 for storages at 5 °C and 20 °C, and after day 30 for 37 °C storage as observed in the non overlapping 95% CI from the average titres of previous time points. This study showed that if temperature excursion occurs above the recommended temperature range (4–8 °C) during storage or transport, the vaccine should remain effective and can still be used in vaccination programs.

Short- and Long-Lived Autoantibody-Secreting Cells in Autoimmune Neurological Disorders.

As B cells differentiate into antibody-secreting cells (ASCs), short-lived plasmablasts (SLPBs) are produced by a primary extrafollicular response, followed by the generation of memory B cells and long-lived plasma cells (LLPCs) in germinal centers (GCs). Generation of IgG4 antibodies is T helper type 2 (Th2) and IL-4, -13, and -10-driven and can occur parallel to IgE, in response to chronic stimulation by allergens and helminths. Although IgG4 antibodies are non-crosslinking and have limited ability to mobilize complement and cellular cytotoxicity, when self-tolerance is lost, they can disrupt ligand-receptor binding and cause a wide range of autoimmune disorders including neurological autoimmunity. In myasthenia gravis with predominantly IgG4 autoantibodies against muscle-specific kinase (MuSK), it has been observed that one-time CD20+ B cell depletion with rituximab commonly leads to long-term remission and a marked reduction in autoantibody titer, pointing to a short-lived nature of autoantibody-secreting cells. This is also observed in other predominantly IgG4 autoantibody-mediated neurological disorders, such as chronic inflammatory demyelinating polyneuropathy and autoimmune encephalitis with autoantibodies against the Ranvier paranode and juxtaparanode, respectively, and extends beyond neurological autoimmunity as well. Although IgG1 autoantibody-mediated neurological disorders can also respond well to rituximab induction therapy in combination with an autoantibody titer drop, remission tends to be less long-lasting and cases where titers are refractory tend to occur more often than in IgG4 autoimmunity. Moreover, presence of GC-like structures in the thymus of myasthenic patients with predominantly IgG1 autoantibodies against the acetylcholine receptor and in ovarian teratomas of autoimmune encephalitis patients with predominantly IgG1 autoantibodies against the N‐methyl‐d‐aspartate receptor (NMDAR) confers increased the ability to generate LLPCs. Here, we review available information on the short-and long-lived nature of ASCs in IgG1 and IgG4 autoantibody-mediated neurological disorders and highlight common mechanisms as well as differences, all of which can inform therapeutic strategies and personalized medical approaches.

Fluorophore labelled BVDV: a novel tool for the analysis of infection dynamics

Genetic labelling of viruses with a fluorophore allows to study their life cycle in real time, without the need for fixation or staining techniques. Within the family Flaviviridae, options for genetic labelling of non-structural proteins exist. Yet, no system to genetically label structural proteins has been put forward to date. Taking advantage of a previously described site within the structural protein E2, a fluorophore was introduced into a cytopathogenic (cpe) BVDV-1 virus (BVDVE2_fluo). This insertion was well tolerated, resulting in a 2-fold drop in titer compared to the parental virus, and remained stably integrated into the genome for more than 10 passages. The fluorophore E2 fusion protein was readily detectable in purified virus particles by Western blot and fluorescence microscopy and the particle integrity and morphology was confirmed by cryo electron microscopy. The same integration site could also be used to label the related Classical swine fever virus. Also, BVDVE2_fluo particles bound to fluorophore labelled CD46 expressing cells could be resolved in fluorescence microscopy. This underlines the applicability of BVDVE2_fluo as a tool to study the dynamics of the whole life cycle of BVDV in real time.

The Pre-Transplant Drop in Panel-Reactive Antibodies Titer Evaluated Using Complement-Dependent Cytotoxicity (PRA-CDC) and the Risk of Early Acute Rejection in Sensitized Kidney Transplant Recipients.

Background: The panel-reactive antibodies that use the complement-dependent cytotoxicity test (PRA-CDC) are still a standard method for monitoring the degree of immunization in kidney transplant candidates on active waiting lists in some countries, including Poland. The aim of this study was to analyze the relationship between the maximum and the last pre-transplant PRA titer on the percentage of positive cross-matches and rate of early acute rejection episodes. Material and methods: The retrospective analysis included 528 patients from two transplant centers. All patients were divided into three groups, depending on their peak and last pre-transplant PRA titers. There were 437 (82.8%) patients with peak PRA <20% (non-sensitized group, non-ST) and 91 (17.2%) patients with peak PRA >20%. Among the latter group, 38 had maintained PRA level >20% at the time of transplantation (sensitized patients, ST), whereas 53 had pre-transplant PRA ≤20% (previously sensitized patients, prev-ST). Results: The percentages of positive crossmatches were 76.9% in ST and 53.7% in prev-ST groups versus 18.4 in non-ST group (both p < 0.001). The acute rejection rates were 18.9, 17.6 and 6.8%, respectively (p < 0.001 for ST or prev-ST versus non-ST). The pre-transplant PRA titer drop did not decrease the risk of early acute rejection [OR = 1.09 (95% CI: 0.31–3.85)] in a multiple logistic regression analysis. The occurrences of primary graft non-function and delayed graft function were similar in all study groups. Conclusions: Previously immunized kidney transplant candidates even with substantial decrease in pre-transplant PRA-CDC levels are still at high immunological risk when compared with non-immunized patients, and they should receive lymphocyte-depleting induction therapy.

288. Development of Cocal Glycoprotein Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T Cells

Lentiviral vectors (LVs) are routinely used for stable gene transfer and have demonstrated great promise in hematopoietic stem cell gene therapy and also immunotherapy using genetically modified T cells. LVs are commonly pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G), which confers broad tropism to the vector and allows for vector concentration by centrifugation. However, the use of VSV-G has several limitations, such as susceptibility to inactivation by human serum complement making it unsuitable for in vivo delivery. Furthermore, VSV-G is toxic when constitutively expressed, which has impeded efforts to generate stable producer cell lines. In this study, we first validate the use of cocal vesiculovirus envelope to pseudotype LVs by demonstrating that cocal LVs transduce hematopoietic stem cells and CD4+ T cells more efficiently than VSV-G LVs. We also provide evidence that cocal and VSV-G envelopes use the same receptor for cell entry. We then describe the development of two high-titer, cocal-pseudotyped, LV producer cell lines for a GFP reporter and for a WT1 tumor-specific T cell receptor (TCR). The different 3rd generation lentiviral helper genes were sequentially introduced in HEK293T cells by co-transfection with plasmids encoding antibiotic resistance genes followed by selection to allow for stable protein expression. Cells expressing the cocal envelope produced over 10-times more infectious LV particles as compared to VSV-G expressing cells. High-titer cocal producer cells were isolated by screening for best single clones, which were capable of generating concentrated titers above 108 infectious units per mL. We found that these producer cells were stable after serial passages for over 3 months, with no drop in titer detected over time. The resulting GFP and WT1-TCR vectors performed at least as well as identical vectors made with our standard transient transfection protocol for the transduction of CD34+ and CD4+ T cells, respectively. Cocal LV producer cells were also adapted for growth in suspension, serum-free culture, which will facilitate efforts for the scaling up of vector production. In summary, we have successfully developed two independent LV producer cells lines with clinically usable titers. The broad applicability of our cocal packaging cell line offers a promising tool toward the generation of large-scale, clinical grade LV.

Hatchery Spray Cabinet Administration Does Not Damage Avian Coronavirus Infectious Bronchitis Virus Vaccine Based on Analysis by Electron Microscopy and Virus Titration.

studies in our laboratory showed that the Arkansas-Delmarva Poultry Industry (Ark-DPI) vaccine given to 1-day-old chickens by hatchery spray cabinet replicated poorly and failed to adequately protect broilers against homologous virus challenge, whereas the same vaccine given by eye-drop did replicate and the birds were protected following homologous virus challenge. To determine if mechanical damage following spray application plays a role in failure of the Ark-DPI vaccine, we examined the morphology of three Ark-DPI vaccines from different manufacturers using an electron microscope and included a Massachusetts (Mass) vaccine as control. One of the Ark-DPI vaccines (vaccine A) and the Mass vaccine had significantly (P < 0.005) fewer spikes than the other two Ark-DPI vaccines. We also found that the Ark-DPI and Mass vaccines had significantly (P < 0.005) fewer spike proteins per virus particle when compared to their respective challenge viruses. This observation is interesting and may provide some insight into the mechanism behind infectious bronchitis virus attenuation. No obvious differences were observed in virus morphology and no consistent trend in the number of spikes per virion was found in before- and after-spray samples. We also determined the vaccine titer before and after spray in embryonated eggs and found that both Ark-DPI and Mass vaccines had a similar drop in titer, 0.40 logi and 0.310 logi, respec10ively. Based on these data, it appears that mechanical damage to the Ark-DPI vaccine is not occurring when delivered by a hatchery spray cabinet, suggesting that some other factor is contributing to the failure of that vaccine when given by that method.

The significance of fertile pigs vaccination against porcine parvovirus infection in the prevention of intrauterine infection and formation of colostrum piglets immunity

The aim of this study was to come closer to the knowledge of piglets protection during intrauterine life, as well as formation of colostrum immunity against Porcine Parvovirus Infection (PPV), on the basis of detailed antibody titer analysis from the blood serum of pigs 7 days before previous weaning, 30 days before mating and their piglets during first 3 days of life. The research included 60 fertile pigs and 300 of their offspring. For that purpose we have examined antibody titer specific for PPV in blood serum of vaccinated fertile pigs on 70th and 113th day of gestation, and in the blood serum of piglets originated from itemized fertile pigs during first day of life, before colostrums consummation and also during 3rd day of life. On the 70th day of gestation, in the fertile pigs blood serum, average antibody titer specific for PPV, value of 12.60 was determined, what represents adequate level for solid protection against PPV infection. This was confirmed undoubtedly by examination results of antibodies in the blood serum of piglets before colostrum consummation, which was 100% negative. Titer drop in the blood serum of fertile pigs, on 113th day of gestation (on the level of 8.7) came as a result of specific antibodies transfer from the mother?s blood flow to the colostrum. The above-mentioned is supported by the fact that on the 3rd day of life there was confirmed high average body titer level (13.37) in newborn piglets body serum has been confirmed. The principle of fertile pigs vaccination 7 days before weaning and one month before gilts insemination is an efficient measure which prevents intrauterine infection occurrence during the entire gestation process. The level of antibodies specific for PPV which is determined in gilts blood serum after vaccination can be a result of both primary and secondary immune response (animal infected before first vaccination), as well as absence of gilts revaccination, what is usually recommended by producers, may not always be regarded as a failure.

ABO-Incompatible Kidney Transplantation Enabled by Non-Antigen-Specific Immunoadsorption

ABO-incompatible kidney transplantation performed after desensitization with antigen-specific immunoadsorption (IA) results in good outcomes. However, a unique single-use IA device is required, which creates high costs. From August 2005 to August 2010, 19 patients were desensitized for ABO-incompatible living donor kidney transplantation. Six patients treated with a single-use antigen-specific IA device and 12 patients treated with a reusable non-antigen-specific IA device were analyzed. Six patients who received antigen-specific IA had a median of 5 IA treatments and 12 patients with non-antigen-specific IA had a median of 6 IA treatments preoperatively. Median average titer drop in Coombs technique was 1.2 in antigen-specific IA and 1.7 in non-antigen-specific IA. In two patients with antigen-specific IA and four patients with non-antigen-specific IA, additional plasmapheresis treatments were necessary for recipient desensitization. Despite six treatments with antigen-specific IA and 12 plasmapheresis treatments, one patient with a starting isoagglutinin titer of 1:1024 (Coombs) could not be transplanted. The 18-month graft survival rate for the 17 ABO-incompatible living donor kidney transplants was 100%. One male recipient who was desensitized with antigen-specific IA died 44 months after transplantation from sudden cardiac death with a serum creatinine of 1.2 mg/dL. At last follow-up, a median of 13 months after transplantation, median serum creatinine for 16 patients was 1.5 mg/dL, median glomerular filtration rate as estimated by the modification of diet in renal disease formula 54 mL/min/1.73 m, and median urinary protein-to-creatinine ratio 0.1, with no differences between treatments. A reusable non-antigen-specific IA device allows high number of treatments at reasonable cost, and at the same time might deplete human leukocyte antigen-alloantibodies.

Development of an Anti-SalmonellaPhage Cocktail with Increased Host Range

Salmonella shedding in many livestock species can increase significantly after transport and lairage. Preprocessing increases in shedding can amplify the amount of Salmonella that enters the processing facility and the likelihood of end-product contamination. We previously produced an anti-Salmonella phage cocktail that reduced colonization in swine when the pigs were exposed to an environment heavily contaminated with Salmonella, similar to what might be seen in a transport trailer or processing facility holding pen. The aim of this study was to increase the efficacy of the phage treatment by (1) expanding the host-range of the cocktail and (2) developing a more cost-effective microencapsulation technique. We collected samples from wastewater treatment facilities and isolated 20 distinct phages belonging to either the Siphoviridae or Myoviridae families. From this library we identified 10 phages that together lysed a mixed culture of Salmonella enterica Typhimurium, Enteriditis, and Kentucky--three serovars commonly associated with meat and poultry products. The phages were microencapsulated using two sodium-alginate-based methods that only reduced the cocktail titer by 1.0-1.5 logs (premicroencapsulation: 10.4 log(10) PFU/mL; postmicroencapsulation method one: 9.2 log(10) PFU/mL; postmicroencapsulation method two: 8.9 log(10) PFU/mL). Microencapsulated phages remained stable at both 4°C and 22°C for up to 14 days with no appreciable drop in titer (mean titer: 8.9 log(10) PFU/mL). These data indicate that phage cocktails with wider host ranges are possible and a cost-effective microencapsulation process can protect the phages over an extended period, making simultaneous treatment of large numbers of animals with feed- or water-based delivery possible.

Adenovirus Virion Stability and the Viral Genome: Size Matters

Ongoing investigations into the fundamental aspects of virus biology continue to provide crucial insights for the development of more effective virus-based therapeutics. Within the field of gene therapy, these discoveries have translated into virus vectors with improved tissue tropism, prolonged transgene expression, and enhanced safety. However, radical modifications to the viral genome or capsid constituents can have marked effects on virion stability. In particular, recent insights into the role of viral genomes in contributing to the physical stability of the virion highlight the need to reconsider this heretofore underappreciated aspect of vector design. Here we discuss the impact of the genome on virion stability, with particular emphasis on the development of adenovirus (Ad)-based vectors.

O13 Mumps virus detection by PCR and culture during an outbreak in a highly unvaccinated population

Objective: Rabies virus has the highest case to fatality ratio of any infectious disease; however, vaccines and immunoglobulins are available for preand post-exposure prophylaxis. Due to the risk of possible hypersensitivity reactions, repeated booster doses of rabies vaccine should be administered only when necessary to people at risk of exposure. It is recommend that antibody testing be performed 2 years following primary immunization, followed by the administration of a booster dose if the neutralizing antibody titre falls below 0.5 International Units per ml (IU/ml). The objective of this work was to determine if the recommended testing interval following primary immunization is sufficient to maintain adequate antibody titres in healthy adults. Methods: During the establishment of the rabies laboratory at the NML in 2007, all employees determined to be at risk of exposure were vaccinated against rabies virus. Rabies vaccine was administered in 3 doses on days 0, 7, and 21. Antibody levels were monitored one month post-vaccination and subsequently every 6 months. Data from this testing were analyzed to determine if the vaccinated employees maintained adequate antibody titres for a minimum of 2 years following primary immunization. Results: The antibody titres of 12 employees were assessed 1 month after vaccination and subsequently every 6 months. All 12 employees demonstrated an acceptable antibody titre 1 month after vaccination, although the titres of 2 of the 12 employees were very low (0.5 IU/ml and 0.65 IU/ml). These 2 employees received a booster dose of vaccine immediately. Two other employees demonstrated a substantial drop in titre when subsequently tested 6 months later, resulting in a decision to administer a booster dose of vaccine to these employees who work directly with rabies virus. The antibody titre of these 4 employees has remained high throughout the follow-up testing intervals. Conclusions: Had the recommended testing interval of 2 years been followed, the antibody levels of 4 of the 12 employees may have potentially fallen below the acceptable level of 0.5 IU/ml. This indicates that some healthy individuals may not maintain an antibody titre of 0.5 IU/ml for at least 2 years following primary immunization. All persons at risk of exposure, especially those persons working directly with the live virus, should be monitored carefully. It may be advisable to administer a booster dose of vaccine 1 year following the primary vaccine series to reinforce antibody levels in individuals receiving pre-exposure vaccination for rabies virus.

O12 Antibody response to rabies vaccine in healthy adults following primary immunization and the importance of occupational health surveillance programs

Objective: Rabies virus has the highest case to fatality ratio of any infectious disease; however, vaccines and immunoglobulins are available for preand post-exposure prophylaxis. Due to the risk of possible hypersensitivity reactions, repeated booster doses of rabies vaccine should be administered only when necessary to people at risk of exposure. It is recommend that antibody testing be performed 2 years following primary immunization, followed by the administration of a booster dose if the neutralizing antibody titre falls below 0.5 International Units per ml (IU/ml). The objective of this work was to determine if the recommended testing interval following primary immunization is sufficient to maintain adequate antibody titres in healthy adults. Methods: During the establishment of the rabies laboratory at the NML in 2007, all employees determined to be at risk of exposure were vaccinated against rabies virus. Rabies vaccine was administered in 3 doses on days 0, 7, and 21. Antibody levels were monitored one month post-vaccination and subsequently every 6 months. Data from this testing were analyzed to determine if the vaccinated employees maintained adequate antibody titres for a minimum of 2 years following primary immunization. Results: The antibody titres of 12 employees were assessed 1 month after vaccination and subsequently every 6 months. All 12 employees demonstrated an acceptable antibody titre 1 month after vaccination, although the titres of 2 of the 12 employees were very low (0.5 IU/ml and 0.65 IU/ml). These 2 employees received a booster dose of vaccine immediately. Two other employees demonstrated a substantial drop in titre when subsequently tested 6 months later, resulting in a decision to administer a booster dose of vaccine to these employees who work directly with rabies virus. The antibody titre of these 4 employees has remained high throughout the follow-up testing intervals. Conclusions: Had the recommended testing interval of 2 years been followed, the antibody levels of 4 of the 12 employees may have potentially fallen below the acceptable level of 0.5 IU/ml. This indicates that some healthy individuals may not maintain an antibody titre of 0.5 IU/ml for at least 2 years following primary immunization. All persons at risk of exposure, especially those persons working directly with the live virus, should be monitored carefully. It may be advisable to administer a booster dose of vaccine 1 year following the primary vaccine series to reinforce antibody levels in individuals receiving pre-exposure vaccination for rabies virus.

Rituximab for the Treatment of High Titer Acquired Factor VIII Inhibitor with Long Term Follow up

Acquired factor VIII inhibitors are auto antibodies directed against factor VIII (FVIII) and have a reported incidence of 0.2–1.0 per million individuals per year. It is a rare cause of serious bleeding associated with high mortality. Individuals with high titer antibodies (> 100 Bethesda units (BU)), often have difficulty achieving a complete sustained remission although different strategies have been advocated for these patients. Rituximab has been demonstrated to show efficacy in patients with acquired hemophilia. In this study we retrospectively studied three patients with high titer acquired FVIII inhibitors with long term follow up. Two of the patients were resistant to steroids and Intravenous Immunoglobulin, one patient had received steroids and Cyclophosphamide prior to rituximab therapy. Patients ages were 36, 45 and 48 years, two were women and one male. One of the women has a h/o pregnancy about 8 months ago and other two were thought to have idiopathic inhibitors. The patient's inhibitor titers ranged from 124–800 BU at the time of treatment with Rituximab. All received 4 weekly infusion of rituximab at 375mg/m2. No significant treatment related complication was noted. One patient did not respond to treatment. Two patients had a decline in inhibitor titers. One patient had a gradual drop in titer from 124 BU to 0.00 BU over 18 months. The other responder had a drop in titer from 800 BU to 41 BU in 16 months and then relapsed with significant bleeding and anti factor 8 titers of 700 BU and was retreated with rituximab and had a gradual drop in inhibitor titers to 50 BU over 8 months. The two patients have been followed for 28 and 30 months respectively. The non responder continues to have high tires of antibodies and has been followed for 25 months.We conclude that in patients with high titer of acquired inhibitors rituximab alone may produce a drop in inhibitor titer but is not sufficient to achieve a complete response. Rituximab in combination with other therapies or novel agents may provide better results,

The survivability of Bacillus anthracis (Sterne strain) in processed liquid eggs

In this study, we investigated the survival and inactivation kinetics of a surrogate strain of Bacillus anthracis (Sterne strain) in whole egg (WE), egg white (EW), sugared egg yolk (YSU), and salted egg yolk (YSA) at low (−20, 0, and 5 °C), moderate (15, 20, 25, 30, 35, and 40 °C), and high storage temperatures (45, 50, 55, and 60 °C). Outgrowth of the spores was measured as lag phase duration (LPD). Replication of vegetative cells was measured in terms of growth rate (GR) and maximum population density (MPD). Spore inactivation was recorded as inactivation rate and percent reduction in viable count. In general, spore viability decreased at low and high temperatures and increased at moderate temperatures. At 0 and 5 °C, a 60–100% reduction in spore viability was seen within 2–3 weeks in WE and YSU, 0–30% in YSA, and 50–100% in EW. At −20 °C, however, no drop in spore titer was observed in YSU and EW but a 20% drop in titer was seen in YSA and 50% in WE within 2–3 weeks. At high temperatures, WE, EW, and YSA produced a 20–50% drop in the spore titer within 1–4 h whereas YSU showed 100% inactivation within 0.75 h. At moderate storage temperatures, as the temperature increased from 15 to 40 °C, LPD decreased from 13.5 to 0.75 h and MPD reached 0.27–2.2 × 10 9 CFU/ml in YSU and WE, respectively. Markedly lower growth was observed in YSA (LPD = 24–270 h, MPD = 9 × 10 5 CFU/ml) and spores were inactivated completely within 1–6 h in EW. The survivability and inactivation data of B. anthracis in liquid egg products reported in this investigation will be helpful in developing risk assessment models on food biosecurity.