Efficacy assessment of a triple anthrax chimeric antigen as a vaccine candidate in guinea pigs: challenge test with Bacillus anthracis 17 JB strain spores.

CONTEXT: Bacillus anthracis secretes a tripartite toxin comprising protective antigen (PA), edema factor (EF), and lethal factor (LF). The human anthrax vaccine is mainly composed of the anthrax protective antigen (PA). Considerable efforts are being directed towards improving the efficacy of vaccines because the use of commercial anthrax vaccines (human/veterinary) is associated with several limitations. OBJECTIVE: In this study, a triple chimeric antigen referred to as ELP (gene accession no: MT590758) comprising highly immunogenic domains of PA, LF, and EF was designed, constructed, and assessed for the immunization capacity against anthrax in a guinea pig model. MATERIALS AND METHODS: Immunization was carried out considering antigen titration and immunization protocol. The immunoprotective efficacy of the ELP was evaluated in guinea pigs and compared with the potency of veterinary anthrax vaccine using a challenge test with B. anthracis 17JB strain spores. RESULTS: The results demonstrated that the ELP antigen induced strong humoral responses. The T-cell response of the ELP was found to be similar to PA, and showed that the ELP could protect 100%, 100%, 100%, 80% and 60% of the animals from 50, 70, 90, 100 and 120 times the minimum lethal dose (MLD, equal 5 × 105 spore/ml), respectively, which killed control animals within 48 h. DISCUSSION AND CONCLUSIONS: It is concluded that the ELP antigen has the necessary requirement for proper immunization against anthrax and it can be used to develop an effective recombinant vaccine candidate against anthrax.

Assessment and utility of 2 Chlamydia trachomatis Pgp3 serological assays for seroprevalence studies among women in the United States.

Two plasmid gene protein (Pgp3)-based serological assays, the Pgp3-ELISA and multiplex bead assay (Pgp3-MBA), were compared and used to estimate seropositivity of Chlamydia trachomatis (CT) among females 14 to 39 years old participating in the National Health and Nutrition Examination Survey between 2013-2016. Of the 2,201 specimens tested, 502 (29.5%, 95% CI 27.6-31.5) were positive using Pgp3-ELISA and 624 (28.4%, 95% CI 26.5-30.3) were positive using Pgp3-MBA. The overall agreement between the assays was 87.7%. Corresponding nucleic acid amplification test (NAAT) results were available for 1,725 specimens (from women 18-39 years old); of these, 42 (2.4%, 95% CI 1.8-3.3) were CT NAAT-positive. Most of the CT NAAT-positive specimens had corresponding positive serological assay results; 33 (78.6%, 95% CI 62.8-89.2) were Pgp3-ELISA-positive and 36 (85.7%, 95% CI 70.8-94.1) were Pgp3-MBA-positive. Although Pgp3-ELISA and Pgp3-MBA demonstrated equivalent performance in this study, an advantage of the Pgp3-MBA over Pgp3-ELISA is that it is well suited for high sample throughput applications.

A new recombinant F1 antigen as a cost and time-effective tool for plague diagnosis.

The Yersinia pestis capsular antigen F1 is widely used in plague laboratory diagnosis. Here, we describe the production of an F1 recombinant protein within reduced time and biosafety requirements. Its evaluation in hemagglutination tests indicated that the recombinant F1 can replace the conventional F1 protein for plague diagnosis.

High protection of mice against Brucella abortus by oral immunization with recombinant probiotic Lactobacillus casei vector vaccine, expressing the outer membrane protein OMP19 of Brucella species.

Brucellosis is a zoonotic disease threatening the public health and hindering the trade of animals and their products, which has a negative impact on the economic development of a country. Vaccination is the most effective way to control brucellosis. The recombinant vector vaccines are promising candidates for immunization in humans and animals. In this study, the gene encoding OMP19 antigen was primarily amplified and cloned into an expression vector called pT1NX, and then transformed to L. casei cell via electroporation technique. The expression was confirmed using specific antibody against the recombinant protein via immunological screening tests such as western blot and immunofluorescence assay. Finally, recombinant L. casei was orally fed to mice and the results were further recorded, indicating that the mice group which received OMP19 through L. casei based vaccine represented a very good general and mucosal immune responses protective against challenges with virulent B. abortus 544 strain compared with negative control recipient groups. Therefore, the vaccine produced in this research plan can be a very good candidate for protection against brucellosis.

Assessment of IgA anti-PT and IgG anti-ACT reflex testing to improve Bordetella pertussis serodiagnosis in recently vaccinated subjects.

OBJECTIVES: Quantifying IgG antibodies to pertussis toxin (PT) is the most specific and sensitive method for the serodiagnosis of a Bordetella pertussis infection. Since PT is a component of acellular pertussis vaccines, anti-PT IgG is also induced by vaccination, precluding pertussis serodiagnosis based exclusively on anti-PT IgG in recently vaccinated subjects. Here, we aim to identify additional B. pertussis-specific serological markers that can discriminate between infection and recent vaccination. METHODS: The clinical usefulness of measuring IgA directed to the vaccine antigen PT and IgG directed to non-vaccine antigens (Fim2/3, LPS, ACT, CatACT) was evaluated in nine well characterized subject groups, aged 10-89 years (n = 390). Serum anti-PT IgG levels (>125 IU/mL) served as an indicator for a recent B. pertussis infection. Comparing symptomatic pertussis-infected subjects (n = 140) with recently vaccinated, non-infected subjects (n = 100) revealed the optimal cut-off, accuracy, sensitivity and specificity for each single parameter. RESULTS: For pertussis diagnosis in recently vaccinated subjects, the measurement of anti-PT IgA (cut-off 15 IU/mL) and anti-ACT IgG (cut-off 15 U/mL) resulted in accuracies of 95% (91.5-97.1) and 87.5% (82.7-91.1), sensitivities of 92.9% (87.4-96.0) and 83.6% (76.5-88.8) and specificities of 98% (93.0-99.4) and 93% (86.3-96.6), respectively. Comparing anti-PT IgA levels between the youngest (10-19 years, n = 38) and oldest (70-89 years, n = 17) age groups revealed an age-dependent increase in antibody levels in pertussis-infected subjects (p < 0.0001). CONCLUSIONS: Reflex testing of anti-PT IgA and anti-ACT IgG improves pertussis serodiagnosis in recently vaccinated symptomatic subjects with elevated anti-PT IgG levels. Furthermore, both markers can discriminate between vaccination and recent infection in pertussis serosurveillance studies.

Assessment of the immunogenicity of the leptospiral LipL32, LigAni, and LigBrep recombinant proteins in the sheep model.

Veterinary leptospirosis vaccines are composed of bacterins and present limitations, for example, the need for bacteriological culture and serovar-dependent immunity. Recombinant antigens represent a promising alternative. LigAni, LigBrep, and LipL32 proteins have been shown to promote a protective immune response against the homologous challenge in hamsters. Therefore, the next step is to evaluate the immunological properties of these immunogens in the actual hosts, as ruminants, which has never been performed before. The objective of this study was to evaluate the immunogenicity and potential adverse effects of the recombinant proteins LigAni, LigBrep, and LipL32 in the ovine model. For this, 16 Santa Inês sheep were allocated into three groups: two experimental (Groups A and B) and one control group (Group C). Group A was inoculated with a formulation containing the recombinant proteins in combination with the aluminum hydroxide adjuvant; Group B was inoculated with a formulation containing the recombinant proteins in combination with the Montanide adjuvant; and Group C was inoculated with adjuvants only. The results revealed that formulations containing the recombinant proteins induced total IgG seroconversion and led to a significant increase in antibody titers in the sheep model. Besides, there were no clinical changes or adverse effects. Thus, LigAni, LigBrep, and LipL32 proteins elicited a significant humoral immune response with elevated serum IgG levels, demonstrating that they possess the immunogenic and safety characteristics necessary to sustain their potential use as leptospirosis vaccines in the ruminant model.

Persistent stimulation with Mycobacterium tuberculosis antigen impairs the proliferation and transcriptional program of hematopoietic cells in bone marrow.

Mycobacterium tuberculosis (M. tuberculosis) persistent infection might cause the dysfunction of hematopoiesis. To investigate whether M. tuberculosis persistent antigen stimulation impairs the proliferation and differentiation of hematopoietic stem and progenitor cells characterized as lineage- c-Kit+ (LK cells), C57BL/6 mice were primed with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and boosted with a cocktail of M. tuberculosis antigens ESAT6, CFP10 and Mtb10.4-HspX (MH) along with adjuvant N, N'-dimethyl-N, N'-dioctadecylammonium bromide (DDA) plus polyinosinic-polycytidylic acid (Poly I:C) weekly for 12 or 22 weeks. The cytokine production by splenic T cells, proliferation of LK cells and transcriptional events during differentiation of bone marrow (BM) c-Kit+ cells were investigated. Meanwhile, the mice were treated with interleukin 2 (IL-2) and the therapeutic effects were analyzed. We found that antigen specific interferon-γ (IFN-γ) production by splenic CD4+ T cells increased following antigen stimulation for 12 weeks, but it declined after continuous stimulation for 22 weeks. The long-term exposure of mice to M. tuberculosis antigen compromised the proliferation of LK cells. Moreover, the expression of transcription factors in the c-Kit+ cells was adjusted, with up-regulation of IRF8 and Batf2 involved in myeloid differentiation and down-regulation of NOTCH1 and GATA2 participated in T-cell lineage commitment. The concentrations of IFN-γ in BM of the persistent antigen group were higher than that in sham control at the 12th week, while the concentrations of IL-2 in BM of the persistent antigen group were lower compared with the transient antigen stimulation control. Following IL-2 treatment, the concentrations of IL-2 in BM increased while IFN-γ got declined. IL-2 treatment could restore the expression levels of those transcription factors and the proliferating activity of LK cells impaired by persistent antigen stimulation. Our results indicate that M. tuberculosis antigen persistent stimulation decreases the proliferating activity of LK cells, promotes myelopoietic differentiation, and represses lymphopoietic differentiation as a consequence of elevated IFN-γ production. IL-2 supplementation contributes to maintaining the homeostasis of hemopoiesis.

Molecular characterization of Streptococcus pneumoniae in children living in southwest China and assessment of a potential protein vaccine, rPfbA.

BACKGROUND: Few children living in southwest China are vaccinated against Streptococcus pneumoniae (S. pneumoniae), which is an important pathogen in causing high morbidity and high mortality. This study aimed the molecular characterization of S. pneumoniae strains isolated from children and a new vaccine strategy based on a potential protein antigen. METHODS: Molecular characterizations, including serotype, virulence gene and pilus analyses, were performed using PCR. Seven housekeeping genes were sequenced to identify the sequence types (STs), and antibiotic resistance was analysed using the microdilution broth method. In addition, we evaluated the protective effects of recombinant plasmin- and fibronectin-binding protein A (rPfbA) in murine pneumococcal infection models by challenge and passive transfer analyses, and assessed cytokine changes after immunization. RESULTS: The prevalent serotypes were 19F (31.4%), 19A (21.6%), 6B (13.7%), 14 (11.8%) and 23F (9.8%), and the coverage rates of the 13-valent pneumococcal conjugate vaccine (PCV13) were high in 93.3% of the isolates. The predominant STs were ST271 (23.5%), ST320 (21.6%) and ST876 (11.8%). Most of the S. pneumoniae isolates were resistant to erythromycin (95.1%) and clindamycin (90.2%). The molecular distributions and antibiotic resistance rates of the S. pneumoniae isolates differed between the plateau and the basin regions. More than 93% of the S. pneumoniae isolates carried ply, cbpA, phtD and nanA, and over half of the isolates carried pilus-1, pilus-2 and pfbA. Mucosal immunization with rPfbA induced pneumococcal specific antibody responses which provided to eliminate colonization in lung and nasopharynx, and protection against pneumococcal challenge. CONCLUSION: Vaccine strategies based on epidemiological surveillance can be more adaptive to specific areas, reduce costs and protect against changing antigenic sites. We advise that children currently living in southwest China be vaccinated with PCV13.

Biological feasibility and importance of a gonorrhea vaccine for global public health.

There is a growing public health interest in controlling sexually transmitted infections (STIs) through vaccination due to increasing recognition of the global disease burden of STIs and the role of STIs in women's reproductive health, adverse pregnancy outcomes, and the health and well-being of neonates. Neisseria gonorrhoeae has historically challenged vaccine development through the expression of phase and antigenically variable surface molecules and its capacity to cause repeated infections without inducing protective immunity. An estimated 78 million new N. gonorrhoeae infections occur annually and the greatest disease burden is carried by low- and middle-income countries (LMIC). Current control measures are clearly inadequate and threatened by the rapid emergence of antibiotic resistance. The gonococcus now holds the status of "super-bug" as there is currently no single reliable monotherapy for empirical treatment of gonorrhea. The problem of antibiotic resistance has elevated treatment costs and necessitated the establishment of large surveillance programs to track the spread of resistant strains. Here we review the need for a gonorrhea vaccine with respect to global disease burden and related socioeconomic and treatment costs, with an emphasis on the impact of gonorrhea on women and newborns. We also highlight the challenge of estimating the impact of a gonorrhea vaccine due to the need for more data on the burden of gonococcal pelvic inflammatory disease and related sequelae and of gonorrhea-associated adverse pregnancy outcomes and the problem of empirical diagnosis and treatment of STIs in LMIC. There is also a lack of clinical and basic science research in the area of gonococcal/chlamydia coinfection, which occurs in a high percentage of individuals with gonorrhea and should be considered when testing the efficacy of gonorrhea vaccines. Finally, we review recent research that suggests a gonorrhea vaccine is feasible and discuss challenges and research gaps in gonorrhea vaccine development.

Status of vaccine research and development for Helicobacter pylori.

Gastric adenocarcinoma is globally the third leading cause of death due to malignancy, with the bulk of this disease burden being suffered by low and middle income countries (LMIC), especially in Asia. The majority of these cancers develop as a result of a chronic gastritis that arises in response to infection with the stomach-dwelling bacterium, Helicobacter pylori. A vaccine against this pathogen would therefore be a powerful tool for preventing gastric adenocarcinoma. However, notwithstanding a proof-of-concept that vaccination can protect children from acquisition of H. pylori infection, there are currently no advanced vaccine candidates with only a single vaccine in Phase I clinical trial. Further, the development of a vaccine against H. pylori is not a current strategic priority of major pharmaceutical companies despite the large global disease burden. Given the involvement of such companies is likely to be critical for late stage development, there is therefore a need for an increased appreciation of the burden of this disease in LMIC and more investment to reinvigorate research in H. pylori vaccine Research and Development.

Assessment of an Antibody-in-Lymphocyte Supernatant Assay for the Etiological Diagnosis of Pneumococcal Pneumonia in Children.

New diagnostic tests for the etiology of childhood pneumonia are needed. We evaluated the antibody-in-lymphocyte supernatant (ALS) assay to detect immunoglobulin (Ig) G secretion from ex vivo peripheral blood mononuclear cell (PBMC) culture, as a potential diagnostic test for pneumococcal pneumonia. We enrolled 348 children with pneumonia admitted to Patan Hospital, Kathmandu, Nepal between December 2015 and September 2016. PBMCs sampled from participants were incubated for 48 h before harvesting of cell culture supernatant (ALS). We used a fluorescence-based multiplexed immunoassay to measure the concentration of IgG in ALS against five conserved pneumococcal protein antigens. Of children with pneumonia, 68 had a confirmed etiological diagnosis: 12 children had pneumococcal pneumonia (defined as blood or pleural fluid culture-confirmed; or plasma CRP concentration ≥60 mg/l and nasopharyngeal carriage of serotype 1 pneumococci), and 56 children had non-pneumococcal pneumonia. Children with non-pneumococcal pneumonia had either a bacterial pathogen isolated from blood (six children); or C-reactive protein <60 mg/l, absence of radiographic consolidation and detection of a pathogenic virus by multiplex PCR (respiratory syncytial virus, influenza viruses, or parainfluenza viruses; 23 children). Concentrations of ALS IgG to all five pneumococcal proteins were significantly higher in children with pneumococcal pneumonia than in children with non-pneumococcal pneumonia. The concentration of IgG in ALS to the best-performing antigen discriminated between children with pneumococcal and non-pneumococcal pneumonia with a sensitivity of 1.0 (95% CI 0.73-1.0), specificity of 0.66 (95% CI 0.52-0.78) and area under the receiver-operating characteristic curve (AUROCC) 0.85 (95% CI 0.75-0.94). Children with pneumococcal pneumonia were older than children with non-pneumococcal pneumonia (median 5.6 and 2.0 years, respectively, p < 0.001). When the analysis was limited to children ≥2 years of age, assay of IgG ALS to pneumococcal proteins was unable to discriminate between children with pneumococcal pneumonia and non-pneumococcal pneumonia (AUROCC 0.67, 95% CI 0.47-0.88). This method detected spontaneous secretion of IgG to pneumococcal protein antigens from cultured PBMCs. However, when stratified by age group, assay of IgG in ALS to pneumococcal proteins showed limited utility as a test to discriminate between pneumococcal and non-pneumococcal pneumonia in children.

Helicobacter pylori seroprevalence in Spain: influence of adult and childhood sociodemographic factors.

Helicobacter pylori (H. pylori) chronic infection causes severe digestive diseases, including gastric cancer, and certain strains entail a higher risk. Risk factors for this infection are still not fully understood. The aim of this study was to describe the association of adult and childhood sociodemographic factors with the seroprevalence of H. pylori, and with CagA and VacA antigen-specific seropositivity among H. pylori-seropositive individuals in the Spanish adult population. Serum antibody reactivity to H. pylori proteins was evaluated using multiplex serology in 2555 population-based controls enrolled in the MCC-Spain study, a multicase-control study recruiting participants from 2008 to 2013 in different areas of Spain. H. pylori seroprevalence was defined as seropositivity against at least four bacterial proteins. Information on sociodemographics, lifestyles, and environmental exposures was collected through personal interviews. Prevalence ratios and 95% confidence intervals were estimated using Poisson regression models to assess the association of lifetime sociodemographic factors with H. pylori seroprevalence and with seropositivity for CagA and VacA. H. pylori seroprevalence was 87.2%. Seropositivity was statistically significantly higher in men, increased with age, BMI, and number of siblings, and decreased with education and socioeconomic family level at birth. Among H. pylori-seropositive individuals, seropositivity was 53.3% for CagA, 61.4% for VacA, and 38.8% for both CagA and VacA. Ever smokers had lower seroprevalence for CagA and VacA than never smokers. H. pylori seroprevalence among this Spanish adult population was high and one third of the population was seropositive for two well-known markers of gastric cancer risk: CagA and VacA. Sex, age, education, and BMI were associated with H. pylori seroprevalence.

Antigenic variation in the Lyme spirochete: detailed functional assessment of recombinational switching at vlsE in the JD1 strain of Borrelia burgdorferi.

Borrelia burgdorferi is a causative agent of Lyme disease and establishes long-term infection in mammalian hosts. Persistence is promoted by the VlsE antigenic variation system, which generates combinatorial diversity of VlsE through unidirectional, segmental gene conversion from an array of silent cassettes. Here we explore the variants generated by the vls system of strain JD1, which has divergent sequence and structural elements from the type strain B31, the only B. burgdorferi strain in which recombinational switching at vlsE has been studied in detail. We first completed the sequencing of the vls region in JD1, uncovering a previously unreported 114 bp inverted repeat sequence upstream of vlsE. A five-week infection of WT and SCID mice was used for PacBio long read sequencing along with our recently developed VAST pipeline to analyze recombinational switching at vlsE from 40,000 sequences comprising 226,000 inferred recombination events. We show that antigenic variation in B31 and JD1 is highly similar, despite the lack of 17 bp direct repeats in JD1, a somewhat different arrangement of the silent cassettes, divergent inverted repeat sequences and general divergence in the vls sequences. We also present data that strongly suggest that dimerization is required for in vivo functionality of VlsE.

Monitoring and detection of leprosy patients in Southwest China: A retrospective study, 2010-2014.

More than 100 counties, mainly in southwest China, report incidence rates of leprosy >1/100,000. The current study analysed the epidemiology of leprosy in southwest China to improve our understanding of the transmission pattern and improve control programs. 207 counties were selected in southwest China. Leprosy patients and their household contacts were recruited. The data from the medical interview and the serological antileprosy antibody of the leprosy patients were analysed. A total of 2,353 new cases of leprosy were interviewed. The distribution of leprosy patients was partly associated with local natural and economic conditions, especially several pocket areas. A total of 53 from 6643 household contacts developed leprosy, and the incidence rate of leprosy in the household contacts was 364/100,000 person-years. We found that NDO-BSA attained higher positive rates than MMP-II and LID-1 regardless of clinical types, disability and infection time in leprosy patients. By means of combination of antigens, 88.4% patients of multibacillary leprosy were detected, in contrast to 59.9% in paucibacillary leprosy. Household contacts should be given close attention for the early diagnosis, disruption of disease transmission and precise control. Applications of serology for multi-antigens were recommended for effective coverage and monitoring in leprosy control.

Global epidemiology of serogroup B meningococcal disease and opportunities for prevention with novel recombinant protein vaccines.

BACKGROUND: Meningococcal disease (MD) is a major cause of meningitis and sepsis worldwide, with a high case fatality rate and frequent sequelae. Neisseria meningitidis serogroups A, B, C, W, X and Y are responsible for most of these life-threatening infections, and its unpredictable epidemiology can cause outbreaks in communities, with significant health, social and economic impact. Currently, serogroup B is the main cause of MD in Europe and North America and one of the most prevalent serogroups in Latin America. Mass vaccination strategies using polysaccharide vaccines have been deployed since the 1970s and the use of conjugate vaccines has controlled endemic and epidemic disease caused by serogroups A, C, W and Y and more recently serogroup B using geographically-specific outer membrane vesicle based vaccines. Two novel protein-based vaccines are a significant addition to our armamentarium against N. meningitidis as they provide broad coverage against highly diverse strains in serogroup B and other groups. Early safety, effectiveness and impact data of these vaccines are encouraging. These novel serogroup B vaccines should be actively considered for individuals at increased risk of disease and to control serogroup B outbreaks occurring in institutions or specific regions, as they are likely to save lives and prevent severe sequelae. Incorporation into national programs will require thorough country-specific analysis.

Multiplex serology of Helicobacter pylori antigens in detection of current infection and atrophic gastritis - A simple and cost-efficient method.

INTRODUCTION: Helicobacter pylori express a large array of antigens, each of which is duly responsible for successful colonization and pathogenesis. Here, we have studied host serum antibody responses to four of its immunodominant antigens in association with the infection status and the resulting clinical outcomes. METHODS: For this purpose, four individual H. pylori proteins (UreB, CagA, Tip-α and HP0175) were produced in recombinant forms. Serum antibody responses of 246 (75 GC and 171 NUD) patients, against the above antigens, were evaluated by multiplex immunoblotting. The associations between the resulting data and the infection status, as well as clinical outcomes were evaluated using logistic regression models. RESULTS: Serum antibodies to all four recombinant antigens increased the chances of detecting screening ELISA-positive subjects, in an escalating dose-dependent manner, ranging from 2.6 (1.5-4.7) for HP0175 to 14.3 for UreB (4.3-50.7), exhibiting the lowest and highest odds ratios, respectively (PAdj ≤ 0.001), such that 98.2% of the subjects with antibodies to all four antigens, were also positive by the screening ELISA (P < 0.0001). Among the screening ELISA-positive subjects, the three antigens of CagA, Tip-α, and HP0175 were able to segregate current from past H. pylori infection (P < 0.05). Accordingly, subjects with antibodies to one or more antigen(s) were at 5.4 (95% CI: 1.8-16.4) folds increased chances of having current infection, as compared to triple negatives (PAdj = 0.003). In reference to the clinical outcomes, those with serum antibodies to CagA were more prevalent among gastric cancer, as compared to NUD patients (ORAdj: 5.4, 95% CI: 2.4-12.2, PAdj < 0.0001). When NUD patients were categorized according to their histopathologic status, multiple antigen analysis revealed that subjects with serum antibodies to one or more of the 3 current infection-positive antigens (CagA, Tip-α, and HP0175) were at 9.7 (95% CI: 2.1-44.9, P = 0.004) folds increased risk of atrophic gastritis, in reference to triple negatives. CONCLUSION: The non-invasive multiplex serology assay, presented here, was able to not only detect subjects with current H. pylori infection, it could also screen dyspeptic patients for the presence of gastric atrophy. This simple and cost-efficient method can supplement routine screening ELISAs, to increase the chances of detecting current infections as well as atrophic gastritis.

Bivalent rLP2086 (Trumenba®): Development of a well-characterized vaccine through commercialization.

The phrase "Process is the Product" is often applied to biologics, including multicomponent vaccines composed of complex components that evade complete characterization. Vaccine production processes must be defined and locked early in the development cycle to ensure consistent quality of the vaccine throughout scale-up, clinical studies, and commercialization. This approach of front-loading the development work helped facilitate the accelerated approval of the Biologic License Application for the well-characterized vaccine bivalent rLP2086 (Trumenba®, Pfizer Inc) in 2014 under Breakthrough Therapy Designation. Bivalent rLP2086 contains two rLP2086 antigens and is licensed for the prevention of meningococcal meningitis disease caused by Neisseria meningitidis serogroup B in individuals 10-25years of age in the United States. This paper discusses the development of the manufacturing process of the two antigens for the purpose of making it amenable to any manufacturing facility. For the journey to commercialization, the operating model used to manage this highly accelerated program led to a framework that ensured "right the first time" execution, robust process characterization, and proactive process monitoring. This framework enabled quick problem identification and proactive resolutions, resulting in a robust control strategy for the commercial process.

The Impact of Nucleotide Sequence Analysis on Meningococcal Vaccine Development and Assessment.

Since it became available as a routine tool in biology, the determination and analysis of nucleotide sequences has been applied to the design of vaccines and the investigation of their effectiveness. As vaccination is primarily concerned with the interaction of biological molecules with the immune system, the utility of sequence data is not immediately obvious and, indeed, nucleotide sequence data are most effective when used to complement more conventional immunological approaches. Here, the impact of sequencing on the field of vaccinology will be illustrated with reference to the development and implementation of vaccines against Neisseria meningitidis (the meningococcus) over the 30-year period from the late-1980s to the late-2010s. Nucleotide sequence-based studies have been important in the fight against this aggressive pathogen largely because of its high genetic and antigenic diversity, properties that were only fully appreciated because of sequence-based studies. Five aspects will be considered, the use of sequence data to: (i) discover vaccine antigens; (ii) assess the diversity and distribution of vaccine antigens; (iii) determine the evolutionary and population biology of the organism and their implications for immunization; and (iv) develop molecular approaches to investigate pre- and post-vaccine pathogen populations to assess vaccine impact. One of the great advantages of nucleotide sequence data has been its scalability, which has meant that increasingly large data sets have been available, which has proved invaluable in the investigation of an organism as diverse and enigmatic as the meningococcus.

Longitudinal assessment of anti-PGL-I serology in contacts of leprosy patients in Bangladesh.

BACKGROUND: Despite elimination efforts, the number of Mycobacterium leprae (M. leprae) infected individuals who develop leprosy, is still substantial. Solid evidence exists that individuals living in close proximity to patients are at increased risk to develop leprosy. Early diagnosis of leprosy in endemic areas requires field-friendly tests that identify individuals at risk of developing the disease before clinical manifestation. Such assays will simultaneously contribute to reduction of current diagnostic delay as well as transmission. Antibody (Ab) levels directed against the M.leprae-specific phenolic glycolipid I (PGL-I) represents a surrogate marker for bacterial load. However, it is insufficiently defined whether anti-PGL-I antibodies can be utilized as prognostic biomarkers for disease in contacts. Particularly, in Bangladesh, where paucibacillary (PB) patients form the majority of leprosy cases, anti-PGL-I serology is an inadequate method for leprosy screening in contacts as a directive for prophylactic treatment. METHODS: Between 2002 and 2009, fingerstick blood from leprosy patients' contacts without clinical signs of disease from a field-trial in Bangladesh was collected on filter paper at three time points covering six years of follow-up per person. Analysis of anti-PGL-I Ab levels for 25 contacts who developed leprosy during follow-up and 199 contacts who were not diagnosed with leprosy, was performed by ELISA after elution of bloodspots from filter paper. RESULTS: Anti-PGL-I Ab levels at intake did not significantly differ between contacts who developed leprosy during the study and those who remained free of disease. Moreover, anti-PGL-I serology was not prognostic in this population as no significant correlation was identified between anti-PGL-I Ab levels at intake and the onset of leprosy. CONCLUSION: In this highly endemic population in Bangladesh, no association was observed between anti-PGL-I Ab levels and onset of disease, urging the need for an extended, more specific biomarker signature for early detection of leprosy in this area. TRIAL REGISTRATION: ClinicalTrials.gov ISRCTN61223447.