Stephen Taylor

Anti-group B Streptococcus antibody in infants born to mothers with human immunodeficiency virus (HIV) infection.

Highlights • HIV-infected women have lower anti-GBS surface binding antibody concentrations than uninfected controls with reduced antibody-mediated deposition of complement C3b/iC3b onto GBS bacteria.• HIV-exposed, uninfected infants have a lower concentration of antibody binding to the surface of GBS bacteria as well as a reduction in antibody-mediated deposition of complement C3b/iC3b onto the surface of GBS strains at birth compared to HIV-unexposed infants.• As a result, HIV-exposed uninfected infants may be at increased risk of early and late onset GBS disease compared to unexposed infants.

Identification of vaccine antigens using integrated proteomic analyses of surface immunogens from serogroup B Neisseria meningitidis

UNLABELLEDnMeningococcal surface proteins capable of evoking a protective immune response are candidates for inclusion in protein-based vaccines against serogroup B Neisseria meningitidis (NmB). In this study, a 2-dimensional (2-D) gel-based platform integrating surface and immune-proteomics was developed to characterize NmB surface protein antigens. The surface proteome was analyzed by differential 2-D gel electrophoresis following treatment of live bacteria with proteinase K. Alongside, proteins recognized by immune sera from mice challenged with live meningococci were detected using 2-D immunoblots. In combination, seventeen proteins were identified including the well documented antigens PorA, OpcA and factor H-binding protein, previously reported potential antigens and novel potential immunogens. Results were validated for the macrophage infectivity potentiator (MIP), a recently proposed NmB vaccine candidate. MIP-specific antisera bound to meningococci in whole-cell ELISA and facilitated opsonophagocytosis and deposition of complement factors on the surface of meningococcal isolates of different serosubtypes. Cleavage by proteinase K was confirmed in western blots and shown to occur in a fraction of the MIP expressed by meningococci suggesting transient or limited surface exposure. These observations add knowledge for the development of a protein NmB vaccine. The proteomic workflow presented here may be used for the discovery of vaccine candidates against other pathogens.nnnBIOLOGICAL SIGNIFICANCEnThis study presents an integrated proteomic strategy to identify proteins from N. meningitidis with desirable properties (i.e. surface exposure and immunogenicity) for inclusion in subunit vaccines against bacterial meningitis. The effectiveness of the method was demonstrated by the identification of some of the major meningococcal vaccine antigens. Information was also obtained about novel potential immunogens as well as the recently described potential antigen macrophage infectivity potentiator which can be useful for its consideration as a vaccine candidate. Additionally, the proteomic strategy presented in this study provides a generic 2-D gel-based platform for the discovery of vaccine candidates against other bacterial infections.

Safe laboratory handling of Neisseria meningitidis.

Transmission of Neisseria meningitidis mainly occurs by formation of aerosols generated in the nasopharynx. Working with meningococci in vitro and performing manipulations where aerosols may be generated may result in laboratory acquired infections if appropriate safety precautions are not taken. This review details the practical aspects and experience of safe working with N. meningitidis in the laboratory. The specific risk factor for laboratory-acquired infection is exposure to aerosols containing meningococci. Prevention should therefore focus on the use of microbiology safety cabinets during manipulation of meningococci and this should be reflected in local risk assessments, supported by safe practices of work appropriate facilities and equipment, training/competency and immunisation policies.

Association between functional antibody against Group B Streptococcus and maternal and infant colonization in a Gambian cohort.

Highlights • As maternally-derived anti-GBS antibody increases infant colonization risk decreases.• There is a serotype-specific threshold above which an infant is uncolonised with GBS.• Higher anti-GBS antibody is associated with infant clearance of GBS between birth and 3 months.

Relationships between mucosal antibodies, non-typeable haemophilus influenzae (NTHi) infection and airway inflammation in COPD

Non-typeable Haemophilus influenzae (NTHi) is a key pathogen in COPD, being associated with airway inflammation and risk of exacerbation. Why some patients are susceptible to colonisation is not understood. We hypothesised that this susceptibility may be due to a deficiency in mucosal humoral immunity. The aim of our study (NCT01701869) was to quantify the amount and specificity of antibodies against NTHi in the lungs and the associated risk of infection and inflammation in health and COPD. Phlebotomy, sputum induction and bronchoscopy were performed on 24 mild-to-moderate COPD patients and 8 age and smoking-matched controls. BAL (Bronchoalveolar lavage) total IgG1, IgG2, IgG3, IgM and IgA concentrations were significantly increased in COPD patients compared to controls. NTHi was detected in the lungs of 7 of the COPD patients (NTHi+ve—29%) and these patients had a higher median number of previous exacerbations than NTHi-ve patients as well as evidence of increased systemic inflammation. When comparing NTHi+ve versus NTHi-ve patients we observed a decrease in the amount of both total IgG1 (p = 0.0068) and NTHi-specific IgG1 (p = 0.0433) in the BAL of NTHi+ve patients, but no differences in total IgA or IgM. We observed no evidence of decreased IgG1 in the serum of NTHi+ve patients, suggesting this phenomenon is restricted to the airway. Furthermore, the NTHi+ve patients had significantly greater levels of IL-1β (p = 0.0003), in BAL than NTHi-ve COPD patients.This study indicates that the presence of NTHi is associated with reduced levels and function of IgG1 in the airway of NTHi-colonised COPD patients. This decrease in total and NTHI-specific IgG1 was associated with greater systemic and airway inflammation and a history of more frequent exacerbations and may explain the susceptibility of some COPD patients to the impacts of NTHi.

Bordetella pertussis isolates vary in their interactions with human complement components

Whooping cough is a re-emerging respiratory tract infection. It has become clear that there is a need for better understanding of protective immune responses and variation between Bordetella pertussis strains to aid the development of improved vaccines. In order to survive in the host, B. pertussis has evolved mechanisms to evade complement-mediated killing, including the ability to bind complement-regulatory proteins. Here we evaluate the variation in interactions with the complement system among recently isolated strains. Isolates whose genomes appear highly similar and cluster together on a SNP-based dendrogram were found to vary significantly in resistance to complement-mediated killing and in the deposition of C3b/iC3b, C5b-9 and C1 esterase inhibitor (C1-INH). The key role of Vag8 as a receptor for C1-INH was confirmed and its expression was shown to vary in a panel of isolates. A Vag8 knockout mutant showed increased sensitivity to complement-mediated killing. Antibodies in convalescent sera blocked C1-INH binding to B. pertussis and may play an important role in natural immunity.

A Flow Cytometry Method for Rapidly Assessing Mycobacterium tuberculosis Responses to Antibiotics with Different Modes of Action

ABSTRACT Current methods for assessing the drug susceptibility of Mycobacterium tuberculosis are lengthy and do not capture information about viable organisms that are not immediately culturable under standard laboratory conditions as a result of antibiotic exposure. We have developed a rapid dual-fluorescence flow cytometry method using markers for cell viability and death. We show that the fluorescent marker calcein violet with an acetoxy-methyl ester group (CV-AM) can differentiate between populations of M. tuberculosis growing at different rates, while Sytox green (SG) can differentiate between live and dead mycobacteria. M. tuberculosis was exposed to isoniazid or rifampin at different concentrations over time and either dual stained with CV-AM and SG and analyzed by flow cytometry or plated to determine the viability of the cells. Although similar trends in the loss of viability were observed when the results of flow cytometry and the plate counting methods were compared, there was a lack of correlation between these two approaches, as the flow cytometry analysis potentially captured information about cell populations that were unable to grow under standard conditions. The flow cytometry approach had an additional advantage in that it could provide insights into the mode of action of the drug: antibiotics targeting the cell wall gave a flow cytometry profile distinct from those inhibiting intracellular processes. This rapid drug susceptibility testing method could identify more effective antimycobacterials, provide information about their potential mode of action, and accelerate their progress to the clinic.

Seroprevalence of Antibody-Mediated, Complement-Dependent Opsonophagocytic Activity against Neisseria meningitidis Serogroup B in England

ABSTRACT The correlate of protection for the licensure of meningococcal vaccines is serum bactericidal activity. However, evidence indicates that a complex situation and other mechanisms, such as antibody-mediated, complement-dependent opsonophagocytosis (OP), may play a role in protection and should be investigated in order to understand immunity to this disease. In this study, a high-throughput flow cytometric opsonophagocytic assay (OPA) was optimized. The assay measures the presence of killed fluorescently labeled Neisseria meningitidis within human granulocytes (differentiated HL60 cells) by flow cytometry, using IgG-depleted pooled human plasma as an exogenous source of complement. This method was found to be reliable and correlated with the results of an opsonophagocytic killing assay. The OPA was used to measure OP activity in 1,878 serum samples from individuals ranging from 0 to 99 years of age against N. meningitidis strain NZ98/254 (B:4:P1.7-2,4). The levels of OP activity in individual serum samples varied greatly. OP activity showed an initial peak in the 6- to 12-month age group corresponding to a peak in disease incidence. The OP activity dropped in childhood until the late teenage years, although there was still a higher percentage of individuals with OP activity than with protective bactericidal antibody titers. OP activity reached a peak in the 30- to 39-year age group and then declined. This later peak in OP activity did not coincide with the young adults in whom peak serum bactericidal activity and disease incidence occurred. The demonstration of OP activity when disease incidence is low and when protective bactericidal antibody titers are not detected may indicate a role for OP in protection from meningococcal disease in these age groups.

Antibody-mediated complement C3b/iC3b binding to Group B Streptococcus in paired mother and baby serum samples in a refugee population on the Thailand-Myanmar border.

ABSTRACT Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal sepsis and meningitis. In this study, we determined antibody-mediated deposition of complement C3b/iC3b onto the bacterial cell surface of GBS serotypes Ia, Ib, II, III, and V. This was determined for 520 mother and umbilical cord serum sample pairs obtained at the time of birth from a population on the Thailand-Myanmar border. Antibody-mediated deposition of complement C3b/iC3b was detected to at least one serotype in 91% of mothers, despite a known carriage rate in this population of only 12%. Antibody-mediated C3b/iC3b deposition corresponded to known carriage rates, with the highest levels of complement deposition observed onto the most prevalent serotype (serotype II) followed by serotypes Ia, III, V, and Ib. Finally, neonates born to mothers carrying serotype II GBS at the time of birth showed higher antibody-mediated C3b/iC3b deposition against serotype II GBS than neonates born to mothers with no serotype II carriage. Assessment of antibody-mediated C3b/iC3b deposition against GBS may provide insights into the seroepidemiology of anti-GBS antibodies in mothers and infants in different populations.

SIgA, TGF-β1, IL-10, and TNFα in Colostrum Are Associated with Infant Group B Streptococcus Colonization

Background Group B Streptococcus (GBS) is a major cause of mortality and morbidity in infants and is associated with transmission from a colonized mother at birth and via infected breastmilk. Although maternal/infant colonization with GBS is common, the majority of infants exposed to GBS remain unaffected. The association between breastmilk immune factors and infant colonization and disease prevention has not been elucidated. Objectives We have investigated the association between SIgA and cytokines in breastmilk and infant GBS colonization and clearance. Methods Mother/infant GBS colonization was determined in a prospective cohort of 750 Gambian mother/infant pairs followed to day 89 of life. Anti-GBS secretory IgA bound to the surface of whole bacteria was assessed by flow cytometry and a panel of 12 cytokines quantified by mesoscale discovery in colostrum, breastmilk and serum. Results Compared with infants receiving low anti-GBS SIgA in colostrum, infants receiving high anti-GBS SIgA were at decreased risk of GBS colonization for serotypes III and V. Infants colonized at day 6 were twice as likely to receive colostrum with high TGF-β1, TNFα, IL10, and IL-6 compared to uncolonized infants. Infants receiving high colostral TGF-β1, TNFα, and IL-6 had two-fold enhanced GBS clearance between birth and day 89. Conclusion Our results suggest that the infant GBS colonization risk diminishes with increasing anti-GBS SIgA antibody in breastmilk and that key maternally derived cytokines might contribute to protection against infant colonization. These findings might be leveraged to develop interventions including maternal vaccination that may reduce infant GBS colonization.