From genome to structure and beyond

The genomic era has completely changed the vaccinology landscape. Starting from the genomic repertoire of microbial pathogens it is possible to identify novel antigens expected to induce a potent immunoresponse. Identified antigens can be expressed in different forms, as recombinant proteins, as fusion proteins, in multimeric forms on nanoparticles or into outer membrane to resemble their natural conformation. Immune response induced by the expressed antigens is then analyzed using novel in vitro and in vivo models and immunogenicity and stability improved by structure-based design. Moreover, to accelerate product development, an in-depth analysis of the physical-chemical properties of the antigens is applied starting from early development with the aim to identify the critical attributes to be monitored and controlled during vaccine production and lifecycle, to ensure safety and efficacy of the vaccine product. It is expected that the availability of new and powerful technologies, the rational based design of new vaccines from antigen discovery to formulations, product characterization, immunogenicity in animal models and in humans, registration and lifecycle plans, and proactive interaction with Regulatory Agencies, will result in a fast-track path. The rapid evolution in the field and the new learnings may address most of the present and future challenges in vaccine design and development

The Development of a Vaccine Against Meningococcus B Using Reverse Vaccinology

The discovery of vaccine antigens through whole genome sequencing (WGS) contrasts with the classical hypothesis-driven laboratory-based analysis of microbes to identify components to elicit protective immunity. This radical change in scientific direction and action in vaccine research is captured in the term reverse vaccinology. The complete genome sequence of an isolate of Neisseria meningitidis serogroup B (MenB) was systematically analyzed to identify proteins predicted to be secreted or exported to the outer membrane. This identified hundreds of genes coding for potential surface-exposed antigens. These were amplified, cloned in expression vectors and used to immunize mice. Antisera against 350 recombinant antigens were obtained and analyzed in a panel of immunological assays from which 28 were selected as potentially protective based on the -antibody dependent, complement mediated- serum bactericidal activity assay. Testing of these candidate vaccine antigens, using a large globally representative strain collection of Neisseria species isolated from cases of disease and carriage, indicated that no single component would be sufficient to induce broad coverage and that a “universal” vaccine should contain multiple antigens. The final choice of antigens to be included was based on cross-protective ability, assayed by serum bactericidal activity and maximum coverage of the extensive antigenic variability of MenB strains. The resulting multivalent vaccine formulation selected consisted of three recombinant antigens (Neisserial Heparin Binding Antigen or NHBA, Factor H binding protein or fHbp and Neisseria Adhesin A or NadA). To improve immunogenicity and potential strain coverage, an outer membrane vesicle component obtained from the epidemic New Zealand strain (OMVNz) was added to the formulation to create a four component vaccine, called 4CMenB. A series of phase 2 and 3 clinical trials were conducted to evaluate safety and tolerability and to estimate the vaccine effectiveness of human immune responses at different ages and how these were affected by various factors including concomitant vaccine use and lot-to-lot consistency. 4CMenB was approved in Europe in 2013 and introduced in the National Immunization Program in the UK starting from September 2015 when the vaccine was offered to all newborns using a 2, 4, and 12 months schedule., The effectiveness against invasive MenB disease measured at 11 months after the study start and 5 months after the second vaccination was 83% and there have been no safety concerns

KDEL Receptor (Erd2p)-mediated Retrograde Transport of the Cholera Toxin A Subunit from the Golgi Involves COPI, p23, and the COOH Terminus of Erd2p

A cholera toxin mutant (CTX–K63) unable to raise cAMP levels was used to study in Vero cells the retrograde transport of the toxin A subunit (CTX-A–K63), which possesses a COOH-terminal KDEL retrieval signal. Microinjected GTP-γ-S inhibits the internalization as well as Golgi–ER transport of CTX-A–K63. The appearance of CTX-A–K63 in the Golgi induces a marked dispersion of Erd2p and p53 but not of the Golgi marker giantin. Erd2p is translocated under these conditions most likely to the intermediate compartment as indicated by an increased colocalization of Erd2p with mSEC13, a member of the mammalian coat protein II complex. IgGs as well as Fab fragments directed against Erd2p, β-COP, or p23, a new member of the p24 protein family, inhibit or block retrograde transport of CTX-A–K63 from the Golgi without affecting its internalization or its transport to the Golgi. Anti-Erd2p antibodies do not affect the binding of CTX-A to Erd2p, but inhibit the CTX-K63–induced translocation of Erd2p and p53

1H, 13C and 15N assignment of the C-terminal domain of GNA2132 from Neisseria meningitidis

GNA2132 (Genome-derived Neisseria Antigen 2132) is a surface-exposed lipoprotein discovered by reverse vaccinology and expressed by genetically diverse Neisseria meningitidis strains (Pizza et al. 2000). The protein induces bactericidal antibodies against most strains of Meningococccus and has been included in a multivalent recombinant vaccine against N. meningitidis serogroup B. Structure determination of GNA2132 is important for understanding the antigenic properties of the protein in view of increased efficiency vaccine development. We report practically complete 1H, 13C and 15N assignment of the detectable spectrum of a highly conserved C-terminal region of GNA2132 (residues 245–427) in micellar solution, a medium used to improve the spectral quality. The first 32 residues of our construct up to residue 277 were not visible in the spectrum, presumably because of line broadening due to solvent and/or conformational exchange. Secondary structure predictions based on chemical shift information indicate the presence of an all β-protein with eight β strands

Auto ADP-ribosylation of NarE, a Neisseria meningitidis ADP-ribosyltransferase, regulates its catalytic activities

NarE is an arginine-specific mono-ADPribosyltransferase identified in Neisseria meningitidis that requires the presence of iron in a structured cluster for its enzymatic activities. In this study, we show that NarE can perform auto-ADP-ribosylation. This automodification occurred in a time- and NADconcentration- dependent manner; was inhibited by novobiocin, an ADP-ribosyltransferase inhibitor; and did not occur when NarE was heat inactivated. No reduction in incorporation was evidenced in the presence of high concentrations of ATP, GTP, ADP-ribose, or nicotinamide, which inhibits NAD-glycohydrolase, impeding the formation of free ADP-ribose. Based on the electrophoretic profile of NarE on auto-ADP-ribosylation and on the results of mutagenesis and mass spectrometry analysis, the auto-ADP-ribosylation appeared to be restricted to the addition of a single ADP-ribose. Chemical stability experiments showed that the ADP-ribosyl linkage was sensitive to hydroxylamine, which breaks ADP-ribose-arginine bonds. Sitedirected mutagenesis suggested that the auto-ADP-ribosylation site occurred preferentially on the R7 residue, which is located in the region I of the ADP-ribosyltransferase family. After auto-ADP-ribosylation, NarE showed a reduction in ADP-ribosyltransferase activity, while NAD-glycohydrolase activity was increased. Overall, our findings provide evidence for a novel intramolecular mechanism used by NarE to regulate its enzymatic activities.—Picchianti, M., Del Vecchio, M., Di Marcello, F., Biagini, M., Veggi, D., Norais N., Rappuoli, R., Pizza, M., Balducci, E. Auto ADP-ribosylation of NarE, a Neisseria meningitidis ADP-ribosyltransferase, regulates its catalytic activities. FASEB J

Solution Structure of the Immunodominant Domain of Protective Antigen GNA1870 of Neisseria meningitidis

GNA1870, a 28-kDa surface-exposed lipoprotein of Neisseria meningitidis recently discovered by reverse vaccinology, is one of the most potent antigens of Meningococcus and a promising candidate for a universal vaccine against a devastating disease. Previous studies of epitope mapping and genetic characterization identified residues critical for bactericidal response within the C-terminal domain of the molecule. To elucidate the conformation of protective epitopes, we used NMR spectroscopy to obtain the solution structure of the immunodominant 18-kDa C-terminal portion of GNA1870. The structure consists of an eight-stranded antiparallel beta-barrel overlaid by a short alpha-helix with an unstructured N-terminal end. Residues previously shown to be important for antibody recognition were mapped on loops facing the same ridge of the molecule. The sequence similarity of GNA1870 with members of the bacterial transferrin receptor family allows one to predict the folding of this class of well known bacterial antigens, providing the basis for the rational engineering of high affinity B cell epitopes

Recent advances in meningococcal B disease prevention: real-world evidence from 4CMenB vaccination.

Abstract Objectives 4CMenB is a broadly protective vaccine against invasive meningococcal capsular group B disease (MenB IMD). Licensed worldwide based on immunogenicity and safety data, effectiveness and impact data are now available. We comprehensively reviewed all available real-world evidence gathered from use of 4CMenB since licensure. Results Data from 7 countries provide evidence of effectiveness and impact across different healthcare settings and age-groups, including national/regional immunization programs, observational studies and outbreak control. At least 2 4CMenB doses reduced MenB IMD by 50%-100% in 2-month to 20-year-olds depending on length of follow-up. Estimates of vaccine effectiveness in fully vaccinated cohorts ranged from 59%-100%. The safety profile of 4CMenB administered in real-world settings was consistent with pre-licensure clinical trial data. Conclusion MenB IMD is an uncommon but life-threatening disease with unpredictable epidemiology. The substantial body of data demonstrating 4CMenB effectiveness and impact supports its use in IMD prevention. The results reinforce the importance of direct protection of the highest risk groups; infants/young children and adolescents. Direct protection via routine infant immunization with catch-up in young children and routine adolescent vaccination could be the preferred option for MenB disease control. A Video Abstract linked to this article is available on Figshare: https://doi.org/10.6084/m9.figshare.14546790

NadA3 structures reveal undecad coiled coils and LOX1 binding regions competed by meningococcus B vaccine-elicited human antibodies

Neisseria meningitidis serogroup B (MenB) is a major cause of sepsis and invasive meningococcal disease. A multicomponent vaccine, 4CMenB, is approved for protection against MenB. Neisserial adhesin A (NadA) is one of the main vaccine antigens, acts in host cell adhesion, and may influence colonization and invasion. Six major genetic variants of NadA exist and can be classified into immunologically distinct groups I and II. Knowledge of the crystal structure of the 4CMenB vaccine component NadA3 (group I) would improve understanding of its immunogenicity, folding, and functional properties and might aid antigen design. Here, X-ray crystallography, biochemical, and cellular studies were used to deeply characterize NadA3. The NadA3 crystal structure is reported; it revealed two unexpected regions of undecad coiled-coil motifs and other conformational differences from NadA5 (group II) not predicted by previous analyses. Structure-guided engineering was performed to increase NadA3 thermostability, and a second crystal structure confirmed the improved packing. Functional NadA3 residues mediating interactions with human receptor LOX-1 were identified. Also, for two protective vaccine-elicited human monoclonal antibodies (5D11, 12H11), we mapped key NadA3 epitopes. These vaccine-elicited human MAbs competed binding of NadA3 to LOX-1, suggesting their potential to inhibit host-pathogen colonizing interactions. The data presented provide a significant advance in the understanding of the structure, immunogenicity and function of NadA, one of the main antigens of the multicomponent meningococcus B vaccine.IMPORTANCE The bacterial microbe Neisseria meningitidis serogroup B (MenB) is a major cause of devastating meningococcal disease. An approved multicomponent vaccine, 4CMenB, protects against MenB. Neisserial adhesin A (NadA) is a key vaccine antigen and acts in host cell-pathogen interactions. We investigated the 4CMenB vaccine component NadA3 in order to improve the understanding of its immunogenicity, structure, and function and to aid antigen design. We report crystal structures of NadA3, revealing unexpected structural motifs, and other conformational differences from the NadA5 orthologue studied previously. We performed structure-based antigen design to engineer increased NadA3 thermostability. Functional NadA3 residues mediating interactions with the human receptor LOX-1 and vaccine-elicited human antibodies were identified. These antibodies competed binding of NadA3 to LOX-1, suggesting their potential to inhibit host-pathogen colonizing interactions. Our data provide a significant advance in the overall understanding of the 4CMenB vaccine antigen NadA

Molecular determinants of surface colonisation in diarrhoeagenic Escherichia coli (DEC): from bacterial adhesion to biofilm formation

peer-reviewedEscherichia coli is primarily known as a commensal colonising the gastrointestinal tract of infants very early in life but some strains being responsible for diarrhoea, which can be especially severe in young children. Intestinal pathogenic E. coli include six pathotypes of diarrhoeagenic E. coli (DEC), namely, the (i) enterotoxigenic E. coli, (ii) enteroaggregative E. coli, (iii) enteropathogenic E. coli, (iv) enterohemorragic E. coli, (v) enteroinvasive E. coli and (vi) diffusely adherent E. coli. Prior to human infection, DEC can be found in natural environments, animal reservoirs, food processing environments and contaminated food matrices. From an ecophysiological point of view, DEC thus deal with very different biotopes and biocoenoses all along the food chain. In this context, this review focuses on the wide range of surface molecular determinants acting as surface colonisation factors (SCFs) in DEC. In the first instance, SCFs can be broadly discriminated into (i) extracellular polysaccharides, (ii) extracellular DNA and (iii) surface proteins. Surface proteins constitute the most diverse group of SCFs broadly discriminated into (i) monomeric SCFs, such as autotransporter (AT) adhesins, inverted ATs, heat-resistant agglutinins or some moonlighting proteins, (ii) oligomeric SCFs, namely, the trimeric ATs and (iii) supramolecular SCFs, including flagella and numerous pili, e.g. the injectisome, type 4 pili, curli chaperone-usher pili or conjugative pili. This review also details the gene regulatory network of these numerous SCFs at the various stages as it occurs from pre-transcriptional to post-translocational levels, which remains to be fully elucidated in many cases

Enhancing a multi-purpose artificial urine for culture and gene expression studies of uropathogenic Escherichia coli strains

Aims: The main objective of this study was to modify a recently reported multi-purpose artificial urine (MP-AU) for culture and gene expression studies of uropathogenic Escherichia coli (UPEC) strains. Methods and results: We used liquid chromatography mass spectrometry (LC-MS) to identify and adjust the metabolic profile of MP-AU closer to that of pooled human urine (PHU). Modification in this way facilitated growth of UPEC strains with growth rates similar to those obtained in PHU. Transcriptomic analysis of UPEC strains cultured in enhanced artificial urine (enhanced AU) and PHU showed that the gene expression profiles are similar, with less than 7% of genes differentially expressed between the two conditions. Conclusions: Enhancing an MP-AU with metabolites identified in PHU allows the enhanced AU to be used as a substitute for the culture and in vitro gene expression studies of UPEC strains