Traditionally, vaccines have been developed empirically by isolating, inactivating, and injecting the microorganisms (or portions of them) that cause disease (Table 1; Rappuoli, 2014). Two decades ago, genome sequencing revolutionized this process, allowing for the discovery of novel vaccine antigens starting directly from genomic information. The process was named "reverse vaccinology" to underline that vaccine design was possible starting from sequence information without the need to grow pathogens (Rappuoli, 2000). Indeed, a vaccine against meningococcus B, the first deriving from reverse vaccinology, has recently been licensed (Serruto et al., 2012;O'Ryan et al., 2014). Today, a new wave of technologies in the fields of human immunology and structural biology provide the molecular information that allows for the discovery and design of vaccines against respiratory syncytial virus (RSV) and human CMV (HCMV) that have been impossible thus far and to propose universal vaccines to tackle influenza and HIV infections. Here, we provide our perspective (summarized in Table 1) of how several new advances, some of which have been partially discussed elsewhere (Burton, 2002;Dormitzer et al., 2012;Haynes et al., 2012), can be synergized to become the engine driving what might be considered a new era in vaccinology, an era in which we perform "reverse vaccinology 2.0." Several technological breakthroughs over the past decade have potentiated vaccine design. First, the greatly enhanced ability to clone human B cells and then to produce the corresponding recombinant mAbs or antigen-binding fragments (Fab's) has provided access to an enormously rich set of reagents that allows for the proper evaluation of the protective human immune response to any given immunogen upon immunization or infection. A fundamental step for the success of this approach has been the growing capacity to select the most favorable donors for the isolation of the most potent antibodies (Abs) through extensive examination of serum-functional Ab responses. Second, conformational epitope mapping studies, performed via improved structural biology tools for the three-dimensional characterization of Fab's complexed with their target antigens , can now readily yield the atomic details of protective epitopes recognized by broadly neutralizing Abs (NAbs [bNAbs]). Third, new computational approaches, informed by such structural and immunological data, have enabled the rational design of novel immunogens to specifically elicit a focused immune response targeting the most desirable protective epitopes (Liljeroos et al., 2015). In addition to these advances, a great improvement in RNA sequencing technology has allowed for a massive analysis of the B cell repertoire, providing an accurate overview of the Ab maturation process generated by an infection or vaccination and driving new strategies aimed at priming the B cell precursors expressing germline-encoded Abs in an effective way before initiation of any somatic mutation. Human B cell technologies to identify fu...

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Chlamydia pneumoniae, a human pathogen causing respiratory infections and probably contributing to the development of atherosclerosis and heart disease, is an obligate intracellular parasite which for replication needs to productively interact with and enter human cells. Because of the intrinsic difficulty in working with C. pneumoniae and in the absence of reliable tools for its genetic manipulation, the molecular definition of the chlamydial cell surface is still limited, thus leaving the mechanisms of chlamydial entry largely unknown. In an effort to define the surface protein organization of C. pneumoniae, we have adopted a combined genomicproteomic approach based on (i) in silico prediction from the available genome sequences of peripherally located proteins, (ii) heterologous expression and purification of selected proteins, (iii) production of mouse immune sera against the recombinant proteins to be used in Western blotting and fluorescence-activated cell sorter (FACS) analyses for the identification of surface antigens, and (iv) mass spectrometry analysis of two-dimensional electrophoresis (2DE) maps of chlamydial protein extracts to confirm the presence of the FACS-positive antigens in the chlamydial cell. Of the 53 FACS-positive sera, 41 recognized a protein species with the expected size on Western blots, and 28 of the 53 antigens shown to be surface-exposed by FACS were identified on 2DE maps of elementary-body extracts. This work represents the first systematic attempt to define surface protein organization in C. pneumoniae.Chlamydia pneumoniae is an obligate intracellular bacterium and a common human pathogen (48). It is a significant cause of pneumonia in both hospital and outpatient settings, accounting for approximately 7 to 10% of cases of community-acquired pneumonia among adults. C. pneumoniae has also been associated with atherosclerotic and cardiovascular disease, as suggested by results of seroepidemiologic studies, detection of the organism in atherosclerotic plaque specimens, experimental in vitro cell culture studies, animal model studies, and two small secondary prevention antibiotic treatment trials (12,13,15,19,20,28,45).Like all obligate intracellular parasites, for its survival and propagation C. pneumoniae must accomplish several essential tasks which include adhering to and entering host cells, creating an intracellular niche for replication, exiting host cells for subsequent invasion of neighboring cells, and also avoiding host defense mechanisms. To carry out all these functions, C. pneumoniae has developed a unique biphasic life cycle involving two developmental forms, a spore-like infectious form (elementary bodies [EBs]) and an intracelluar replicative form (reticulate bodies [RBs]). Adhesion, host cell colonization capabilities, and the ability to cope with host defense mechanisms when outside the cell presumably rely in large part on EB surface organization.Because of the intrinsic difficulty in working with C. pneumoniae and the lack of adequate methods for its genetic ma...

4CMenB is the first broad coverage vaccine for the prevention of invasive meningococcal disease caused by serogroup B strains. To gain a comprehensive picture of the antibody response induced upon 4CMenB vaccination and to obtain relevant translational information directly from human studies, we have isolated a panel of human monoclonal antibodies from adult vaccinees. Based on the Ig-gene sequence of the variable region, 37 antigen-specific monoclonal antibodies were identified and produced as recombinant Fab fragments, and a subset also produced as full length recombinant IgG1 and functionally characterized. We found that the monoclonal antibodies were cross-reactive against different antigen variants and recognized multiple epitopes on each of the antigens. Interestingly, synergy between antibodies targeting different epitopes enhanced the potency of the bactericidal response. This work represents the first extensive characterization of monoclonal antibodies generated in humans upon 4CMenB immunization and contributes to further unraveling the immunological and functional properties of the vaccine antigens. Moreover, understanding the mechanistic nature of protection induced by vaccination paves the way to more rational vaccine design and implementation.

SummaryNeutral endopeptidase (NEP; EC. 3.4.24.11) is a type 2 cell surface metalloprotease known by a variety of eponyms, including enkephalinase, common acute lymphoblastic leukemia antigen, and CD10. Identified substrates are largely neural or humoral oligopeptide agonists, and the enzyme functions to terminate signaling by degrading the ligand, analogously to acetylcholine/acetylcholinesterase. Targeted disruption of the NEP locus in mice results in enhanced lethality to endotoxin shock with a pronounced gene dosage effect. The site(s) of action appears downstream from release of tumor necrosis factor and interleukin-1 since NEP-deficient animals demonstrate increased sensitivity to these mediators as well. This unexpected finding indicates an important protective role for NEP in septic shock. p eptide signaling molecules are controlled at the level of synthesis and release. Another level of control can exist through their degradation, analogous to the elimination of acetylcholine by acetylcholinesterase. Neutral endopeptidase (NEP) appears particularly well suited as a model system for studies of proteolytic control of biologically active peptides (1). NEP is a member of a newly recognized family of metalloproteinases that includes the endothelin-converting enzyme and the Kjell blood group antigen (2). The NEP enzyme is colocalized with peptidergic neurons in the central nervous system, along mucosal epithelial surfaces, and in the brush border membrane of the kidney. Pre-B cell expression of NEP was first noted as the common acute lymphoblastic leukemia antigen, a marker for a childhood leukemia (3, 4). Inflammatory cells such as the PMN are also positive for NEP expression (5).Highly specific, rationally designed NEP inhibitors such as thiorphan (6) have been used as pharmacologic tools, and they potentiate the activity of particular peptide agonists, in some cases, by several orders of magnitude. Such studies have identified two broad classes of substrates subject to NEP control in vivo, neuropeptides including enkephalins, tachykinins, and bombesin-like peptides (7-9), and humoral mediators including bradykinin, atriopeptin, and endothelins (10-12).In the present report, we characterize mice that bear a targeted disruption of the NEP gene in two models of shock. The models were chosen because of the wide variety of potential NEP substrates that may be present in septic shock. Materials and MethodsGene Targeting of the NEP Locus. A murine Sv 129 genomic library was screened with the human NEP cDNA, resulting in multiple overlapping clones. A 6.5-kb genomic fragment containing exons 10-14 was digested with Bglll, and the 0.6-kb fragment was replaced with a neomycin resistance cassette driven by the glucokinase promoter.The targeting fragment was subcloned into the pPNT vector (13) for positive/negative selection with geneticin and gancyclovir. Electroporation into J1 ES cells (,~107 cells, 16 #g targeting vector, 400 V at 25/tF) was followed by plating on 75 cm 2 of neomycinresistant primary embryonic fibr...

SummaryBackgroundApproximately 164,000 deaths yearly are due to shigellosis, primarily in developing countries. Thus, a safe and affordable Shigella vaccine is an important public health priority. The GSK Vaccines Institute for Global Health (GVGH) developed a candidate Shigella sonnei vaccine (1790GAHB) using the Generalized Modules for Membrane Antigens (GMMA) technology. The paper reports results of 1790GAHB Phase 1 studies in healthy European adults.MethodsTo evaluate the safety and immunogenicity profiles of 1790GAHB, we performed two parallel, phase 1, observer-blind, randomized, placebo-controlled, dose escalation studies in France (“study 1”) and the United Kingdom (“study 2”) between February 2014 and April 2015 (ClinicalTrials.gov, number NCT02017899 and NCT02034500, respectively) in 18–45 years old subjects (50 in study 1, 52 in study 2). Increasing doses of Alhydrogel adsorbed 1790, expressed by both O Antigen (OAg) and protein quantity, or placebo were given either by intramuscular route (0.059/1, 0.29/5, 1.5/25, 2.9/50, 5.9/100 μg of OAg/μg of protein; study 1) or by intradermal (ID), intranasal (IN) or intramuscular (IM) route of immunization (0.0059/0.1, 0.059/1, 0.59/10 μg ID, 0.29/5, 1.2/20, 4.8/80 μg IN and 0.29/5 μg IM, respectively; study 2). In absence of serologic correlates of protection for Shigella sonnei, vaccine induced immunogenicity was compared to anti-LPS antibody in a population naturally exposed to S. sonnei.FindingsVaccines were well tolerated in both studies and no death or vaccine related serious adverse events were reported. In study 1, doses ≥ 1.5/25 μg elicited serum IgG median antibody greater than median level in convalescent subjects after the first dose. No vaccine group in study 2 achieved median antibody greater than the median convalescent antibody.InterpretationIntramuscularly administered Shigella sonnei GMMA vaccine is well tolerated, up to and including 5.9/100 μg and induces antibody to the OAg of at least the same magnitude of those observed following natural exposure to the pathogen. Vaccine administered by ID or IN, although well tolerated, is poorly immunogenic at the doses delivered. The data support the use of the GMMA technology for the development of intramuscular multivalent Shigella vaccines.

For broad protection against infection by viruses such as influenza or HIV, vaccines should elicit antibodies that bind conserved viral epitopes, such as the receptor-binding site (RBS). RBS-directed antibodies have been described for both HIV1–3 and influenza virus4–8, and the design of immunogens to elicit them is a goal of vaccine research in both fields. Residues in the RBS of influenza virus hemagglutinin (HA) determine a preference for the avian or human receptor, α -2,3-linked sialic acid and α -2,6-linked sialic acid, respectively9,10. Transmission of an avian-origin virus between humans generally requires one or more mutations in the sequences encoding the influenza virus RBS to change the preferred receptor from avian to human9,11,12, but passage of a human-derived vaccine candidate in chicken eggs can select for reversion to avian receptor preference13–15. For example, the X-181 strain of the 2009 new pandemic H1N1 influenza virus, derived from the A/California/07/2009 isolate and used in essentially all vaccines since 2009, has arginine at position 226, a residue known to confer preference for an α -2,3 linkage in H1 subtype viruses13,14; the wild-type A/California/07/2009 isolate, like most circulating human H1N1 viruses, has glutamine at position 226. We describe, from three different individuals, RBS-directed antibodies that recognize the avian-adapted H1 strain in current influenza vaccines but not the circulating new pandemic 2009 virus; Arg226 in the vaccine-strain RBS accounts for the restriction. The polyclonal sera of the three donors also reflect this preference. Therefore, when vaccines produced from strains that are never passaged in avian cells become widely available, they may prove more capable of eliciting RBS-directed, broadly neutralizing antibodies than those produced from egg-adapted viruses, extending the established benefits of current seasonal influenza immunizations.

Protection against influenza is mediated by neutralizing antibodies, and their induction at high and sustained titers is key for successful vaccination. Optimal B cells activation requires delivery of help from CD4 + T lymphocytes. In lymph nodes and tonsils, T-follicular helper cells have been identified as the T cells subset specialized in helping B lymphocytes, with interleukin-21 (IL-21) and inducible costimulatory molecule (ICOS1) playing a central role for this function. We followed the expansion of antigen-specific IL-21 + CD4 + T cells upon influenza vaccination in adults. We show that, after an overnight in vitro stimulation, influenza-specific IL-21 + CD4 + T cells can be measured in human blood, accumulate in the CXCR5 − ICOS1 + population, and increase in frequency after vaccination. The expansion of influenza-specific ICOS1 + IL-21 + CD4 + T cells associates with and predicts the rise of functionally active antibodies to avian H5N1. We also show that blood-derived CXCR5 − ICOS1 + CD4 + T cells exert helper function in vitro and support the differentiation of influenza specific B cells in an ICOS1-and IL-21-dependent manner. We propose that the expansion of antigen-specific ICOS1 + IL-21 + CD4 + T cells in blood is an early marker of vaccine immunogenicity and an important immune parameter for the evaluation of novel vaccination strategies.CD4 help | predictivity | humoral response T o confer protection, human vaccines rely on the induction of neutralizing antibodies and on the generation of a pool of memory lymphocytes able to mount an accelerated response upon encounter with the target pathogen. In recent years, novel vaccines, adjuvants, and delivery systems that are able to improve vaccine immunogenicity while reducing their reactogenicity have been developed. As vaccines are given to healthy subjects, their development is a challenging endeavor that requires extensive studies to assess safety, immunogenicity, and clinical efficacy. To accelerate the screening of novel candidates, research has focused on the identification of early biomarkers, molecular and transcriptional signatures predicting vaccine efficacy (1). Predictors should be easy to test in large clinical trials and have a clear mechanistic relationship with the correlates or surrogates of protection taken as the study endpoint. We have previously shown that an early postvaccination increase in the number of vaccine-specific CD4 + T cells is correlated in a predictive manner with the rise and long-term maintenance of protective antibody titers to avian influenza (2). The aim of the present study was to characterize the CD4 + T cells subset responsible for this function.T follicular helper (Tfh) cells have been identified in lymph nodes and tonsils as the CD4 + T cells subpopulation specialized in providing help to B cells (3-11). The recent identification of a circulating counterpart of this T cells subset in blood led us to investigate whether vaccine-specific IL-21 + CD4 + T cells are detectable in human blood, if their frequency is...