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The development of vaccines for prevention of diseases caused by pathogenic species can encounter major obstacles if high sequence diversity is observed between individual strains. Therefore, development might be restricted either to conserved antigens, which are often rare, or to multivalent vaccines, which renders the production more costly and cumbersome. In light of this complexity, we applied a structure-based surface shaping approach for the development of a Lyme borreliosis (LB) vaccine suitable for the United States and Europe. The surface of the C-terminal fragment of outer surface protein A (OspA) was divided into distinct regions, based primarily on binding sites of monoclonal antibodies (MAbs). In order to target the six clinically most relevant OspA serotypes (ST) in a single protein, exposed amino acids of the individual regions were exchanged to corresponding amino acids of a chosen OspA serotype. Six chimeric proteins were constructed, and, based on their immunogenicity, four of these chimeras were tested in mouse challenge models. Significant protection could be demonstrated for all four proteins following challenge with infected ticks (OspA ST1, OspA ST2, and OspA ST4) or with in vitro-grown spirochetes (OspA ST1 and OspA ST5). Two of the chimeric proteins were linked to form a fusion protein, which provided significant protection against in vitro-grown spirochetes (OspA ST1) and infected ticks (OspA ST2). This article presents the proof-of-concept study for a multivalent OspA vaccine targeting a wide range of pathogenic LB Borrelia species with a single recombinant antigen for prevention of Lyme borreliosis.

Summary Background Chikungunya disease, which results in incapacitating arthralgia, has been reported worldwide. We developed a live-attenuated chikungunya virus (CHIKV) vaccine candidate designed for active immunisation of the general population living in endemic regions, as well as serving as a prophylactic measure for travellers to endemic areas. Methods This single-blind, randomised, dose-escalation, phase 1 study investigated as primary outcome safety of a live-attenuated CHIKV vaccine candidate. At two professional clinical trial centres in Illinois and Alabama, USA, healthy volunteers aged 18–45 years were randomly assigned (1:1:2) to one of three escalating dose groups (low dose 3·2 × 103 per 0·1 mL; medium dose 3·2 × 104 per 1 mL; or high dose 3·2 × 105 50% tissue culture infection dose per 1 mL) and received a single-shot immunisation on day 0. Individuals in all groups were revaccinated with the highest dose on either month 6 or 12, and followed up for 28 days after revaccination. The safety analysis included all individuals who received the single vaccination; the immunogenicity analysis, which was a secondary outcome, included all individuals who completed the study without major protocol deviations (per-protocol population). The study is registered with ClinicalTrials.gov , NCT03382964 , and is complete. Findings The study was done between March 5, 2018, and Jul 23, 2019, with 120 adults recruited and enrolled between March 5 and June 21, 2018, and assigned to receive a low (n=31), medium (n=30), or high (n=59) dose of the vaccine. The vaccine was safe in the high-dose group and well tolerated in the low-dose and medium-dose groups. Four (7%) of 59 vaccinees in the high-dose group reported any local reaction, and 11 (36%), 12 (40%), and 40 (68%) volunteers in the low-dose, medium-dose, and high-dose groups, respectively, reported any solicited systemic reaction. No vaccine-related serious adverse events were reported. Data up to month 12 after a single immunisation of the 120 healthy volunteers showed a good immunogenicity profile with 100% seroconversion rates achieved at day 14 (103 [100%] of 103) and sustained for 1 year across all dose groups. Mean peak antibody titres at day 28 ranged from 592·6 to 686·9 geometric mean titres from the low-dose to high-dose groups, respectively. A single vaccination was sufficient to induce sustaining high-titre neutralising antibodies, as shown by the absence of an anamnestic response after any revaccination ranging from 94% to 100% of participants. Following revaccination, vaccinees were protected from vaccine-induced viraemia. Interpretation A novel live-attenuated CHIKV vaccine was well tolerated and highly immunogenic in an adult population and could be an effective intervention for prophylaxis of chikungunya disease worldwide. Funding Valneva, Vienna, Austria; Coalition for Epidemic Preparedness Innovation and EU Horizon 2020.

Background: Chikungunya disease has been reported worldwide resulting in incapacitating arthralgia. We developed a live-attenuated Chikungunya virus vaccine candidate designed for active immunization of the general population living in endemic regions, as well as serving as a prophylactic measure for travelers to endemic areas. Methods: An observer-blinded, randomized, dose-escalation phase 1 clinical study investigated safety, immunogenicity and antibody persistence. Healthy volunteers aged 18 to 45 years were randomly assigned 1:1:2 to one of three escalating dose groups (low 3·2×103/0·1mL, medium 3·2×104 /1mL or high dose 3·2×105 TCID50/1mL) and received a single-shot immunization on Day 0. Half of individuals in Group H were challenged with the highest dose on Month 6 and followed up for 28 days post-challenge. Findings: Data up to Month 6 after a single immunization of 120 healthy volunteers showed an excellent immunogenicity profile with 100% seroconversion rates already achieved at Day 14 in all dose groups. Mean peak antibody titers at Day 28 range from 592·6 to 686·9 GMT from Groups L to H, respectively, with maximum titers reaching 2560. A single vaccination is sufficient to induce sustaining high titer neutralizing antibodies, as demonstrated by the absence of an anamnestic response following challenge and the development of sterilizing immunity (96·2% of participants). The vaccine was generally safe and well tolerated in the low and medium dose group, with both doses demonstrating a superior reactogenicity profile compared to the high dose group. Following challenge, vaccinees were protected from vaccine induced viremia. No adverse events of special interest and no vaccine related serious adverse events were reported. Interpretation: A novel live-attenuated CHIKV vaccine was well tolerated and highly immunogenic in an adult population and could be an effective intervention for prophylaxis of Chikungunya disease worldwide supporting further development. Trial Registration: https://clinicaltrials.gov NCT03382964. Funding Statement: Valneva Austria GmbH, Vienna, Austria. Declaration of Interests: NW, RH, OZ, AF, NB, KL, MS, UL, AM, AP, SEL, KD and WB are Valneva employees and own stock and share options in Valneva. All other authors declare that they have no competing interests. Ethics Approval Statement: Ethics committee approval has been obtained prior any study related assessments occurred. The study was compliant with the current International Council on Harmonisation/Good Clinical Practice guidelines and in accordance with the principles set forth in the Declaration of Helsinki. Throughout the study an independent data safety monitoring board consisting of four external medical experts performed periodic reviews of accruing safety information. All enrolled individuals provided their written informed consent prior to any study-related procedure.

The etiological diagnosis of infection by Streptococcus pneumoniae in children is difficult, and the use of indirect techniques is frequently warranted. We aimed to study the use of pneumococcal proteins for the serological diagnosis of pneumococcal infection in children with pneumonia. We analyzed paired serum samples from 13 Brazilian children with invasive pneumococcal pneumonia (positive control group) and 23 Finnish children with viral pharyngitis (negative control group), all aged

Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are common causative agents of respiratory infections. Pneumococcal conjugate vaccines have been introduced recently, but their effect on the natural immunity against protein antigens from these pathogens has not been elucidated.This was an age-matched observational controlled study that evaluated the influence of 10-valent pneumococcal conjugate vaccines on the levels of antibodies and frequencies of antibody responses against proteins from S. pneumoniae, H. influenzae and M. catarrhalis in serum samples of children with community-acquired pneumonia. Eight pneumococcal proteins (pneumolysin, choline-binding protein A, pneumococcal surface protein A families 1 and 2, pneumococcal choline-binding protein A, pneumococcal histidine triad protein D, serine/threonine protein kinase, protein required for cell wall separation of group B streptococcus), 3 proteins from H. influenzae (including protein D) and 5 M. catarrhalis proteins were investigated.The study group comprised 38 vaccinated children and 114 age-matched controls (median age: 14.5 vs. 14.6 months, respectively; P = 0.997), all with community-acquired pneumonia. There was no difference on clinical baseline characteristics between vaccinated and unvaccinated children. Vaccinated children had significantly lower levels of antibodies against 4 of the studied pneumococcal antigens (P = 0.048 for Ply, P = 0.018 for pneumococcal surface protein A, P = 0.001 for StkP and P = 0.028 for PcsB) and higher levels of antibodies against M. catarrhalis (P = 0.015). Nevertheless, the vaccination status did not significantly affect the rates of antibody responses against S. pneumoniae, H. influenzae and M. catarrhalis.In spite of the differences that have been found on the level of natural antibodies, no effect from pneumococcal vaccination was observed on the rate of immune responses associated with community-acquired pneumonia against protein antigens from S. pneumoniae, H. influenzae and M. catarrhalis.

Lyme borreliosis (LB) is the most common vector-borne disease in the northern hemisphere and there is no vaccine available for disease prevention. The majority of LB cases in Europe are caused by four different Borrelia species expressing six different OspA serotypes, whereas in the US only one of these serotypes is present. Immunization with the outer surface protein A (OspA) can prevent infection and the C-terminal part of OspA is sufficient for protection against infection transmitted by Ixodes ticks. Here we show that the order of the stabilized monomeric OspA fragments making up the heterodimers in our LB vaccine does not influence the induced immunogenicity and protection. Using bioinformatics analysis (surface electrostatics), we have designed an improved version of an LB vaccine which has an increased immunogenicity for OspA serotype 3 and an optimized expression and purification profile. The OspA heterodimers were highly purified with low amounts of endotoxin, host cell proteins and host cell DNA. All three proteins were at least 85% triacylated which ensured high immunogenicity. The LB vaccine presented here was designed, produced and characterized to a level which warrants further development as a second generation human LB vaccine.

Chikungunya virus (CHIKV) is rapidly spreading across the globe, and millions are infected. Morbidity due to this virus is a serious threat to public health, but at present, there is no vaccine against this debilitating disease. We have recently developed a number of vaccine candidates, and here we have evaluated 3 of them in a nonhuman primate model. A single immunization with an attenuated strain of CHIKV (Δ5nsP3), a homologous prime-boost immunization with a DNA-launched RNA replicon encoding CHIKV envelope proteins (DREP-E), and a DREP-E prime followed by a recombinant modified vaccinia virus Ankara encoding CHIKV capsid and envelope (MVA-CE) boost all induced protection against WT CHIKV infection. The attenuated Δ5nsP3 virus proved to be safe and did not show any clinical signs typically associated with WT CHIKV infections such as fever, skin rash, lymphopenia, or joint swelling. These vaccines are based on an East/Central/South African strain of Indian Ocean lineage, but they also generated neutralizing antibodies against an isolate of the Asian genotype that now is rapidly spreading across the Americas. These results form the basis for clinical development of an efficacious CHIKV vaccine that generates both humoral and cellular immunity with long-term immunological memory.

We have previously shown that the Outer surface protein A (OspA) based Lyme borreliosis vaccine VLA15 induces protective immunity in mice. Herein, we report the induction of protective immunity by VLA15 with mouse models using ticks infected with B. burgdorferi (OspA serotype 1), B. afzelii (OspA serotype 2) and B. bavariensis (OspA serotype 4) or with in vitro grown B. garinii (OspA serotype 5 and 6) for challenge. For B. garinii (OspA serotype 3), we have developed a growth inhibition assay using chicken complement and functional antibodies targeting B. garinii (OspA serotype 3) could be demonstrated after immunization with VLA15. Furthermore, following three priming immunizations, a booster dose was administered five months later and the induction of immunological memory could be confirmed. Thus, the antibody titers after the booster dose were increased considerably compared to those after primary immunization. In addition, the half-lives of anti-OspA serotype specific antibodies after administration of the booster immunization were longer than after primary immunization. Taken together, we could show that VLA15 induced protection in mice against challenge with four different clinically relevant Borrelia species (B. burgdorferi, B. afzelii, B. garinii and B. bavariensis) expressing five of the six OspA serotypes included in the vaccine. The protection data is supported by functional assays showing efficacy against spirochetes expressing any of the six OspA serotypes (1 to 6). To our knowledge, this is the first time a Lyme borreliosis vaccine has been able to demonstrate such broad protection in preclinical studies. These new data provide further promise for the clinical development of VLA15 and supports our efforts to provide a new Lyme borreliosis vaccine available for global use.

Nosocomial infections, also called "hospital acquired infections," occur worldwide and affect both developed and resource-poor countries, thus having a major impact on their health care systems. Klebsiella pneumoniae, which is an opportunistic Gram-negative pathogen, is responsible for causing pneumonia, urinary tract infections and septicemia in immune compromised hosts such as neonates. Unfortunately, there is no vaccine or mAb available for prophylactic or therapeutic use against K. pneumoniae infections. For this reason, we sought for a protein-based subunit vaccine capable of combating K. pneumoniae infections, by applying our ANTIGENome technology for the identification of potential vaccine candidates, focusing on conserved protein antigens present in strains with different serotypes. We identified numerous novel immunogenic proteins using genomic surface display libraries and human serum antibodies from donors exposed to or infected by K. pneumoniae. Vaccine candidate antigens were finally selected based on animal protection in a murine lethal-sepsis model. The protective and highly conserved antigens identified in this study are promising candidates for the development of a protein-based vaccine to prevent infection by K. pneumoniae.

Objective: Community-acquired pneumonia is an important cause of morbidity in childhood, but the detection of its causative agent remains a diagnostic challenge. The authors aimed to evaluate the role of the chest radiograph to identify cases of community-aquired pneumonia caused by typical bacteria. Methods: The frequency of infection by Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis was compared in non-hospitalized children with clinical diagnosis of community acquired pneumonia aged 2–59 months with or without radiological confirmation (n = 249 and 366, respectively). Infection by S. pneumoniae was diagnosed by the detection of a serological response against at least one of eight pneumococcal proteins (defined as an increase ≥2-fold in the IgG levels against Ply, CbpA, PspA1 and PspA2, PhtD, StkP-C, and PcsB-N, or an increase ≥1.5-fold against PcpA). Infection by H. influenzae and M. catarrhalis was defined as an increase ≥2-fold on the levels of microbe-specific IgG. Results: Children with radiologically confirmed pneumonia had higher rates of infection by S. pneumoniae. The presence of pneumococcal infection increased the odds of having radiologically confirmed pneumonia by 2.8 times (95% CI: 1.8–4.3). The negative predictive value of the normal chest radiograph for infection by S. pneumoniae was 86.3% (95% CI: 82.4–89.7%). There was no difference on the rates of infection by H. influenzae and M. catarrhalis between children with community-acquired pneumonia with and without radiological confirmation. Conclusions: Among children with clinical diagnosis of community-acquired pneumonia submitted to chest radiograph, those with radiologically confirmed pneumonia present a higher rate of infection by S. pneumoniae when compared with those with a normal chest radiograph. Resumo: Objetivos: O objetivo deste estudo foi avaliar o papel do raio-X de tórax na identificação de casos de pneumonia adquirida na comunidade (PAC) causada por agentes bacterianos. Métodos: A frequência de infecção por Streptococcus pneumoniae, Haemophilus influenzae e Moraxella catarrhalis em crianças com PAC não hospitalizadas foi comparada com a presença de confirmação radiológica da pneumonia (n = 249 crianças com pneumonia radiologicamente confirmada e 366 crianças com raio X de tórax normal). Infecção por S. pneumoniae foi diagnosticada com base na resposta sorológica a pelo menos uma dentre oito proteínas pneumocócicas investigadas (aumento ≥2 vezes nos níveis de IgG em relação a Ply, CbpA, PspA1 e 2, PhtD, StkP-C e PcsB-N ou aumento≥1,5 vezes em relação aPcpA). Infecção por H. influenzae e M. catarrhalis foi definida por aumento≥2 vezes nos níveis de IgG específica a antígenos de cada agente. Resultados: Crianças com pneumonia radiologicamente confirmada apresentaram maior taxa de infecção pelo pneumococo. Além disso, a presença de infecção pneumocócica foi um fator preditor de pneumonia radiologicamente confirmada, aumentando sua chance de detecção em 2,8 vezes (IC 95%: 1,8-4,3). O valor preditivo negative do raio X normal para a infecção por S. pneumoniae foi 86,3% (IC95%: 82,4%-89,7%). Não houve diferença nas frequências de infecção por H. influenzae e M. catarrhalis entre crianças com PAC com ou sem confirmação radiológica. Conclusões: Crianças com diagnóstico clínico de PAC submetidas a um raio X de tórax que apresentam confirmação radiológica tem maior taxa de infecção por S. pneumoniae, comparado às crianças com raio X normal. Keywords: Bacterial infection, Etiology, Lower respiratory tract infection, Radiological study, Serological tests, Palavras-chave: Infecção bacteriana, Etiologia, Infecção do trato respiratório inferior, Estudo radiológico, Testes sorológicos

Bahia State Agency for Research Funding (FAPESB), Brazil; Brazilian Council for Scientific and Technological Development (CNPq), Brazil; and in part by National Institute for Health

Moraxella catarrhalis, a Gram-negative bacterium, is an important respiratory pathogen causing acute otitis media and exacerbations of chronic obstructive pulmonary disease. Adhesion of the pathogen to human epithelial cells is mediated via bacterial membrane adhesin proteins. To identify the surface proteome of Moraxella catarrhalis, we applied different membrane protein extraction methods in combination with different proteomic technologies. Proteins from preparations of outer membrane vesicles and from carbonate extractions were analyzed using either a gel-based nano-HPLC-MS/MS technique or 2D-LC-MS/MS. Furthermore, because glycosaminoglycans (GAGs) play an important role for microbial entry into human cells, the GAG-binding membranome of Moraxella catarrhalis was investigated using a glycan-based pull-down approach. By these means, potential vaccine protein candidates that were previously selected by the ANTIGENome technology were confirmed, but importantly also novel proteins were identified as candidates.

The majority of vaccine candidates in clinical development are highly purified proteins and peptides relying on adjuvants to enhance and/or direct immune responses. Despite the acknowledged need for novel adjuvants, there are still very few adjuvants in licensed human vaccines. A vast number of adjuvants have been tested pre-clinically using different experimental conditions, rendering it impossible to directly compare their activity. We performed a head-to-head comparison of five different adjuvants Alum, MF59®, GLA-SE, IC31® and CAF01 in mice and combined these with antigens from M. tuberculosis, influenza and chlamydia to test immune-profiles and efficacy in infection models using standardized protocols. Regardless of antigen, each adjuvant had a unique immunological signature suggesting that the adjuvants have potential for different disease targets. Alum increased antibody titers; MF59® induced strong antibody and IL-5 responses; GLA-SE induced antibodies and Th1; CAF01 showed a mixed Th1/Th17 profile and IC31® induced strong Th1 responses. MF59® and GLA-SE were strong inducers of influenza HI titers while CAF01, GLA-SE and IC31® enhanced protection to TB and chlamydia. Importantly, this is the first extensive attempt to categorize clinical-grade adjuvants based on their immune profiles and protective efficacy to inform a rational development of next generation vaccines for human use.

The three Borrelia species, Borrelia afzelii, Borrelia burgdorferi and Borrelia garinii are the main species causing the most common tick-borne zoonosis, Lyme borreliosis. By applying a genomic approach relying on human antibodies we have identified 122 antigenic Borrelia proteins associated with Lyme borreliosis, including already known and published protective antigens. The heterogeneity of the Borrelia species causing Lyme borreliosis makes the search for conserved antigens providing broad protection challenging. Using several in vitro assays we narrowed down the selection to 15 vaccine candidates. These antigens were further analyzed for antigenicity and cross-reactivity using sera from mice infected with the three pathogenic Borrelia species. All antigens analyzed showed a high degree of cross-reactivity between the three Borrelia species, essential for providing cross-protection. We also investigated whether mice infected with B. afzelii through tick exposure are primed to mount cytokine responses. For a selection of these antigens, we observed preferentially a pro-inflammatory response in C3H/HeN mice, while in contrast also a type 2 T cell response was seen in the Borrelia-resistant mouse strain BALB/c. Thus, antigens mounting a type 2 or mixed type 2/type 1 T cell response might be preferred vaccine candidates for evaluation in animal models of Lyme borreliosis.

Indwelling central venous catheters are a common and important source of nosocomial Staphylococcus epidermidis and S. aureus infections, causing increased morbidity and mortality during hospitalization. A model was developed to reflect the clinical situation of catheter colonization by transient hematogeneously spread staphylococci, in order to investigate potential vaccine candidates. Rats were cannulated in the right jugular vein, followed by challenge through the tail vein with either S. epidermidis RP62a, or S. aureus Becker. At 24 hr post challenge, colonizing bacteria were found to be present on the catheter in an early biofilm, as evidenced by the presence of polysaccharide intercellular adhesin (PIA). For vaccination studies, rats were first immunized, surgically cannulated, and then challenged via the tail vein. At 24 hr post challenge, the catheters were harvested and cultured on mannitol salt agar plates. The catheters were scored as positive if there was outgrowth of bacterial colonies, and negative if no colonies were observed. A S. epidermidis antigen (SERP0630, MenD), and a S. aureus antigen (SACOL1138, iron regulated surface determinant B, IsdB) were found to have significant protective activity in this model, compared to mock immunized controls. Using SERP0630 as the test immunogen, it was also determined that a single vaccination of rats after cannulation was sufficient for significant catheter protection. This model may be used to evaluate antigens for protective activity against transient hematogenous spread of staphylococci resulting in catheter colonization and early biofilm formation.

Protein C deletion mutants of West Nile virus (WNV) were evaluated for their potential use as live virus vaccine candidates in vivo. Double and triple mutants carrying small deletions and second-site point mutations, as well as mutants with large deletions of 36 and 37 amino acid residues were tested in a stringent mouse challenge model. The mutant viruses were found to be non-pathogenic and to induce protective immunity in a wide range of inoculation doses (101–106 FFU). Furthermore, the effects of combining three different previously identified resuscitating point mutations, as well as the combination of a large protein C deletion with a deletion mutation in the 3′ non-coding region were studied. The data indicate that the production of safe and efficacious WNV live vaccines based on protein C deletion mutations is feasible.

Streptococcus pyogenes is one of the most common human pathogens and possesses diverse mechanisms to evade the human immune defence. One example of its immune evasion is the degradation of the chemokine IL (interleukin)-8 by ScpC, a serine proteinase that prevents the recruitment of neutrophils to an infection site. By applying the ANTIGENome technology and using human serum antibodies, we identified Spy0416, annotated as ScpC, as a prominent antigen that induces protective immune responses in animals. We demonstrate here for the first time that the recombinant form of Spy0416 is capable of IL-8 degradation in vitro in a concentration- and time-dependent manner. Mutations in the conserved amino acid residues of the catalytic triad of Spy0416 completely abolished in vitro activity. However, the isolated predicted proteinase domain does not exhibit IL-8-degrading activity, but is dependent on the presence of the C-terminal region of Spy0416. Binding to IL-8 is mainly mediated by the catalytic domain. However, the C-terminal region modulates substrate binding, indicating that the proteolytic activity is amenable to regulation via the non-catalytic regions. The specificity for human substrates is not restricted to IL-8, since we also detected in vitro protease activity for another CXC chemokine GRO-alpha (growth-related oncogene alpha), but not for NAP-2 (neutrophil-activating protein 2), SDF (stromal-cell-derived factor)-1alpha, PF-4 (platelet factor 4), I-TAC (interferon-gamma-inducible T-cell alpha-chemoattractant), IP-10 (interferon-gamma-inducible protein 10) and MCP-1 (monocyte chemoattractant protein 1). The degradation of two human CXC chemokines in vitro, the high sequence conservation, the immunogenicity of the protein in humans and the shown protection in animal studies suggest that Spy0416 is a promising vaccine candidate for the prevention of infections by S. pyogenes.

Helicobacter pylori is the most prevalent human pathogen and although, it remains silent in most individuals for lifetime, colonization may develop into severe gastric and duodenal conditions. Rapidly developing resistance to antibiotic treatment urgently calls for the development of effective vaccines. We determined the ANTIGENome of two clinical isolates of H. pylori, KTH-Ca1 and KTH-Du, derived from patients with gastric cancer and duodenal ulcer, respectively. Using disease-relevant human sera from well-characterized donors we identified 124 annotated ORFs and 54 non-annotated peptides as antigens. Through in vitro validation assays we selected the 20 most promising vaccine candidates. Importantly, two candidates represent proteins that were previously shown to provide protection in models of H. pylori infection. One of the most frequently selected and conserved protein, the siderophore-dependent transporter HP1341, was confirmed to show high reactivity with human serum IgGs. These analyses provide the means to identify novel antigens for the selection of vaccine candidates, as well as disease associated biomarkers.

Pneumococcus is one of the most important human pathogens that causes life-threatening invasive diseases, especially at the extremities of age. Capsular polysaccharides (CPSs) are known to induce protective antibodies; however, it is not feasible to develop CPS-based vaccines that cover all of the 90 disease-causing serotypes. We applied a genomic approach and described the antibody repertoire for pneumococcal proteins using display libraries expressing 15–150 amino acid fragments of the pathogen's proteome. Serum antibodies of exposed, but not infected, individuals and convalescing patients identified the ANTIGENome of pneumococcus consisting of ∼140 antigens, many of them surface exposed. Based on several in vitro assays, 18 novel candidates were preselected for animal studies, and 4 of them showed significant protection against lethal sepsis. Two lead vaccine candidates, protein required for cell wall separation of group B streptococcus (PcsB) and serine/threonine protein kinase (StkP), were found to be exceptionally conserved among clinical isolates (>99.5% identity) and cross-protective against four different serotypes in lethal sepsis and pneumonia models, and have important nonredundant functions in bacterial multiplication based on gene deletion studies. We describe for the first time opsonophagocytic killing activity for pneumococcal protein antigens. A vaccine containing PcsB and StkP is intended for the prevention of infections caused by all serotypes of pneumococcus in the elderly and in children.

Iron scavenging from the host is essential for the growth of pathogenic bacteria. In this study, we further characterized two staphylococcal cell wall proteins previously shown to bind hemoproteins. HarA and IsdB harbor homologous ligand binding domains, the so called NEAT domain (for “near transporter”) present in several surface proteins of gram-positive pathogens. Surface plasmon resonance measurements using glutathione S -transferase (GST)-tagged HarAD1, one of the ligand binding domains of HarA, and GST-tagged full-length IsdB proteins confirmed high-affinity binding to hemoglobin and haptoglobin-hemoglobin complexes with equilibrium dissociation constants ( K D ) of 5 to 50 nM. Haptoglobin binding could be detected only with HarA and was in the low micromolar range. In order to determine the fold of this evolutionarily conserved ligand binding domain, the untagged HarAD1 protein was subjected to nuclear magnetic resonance spectroscopy, which revealed an eight-stranded, purely antiparallel β-barrel with the strand order (-β1 ↓ -β2 ↑ -β3 ↓ -β6 ↑ -β5 ↓ -β4 ↑ -β7 ↓ -β8 ↑ ), forming two Greek key motifs. Based on structural-homology searches, the topology of the HarAD1 domain resembles that of the immunoglobulin (Ig) fold family, whose members are involved in protein-protein interactions, but with distinct structural features. Therefore, we consider that the HarAD1/NEAT domain fold is a novel variant of the Ig fold that has not yet been observed in other proteins.

We evaluated the effects of combining different numbers of pneumococcal antigens, pre-existing antibody levels, sampling interval, age, and duration of illness on the detection of IgG responses against eight Streptococcus pneumoniae proteins, three Haemophilus influenzae proteins, and five Moraxella catarrhalis proteins in 690 children aged

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of midges (Culicoides spp.). IgE-mediated reactions are often involved in the pathogenesis of this disease. IBH does not occur in Iceland due to the absence of Culicoides, but it occurs with a high frequency in Icelandic horses exported to mainland Europe, where Culicoides are present. We hypothesize that immunization with the Culicoides allergens before export could reduce the incidence of IBH in exported Icelandic horses. The aim of the present study was therefore to compare intradermal and intralymphatic vaccination using four purified recombinant allergens, in combination with a Th1 focusing adjuvant. Twelve horses were vaccinated three times with 10μg of each of the four recombinant Culicoides nubeculosus allergens. Six horses were injected intralymphatically, three with and three without IC31(®), and six were injected intradermally, in the presence or absence of IC31(®). Antibody responses were measured by immunoblots and ELISA, potential sensitization in a sulfidoleukotriene release test and an intradermal test, cytokine and FoxP3 expression with real time PCR following in vitro stimulation of PBMC. Immunization with the r-allergens induced a significant increase in levels of r-allergen-specific IgG1, IgG1/3, IgG4/7, IgG5 and IgG(T). Application of the r-allergens in IC31(®) adjuvant resulted in a significantly higher IgG1, IgG1/3, IgG4/7 allergen-specific response. Intralymphatic injection was slightly more efficient than intradermal injection, but the difference did not reach significance. Testing of the blocking activity of the sera from the horses immunized intralymphatically with IC31(®) showed that the generated IgG antibodies were able to partly block binding of serum IgE from an IBH-affected horse to these r-allergens. Furthermore, IgG antibodies bound to protein bands on blots of C. nubeculosus salivary gland extract. No allergen-specific IgE was induced and there was no indication of induction of IgE-mediated reactions, as horses neither responded to Culicoides extract stimulation in a sulfidoleukotriene release test, nor developed a relevant immediate hypersensitivity reaction to the recombinant allergens in skin test. IL-4 expression was significantly higher in horses vaccinated intralymphatically without IC31(®), as compared to horses intradermally vaccinated with IC31(®). Both routes gave higher IL-10 expression with IC31(®). Both intralymphatic and intradermal vaccination of horses with recombinant allergens in IC31(®) adjuvant induced an immune response without adverse effects and without IgE production. The horses were not sensitized and produced IgG that could inhibit allergen-specific IgE binding. We therefore conclude that both the injection routes and the IC31(®) adjuvant are strong candidates for further development of immunoprophylaxis and therapy in horses.

The main Borrelia species causing Lyme borreliosis in Europe and Asia are Borrelia afzelii, B. garinii, B. burgdorferi and B. bavariensis. This is in contrast to the United States, where infections are exclusively caused by B. burgdorferi. Until to date the genome sequences of four B. afzelii strains, of which only two include the numerous plasmids, are available. In order to further assess the genetic diversity of B. afzelii, the most common species in Europe, responsible for the large variety of clinical manifestations of Lyme borreliosis, we have determined the full genome sequence of the B. afzelii strain K78, a clinical isolate from Austria. The K78 genome contains a linear chromosome (905,949 bp) and 13 plasmids (8 linear and 5 circular) together presenting 1,309 open reading frames of which 496 are located on plasmids. With the exception of lp28-8, all linear replicons in their full length including their telomeres have been sequenced. The comparison with the genomes of the four other B. afzelii strains, ACA-1, PKo, HLJ01 and Tom3107, as well as the one of B. burgdorferi strain B31, confirmed a high degree of conservation within the linear chromosome of B. afzelii, whereas plasmid encoded genes showed a much larger diversity. Since some plasmids present in B. burgdorferi are missing in the B. afzelii genomes, the corresponding virulence factors of B. burgdorferi are found in B. afzelii on other unrelated plasmids. In addition, we have identified a species specific region in the circular plasmid, cp26, which could be used for species determination. Different non-coding RNAs have been located on the B. afzelii K78 genome, which have not previously been annotated in any of the published Borrelia genomes.

Anti-cytokine autoantibodies have been widely reported to be present in human plasma, both in healthy subjects and in patients with underlying autoimmune conditions, such as autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) or thymic epithelial neoplasms. While often asymptomatic, they can cause or facilitate a wide range of diseases including opportunistic infections. The potential therapeutic value of specific neutralizing anti-cytokine autoantibodies has not been thoroughly investigated. Here we used mammalian cell display to isolate IL17A-specific antibodies from a thymoma patient with proven high-titer autoantibodies against the same. We identified 3 distinct clonotypes that efficiently neutralized IL17A in a cell-based in vitro assay. Their potencies were comparable to those of known neutralizing antibodies, including 2, AIN457 (secukinumab) and ixekizumab that are currently in clinical development for the treatment of various inflammatory disorders. These data clearly demonstrate that the human autoantibody repertoire can be mined for antibodies with high therapeutic potential for clinical development.

A novel approach for the identification of protein antigens from bacterial pathogens was previously developed in our laboratory that combines the advantages of full genome coverage and serological antigen identification. We have applied this technology to several bacterial pathogens of the genera Staphylococcus and Streptococcus and have, as a result, defined the "antigenome" of these pathogens. This catalogue defines the most relevant antigenic proteins that are targeted by the human immune system, including their antibody binding sites. The antigenome technology offers an integrated approach for antigen validation in order to select the most promising candidates for the development of subunit vaccines against the targeted bacterial diseases. Using this technology, novel protective antigens were discovered from several important human pathogens.

Streptococcus agalactiae is a major cause of bacterial pneumonia, sepsis, and meningitis in human neonates. During the course of infection, S. agalactiae adheres to a variety of epithelial cells but the underlying mechanisms are only poorly understood. The present report demonstrates the importance of the fibrinogen receptor FbsA for the streptococcal adherence and invasion of epithelial cells. Deletion of the fbsA gene in various S. agalactiae strains substantially reduced their binding of soluble fibrinogen and their adherence to and invasion of epithelial cells, indicating a role of FbsA in these different processes. The adherence and invasiveness of an fbsA deletion mutant were partially restored by reintroducing the fbsA gene on an expression vector. Heterologous expression of fbsA in Lactococcus lactis enabled this bacterium to adhere to but not to invade epithelial cells, suggesting that FbsA is a streptococcal adhesin. Flow cytometry experiments revealed a dose-dependent binding of FbsA to the surface of epithelial cells. Furthermore, tissue culture experiments exhibited an intimate contact of FbsA-coated latex beads with the surfaces of human epithelial cells. Finally, host cell adherence and invasion were significantly blocked in competition experiments with either purified FbsA protein or a monoclonal antibody directed against the fibrinogen-binding epitope of FbsA. Taken together, our studies demonstrate that FbsA promotes the adherence of S. agalactiae to epithelial cells but that FbsA does not mediate the bacterial invasion into host cells. Our results also indicate that fibrinogen-binding epitopes within FbsA are involved in the adherence of S. agalactiae to epithelial cells.

The availability of complete genome sequences of pathogens has dramatically changed the scope for developing improved and novel vaccines by increasing the speed of target identification. Genomics-based technologies have many advantages, compared to conventional approaches, which are time-consuming and usually identify only abundant antigens that are expressible under in vitro culture conditions. This review focuses on recent reports of genomics-based strategies that can be applied to most pathogens and that exploit genome sequence information in alliance with adjunct technologies, including bioinformatics, expression analyses, random mutagenesis or protein/peptide-based selection methods. Despite the caveats that are associated with the individual approaches, these technologies have already made major contributions to the identification and selection of novel vaccine candidates to combat bacterial infections.

Taking advantage of whole genome sequences of bacterial pathogens in many thriving diseases with global impact, we developed a comprehensive screening procedure for the identification of putative vaccine candidate antigens. Importantly, this procedure relies on highly representative small-fragment genomic libraries that are expressed to display frame-selected epitope-size peptides on a bacterial cell surface and to interact directly with carefully selected disease-relevant high-titer sera. Here we describe the generation of small-fragment genomic libraries of Gram-positive and Gram-negative clinically significant pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, and Streptoococcus pneumoniae, Enterococcus faecalis, Helicobacter pylori, Chlamydia pneumoniae, the enterotoxigenic Escherichia coli, and Campylobacter jejuni. Large-scale sequencing revealed that the libraries, which provide an average of 20-fold coverage, were random and, as demonstrated with two S. aureus libraries, highly representative. Consistent with the comprehensive nature of this approach is the identification of epitopes that reside in both annotated and putatively novel open reading frames. The use of these libraries therefore allows for the rapid and direct identification of immunogenic epitopes with no apparent bias or difficulty that often associate with conventional expression methods.

For the design of potent subunit vaccines, it is of paramount importance to identify all antigens immunologically recognized by a patient population infected with a pathogen. We have developed a rapid and efficient procedure to identify such commonly recognized antigens, and here we provide a comprehensive in vivo antigenic profile of Staphylococcus aureus , an important human pathogen. S. aureus peptides were displayed on the surface of Escherichia coli via fusion to one of two outer membrane proteins (LamB and FhuA) and probed with sera selected for high Ab titer and opsonic activity. A total of 60 antigenic proteins were identified, most of which are located or predicted to be located on the surface of the bacterium or secreted. The identification of these antigens and their reactivity with individual sera from patients and healthy individuals greatly facilitate the selection of promising vaccine candidates for further evaluation. This approach, which makes use of whole genome sequence information, has the potential to greatly accelerate and facilitate the formulation of novel vaccines and is applicable to any pathogen that induces Abs in humans and/or experimental animals.

Dry tetanus toxoid (TTx) patches were formulated without any adjuvant, with excipients to impart antigen stabilization and to enhance skin delivery. The booster effects of the TTx patches were assessed using a guinea pig model. The study revealed significant rises in TTx IgG titers induced by the TTx patches after a low-dose subcutaneous (s.c.) prime with TTx adsorbed to aluminum hydroxide. The TTx patch can therefore be considered an effective alternative to a subcutaneous booster.

Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are pathogens commonly associated with infectious diseases in childhood. This study aimed to develop a fluorescent multiplexed bead-based immunoassay (FMIA) using recombinant proteins for the quantitation of serum IgG antibodies against these bacteria. Eight pneumococcal proteins (Ply, CbpA, PspA1, PspA2, PcpA, PhtD, SP1732-3 and SP2216-1), 3 proteins of H. influenzae (NTHi Protein D, NTHi0371-1, NTHi0830), and 5 proteins of M. catarrhalis (MC Omp CD, MC_RH4_2506, MC_RH4_1701, MC_RH4_3729-1, MC_RH4_4730) were used to develop the FMIA. Optimal coupling concentrations for each protein, comparison of singleplex and multiplex assays, specificity, reproducibility, and correlation to ELISA for six pneumococcal antigens were determined for validation. FMIA was then used to analyze acute and convalescent paired serum samples of 50 children with non-severe pneumonia. The coupling concentrations varied for different antigens, ranging from 1.6 to 32μg of protein/million beads. Correlation between singleplexed and multiplexed assays was excellent, with R≥0.987. The FMIA was specific, reaching >92% homologous inhibition for all specificities; heterologous inhibition ≥20% was found only in six cases. The assay was repeatable, with averages of intra-assay variation ≤10.5%, day-to-day variation ≤9.7% and variation between technicians ≤9.1%. Comparison with ELISA for pneumococcal antigens demonstrated good correlation with R ranging from 0.854 (PspA2) to 0.976 (PcpA). The samples from children showed a wide range of antibody concentrations and increases in convalescent samples. In conclusion, the FMIA was sensitive, specific, and repeatable, using small amounts of recombinant proteins and sera to detect antibodies against S. pneumoniae, H. influenzae and M. catarrhalis. The methodology would be suitable for studies investigating etiological diagnosis and in experimental vaccine studies.

There is currently no Lyme borreliosis vaccine available for humans, although it has been shown that the disease can be prevented by immunization with an OspA-based vaccine (LYMErix). Outer surface protein A (OspA) is one of the dominant antigens expressed by the spirochetes when present in a tick. The Borrelia species causing Lyme borreliosis in Europe express different OspA serotypes on their surface, B. burgdorferi (serotype 1), B. afzelii (serotype 2), B. garinii (serotypes, 3, 5 and 6) and B. bavariensis (serotype 4), while only B. burgdorferi is present in the US. In order to target all these pathogenic Borrelia species, we have designed a multivalent OspA-based vaccine. The vaccine includes three proteins, each containing the C-terminal half of two OspA serotypes linked to form a heterodimer. In order to stabilize the C-terminal fragment and thus preserve important structural epitopes at physiological temperature, disulfide bonds were introduced. The immunogenicity was increased by introduction of a lipidation signal which ensures the addition of an N-terminal lipid moiety. Three immunizations with 3.0 µg adjuvanted vaccine protected mice from a challenge with spirochetes expressing either OspA serotype 1, 2 or 5. Mice were protected against both challenge with infected ticks and in vitro grown spirochetes. Immunological analyses (ELISA, surface binding and growth inhibition) indicated that the vaccine can provide protection against the majority of Borrelia species pathogenic for humans. This article presents the approach which allows for the generation of a hexavalent vaccine that can potentially protect against a broad range of globally distributed Borrelia species causing Lyme borreliosis.

In yeast, anaphase depends on cohesin cleavage. How anaphase is controlled in vertebrates is unknown because their cohesins dissociate from chromosomes before anaphase. We show that residual amounts of the cohesin SCC1 remain associated with human centromeres until the onset of anaphase when a similarly small amount of SCC1 is cleaved. In Xenopus extracts, SCC1 cleavage depends on the anaphase-promoting complex and separin. Separin immunoprecipitates are sufficient to cleave SCC1, indicating that separin is associated with a protease activity. Separin activation coincides with securin destruction and partial separin cleavage, suggesting that several mechanisms regulate separin activity. We propose that in vertebrates, a cleavage-independent pathway removes cohesin from chromosome arms during prophase, whereas a separin-dependent pathway cleaves centromeric cohesin at the metaphase–anaphase transition.

Many cytokine receptors employ Janus protein tyrosine kinases (Jaks) and signal transducers and activators of transcription (Stats) for nuclear signaling. Here, we have established yeast strains in which an autoactivated Jak2 kinase induces tyrosine phosphorylation, dimerization, nuclear translocation, and DNA binding of a concomitantly expressed Stat5 protein. Transcriptional activity of Stat5 on a stably integrated, Stat-dependent reporter gene required the C-terminal fusion of the VP16 transactivation domain. In such yeast strains, the interaction between Jak2 and Stat5 was analyzed without interference by other mammalian proteins involved in regulating Jak-Stat signaling, and mutant versions of both proteins were analyzed for their ability to productively interact. Complexes between Jak2 and Stat5 were found to be stable under stringent co-immunoprecipitation conditions. Deletion of the Jak homology regions 2-7 (JH2-JH7) of Jak2, leaving only the kinase domain (JH1) intact, reduced the ability of the kinase to phosphorylate Stat5, whereas deletion of the JH2 domain caused an increased enzymatic activity. A site-directed R618K mutation in the Stat5 SH2 domain abolished the phosphorylation by Jak2, while deletion of the C terminus led to Stat5 hyperphosphorylation. A single phosphotyrosine-SH2 domain interaction was sufficient for the dimerization of Stat5, but such dimers bound to DNA very inefficiently. Together, our data show that yeast cells are appropriate tools for studying Jak-Stat or Stat-Stat interactions. Our mutational analysis suggests that the Stat5 SH2 domain is essential for the interaction with Jak2 and that the kinase domain of Jak2 is sufficient for Jak2-Stat5 interaction. Therefore, the Jak kinase domain may be all that is needed to cause Stat phosphorylation in situations where receptor docking is dispensable.

Changes in gene expression are necessary for an adaptive response of cells to immunological stimuli and thus for their proper function in the context of the immune system. Regulatory inputs usually originate from cell surface receptors and in many cases affect the transcription rates of specific genes by modulating the activity of transcription factors. The Jak-Stat signalling paradigm has received large attention by molecular immunologists because it applies to nuclear signalling by all cytokine receptors. In its simplest form it requires only two protein components downstream of the receptor: Janus family protein tyrosine kinases (Jaks) which are usually receptor-associated, and signal transducer and activator of transcription (Stat) family transcription factors which carry the receptor-generated signal to the nucleus and stimulate gene expression. Here we give a brief overview of both recent progress and open questions concerning the Jak and Stat molecules, their regulation, and the biological implications of their activity.

Moraxella catarrhalis is one of the three most common causative bacterial pathogens of otitis media, however no effective vaccine against M. catarrhalis has been developed so far. To identify M. catarrhalis vaccine candidate antigens, we used carefully selected sera from children with otitis media and healthy individuals to screen small-fragment genomic libraries that are expressed to display frame-selected peptides on a bacterial cell surface. This ANTIGENome technology led to the identification of 214 antigens, 23 of which were selected by in vitro or in vivo studies for additional characterization. Eight of the 23 candidates were tested in a Moraxella mouse pulmonary clearance model, and 3 of these antigens induced significantly faster bacterial clearance compared to adjuvant or to the previously characterized antigen OmpCD. The most significant protection data were obtained with the antigen MCR_1416 (Msp22), which was further investigated for its biological function by in vitro studies suggesting that Msp22 is a heme binding protein. This study comprises one of the most exhaustive studies to identify potential vaccine candidate antigens against the bacterial pathogen M. catarrhalis.

The versatility of the surface of Borrelia, the causative agent of Lyme borreliosis, is very important in host-pathogen interactions allowing bacteria to survive in ticks and to persist in a mammalian environment. To identify the surface proteome of Borrelia, we have performed a large comparative proteomic analysis on the three most important pathogenic Borrelia species, namely B. burgdorferi (strain B31), B. afzelii (strain K78), and B. garinii (strain PBi). Isolation of membrane proteins was performed by using three different approaches: (i) a detergent-based fractionation of outer membrane proteins; (ii) a trypsin-based partial shedding of outer cell surface proteins; (iii) biotinylation of membrane proteins and preparation of the biotin-labelled fraction using streptavidin. Proteins derived from the detergent-based fractionation were further sub-fractionated by heparin affinity chromatography since heparin-like molecules play an important role for microbial entry into human cells. All isolated proteins were analysed using either a gel-based liquid chromatography (LC)-MS/MS technique or by two-dimensional (2D)-LC-MS/MS resulting in the identification of 286 unique proteins. Ninety seven of these were found in all three Borrelia species, representing potential targets for a broad coverage vaccine for the prevention of Lyme borreliosis caused by the different Borrelia species.

The preformulation of a trivalent recombinant protein-based vaccine candidate for protection against Streptococcus pneumoniae is described both in the presence and in the absence of aluminum salt adjuvants. The biophysical properties of the three protein-based antigens, fragments of pneumococcal surface adhesion A (PsaA), serine–threonine protein kinase (StkP), and protein required for cell wall separation of group B streptococcus (PcsB), were studied using several spectroscopic and light scattering techniques. An empirical phase diagram was constructed to assess the overall conformational stability of the three antigens as a function of pH and temperatures. A variety of excipients were screened on the basis of their ability to stabilize each antigen using intrinsic fluorescence spectroscopy and circular dichroism spectroscopy. Sorbitol, sucrose, and trehalose stabilized the three proteins in solution. The addition of manganese also showed a drastic increase in the thermal stability of SP1650 in solution. The adsorption and desorption processes of each of the antigens to aluminum salt adjuvants were evaluated, and the stability of the adsorbed proteins was then assessed using intrinsic fluorescence spectroscopy and Fourier transform infrared spectroscopy. All the three proteins showed good adsorption to Alhydrogel. PsaA was destabilized when adsorbed onto Alhydrogel® and adding sodium phosphate showed a stabilizing effect. PcsB was found to be stabilized when adsorbed to Alhydrogel®, and no destabilizing or stabilizing effects were seen in the case of StkP. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:3078–3090, 2012

Here we report the annotated genome sequence of Moraxella catarrhalis strain RH4, a seroresistant-lineage strain isolated from the blood of an infected patient. This genome sequence will allow us to gain further insight into the genetic diversity of clinical M. catarrhalis isolates and will facilitate study of M. catarrhalis pathogenesis.