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Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have emerged as a novel and flexible vaccine platform. OMVs can be decorated with foreign antigens and carry potent immunostimulatory components. Therefore, after their purification from the culture supernatant, they are ready to be formulated for vaccine use. It has been extensively demonstrated that immunization with engineered OMVs can elicit excellent antibody responses against the heterologous antigens. However, the definition of the conditions necessary to reach the optimal antibody titers still needs to be investigated. Here, we defined the protein concentrations required to induce antigen-specific antibodies, and the amount of antigen and OMVs necessary and sufficient to elicit saturating levels of antigen-specific antibodies. Since not all antigens can be expressed in OMVs, we also investigated the effectiveness of vaccines in which OMVs and purified antigens are mixed together without using any procedure for their physical association. Our data show that in most of the cases OMV–antigen mixtures are very effective in eliciting antigen-specific antibodies. This is probably due to the capacity of OMVs to “absorb” antigens, establishing sufficiently stable interactions that allow antigen–OMV co-presentation to the same antigen presenting cell. In those cases when antigen–OMV interaction is not sufficiently stable, the addition of alum to the formulation guarantees the elicitation of high titers of antigen-specific antibodies.

Human papillomaviruses (HPVs) are a large family of viruses with a capsid composed of the L1 and L2 proteins, which bind to receptors of the basal epithelial cells and promote virus entry. The majority of sexually active people become exposed to HPV and the virus is the most common cause of cervical cancer. Vaccines are available based on the L1 protein, which self-assembles and forms virus-like particles (VLPs) when expressed in yeast and insect cells. Although very effective, these vaccines are HPV type-restricted and their costs limit broad vaccination campaigns. Recently, vaccine candidates based on the conserved L2 epitope from serotypes 16, 18, 31, 33, 35, 6, 51, and 59 were shown to elicit broadly neutralizing anti-HPV antibodies. In this study, we tested whether E. coli outer membrane vesicles (OMVs) could be successfully decorated with L2 polytopes and whether the engineered OMVs could induce neutralizing antibodies. OMVs represent an attractive vaccine platform owing to their intrinsic adjuvanticity and their low production costs. We show that strings of L2 epitopes could be efficiently expressed on the surface of the OMVs and a polypeptide composed of the L2 epitopes from serotypes 18, 33, 35, and 59 provided a broad cross-protective activity against a large panel of HPV serotypes as determined using pseudovirus neutralization assay. Considering the simplicity of the OMV production process, our work provides a highly effective and inexpensive solution to produce universal anti-HPV vaccines.

The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespective of economical and climatic conditions. Outer membrane vesicles (OMVs) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMVs can be used as vaccines to induce potent immune responses against the associated proteins. Here, we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in vaccinated mice, resulting in the production of neutralizing antibodies (nAbs) with a titre higher than 1:300. The immunity induced by the vaccine is sufficient to protect the animals from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with the RBM of the Omicron BA.1 variant and that such engineered OMVs induce nAbs against Omicron BA.1 and BA.5, as measured using the pseudovirus neutralization infectivity assay. Importantly, we show that the RBM438–509 ancestral-OMVs elicited antibodies which efficiently neutralize in vitro both the homologous ancestral strain, the Omicron BA.1 and BA.5 variants with a neutralization titre ranging from 1:100 to 1:1500, suggesting its potential use as a vaccine targeting diverse SARS-CoV-2 variants. Altogether, given the convenience associated with the ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.

A growing body of evidence supports the notion that the gut microbiome plays an important role in cancer immunity. However, the underpinning mechanisms remain to be fully elucidated. One attractive hypothesis envisages that among the T cells elicited by the plethora of microbiome proteins a few exist that incidentally recognize neo-epitopes arising from cancer mutations (“molecular mimicry (MM)” hypothesis). To support MM, the human probiotic Escherichia coli Nissle was engineered with the SIINFEKL epitope (OVA-E.coli Nissle) and orally administered to C57BL/6 mice. The treatment with OVA-E.coli Nissle, but not with wild type E. coli Nissle, induced OVA-specific CD8+ T cells and inhibited the growth of tumors in mice challenged with B16F10 melanoma cells expressing OVA. The microbiome shotgun sequencing and the sequencing of TCRs from T cells recovered from both lamina propria and tumors provide evidence that the main mechanism of tumor inhibition is mediated by the elicitation at the intestinal site of cross-reacting T cells, which subsequently reach the tumor environment. Importantly, the administration of Outer Membrane Vesicles (OMVs) from engineered E. coli Nissle, as well as from E. coli BL21(DE3)ΔompA, carrying cancer-specific T cell epitopes also elicited epitope-specific T cells in the intestine and inhibited tumor growth. Overall, our data strengthen the important role of MM in tumor immunity and assign a novel function of OMVs in host-pathogen interaction. Moreover, our results pave the way to the exploitation of probiotics and OMVs engineered with tumor specific-antigens as personalized mucosal cancer vaccines.

Over 18 million disease cases and half a million deaths worldwide are estimated to be caused annually by Group A Streptococcus. A vaccine to prevent GAS disease is urgently needed. SpyCEP (Streptococcus pyogenes Cell-Envelope Proteinase) is a surface-exposed serine protease that inactivates chemokines, impairing neutrophil recruitment and bacterial clearance, and has shown promising immunogenicity in preclinical models. Although SpyCEP structure has been partially characterized, a more complete and higher resolution understanding of its antigenic features would be desirable prior to large scale manufacturing. To address these gaps and facilitate development of this globally important vaccine, we performed immunogenicity studies with a safety-engineered SpyCEP mutant, and comprehensively characterized its structure by combining X-ray crystallography, NMR spectroscopy and molecular dynamics simulations. We found that the catalytically-inactive SpyCEP antigen conferred protection similar to wild-type SpyCEP in a mouse infection model. Further, a new higher-resolution crystal structure of the inactive SpyCEP mutant provided new insights into this large chemokine protease comprising nine domains derived from two non-covalently linked fragments. NMR spectroscopy and molecular simulation analyses revealed conformational flexibility that is likely important for optimal substrate recognition and overall function. These combined immunogenicity and structural data demonstrate that the full-length SpyCEP inactive mutant is a strong candidate human vaccine antigen. These findings show how a multi-disciplinary study was used to overcome obstacles in the development of a GAS vaccine, an approach applicable to other future vaccine programs. Moreover, the information provided may also facilitate the structure-based discovery of small-molecule therapeutics targeting SpyCEP protease inhibition.

BACKGROUND Little is known about the autoreactive B cells in antineutrophil cytoplasmic antibody–associated (ANCA-associated) vasculitis (AAV). We aimed to investigate tolerance checkpoints of circulating antigen-specific proteinase 3–reactive (PR3+) B cells.METHODS Multicolor flow cytometry in combination with bioinformatics and functional in vitro studies were performed on baseline samples of PBMCs from 154 well-characterized participants of the RAVE trial (NCT00104299) with severely active PR3-AAV and myeloperoxidase-AAV (MPO-AAV) and 27 healthy controls (HCs). Clinical data and outcomes from the trial were correlated with PR3+ B cells (total and subsets).RESULTS The frequency of PR3+ B cells among circulating B cells was higher in participants with PR3-AAV (4.77% median [IQR, 3.98%–6.01%]) than in participants with MPO-AAV (3.16% median [IQR, 2.51%–5.22%]) and participants with AAV compared with HCs (1.67% median [IQR, 1.27%–2.16%], P < 0.001 for all comparisons), implying a defective central tolerance checkpoint in patients with AAV. Only PBMCs from participants with PR3-AAV contained PR3+ B cells capable of secreting PR3-ANCA IgG in vitro, proving they were functionally distinct from those of participants with MPO-AAV and HCs. Unsupervised clustering identified subtle subsets of atypical autoreactive PR3+ memory B cells accumulating through the maturation process in patients with PR3-AAV. PR3+ B cells were enriched in the memory B cell compartment of participants with PR3-AAV and were associated with higher serum CXCL13 levels, suggesting an increased germinal center activity. PR3+ B cells correlated with systemic inflammation (C-reactive protein and erythrocyte sedimentation rate, P < 0.05) and complete remission (P < 0.001).CONCLUSION This study suggests the presence of defective central antigen-independent and peripheral antigen-dependent checkpoints in patients with PR3-AAV, elucidating the selection process of autoreactive B cells.Trial registration ClinicalTrials.gov NCT00104299.Funding The Vasculitis Foundation, the National Institute of Allergy and Infectious Diseases of the NIH, and the Mayo Foundation for Education and Research.

Modification of surface antigens and differential expression of virulence factors are frequent strategies pathogens adopt to escape the host immune system. These escape mechanisms make pathogens a “moving target” for our immune system and represent a challenge for the development of vaccines, which require more than one antigen to be efficacious. Therefore, the availability of strategies, which simplify vaccine design, is highly desirable. Bacterial Outer Membrane Vesicles (OMVs) are a promising vaccine platform for their built-in adjuvanticity, ease of purification and flexibility to be engineered with foreign proteins. However, data on if and how OMVs can be engineered with multiple antigens is limited. In this work, we report a multi-antigen expression strategy based on the co-expression of two chimeras, each constituted by head-to-tail fusions of immunogenic proteins, in the same OMV-producing strain. We tested the strategy to develop a vaccine against Staphylococcus aureus, a Gram-positive human pathogen responsible for a large number of community and hospital-acquired diseases. Here we describe an OMV-based vaccine in which four S. aureus virulent factors, ClfAY338A, LukE, SpAKKAA and HlaH35L have been co-expressed in the same OMVs (CLSH-OMVsΔ60). The vaccine elicited antigen-specific antibodies with functional activity, as judged by their capacity to promote opsonophagocytosis and to inhibit Hla-mediated hemolysis, LukED-mediated leukocyte killing, and ClfA-mediated S. aureus binding to fibrinogen. Mice vaccinated with CLSH-OMVsΔ60 were robustly protected from S. aureus challenge in the skin, sepsis and kidney abscess models. This study not only describes a generalized approach to develop easy-to-produce and inexpensive multi-component vaccines, but also proposes a new tetravalent vaccine candidate ready to move to development.

Abstract Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes‐specific antibody titers and high frequencies of epitope‐specific IFN‐γ‐producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV‐based vaccines.

Abstract Background Staphylococcus aureus is an opportunistic pathogen and a leading cause of nosocomial infections. It can acquire resistance to all the antibiotics that entered the clinics to date, and the World Health Organization defined it as a high-priority pathogen for research and development of new antibiotics. A deeper understanding of the genetic variability of S. aureus in clinical settings would lead to a better comprehension of its pathogenic potential and improved strategies to contrast its virulence and resistance. However, the number of comprehensive studies addressing clinical cohorts of S. aureus infections by simultaneously looking at the epidemiology, phylogenetic reconstruction, genomic characterisation, and transmission pathways of infective clones is currently low, thus limiting global surveillance and epidemiological monitoring. Methods We applied whole-genome shotgun sequencing (WGS) to 184 S. aureus isolates from 135 patients treated in different operative units of an Italian paediatric hospital over a timespan of 3 years, including both methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) from different infection types. We typed known and unknown clones from their genomes by multilocus sequence typing (MLST), Staphylococcal Cassette Chromosome mec (SCCmec), Staphylococcal protein A gene (spa), and Panton-Valentine Leukocidin (PVL), and we inferred their whole-genome phylogeny. We explored the prevalence of virulence and antibiotic resistance genes in our cohort, and the conservation of genes encoding vaccine candidates. We also performed a timed phylogenetic investigation for a potential outbreak of a newly emerging nosocomial clone. Results The phylogeny of the 135 single-patient S. aureus isolates showed a high level of diversity, including 80 different lineages, and co-presence of local, global, livestock-associated, and hypervirulent clones. Five of these clones do not have representative genomes in public databases. Variability in the epidemiology is mirrored by variability in the SCCmec cassettes, with some novel variants of the type IV cassette carrying extra antibiotic resistances. Virulence and resistance genes were unevenly distributed across different clones and infection types, with highly resistant and lowly virulent clones showing strong association with chronic diseases, and highly virulent strains only reported in acute infections. Antigens included in vaccine formulations undergoing clinical trials were conserved at different levels in our cohort, with only a few highly prevalent genes fully conserved, potentially explaining the difficulty of developing a vaccine against S. aureus. We also found a recently diverged ST1-SCCmecIV-t127 PVL− clone suspected to be hospital-specific, but time-resolved integrative phylogenetic analysis refuted this hypothesis and suggested that this quickly emerging lineage was acquired independently by patients. Conclusions Whole genome sequencing allowed us to study the epidemiology and genomic repertoire of S. aureus in a clinical setting and provided evidence of its often underestimated complexity. Some virulence factors and clones are specific of disease types, but the variability and dispensability of many antigens considered for vaccine development together with the quickly changing epidemiology of S. aureus makes it very challenging to develop full-coverage therapies and vaccines. Expanding WGS-based surveillance of S. aureus to many more hospitals would allow the identification of specific strains representing the main burden of infection and therefore reassessing the efforts for the discovery of new treatments and clinical practices.

A large body of data both in animals and humans demonstrates that the gut microbiome plays a fundamental role in cancer immunity and in determining the efficacy of cancer immunotherapy. In this work, we have investigated whether and to what extent the gut microbiome can influence the antitumor activity of neo-epitope-based cancer vaccines in a BALB/c-CT26 cancer mouse model. Similarly to that observed in the C57BL/6-B16 model, Bifidobacterium administration per se has a beneficial effect on CT26 tumor inhibition. Furthermore, the combination of Bifidobacterium administration and vaccination resulted in a protection which was superior to vaccination alone and to Bifidobacterium administration alone, and correlated with an increase in the frequency of vaccine-specific T cells. The gut microbiome analysis by 16S rRNA gene sequencing and shotgun metagenomics showed that tumor challenge rapidly altered the microbiome population, with Muribaculaceae being enriched and Lachnospiraceae being reduced. Over time, the population of Muribaculaceae progressively reduced while the Lachnospiraceae population increased—a trend that appeared to be retarded by the oral administration of Bifidobacterium. Interestingly, in some Bacteroidales, Prevotella and Muribaculacee species we identified sequences highly homologous to immunogenic neo-epitopes of CT26 cells, supporting the possible role of “molecular mimicry” in anticancer immunity. Our data strengthen the importance of the microbiome in cancer immunity and suggests a microbiome-based strategy to potentiate neo-epitope-based cancer vaccines.

Abstract Background The exploitation of the CRISPR/Cas9 machinery coupled to lambda (λ) recombinase-mediated homologous recombination (recombineering) is becoming the method of choice for genome editing in E. coli. First proposed by Jiang and co-workers, the strategy has been subsequently fine-tuned by several authors who demonstrated, by using few selected loci, that the efficiency of mutagenesis (number of mutant colonies over total number of colonies analyzed) can be extremely high (up to 100%). However, from published data it is difficult to appreciate the robustness of the technology, defined as the number of successfully mutated loci over the total number of targeted loci. This information is particularly relevant in high-throughput genome editing, where repetition of experiments to rescue missing mutants would be impractical. This work describes a “brute force” validation activity, which culminated in the definition of a robust, simple and rapid protocol for single or multiple gene deletions. Results We first set up our own version of the CRISPR/Cas9 protocol and then we evaluated the mutagenesis efficiency by changing different parameters including sequence of guide RNAs, length and concentration of donor DNAs, and use of single stranded and double stranded donor DNAs. We then validated the optimized conditions targeting 78 “dispensable” genes. This work led to the definition of a protocol, featuring the use of double stranded synthetic donor DNAs, which guarantees mutagenesis efficiencies consistently higher than 10% and a robustness of 100%. The procedure can be applied also for simultaneous gene deletions. Conclusions This work defines for the first time the robustness of a CRISPR/Cas9-based protocol based on a large sample size. Since the technical solutions here proposed can be applied to other similar procedures, the data could be of general interest for the scientific community working on bacterial genome editing and, in particular, for those involved in synthetic biology projects requiring high throughput procedures.

Human FAT1 is overexpressed on the surface of most colorectal cancers (CRCs) and in particular a 25 amino acid sequence (D8) present in one of the 34 cadherin extracellular repeats carries the epitope recognized by mAb198.3, a monoclonal antibody which partially protects mice from the challenge with human CRC cell lines in xenograft mouse models. Here we present data in immune competent mice demonstrating the potential of the D8-FAT1 epitope as CRC cancer vaccine. We first demonstrated that the mouse homolog of D8-FAT1 (mD8-FAT1) is also expressed on the surface of CT26 and B16F10 murine cell lines. We then engineered bacterial outer membranes vesicles (OMVs) with mD8-FAT1 and we showed that immunization of BALB/c and C57bl6 mice with engineered OMVs elicited anti-mD8-FAT1 antibodies and partially protected mice from the challenge against CT26 and EGFRvIII-B16F10 cell lines, respectively. We also show that when combined with OMVs decorated with the EGFRvIII B cell epitope or with OMVs carrying five tumor-specific CD4+ T cells neoepitopes, mD8-FAT1 OMVs conferred robust protection against tumor challenge in C57bl6 and BALB/c mice, respectively. Considering that FAT1 is overexpressed in both KRAS+ and KRAS− CRCs, these data support the development of anti-CRC cancer vaccines in which the D8-FAT1 epitope is used in combination with other CRC-specific antigens, including mutation-derived neoepitopes.

With the recent implementation of the CRISPR/Cas9 technology as a standard tool for genome editing, laboratories all over the world are undergoing one of the biggest advancements in molecular biology since PCR. The key advantage of this method is its simplicity and universal applicability for species of any phylum. Of particular interest is the extensively studied Gram-negative bacterium Escherichia coli, as it is considered as the workhorse for both research and industrial purposes. Here, we present a simple, robust and effective protocol using the CRISPR/Cas9 system in combination with the λ Red machinery for gene knockout in E. coli. Crucial in our procedure is the use of a double-stranded donor DNA and a curing strategy for removal of the guide RNA encoding plasmid that allows starting a new mutation after only two working days. Our protocol allows multiple, stepwise gene knockout strains with high mutagenesis efficiencies applicable for high-throughput approaches.

IntroductionBacterial outer membrane vesicles (OMVs) are naturally produced by all Gram-negative bacteria and, thanks to their plasticity and unique adjuvanticity, are emerging as an attractive vaccine platform. To test the applicability of OMVs in cancer immunotherapy, we decorated them with either one or two protective epitopes present in the B16F10EGFRvIII cell line and tested the protective activity of OMV immunization in C57BL/6 mice challenged with B16F10EGFRvIII.Materials and methodsThe 14 amino acid B cell epitope of human epidermal growth factor receptor variant III (EGFRvIII) and the mutation-derived CD4+ T cell neo-epitope of kif18b gene (B16-M30) were used to decorate OMVs either alone or in combination. C57BL/6 were immunized with the OMVs and then challenged with B16F10EGFRvIII cells. Immunogenicity and protective activity was followed by measuring anti-EGFRvIII antibodies, M30-specific T cells, tumor-infiltrating cell population, and tumor growth.ResultsImmunization with engineered EGFRvIII-OMVs induced a strong inhibition of tumor growth after B16F10EGFRvIII challenge. Furthermore, mice immunized with engineered OMVs carrying both EGFRvIII and M30 epitopes were completely protected from tumor challenge. Immunization was accompanied by induction of high anti-EGFRvIII antibody titers, M30-specific T cells, and infiltration of CD4+ and CD8+ T cells at the tumor site.ConclusionOMVs can be decorated with tumor antigens and can elicit antigen-specific, protective antitumor responses in immunocompetent mice. The synergistic protective activity of multiple epitopes simultaneously administered with OMVs makes the OMV platform particularly attractive for cancer immunotherapy.

Background: Outer membrane vesicles (OMVs) from Gram-negative bacteria are gaining increasing attention as vaccine platform for their built-in adjuvanticity and for their potential use as carriers of heterologous antigens. These 2 properties offer the opportunity to make highly effective, easy to produce multi-valent vaccines. OMVs can be loaded with foreign antigens by targeting protein expression either to the outer membrane or to the periplasm of the OMV-producing strain. Periplasmic expression is simple and relatively efficient but leads to the accumulation of recombinant antigens in the lumen of OMVs and the ability of OMVs carrying internalized antigens to induce antigen-specific antibody responses has been only marginally investigated and is considered to be sub-optimal. Methods: We have systematically analyzed in qualitative and quantitative terms antibody responses induced by OMVs carrying different heterologous antigens in their lumen. Group A Streptococcus (GAS) Slo, SpyCEP, Spy0269 and Group B Streptococcus (GBS) SAM_1372 were fused to the OmpA leader sequence for secretion and expressed in Escherichia coli. OMVs from the recombinant strains were purified and tested for immunogenicity and protective activity. Results: All proteins were incorporated into the OMVs lumen in their native conformation. Upon mice immunization, OMVs induced high functional antibody titers against the recombinant proteins. Furthermore, immunization with Slo-OMVs and SpyCEP-OMVs protected mice against GAS lethal challenge. Conclusions: The efficiency of antigen delivery to the vesicular lumen via periplasmic expression, and the surprisingly high immunogenicity and protective activity of OMVs carrying internalized recombinant antigens further strengthens the potential of OMVs as vaccine platform.

Background: Outer membrane vesicles (OMVs) are spheroid particles released by all Gram-negative bacteria as a result of the budding out of the outer membrane. Since they carry many of the bacterial surface-associated proteins and feature a potent built-in adjuvanticity, OMVs are being utilized as vaccines, some of which commercially available. Recently, methods for manipulating the protein content of OMVs have been proposed, thus making OMVs a promising platform for recombinant, multivalent vaccines development. Methods: Chlamydia muridarum DO serine protease HtrA, an antigen which stimulates strong humoral and cellular responses in mice and humans, was expressed in Escherichia coli fused to the OmpA leader sequence to deliver it to the OMV compartment. Purified OMVs carrying HtrA (CM rHtrA-OMV) were analyzed for their capacity to induce antibodies capable of neutralizing Chlamydia infection of LLC-MK2 cells in vitro. Results: CM rHtrA-OMV immunization in mice induced antibodies that neutralize Chlamydial invasion as judged by an in vitro infectivity assay. This was remarkably different from what observed with an enzymatically functional recombinant HtrA expressed in, and purified from the E. coli cytoplasm (CM rHtrA). The difference in functionality between anti-CM rHtrA and anti-CM rHtrA-OMV antibodies was associated to a different pattern of protein epitopes recognition. The epitope recognition profile of anti-CM HtrA-OMV antibodies was similar to that induced in mice during Chlamydial infection. Conclusions: When expressed in OMVs HtrA appears to assume a conformation similar to the native one and this results in the elicitation of functional immune responses. These data further support the potentiality of OMVs as vaccine platform.

A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4+ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4+ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.

The human and bovine bacterial pathogen Streptococcus agalactiae (Group B Streptococcus, GBS) expresses a thick polysaccharide capsule that constitutes a major virulence factor and vaccine target. GBS can be classified into ten distinct serotypes differing in the chemical composition of their capsular polysaccharide. However, non-typeable strains that do not react with anti-capsular sera are frequently isolated from colonized and infected humans and cattle. To gain a comprehensive insight into the molecular basis for the loss of capsule expression in GBS, a collection of well-characterized non-typeable strains was investigated by genome sequencing. Genome based phylogenetic analysis extended to a wide population of sequenced strains confirmed the recently observed high clonality among GBS lineages mainly containing human strains, and revealed a much higher degree of diversity in the bovine population. Remarkably, non-typeable strains were equally distributed in all lineages. A number of distinct mutations in the cps operon were identified that were apparently responsible for inactivation of capsule synthesis. The most frequent genetic alterations were point mutations leading to stop codons in the cps genes, and the main target was found to be cpsE encoding the portal glycosyl transferase of capsule biosynthesis. Complementation of strains carrying missense mutations in cpsE with a wild-type gene restored capsule expression allowing the identification of amino acid residues essential for enzyme activity.

ABSTRACT Group B Streptococcus (GBS), in the transition from commensal organisms to pathogens, will encounter diverse host environments and, thus, require coordinated control of the transcriptional responses to these changes. This work was aimed at better understanding the role of two-component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knockout strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1% to 3% of the genome. Interestingly, two sugar phosphotransferase systems appeared to be differentially regulated in the TCS-16 knockout strain (TCS loci were numbered in order of their appearance on the chromosome), suggesting an involvement in monitoring carbon source availability. High-throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for the growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16, with concomitant dramatic upregulation of the adjacent operon, which encodes a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and the data also provide experimental evidence for TCS-17/RgfAC involvement in virulence. IMPORTANCE Two-component systems have been evolved by bacteria to detect environmental changes, and they play key roles in pathogenicity. A comprehensive analysis of TCS in GBS has not been performed previously. In this work, we classify 21 TCS and present evidence for the involvement of two specific TCS in GBS virulence and colonization in vivo. Although pinpointing specific TCS stimuli is notoriously difficult, we used a combination of techniques to identify two systems with different effects on GBS pathogenesis. For one of the systems, we propose that fructose-6-phosphate, a metabolite in glycolysis, is sufficient to induce a regulatory response involving a sugar transport system. Our catalogue and classification of TCS may guide further studies into the role of TCS in GBS pathogenicity and biology.

ABSTRACT Streptolysin O is a potent pore-forming toxin produced by group A Streptococcus. The aims of the present study were to dissect the relative contributions of different structural domains of the protein to hemolytic activity, to obtain a detoxified form of streptolysin O amenable to human vaccine formulation, and to investigate the role of streptolysin O-specific antibodies in protection against group A Streptococcus infection. On the basis of in silico structural predictions, we introduced two amino acid substitutions, one in the proline-rich domain 1 and the other in the conserved undecapeptide loop in domain 4. The resulting streptolysin O derivative showed no toxicity, was highly impaired in binding to eukaryotic cells, and was unable to form organized oligomeric structures on the cell surface. However, it was fully capable of conferring consistent protection in a murine model of group A Streptococcus infection. When we engineered a streptococcal strain to express the double-mutated streptolysin O, a drastic reduction in virulence as well as a diminished capacity to kill immune cells recruited at the infection site was observed. Furthermore, when mice immunized with the toxoid were challenged with the wild-type and mutant strains, protection only against the wild-type strain, not against the strain expressing the double-mutated streptolysin O, was obtained. We conclude that protection occurs by antibody-mediated neutralization of active toxin. IMPORTANCE We present a novel example of structural design of a vaccine antigen optimized for human vaccine use. Having previously demonstrated that immunization of mice with streptolysin O elicits a protective immune response against infection with group A Streptococcus strains of different serotypes, we developed in this study a double-mutated nontoxic derivative that represents a novel tool for the development of protective vaccine formulations against this important human pathogen. Furthermore, the innovative construction of an isogenic strain expressing a functionally inactive toxin and its use in infection and opsonophagocytosis experiments allowed us to investigate the mechanism by which streptolysin O mediates protection against group A Streptococcus. Finally, the ability of this toxin to directly attack and kill host immune cells during infection was studied in an air pouch model, which allowed parallel quantification of cellular recruitment, vitality, and cytokine release at the infection site.

The pilus 2a backbone protein (BP-2a) is one of the most structurally and functionally characterized components of a potential vaccine formulation against Group B Streptococcus. It is characterized by six main immunologically distinct allelic variants, each inducing variant-specific protection. To investigate the molecular determinants driving the variant immunogenic specificity of BP-2a, in terms of single residue contributions, we generated six monoclonal antibodies against a specific protein variant based on their capability to recognize the polymerized pili structure on the bacterial surface. Three mAbs were also able to induce complement-dependent opsonophagocytosis killing of live GBS and target the same linear epitope present in the structurally defined and immunodominant domain D3 of the protein. Molecular docking between the modelled scFv antibody sequences and the BP-2a crystal structure revealed the potential role at the binding interface of some non-conserved antigen residues. Mutagenesis analysis confirmed the necessity of a perfect balance between charges, size and polarity at the binding interface to obtain specific binding of mAbs to the protein antigen for a neutralizing response.

Gram-positive bacteria assemble pili through class C sortase enzymes specialized in polymerizing pilin subunits into covalently linked, high-molecular-weight, elongated structures. Here we report the crystal structures of two class C sortases (SrtC1 and SrtC2) from Group B Streptococcus (GBS) Pilus Island 1. The structures show that both sortases are comprised of two domains: an 8-stranded β-barrel catalytic core conserved among all sortase family members and a flexible N-terminal region made of two α-helices followed by a loop, known as the lid, which acts as a pseudo-substrate. In vitro experiments performed with recombinant SrtC enzymes lacking the N-terminal portion demonstrate that this region of the enzyme is dispensable for catalysis but may have key roles in substrate specificity and regulation. Moreover, in vitro FRET-based assays show that the LPXTG motif common to many sortase substrates is not the sole determinant of sortase C specificity during pilin protein recognition.

Streptococcus pyogenes is a major human pathogen worldwide, responsible for both local and systemic infections. These bacteria express the subtilisin-like protease SpyCEP which cleaves human IL-8 and related chemokines. We show that localization of SpyCEP is growth-phase and strain dependent. Significant shedding was observed only in a strain naturally overexpressing SpyCEP, and shedding was not dependent on SpyCEP autoproteolytic activity. Surface-bound SpyCEP in two different strains was capable of cleaving IL-8. To investigate SpyCEP action in vivo, we adapted the mouse air pouch model of infection for parallel quantification of bacterial growth, host immune cell recruitment and chemokine levels in situ. In response to infection, the predominant cells recruited were neutrophils, monocytes and eosinophils. Concomitantly, the chemokines KC, LIX, and MIP-2 in situ were drastically increased in mice infected with the SpyCEP knockout strain, and growth of this mutant strain was reduced compared to the wild type. SpyCEP has been described as a potential vaccine candidate against S. pyogenes, and we showed that surface-associated SpyCEP was recognized by specific antibodies. In vitro, such antibodies also counteracted the inhibitory effects of SpyCEP on chemokine mediated PMN recruitment. Thus, α-SpyCEP antibodies may benefit the host both directly by enabling opsonophagocytosis, and indirectly, by neutralizing an important virulence factor. The animal model we employed shows promise for broad application in the study of bacterial pathogenesis.

Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is one of the most common causes of life-threatening bacterial infections in infants. In recent years cell surface pili have been identified in several Gram-positive bacteria, including GBS, as important virulence factors and promising vaccine candidates. In GBS, three structurally distinct types of pili have been discovered (pilus 1, 2a and 2b), whose structural subunits are assembled in high-molecular weight polymers by specific class C sortases. In addition, the highly conserved housekeeping sortase A (SrtA), whose main role is to link surface proteins to bacterial cell wall peptidoglycan by a transpeptidation reaction, is also involved in pili cell wall anchoring in many bacteria. Through in vivo mutagenesis, we demonstrate that the LPXTG sorting signal of the minor ancillary protein (AP2) is essential for pilus 2a anchoring. We successfully produced a highly purified recombinant SrtA (SrtA(ΔN40)) able to specifically hydrolyze the sorting signal of pilus 2a minor ancillary protein (AP2-2a) and catalyze in vitro the transpeptidation reaction between peptidoglycan analogues and the LPXTG motif, using both synthetic fluorescent peptides and recombinant proteins. By contrast, SrtA(ΔN40) does not catalyze the transpeptidation reaction with substrate-peptides mimicking sorting signals of the other pilus 2a subunits (the backbone protein and the major ancillary protein). Thus, our results add further insight into the proposed model of GBS pilus 2a assembly, in which SrtA is required for pili cell wall covalent attachment, acting exclusively on the minor accessory pilin, representing the terminal subunit located at the base of the pilus.

Group A Streptococcus (GAS, Streptococcus pyogenes) is a gram-positive human pathogen responsible for a diverse variety of diseases, including pharyngitis, skin infections, invasive necrotizing fasciitis and autoimmune sequelae. We have recently shown that GAS cell adhesion and biofilm formation is associated with the presence of pili on the surface of these bacteria. GAS pilus proteins are encoded in the FCT (Fibronectin-Collagen-T antigen) genomic region, of which nine different variants have been identified so far. In the present study we undertook a global analysis of GAS isolates representing the majority of FCT-variants to investigate the effect of environmental growth conditions on their capacity to form multicellular communities. For FCT-types 2, 3, 5 and 6 and a subset of FCT-4 strains, we observed that acidification resulting from fermentative sugar metabolism leads to an increased ability of the bacteria to form biofilm on abiotic surfaces and microcolonies on epithelial cells. The higher biofilm forming capacity at low environmental pH was directly associated with an enhanced expression of the genes encoding the pilus components and of their transcription regulators. The data indicate that environmental pH affects the expression of most pilus types and thereby the formation of multicellular cell-adhering communities that assist the initial steps of GAS infection.

Streptococcus agalactiae is the primary colonizer of the anogenital mucosa of up to 30% of healthy women and can infect newborns during delivery and cause severe sepsis and meningitis. Persistent colonization usually involves the formation of biofilm and increasing evidences indicate that in pathogenic streptococci biofilm formation is mediated by pili. Recently, we have characterized pili distribution and conservation in 289 GBS clinical isolates and we have shown that GBS has three pilus types, 1, 2a and 2b encoded by three corresponding pilus islands, and that each strain carries one or two islands. Here we have investigated the capacity of these strains to form biofilms. We have found that most of the biofilm-formers carry pilus 2a, and using insertion and deletion mutants we have confirmed that pilus type 2a, but not pilus types 1 and 2b, confers biofilm-forming phenotype. We also show that deletion of the major ancillary protein of type 2a did not impair biofilm formation while the inactivation of the other ancillary protein and of the backbone protein completely abolished this phenotype. Furthermore, antibodies raised against pilus components inhibited bacterial adherence to solid surfaces, offering new strategies to prevent GBS infection by targeting bacteria during their initial attachment to host epithelial cells.

The human pathogen Group A Streptococcus (Streptococcus pyogenes, GAS) is widely recognized as a major cause of common pharyngitis as well as of severe invasive diseases and non-suppurative sequelae associated with the existence of GAS antigens eliciting host autoantibodies. It has been proposed that a subset of paediatric disorders characterized by tics and obsessive-compulsive symptoms would exacerbate in association with relapses of GAS-associated pharyngitis. This hypothesis is however still controversial. In the attempt to shed light on the contribution of GAS infections to the onset of neuropsychiatric or behavioral disorders affecting as many as 3% of children and adolescents, we tested the antibody response of tic patient sera to a representative panel of GAS antigens. In particular, 102 recombinant proteins were spotted on nitrocellulose-coated glass slides and probed against 61 sera collected from young patients with typical tic neuropsychiatric symptoms but with no overt GAS infection. Sera from 35 children with neither tic disorder nor overt GAS infection were also analyzed. The protein recognition patterns of these two sera groups were compared with those obtained using 239 sera from children with GAS-associated pharyngitis. This comparative analysis identified 25 antigens recognized by sera of the three patient groups and 21 antigens recognized by tic and pharyngitis sera, but poorly or not recognized by sera from children without tic. Interestingly, these antigens appeared to be, in quantitative terms, more immunogenic in tic than in pharyngitis patients. Additionally, a third group of antigens appeared to be preferentially and specifically recognized by tic sera. These findings provide the first evidence that tic patient sera exhibit immunological profiles typical of individuals who elicited a broad, specific and strong immune response against GAS. This may be relevant in the context of one of the hypothesis proposing that GAS antigen-dependent induction of autoantibodies in susceptible individuals may be involved the occurrence of tic disorders.

Streptococcal pullulanases have been recently proposed as key components of the metabolic machinery involved in bacterial adaptation to host niches. By sequence analysis of the Group B Streptococcus (GBS) genome we found a novel putative surface exposed protein with pullulanase activity. We named such a protein SAP. The sap gene is highly conserved among GBS strains and homologous genes, such as PulA and SpuA, have been described in other pathogenic streptococci. The SAP protein contains two N-terminal carbohydrate-binding motifs, followed by a catalytic domain and a C-terminal LPXTG cell wall-anchoring domain. In vitro analysis revealed that the recombinant form of SAP is able to degrade alpha-glucan polysaccharides, such as pullulan, glycogen and starch. Moreover, NMR analysis showed that SAP acts as a type I pullulanase. Studies performed on whole bacteria indicated that the presence of alpha-glucan polysaccharides in culture medium up-regulated the expression of SAP on bacterial surface as confirmed by FACS analysis and confocal imaging. Deletion of the sap gene resulted in a reduced capacity of bacteria to grow in medium containing pullulan or glycogen, but not glucose or maltose, confirming the pivotal role of SAP in GBS metabolism of alpha-glucans. As reported for other streptococcal pullulanases, we found specific anti-SAP antibodies in human sera from healthy volunteers. Investigation of the functional role of anti-SAP antibodies revealed that incubation of GBS in the presence of sera from animals immunized with SAP reduced the capacity of the bacterium to degrade pullulan. Of interest, anti-SAP sera, although to a lower extent, also inhibited Group A Streptococcus pullulanase activity. These data open new perspectives on the possibility to use SAP as a potential vaccine component inducing functional cross-reacting antibodies interfering with streptococcal infections.