Evolution of invasive pneumococcal disease by serotype 3 in adults: a Spanish three-decade retrospective study.

Background: Invasive pneumococcal disease due to serotype 3 (S3-IPD) is associated with high mortality rates and long-term adverse effects. The introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) into the Spanish paediatric immunisation programme has not led to a decrease in the adult S3-IPD. We aimed to analyse the incidence, clinical characteristics and genomics of S3-IPD in adults in Spain. Methods: Adult IPD episodes hospitalized in a Southern Barcelona hospital were prospectively collected (1994-2020). For genomic comparison, S3-IPD isolates from six Spanish hospitals (2008-2020) and historical isolates (1989-1993) were analysed by WGS (Illumina and/or MinION). Findings: From 1994 to 2020, 270 S3-IPD episodes were detected. When comparing pre-PCV (1994-2001) and late-PCV13 (2016-2020) periods, only modest changes in S3-IPD were observed (from 1.58 to 1.28 episodes per 100,000 inhabitants year). In this period, the incidence of the two main lineages shifted from 0.38 to 0.67 (CC180-GPSC12) and from 1.18 to 0.55 (CC260-GPSC83). The overall 30-day mortality remained high (24.1%), though a decrease was observed between the pre-PCV (32.4%; 95.0% CI, 22.0-45.0) and the late-PCV13 period (16.7%; 95.0% CI, 7.5-32.0) (p = 0.06). At the same time, comorbidities increased from 77.3% (95.0% CI, 65.0-86.0) to 85.7% (95.0% CI, 71.0-94.0) (p = 0.69). There were no differences in clinical characteristics or 30-day mortality between the two S3 lineages. Although both lineages were genetically homogeneous, the CC180-GPSC12 lineage presented a higher SNP density, a more open pan-genome, and a major presence of prophages and mobile genetic elements carrying resistance genes. Interpretation: Adult S3-IPD remained stable in our area over the study period despite PCV13 introduction in children. However, a clonal shift was observed. The decrease in mortality rates and the increase in comorbidities suggest a change in clinical management and overall population characteristics. The low genetic variability and absence of clinical differences between lineages highlight the role of the S3 capsule in the disease severity. Funding: This study has been funded by Instituto de Salud Carlos III (ISCIII) "PI18/00339", "PI21/01000", "INT22/00096", "FI22/00279", CIBER "CIBERES-CB06/06/0037", "CIBERINFEC-CB21/13/00009" and MSD grant "IISP 60168".

Cytomegalovirus UL44 protein induces a potent T-cell immune response in mice.

Due to the severity of CMV infection in immunocompromised individuals the development of a vaccine has been declared a priority. However, despite the efforts made there is no yet a vaccine available for clinical use. We designed an approach to identify new CMV antigens able to inducing a broad immune response that could be used in future vaccine formulations. We have used serum samples from 28 kidney transplant recipients, with a previously acquired CMV-specific immune response to identify viral proteins that were recognized by the antibodies present in the patient serum samples by Western blot. A band of approximately 45 kDa, identified as UL44, was detected by most serum samples. UL44 immunogenicity was tested in BALB/c mice that received three doses of the UL44-pcDNA DNA vaccine. UL44 elicited both, a strong antibody response and CMV-specific cellular response. Using bioinformatic analysis we demonstrated that UL44 is a highly conserved protein and contains epitopes that are able to activate CD8 lymphocytes of the most common HLA alleles in the world population. We constructed a UL44 ORF deletion mutant virus that produced no viral progeny, suggesting that UL44 is an essential viral protein. In addition, other authors have demonstrated that UL44 is one of the most abundant viral proteins after infection and have suggested an essential role of UL44 in viral replication. Altogether, our data suggests that UL44 is a potent antigen, and favored by its abundance, it may be a good candidate to include in a vaccine formulation.

Mutation Analysis in Regulator DNA-Binding Regions for Antimicrobial Efflux Pumps in 17,000 Pseudomonas aeruginosa Genomes.

Mutations leading to upregulation of efflux pumps can produce multiple drug resistance in the pathogen Pseudomonas aeruginosa. Changes in their DNA binding regions, i.e., palindromic operators, can compromise pump depression and subsequently enhance resistance against several antibacterials and biocides. Here, we have identified (pseudo)palindromic repeats close to promoters of genes encoding 13 core drug-efflux pumps of P. aeruginosa. This framework was applied to detect mutations in these repeats in 17,292 genomes. Eighty-nine percent of isolates carried at least one mutation. Eight binary genetic properties potentially related to expression were calculated for mutations. These included palindromicity reduction, mutation type, positioning within the repeat and DNA-bending shift. High-risk ST298, ST308 and ST357 clones commonly carried four conserved mutations while ST175 and the cystic fibrosis-linked ST649 clones showed none. Remarkably, a T-to-C transition in the fourth position of the upstream repeat for mexEF-oprN was nearly exclusive of the high-risk ST111 clone. Other mutations were associated with high-risk sublineages using sample geotemporal metadata. Moreover, 1.5% of isolates carried five or more mutations suggesting they undergo an alternative program for regulation of their effluxome. Overall, P. aeruginosa shows a wide range of operator mutations with a potential effect on efflux pump expression and antibiotic resistance.

The challenges of the genome-based identification of antifungal resistance in the clinical routine.

The increasing number of chronic and life-threatening infections caused by antimicrobial resistant fungal isolates is of critical concern. Low DNA sequencing cost may facilitate the identification of the genomic profile leading to resistance, the resistome, to rationally optimize the design of antifungal therapies. However, compared to bacteria, initiatives for resistome detection in eukaryotic pathogens are underdeveloped. Firstly, reported mutations in antifungal targets leading to reduced susceptibility must be extensively collected from the literature to generate comprehensive databases. This information should be complemented with specific laboratory screenings to detect the highest number possible of relevant genetic changes in primary targets and associations between resistance and other genomic markers. Strikingly, some drug resistant strains experience high-level genetic changes such as ploidy variation as much as duplications and reorganizations of specific chromosomes. Such variations involve allelic dominance, gene dosage increments and target expression regime effects that should be explicitly parameterized in antifungal resistome prediction algorithms. Clinical data indicate that predictors need to consider the precise pathogen species and drug levels of detail, instead of just genus and drug class. The concomitant needs for mutation accuracy and assembly quality assurance suggest hybrid sequencing approaches involving third-generation methods will be utilized. Moreover, fatal fast infections, like fungemia and meningitis, will further require both sequencing and analysis facilities are available in-house. Altogether, the complex nature of antifungal resistance demands extensive sequencing, data acquisition and processing, bioinformatic analysis pipelines, and standard protocols to be accomplished prior to genome-based protocols are applied in the clinical setting.

StaR Is a Positive Regulator of Topoisomerase I Activity Involved in Supercoiling Maintenance in Streptococcus pneumoniae.

The DNA topoisomerases gyrase and topoisomerase I as well as the nucleoid-associated protein HU maintain supercoiling levels in Streptococcus pneumoniae, a main human pathogen. Here, we characterized, for the first time, a topoisomerase I regulator protein (StaR). In the presence of sub-inhibitory novobiocin concentrations, which inhibit gyrase activity, higher doubling times were observed in a strain lacking staR, and in two strains in which StaR was over-expressed either under the control of the ZnSO4-inducible PZn promoter (strain ΔstaRPZnstaR) or of the maltose-inducible PMal promoter (strain ΔstaRpLS1ROMstaR). These results suggest that StaR has a direct role in novobiocin susceptibility and that the StaR level needs to be maintained within a narrow range. Treatment of ΔstaRPZnstaR with inhibitory novobiocin concentrations resulted in a change of the negative DNA supercoiling density (σ) in vivo, which was higher in the absence of StaR (σ = -0.049) than when StaR was overproduced (σ = -0.045). We have located this protein in the nucleoid by using super-resolution confocal microscopy. Through in vitro activity assays, we demonstrated that StaR stimulates TopoI relaxation activity, while it has no effect on gyrase activity. Interaction between TopoI and StaR was detected both in vitro and in vivo by co-immunoprecipitation. No alteration of the transcriptome was associated with StaR amount variation. The results suggest that StaR is a new streptococcal nucleoid-associated protein that activates topoisomerase I activity by direct protein-protein interaction.

Unsupervised Machine Learning Organization of the Functional Dark Proteome of Gram-Negative "Superbugs": Six Protein Clusters Amenable for Distinct Scientific Applications.

Uncharacterized proteins have been underutilized as targets for the development of novel therapeutics for difficult-to-treat bacterial infections. To facilitate the exploration of these proteins, 2819 predicted, uncharacterized proteins (19.1% of the total) from reference strains of multidrug Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa species were organized using an unsupervised k-means machine learning algorithm. Classification using normalized values for protein length, pI, hydrophobicity, degree of conservation, structural disorder, and %AT of the coding gene rendered six natural clusters. Cluster proteins showed different trends regarding operon membership, expression, presence of unknown function domains, and interactomic relevance. Clusters 2, 4, and 5 were enriched with highly disordered proteins, nonworkable membrane proteins, and likely spurious proteins, respectively. Clusters 1, 3, and 6 showed closer distances to known antigens, antibiotic targets, and virulence factors. Up to 21.8% of proteins in these clusters were structurally covered by modeling, which allowed assessment of druggability and discontinuous B-cell epitopes. Five proteins (4 in Cluster 1) were potential druggable targets for antibiotherapy. Eighteen proteins (11 in Cluster 6) were strong B-cell and T-cell immunogen candidates for vaccine development. Conclusively, we provide a feature-based schema to fractionate the functional dark proteome of critical pathogens for fundamental and biomedical purposes.

Association between HLA-C alleles and COVID-19 severity in a pilot study with a Spanish Mediterranean Caucasian cohort.

The clinical presentations of COVID-19 may range from an asymptomatic or mild infection to a critical or fatal disease. Several host factors such as elderly age, male gender, and previous comorbidities seem to be involved in the most severe outcomes, but also an impaired immune response that causes a hyperinflammatory state but is unable to clear the infection. In order to get further understanding about this impaired immune response, we aimed to determine the association of specific HLA alleles with different clinical presentations of COVID-19. Therefore, we analyzed HLA Class I and II, as well as KIR gene sequences, in 72 individuals with Spanish Mediterranean Caucasian ethnicity who presented mild, severe, or critical COVID-19, according to their clinical characteristics and management. This cohort was recruited in Madrid (Spain) during the first and second pandemic waves between April and October 2020. There were no significant differences in HLA-A or HLA-B alleles among groups. However, despite the small sample size, we found that HLA-C alleles from group C1 HLA-C*08:02, -C*12:03, or -C*16:01 were more frequently associated in individuals with mild COVID-19 (43.8%) than in individuals with severe (8.3%; p = 0.0030; pc = 0.033) and critical (16.1%; p = 0.0014; pc = 0.0154) disease. C1 alleles are supposed to be highly efficient to present peptides to T cells, and HLA-C*12:03 may present a high number of verified epitopes from abundant SARS-CoV-2 proteins M, N, and S, thereby being allegedly able to trigger an efficient antiviral response. On the contrary, C2 alleles are usually poorly expressed on the cell surface due to low association with ß2-microglobulin (ß2M) and peptides, which may impede the adequate formation of stable HLA-C/ß2M/peptide heterotrimers. Consequently, this pilot study described significant differences in the presence of specific HLA-C1 alleles in individuals with different clinical presentations of COVID-19, thereby suggesting that HLA haplotyping could be valuable to get further understanding in the underlying mechanisms of the impaired immune response during critical COVID-19.

Genomic features of predominant non-PCV13 serotypes responsible for adult invasive pneumococcal disease in Spain.

BACKGROUND: Although pneumococcal conjugate vaccines (PCVs) effectively prevent invasive pneumococcal disease (IPD), serotype replacement has occurred. OBJECTIVES: We studied the pangenome, antibiotic resistance mechanisms and presence of mobile elements in predominant non-PCV13 serotypes causing adult IPD after PCV13 vaccine introduction in Spain. METHODS: We conducted a multicentre study comparing three periods in six Spanish hospitals and analysed through whole genome sequencing representative strains collected in the pre-PCV13, early-PCV13 and late-PCV13 periods. RESULTS: Among 2197 cases of adult IPD identified, 110 pneumococci expressing non-PCV13 capsules were sequenced. Seven predominant serotypes accounted for 42.6% of IPD episodes in the late-PCV13 period: serotypes 8 (14.4%), 12F (7.5%), 9N (5.2%), 11A (4.1%), 22F (3.9%), 24F (3.9%) and 16F (3.6%). All predominant non-PCV13 serotypes were highly clonal, comprising one or two clonal complexes (CC). In general, CC538, CC4048, CC3016F, CC43322F and CC669N, related to predominant non-PCV13 serotypes, were antibiotic susceptible. CC15611A was associated with resistance to co-trimoxazole, penicillin and amoxicillin. CC23024F was non-susceptible to penicillin and resistant to erythromycin, clindamycin, and tetracycline. Six composite transposon structures of the Tn5252-family were found in CC23024F, CC98912F and CC3016F carrying different combinations of erm(B), tet(M), and cat. Pangenome analysis revealed differences in accessory genomes among the different CC, with most variety in CC3016F (23.9%) and more conservation in CC15611A (8.5%). CONCLUSIONS: We identified highly clonal predominant serotypes responsible for IPD in adults. The detection of not only conjugative elements carrying resistance determinants but also clones previously associated with vaccine serotypes (CC15611A and CC23024F) highlights the importance of the accessory genome.

Identification of Promoter Region Markers Associated With Altered Expression of Resistance-Nodulation-Division Antibiotic Efflux Pumps in Acinetobacter baumannii.

Genetic alterations leading to the constitutive upregulation of specific efflux pumps contribute to antibacterial resistance in multidrug resistant bacteria. The identification of such resistance markers remains one of the most challenging tasks of genome-level resistance predictors. In this study, 487 non-redundant genetic events were identified in upstream zones of three operons coding for resistance-nodulation-division (RND) efflux pumps of 4,130 Acinetobacter baumannii isolates. These events included insertion sequences, small indels, and single nucleotide polymorphisms. In some cases, alterations explicitly modified the expression motifs described for these operons, such as the promoter boxes, operators, and Shine-Dalgarno sequences. In addition, changes in DNA curvature and mRNA secondary structures, which are structural elements that regulate expression, were also calculated. According to their influence on RND upregulation, the catalog of upstream modifications were associated with "experimentally verified," "presumed," and "probably irrelevant" degrees of certainty. For experimental verification, DNA of upstream sequences independently carrying selected markers, three for each RND operon, were fused to a luciferase reporter plasmid system. Five out of the nine selected markers tested showed significant increases in expression with respect to the wild-type sequence control. In particular, a 25-fold expression increase was observed with the ISAba1 insertion sequence upstream the adeABC pump. Next, overexpression of each of the three multi-specific RND pumps was linked to their respective antibacterial substrates by a deep A. baumannii literature screen. Consequently, a data flow framework was then developed to link genomic upregulatory RND determinants to potential antibiotic resistance. Assignment of potential increases in minimal inhibitory concentrations at the "experimentally verified" level was permitted for 42 isolates to 7-8 unrelated antibacterial agents including tigecycline, which is overlooked by conventional resistome predictors. Thus, our protocol may represent a time-saving filter step prior to laborious confirmation experiments for efflux-driven resistance. Altogether, a computational-experimental pipeline containing all components required for identifying the upstream regulatory resistome is proposed. This schema may provide the foundational stone for the elaboration of tools approaching antibiotic efflux that complement routine resistome predictors for preventing antimicrobial therapy failure against difficult-to-threat bacteria.

Cross-Recognition of SARS-CoV-2 B-Cell Epitopes with Other Betacoronavirus Nucleoproteins.

The B and T lymphocytes of the adaptive immune system are important for the control of most viral infections, including COVID-19. Identification of epitopes recognized by these cells is fundamental for understanding how the immune system detects and removes pathogens, and for antiviral vaccine design. Intriguingly, several cross-reactive T lymphocyte epitopes from SARS-CoV-2 with other betacoronaviruses responsible for the common cold have been identified. In addition, antibodies that cross-recognize the spike protein, but not the nucleoprotein (N protein), from different betacoronavirus have also been reported. Using a consensus of eight bioinformatic methods for predicting B-cell epitopes and the collection of experimentally detected epitopes for SARS-CoV and SARS-CoV-2, we identified four surface-exposed, conserved, and hypothetical antigenic regions that are exclusive of the N protein. These regions were analyzed using ELISA assays with two cohorts: SARS-CoV-2 infected patients and pre-COVID-19 samples. Here we describe four epitopes from SARS-CoV-2 N protein that are recognized by the humoral response from multiple individuals infected with COVID-19, and are conserved in other human coronaviruses. Three of these linear surface-exposed sequences and their peptide homologs in SARS-CoV-2 and HCoV-OC43 were also recognized by antibodies from pre-COVID-19 serum samples, indicating cross-reactivity of antibodies against coronavirus N proteins. Different conserved human coronaviruses (HCoVs) cross-reactive B epitopes against SARS-CoV-2 N protein are detected in a significant fraction of individuals not exposed to this pandemic virus. These results have potential clinical implications.

Predicted impact of the viral mutational landscape on the cytotoxic response against SARS-CoV-2.

The massive assessment of immune evasion due to viral mutations that increase COVID-19 susceptibility can be computationally facilitated. The adaptive cytotoxic T response is critical during primary infection and the generation of long-term protection. Here, potential HLA class I epitopes in the SARS-CoV-2 proteome were predicted for 2,915 human alleles of 71 families using the netMHCIpan EL algorithm. Allele families showed extreme epitopic differences, underscoring genetic variability of protective capacity between humans. Up to 1,222 epitopes were associated with any of the twelve supertypes, that is, allele clusters covering 90% population. Next, from all mutations identified in ~118,000 viral NCBI isolates, those causing significant epitope score reduction were considered epitope escape mutations. These mutations mainly involved non-conservative substitutions at the second and C-terminal position of the ligand core, or total ligand removal by large recurrent deletions. Escape mutations affected 47% of supertype epitopes, which in 21% of cases concerned isolates from two or more sub-continental areas. Some of these changes were coupled, but never surpassed 15% of evaded epitopes for the same supertype in the same isolate, except for B27. In contrast to most supertypes, eight allele families mostly contained alleles with few SARS-CoV-2 ligands. Isolates harboring cytotoxic escape mutations for these families co-existed geographically within sub-Saharan and Asian populations enriched in these alleles according to the Allele Frequency Net Database. Collectively, our findings indicate that escape mutation events have already occurred for half of HLA class I supertype epitopes. However, it is presently unlikely that, overall, it poses a threat to the global population. In contrast, single and double mutations for susceptible alleles may be associated with viral selective pressure and alarming local outbreaks. The integration of genomic, geographical and immunoinformatic information eases the surveillance of variants potentially affecting the global population, as well as minority subpopulations.

Abundance, Betweenness Centrality, Hydrophobicity, and Isoelectric Points Are Relevant Factors in the Processing of Parental Proteins of the HLA Class II Ligandome.

Adaptive cellular and humoral immune responses to infectious agents require previous recognition of pathogenic peptides bound to human leukocyte antigen (HLA) class II molecules exposed on the surface of the professional antigen-presenting cells. Knowledge of how these peptide ligands are generated is essential to understand the basis for CD4+ T-cell-mediated immunity and tolerance. In this study, a high-throughput mass spectrometry analysis was used to identify more than 16,000 cell peptides bound to several HLA-DR and -DP class II molecules isolated from large amounts of uninfected and virus-infected human cells (ProteomeXchange accession: PXD028006). The analysis of the 1808 parental proteins containing HLA class II ligands revealed that these cell proteins were more acidic, abundant, and highly connected but less hydrophilic than non-parental proteomes. Therefore, the percentage of acidic residues was increased and hydroxyl and polar residues were decreased in the parental proteins for the HLA class II ligandomes versus the non-parental proteomes. This definition of the properties shared by parental proteins that constitute the source of the HLA class II ligandomes can serve as the basis for the development of bioinformatics tools to predict proteins that are most likely recognized by the immune system through the CD4+ helper T lymphocytes in both autoimmunity and infection.

Insights into the functional expansion of the astacin peptidase family in parasitic helminths.

Helminths secrete a plethora of proteins involved in parasitism-related processes such as tissue penetration, migration, feeding and immunoregulation. Astacins, a family of zinc metalloproteases belonging to the peptidase family M12, are one of the most abundantly represented protein families in the secretomes of helminths. Despite their involvement in virulence, very few studies have addressed the role of this loosely defined protein group in parasitic helminths. Herein, we have analysed the predicted proteomes from 154 helminth species and confirmed the expansion of the astacin family in several nematode taxa. The astacin domain associated with up to 110 other domains into 145 unique domain architectures, where CUB and ShK constitute the principal and nearly independent bi-domain frameworks. The presence of co-existing domains suggests promiscuous adaptable functions to several roles. These activities could be related either to substrate specificity or to higher-order functions, such as anti-angiogenesis and immunomodulation, where the astacin domain would play an accessory role. Furthermore, some phylogenetically restricted mutations in the astacin domain affected residues located at the active cleft and binding sub-pockets, suggesting adaptation to different substrate specificities. Altogether, these findings suggest the astacin domain is a highly adaptable module that fulfils multiple proteolytic needs of the parasitic lifestyle. This study contributes to the understanding of helminth-secreted astacins and, ultimately, provides the foundation to guide future investigations about the role of this diverse family of proteins in host-parasite interactions.

Predicted Epitope Abundance Supports Vaccine-Induced Cytotoxic Protection Against SARS-CoV-2 Variants of Concern.

The effect of emerging SARS-CoV-2 variants on vaccine efficacy is of critical importance. In this study, the potential impact of mutations that facilitate escape from the cytotoxic cellular immune response in these new virus variants for the 551 most abundant HLA class I alleles was analyzed. Computational prediction showed that most of these alleles, that cover >90% of the population, contain enough epitopes without escape mutations in the principal SARS-CoV-2 variants. These data suggest that the cytotoxic cellular immune protection elicited by vaccination is not greatly affected by emerging SARS-CoV-2 variants.

Streptococcus pneumoniae: a Plethora of Temperate Bacteriophages With a Role in Host Genome Rearrangement.

Bacteriophages (phages) are viruses that infect bacteria. They are the most abundant biological entity on Earth (current estimates suggest there to be perhaps 1031 particles) and are found nearly everywhere. Temperate phages can integrate into the chromosome of their host, and prophages have been found in abundance in sequenced bacterial genomes. Prophages may modulate the virulence of their host in different ways, e.g., by the secretion of phage-encoded toxins or by mediating bacterial infectivity. Some 70% of Streptococcus pneumoniae (the pneumococcus)-a frequent cause of otitis media, pneumonia, bacteremia and meningitis-isolates harbor one or more prophages. In the present study, over 4000 S. pneumoniae genomes were examined for the presence of prophages, and nearly 90% were found to contain at least one prophage, either defective (47%) or present in full (43%). More than 7000 complete putative integrases, either of the tyrosine (6243) or serine (957) families, and 1210 full-sized endolysins (among them 1180 enzymes corresponding to 318 amino acid-long N-acetylmuramoyl-L-alanine amidases [LytAPPH]) were found. Based on their integration site, 26 different pneumococcal prophage groups were documented. Prophages coding for tRNAs, putative virulence factors and different methyltransferases were also detected. The members of one group of diverse prophages (PPH090) were found to integrate into the 3' end of the host lytASpn gene encoding the major S. pneumoniae autolysin without disrupting it. The great similarity of the lytASpn and lytAPPH genes (85-92% identity) allowed them to recombine, via an apparent integrase-independent mechanism, to produce different DNA rearrangements within the pneumococcal chromosome. This study provides a complete dataset that can be used to further analyze pneumococcal prophages, their evolutionary relationships, and their role in the pathogenesis of pneumococcal disease.

Designing Multi-Antigen Vaccines Against Acinetobacter baumannii Using Systemic Approaches.

Vaccines and monoclonal antibodies are promising approaches for preventing and treating infections caused by multidrug resistant Acinetobacter baumannii. However, only partial protection has been achieved with many previously tested protein antigens, which suggests that vaccines incorporating multiple antigens may be necessary in order to obtain high levels of protection. Several aspects that use the wealth of omic data available for A. baumannii have not been fully exploited for antigen identification. In this study, the use of fractionated proteomic and computational data from ~4,200 genomes increased the number of proteins potentially accessible to the humoral response to 8,824 non-redundant proteins in the A. baumannii panproteome. Among them, 59% carried predicted B-cell epitopes and T-cell epitopes recognized by two or more alleles of the HLA class II DP supertype. Potential cross-reactivity with human proteins was detected for 8.9% of antigens at the protein level and 2.7% at the B-cell epitope level. Individual antigens were associated with different infection types by genomic, transcriptomic or functional analyses. High intra-clonal genome density permitted the identification of international clone II as a "vaccitype", in which 20% of identified antigens were specific to this clone. Network-based centrality measurements were used to identify multiple immunologic nodes. Data were formatted, unified and stored in a data warehouse database, which was subsequently used to identify synergistic antigen combinations for different vaccination strategies. This study supports the idea that integration of multi-omic data and fundamental knowledge of the pathobiology of drug-resistant bacteria can facilitate the development of effective multi-antigen vaccines against these challenging infections.

Immunization with SP_1992 (DiiA) Protein of Streptococcus pneumoniae Reduces Nasopharyngeal Colonization and Protects against Invasive Disease in Mice.

Knowledge-based vaccinology can reveal uncharacterized antigen candidates for a new generation of protein-based anti-pneumococcal vaccines. DiiA, encoded by the sp_1992 locus, is a surface protein containing either one or two repeats of a 37mer N-terminal motif that exhibits low interstrain variability. DiiA belongs to the core proteome, contains several conserved B-cell epitopes, and is associated with colonization and pathogenesis. Immunization with DiiA protein via the intraperitoneal route induced a strong IgG response, including different IgG subtypes. Vaccination with DiiA increased bacterial clearance and induced protection against sepsis, conferring 70% increased survival at 48 h post-infection when compared to the adjuvant control. The immunogenic response and survival rates in mice immunized with a truncated DiiA version lacking 119 N-terminal residues were remarkably lower, confirming the relevance of the repeat zone in the immunoprotection by DiiA. Intranasal immunization of mice with the entire recombinant protein elicited mucosal IgG and IgA responses that reduced bacterial colonization of the nasopharynx, confirming that this protein might be a vaccine candidate for reducing the carrier rate. DiiA constitutes an example of how functionally unannotated proteins may still represent promising candidates that can be used in prophylactic strategies against the pneumococcal carrier state and invasive disease.

Identification and Analysis of Unstructured, Linear B-Cell Epitopes in SARS-CoV-2 Virion Proteins for Vaccine Development.

The efficacy of SARS-CoV-2 nucleic acid-based vaccines may be limited by proteolysis of the translated product due to anomalous protein folding. This may be the case for vaccines employing linear SARS-CoV-2 B-cell epitopes identified in previous studies since most of them participate in secondary structure formation. In contrast, we have employed a consensus of predictors for epitopic zones plus a structural filter for identifying 20 unstructured B-cell epitope-containing loops (uBCELs) in S, M, and N proteins. Phylogenetic comparison suggests epitope switching with respect to SARS-CoV in some of the identified uBCELs. Such events may be associated with the reported lack of serum cross-protection between the 2003 and 2019 pandemic strains. Incipient variability within a sample of 1639 SARS-CoV-2 isolates was also detected for 10 uBCELs which could cause vaccine failure. Intermediate stages of the putative epitope switch events were observed in bat coronaviruses in which additive mutational processes possibly facilitating evasion of the bat immune system appear to have taken place prior to transfer to humans. While there was some overlap between uBCELs and previously validated SARS-CoV B-cell epitopes, multiple uBCELs had not been identified in prior studies. Overall, these uBCELs may facilitate the development of biomedical products for SARS-CoV-2.

The Versatility of Opportunistic Infections Caused by Gemella Isolates Is Supported by the Carriage of Virulence Factors From Multiple Origins.

The molecular basis of the pathogenesis of the opportunistic invasive infections caused by isolates of the Gemella genus remains largely unknown. Moreover, inconsistencies in the current species assignation were detected after genome-level comparison of 16 public Gemella isolates. A literature search detected that, between the two most pathogenic species, Gemella morbillorum causes about twice the number of cases compared to Gemella haemolysans. These two species shared their mean diseases - sepsis and endocarditis - but differed in causing other syndromes. A number of well-known virulence factors were harbored by all species, such as a manganese transport/adhesin sharing 83% identity from oral endocarditis-causing streptococci. Likewise, all Gemellae carried the genes required for incorporating phosphorylcholine into their cell walls and encoded some choline-binding proteins. In contrast, other proteins were species-specific, which may justify the known epidemiological differences. G. haemolysans, but not G. morbillorum, harbor a gene cluster potentially encoding a polysaccharidic capsule. Species-specific surface determinants also included Rib and MucBP repeats, hemoglobin-binding NEAT domains, peptidases of C5a complement factor and domains that recognize extracellular matrix molecules exposed in damaged heart valves, such as collagen and fibronectin. Surface virulence determinants were associated with several taxonomically dispersed opportunistic genera of the oral microbiota, such as Granulicatella, Parvimonas, and Streptococcus, suggesting the existence of a horizontally transferrable gene reservoir in the oral environment, likely facilitated by close proximity in biofilms and ultimately linked to endocarditis. The identification of the Gemella virulence pool should be implemented in whole genome-based protocols to rationally predict the pathogenic potential in ongoing clinical infections caused by these poorly known bacterial pathogens.

Modulation of Natural HLA-B*27:05 Ligandome by Ankylosing Spondylitis-associated Endoplasmic Reticulum Aminopeptidase 2 (ERAP2).

The HLA-B*27:05 allele and the endoplasmic reticulum-resident aminopeptidases are strongly associated with AS, a chronic inflammatory spondyloarthropathy. This study examined the effect of ERAP2 in the generation of the natural HLA-B*27:05 ligandome in live cells. Complexes of HLA-B*27:05-bound peptide pools were isolated from human ERAP2-edited cell clones, and the peptides were identified using high-throughput mass spectrometry analyses. The relative abundance of a thousand ligands was established by quantitative tandem mass spectrometry and bioinformatics analysis. The residue frequencies at different peptide position, identified in the presence or absence of ERAP2, determined structural features of ligands and their interactions with specific pockets of the antigen-binding site of the HLA-B*27:05 molecule. Sequence alignment of ligands identified with species of bacteria associated with HLA-B*27-dependent reactive arthritis was performed. In the absence of ERAP2, peptides with N-terminal basic residues and minority canonical P2 residues are enriched in the natural ligandome. Further, alterations of residue frequencies and hydrophobicity profile at P3, P7, and PΩ positions were detected. In addition, several ERAP2-dependent cellular peptides were highly similar to protein sequences of arthritogenic bacteria, including one human HLA-B*27:05 ligand fully conserved in a protein from Campylobacter jejuni These findings highlight the pathogenic role of this aminopeptidase in the triggering of AS autoimmune disease.