Hpv-specific systemic antibody responses and memory b cells are independently maintained up to 6 years and in a vaccine-specific manner following immunization with cervarix and gardasil in adolescent and young adult women in vaccination programs in Italy

Human papillomavirus (HPV) persistent infections are associated with cervical cancer and other HPV-related diseases and tumors. Thus, the characterization of long lasting immunity to currently available HPV vaccines is important. A total of 149 female subjects vaccinated with Cervarix or Gardasil participated to the study and they were stratified according to age (10–12-year-old and 16–20-year-old). Humoral immune responses (IgG and neutralizing antibody titers, antibody avidity) and circulating memory B cells were analyzed after an average of 4–6 years from the third immunization. The humoral responses against HPV-16 and HPV-18 (and HPV-6 and HPV- 11 for Gardasil) were high in both age groups and vaccines up to six years from the third dose. However, Cervarix induced significantly higher and more persistent antibody responses, while the two vaccines were rather equivalent in inducing memory B cells against HPV-16 and HPV-18. Moreover, the percentage of subjects with vaccine-specific memory B cells was even superior among Gardasil vaccinees and, conversely, Cervarix vaccinated individuals with circulating antibodies, but undetectable memory B cells were found. Finally, a higher proportion of Cervarix-vaccinated subjects displayed cross-neutralizing responses against non-vaccine types HPV-31 and HPV-45. Gardasil and Cervarix may, thus, differently affect long-lasting humoral immunity from both the quantitative and qualitative point of view

Fecal microbiota transfer between young and aged mice reverses hallmarks of the aging gut, eye, and brain

Background: Altered intestinal microbiota composition in later life is associated with inflammaging, declining tissue function, and increased susceptibility to age-associated chronic diseases, including neurodegenerative dementias. Here, we tested the hypothesis that manipulating the intestinal microbiota influences the development of major comorbidities associated with aging and, in particular, inflammation affecting the brain and retina. Methods: Using fecal microbiota transplantation, we exchanged the intestinal microbiota of young (3 months), old (18 months), and aged (24 months) mice. Whole metagenomic shotgun sequencing and metabolomics were used to develop a custom analysis workflow, to analyze the changes in gut microbiota composition and metabolic potential. Effects of age and microbiota transfer on the gut barrier, retina, and brain were assessed using protein assays, immunohistology, and behavioral testing. Results: We show that microbiota composition profiles and key species enriched in young or aged mice are successfully transferred by FMT between young and aged mice and that FMT modulates resulting metabolic pathway profiles. The transfer of aged donor microbiota into young mice accelerates age-associated central nervous system (CNS) inflammation, retinal inflammation, and cytokine signaling and promotes loss of key functional protein in the eye, effects which are coincident with increased intestinal barrier permeability. Conversely, these detrimental effects can be reversed by the transfer of young donor microbiota. Conclusions: These findings demonstrate that the aging gut microbiota drives detrimental changes in the gut–brain and gut–retina axes suggesting that microbial modulation may be of therapeutic benefit in preventing inflammation-related tissue decline in later life. [MediaObject not available: see fulltext.] Graphical abstract: [Figure not available: see fulltext.

Use of bioengineered human commensal gut bacteria-derived microvesicles for mucosal plague vaccine delivery and immunization

Plague caused by the Gram-negative bacterium, Yersinia pestis, is still endemic in parts of the world today. Protection against pneumonic plague is essential to prevent the development and spread of epidemics. Despite this, there are currently no licensed plague vaccines in the western world. Here we describe the means of delivering biologically active plague vaccine antigens directly to mucosal sites of plague infection using highly stable microvesicles (outer membrane vesicles; OMVs) that are naturally produced by the abundant and harmless human commensal gut bacterium Bacteroides thetaiotaomicron (Bt). Bt was engineered to express major plague protective antigens in its OMVs, specifically Fraction 1 (F1) in the outer membrane and LcrV (V antigen) in the lumen, for targeted delivery to the gastrointestinal (GI) and respiratory tracts in a non-human primate (NHP) host. Our key findings were that Bt OMVs stably expresses F1 and V plague antigens, particularly the V antigen, in the correct, immunogenic form. When delivered intranasally V-OMVs elicited substantive and specific immune and antibody responses, both in the serum [immunoglobulin (Ig)G] and in the upper and lower respiratory tract (IgA); this included the generation of serum antibodies able to kill plague bacteria. Our results also showed that Bt OMV-based vaccines had many desirable characteristics, including: biosafety and an absence of any adverse effects, pathology or gross alteration of resident microbial communities (microbiotas); high stability and thermo-tolerance; needle-free delivery; intrinsic adjuvanticity; the ability to stimulate both humoral and cell-mediated immune responses; and targeting of primary sites of plague infection

Fecal microbiota transfer between young and aged mice reverses hallmarks of the aging gut, eye, and brain

Background: Altered intestinal microbiota composition in later life is associated with inflammaging, declining tissue function, and increased susceptibility to age-associated chronic diseases, including neurodegenerative dementias. Here, we tested the hypothesis that manipulating the intestinal microbiota influences the development of major comorbidities associated with aging and, in particular, inflammation affecting the brain and retina. Methods: Using fecal microbiota transplantation, we exchanged the intestinal microbiota of young (3 months), old (18 months), and aged (24 months) mice. Whole metagenomic shotgun sequencing and metabolomics were used to develop a custom analysis workflow, to analyze the changes in gut microbiota composition and metabolic potential. Effects of age and microbiota transfer on the gut barrier, retina, and brain were assessed using protein assays, immunohistology, and behavioral testing. Results: We show that microbiota composition profiles and key species enriched in young or aged mice are successfully transferred by FMT between young and aged mice and that FMT modulates resulting metabolic pathway profiles. The transfer of aged donor microbiota into young mice accelerates age-associated central nervous system (CNS) inflammation, retinal inflammation, and cytokine signaling and promotes loss of key functional protein in the eye, effects which are coincident with increased intestinal barrier permeability. Conversely, these detrimental effects can be reversed by the transfer of young donor microbiota. Conclusions: These findings demonstrate that the aging gut microbiota drives detrimental changes in the gut–brain and gut–retina axes suggesting that microbial modulation may be of therapeutic benefit in preventing inflammation-related tissue decline in later life

A hybrid and poly-polish workflow for the complete and accurate assembly of phage genomes: a case study of ten przondoviruses

Bacteriophages (phages) within the genus Przondovirus are T7-like podoviruses belonging to the subfamily Studiervirinae, within the family Autographiviridae, and have a highly conserved genome organisation. The genomes of these phages range from 37 to 42 kb in size, encode 50–60 genes and are characterised by the presence of direct terminal repeats (DTRs) flanking the linear chromosome. These DTRs are often deleted during short-read-only and hybrid assemblies. Moreover, long-read-only assemblies are often littered with sequencing and/or assembly errors and require additional curation. Here, we present the isolation and characterisation of ten novel przondoviruses targeting Klebsiella spp. We describe HYPPA, a HYbrid and Poly-polish Phage Assembly workflow, which utilises long-read assemblies in combination with short-read sequencing to resolve phage DTRs and correcting errors, negating the need for laborious primer walking and Sanger sequencing validation. Our assembly workflow utilised Oxford Nanopore Technologies for long-read sequencing for its accessibility, making it the more relevant long-read sequencing technology at this time, and Illumina DNA Prep for short-read sequencing, representing the most commonly used technologies globally. Our data demonstrate the importance of careful curation of phage assemblies before publication, and prior to using them for comparative genomics

Microbial dynamics during shelf-life of industrial Ricotta cheese and identification of a Bacillus strain as a cause of a pink discolouration

Dairy products are perishable and have to be preserved from spoilage during the food chain to achieve the desired shelf-life. Ricotta is a typical Italian soft dairy food produced by heat coagulation of whey proteins and is considered to be a light and healthy product. The shelf-life of Ricotta could be extended, as required by the international food trade market; however, heat resistant microflora causes spoilage and poses issues regarding the safety of the product. Next-generation sequencing (NGS) applied to the Ricotta samples defined the composition of the microbial community in-depth during the shelf-life. The analysis demonstrated the predominance of spore-forming bacteria throughout the shelf-life, mostly belonging to Bacillus, Paenibacillus and Clostridium genera. A strain involved in spoilage and causing a pink discolouration of Ricotta was isolated and characterised as Bacillus mycoides/weihenstephanensis. This is the first report of a food discolouration caused by a toxigenic strain belonging to the Bacillus cereus group that resulted the predominant strain in the community of the defective ricotta. These results suggest that the processing of raw materials to eliminate spores and residual microflora could be essential for improving the quality and the safety of the product and to extend the shelf-life of industrial Ricotta

SNP-discovery by RAD-sequencing in a germplasm collection of cultivated and wild grapevine accessions

The discovery and use of genome-wide molecular markers across many individuals is crucial for evaluation of patterns and processes in evolutionary changes. RESTRICTION-SITE ASSOCIATED DNA SEQUENCING (RAD-seq) may be a suitable approach due to its ability to identify and get genotyped several markers simultaneously. A novel protocol of RAD-seq was set-up on 5500 SOLiD™ System introducing a biotinylated adapter. Afterwards the novel RAD-seq protocol was applied to a grapevine germplasm collection of 51 cultivars (Vitis vinifera subsp. sativa) and 45 wild accessions (V. vinifera subsp. sylvestis). The resulting 561’843’350 reads were aligned on the V. vinifera ‘PN40024’ reference genome sequence and almost all the predicted RE sites were covered. UNIFIEDGENOTYPER of Genome Analysis Toolkit was used to identify Single Nucleotide Polymorphisms (SNP). A final dataset of 52’644 good quality SNPs was obtained, among which 32’977 SNPs revealed a Minor Allele Frequence higher than 0.05. Based on the new grapevine gene prediction v2.1, 27’902 SNPs were intergenic, 5’649 missense, 240 nonsense and 3’106 synonymous. The genetic diversity analysis revealed how the RAD-seq markers are able to collect and show some undisclosed differences among wild and cultivated grapevine accessions. Therefore, the RAD-seq is a candidate approach to disclose the relationship between ancestor and domesticated species, helping to clarify the process of domestication

Comparison of PCR versus PCR-free DNA library preparation for characterising the human faecal virome

The human intestinal microbiota is abundant in viruses, comprising mainly bacteriophages, occasionally outnumbering bacteria 10:1 and is termed the virome. Due to their high genetic diversity and the lack of suitable tools and reference databases, the virome remains poorly characterised and is often referred to as “viral dark matter”. However, the choice of sequencing platforms, read lengths and library preparation make study design challenging with respect to the virome. Here we have compared the use of PCR and PCR-free methods for sequence-library construction on the Illumina sequencing platform for characterising the human faecal virome. Viral DNA was extracted from faecal samples of three healthy donors and sequenced. Our analysis shows that most variation was reflecting the individually specific faecal virome. However, we observed differences between PCR and PCR-free library preparation that affected the recovery of low-abundance viral genomes. Using three faecal samples in this study, the PCR library preparation samples led to a loss of lowerabundance vOTUs evident in their PCR-free pairs (vOTUs 128, 6202 and 8364) and decreased the alpha-diversity indices (Chao1 p-value = 0.045 and Simpson p-value = 0.044). Thus, differences between PCR and PCR-free methods are important to consider when investigating “rare” members of the gut virome, with these biases likely negligible when investigating moderately and highly abundant viruses

HPV-Specific Systemic Antibody Responses and Memory B Cells are Independently Maintained up to 6 Years and in a Vaccine-Specific Manner Following Immunization with Cervarix and Gardasil in Adolescent and Young Adult Women in Vaccination Programs in Italy

Humanpapillomavirus(HPV)persistentinfectionsareassociatedwithcervicalcancerandother HPV-related diseases and tumors. Thus, the characterization of long lasting immunity to currently availableHPVvaccinesisimportant. Atotalof149femalesubjectsvaccinatedwithCervarixorGardasil participatedtothestudyandtheywerestratifiedaccordingtoage(10\u201312-year-oldand16\u201320-year-old). Humoral immune responses (IgG and neutralizing antibody titers, antibody avidity) and circulating memoryBcellswereanalyzedafteranaverageof4\u20136yearsfromthethirdimmunization. Thehumoral responses against HPV-16 and HPV-18 (andHPV-6andHPV-11forGardasil) were high in both age groupsandvaccinesuptosixyearsfromthethirddose. However,Cervarixinducedsignificantlyhigher and more persistent antibody responses, while the two vaccines were rather equivalent in inducing memoryBcellsagainstHPV-16andHPV-18. Moreover,thepercentageofsubjectswithvaccine-specific memory B cells was even superior among Gardasil vaccinees and, conversely, Cervarix vaccinated individualswithcirculatingantibodies,butundetectablememoryBcellswerefound. Finally,ahigher proportionofCervarix-vaccinatedsubjectsdisplayedcross-neutralizingresponsesagainstnon-vaccine types HPV-31 and HPV-45. Gardasil and Cervarix may, thus, differ