Local and Systemic Immunity During Five Vaccinations Against SARS-CoV-2 in Zanubrutinib-Treated Patients With Chronic Lymphocytic Leukemia.

Background: Patients with chronic lymphocytic leukemia (CLL) are vulnerable to coronavirus disease 2019 (COVID-19) and are at risk of inferior response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination, especially if treated with the first-generation Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib. We aimed to evaluate the impact of the third-generation BTKi, zanubrutinib, on systemic and mucosal response to SARS-CoV-2 vaccination. Methods: Nine patients with CLL with ongoing zanubrutinib therapy were included and donated blood and saliva during SARS-CoV-2 vaccination, before vaccine doses 3 and 5 and 2 - 3 weeks after doses 3, 4, and 5. Ibrutinib-treated control patients (n = 7) and healthy aged-matched controls (n = 7) gave blood 2 - 3 weeks after vaccine dose 5. We quantified reactivity and neutralization capacity of SARS-CoV-2-specific IgG and IgA antibodies (Abs) in both serum and saliva, and reactivity of T cells activated with viral peptides. Results: Both zanubrutinib- and ibrutinib-treated patients had significantly, up to 1,000-fold, lower total spike-specific Ab levels after dose 5 compared to healthy controls (P < 0.01). Spike-IgG levels in serum from zanubrutinib-treated patients correlated well to neutralization capacity (r = 0.68; P < 0.0001) and were thus functional. Mucosal immunity (specific IgA in serum and saliva) was practically absent in zanubrutinib-treated patients even after five vaccine doses, whereas healthy controls had significantly higher levels (tested in serum after vaccine dose 5) (P < 0.05). In contrast, T-cell reactivity against SARS-CoV-2 peptides was equally high in zanubrutinib- and ibrutinib-treated patients as in healthy control donors. Conclusions: In our small cohort of zanubrutinib-treated CLL patients, we conclude that up to five doses of SARS-CoV-2 vaccination induced no detectable IgA mucosal immunity, which likely will impair the primary barrier defence against the infection. Systemic IgG responses were also impaired, whereas T-cell responses were normal. Further and larger studies are needed to evaluate the impact of these findings on disease protection.

Preliminary landscape of Candidatus Saccharibacteria in the human microbiome.

Introduction: Candidate Phyla Radiation (CPR) and more specifically Candidatus Saccharibacteria (TM7) have now been established as ubiquitous members of the human oral microbiota. Additionally, CPR have been reported in the gastrointestinal and urogenital tracts. However, the exploration of new human niches has been limited to date. Methods: In this study, we performed a prospective and retrospective screening of TM7 in human samples using standard PCR, real-time PCR, scanning electron microscopy (SEM) and shotgun metagenomics. Results: Using Real-time PCR and standard PCR, oral samples presented the highest TM7 prevalence followed by fecal samples, breast milk samples, vaginal samples and urine samples. Surprisingly, TM7 were also detected in infectious samples, namely cardiac valves and blood cultures at a low prevalence (under 3%). Moreover, we observed CPR-like structures using SEM in all sample types except cardiac valves. The reconstruction of TM7 genomes in oral and fecal samples from shotgun metagenomics reads further confirmed their high prevalence in some samples. Conclusion: This study confirmed, through their detection in multiple human samples, that TM7 are human commensals that can also be found in clinical settings. Their detection in clinical samples warrants further studies to explore their role in a pathological setting.

Clinical Microbial Identification of Severe Oral Infections by MALDI-TOF Mass Spectrometry in Stockholm County: an 11-Year (2010 to 2020) Epidemiological Investigation.

Growing evidence suggests that oral infections can modify the course of systemic diseases. To date, epidemiological data on microbial oral infections are scarce. Here, we performed a comprehensive analysis of the trend and microbial diversity in oral infection specimens referred for clinical microbiology analysis from 2010 to 2020. The microbes were isolated by culture and were identified via matrix-assisted laser desorption ionization-time of flight mass spectrometry technology (MALDI-TOF MS) throughout the study period. A total of 1,014 referred samples from dental clinics in Stockholm County with dentoalveolar abscesses and jaw osteomyelitis being the main reason were identified. Overall, the microbial composition was dominated by Firmicutes (51%), followed by Bacteroidetes (19%), Proteobacteria (12%), and Actinobacteria (5%). At the genus level, Streptococcus spp. (36%), Prevotella spp. (18%), and Staphylococcus spp. (11%) were among the most frequently reported. Interestingly, a strong increase in trend was noted for Streptococcus anginosus, Streptococcus mitis, Streptococcus sanguinis, Eikenella corrodens, Actinomyces spp., Aggregatibacter aphrophilus, Staphylococcus epidermidis, and Granulicatella adiacens during the study time (R = 0.66 to 0.89, P < 0.05), and a minor increase was noted for Enterococcus faecalis and Klebsiella spp., whereas steady levels were noted for most of the others. The present study shows the diversity of bacteria that have been involved in dental infections during the last decade in the capital of Sweden, as well as the emerging oral microbiota trend, with clear clinical implications on the oral-systemic link. IMPORTANCE Oral diseases and associated microbes are a risk factor for systemic diseases and can change the courses of these diseases. To date, epidemiological data on microbial oral infections are scarce, and longitudinal reports are lacking. We present for the first time the microbial composition of severe oral bacterial infections determined via the MALDI-TOF mass spectrometry technique in a comprehensive study between 2010 and 2020 (11 years) in Stockholm County. The trend and microbial diversity of oral infections were analyzed on referred clinical microbiological samples and were processed by standardized protocols. Trend increase was noted for Streptococcus anginosus, Streptococcus mitis, Streptococcus sanguinis, Eikenella corrodens, Actinomyces spp., Aggregatibacter aphrophilus, Staphylococcus epidermidis, Granulicatella adiacens, Enterococcus faecalis, and Klebsiella spp. Our results provide new insights into the diversity and trend of oral microbiota that were involved in serious oral infections over the past decade in the capital of Sweden and may influence the oral-systemic link.

Candidate Phyla Radiation, an Underappreciated Division of the Human Microbiome, and Its Impact on Health and Disease.

Candidate phyla radiation (CPR) is an emerging division of the bacterial domain within the human microbiota. Still poorly known, these microorganisms were first described in the environment in 1981 as "ultramicrobacteria" with a cell volume under 0.1 µm3 and were first associated with the human oral microbiota in 2007. The evolution of technology has been paramount for the study of CPR within the human microbiota. In fact, since these ultramicrobacteria have yet to be axenically cultured despite ongoing efforts, progress in imaging technology has allowed their observation and morphological description. Although their genomic abilities and taxonomy are still being studied, great strides have been made regarding their taxonomic classification, as well as their lifestyle. In addition, advancements in next-generation sequencing and the continued development of bioinformatics tools have allowed their detection as commensals in different human habitats, including the oral cavity and gastrointestinal and genital tracts, thus highlighting CPR as a nonnegligible part of the human microbiota with an impact on physiological settings. Conversely, several pathologies present dysbiosis affecting CPR levels, including inflammatory, mucosal, and infectious diseases. In this exhaustive review of the literature, we provide a historical perspective on the study of CPR, an overview of the methods available to study these organisms and a description of their taxonomy and lifestyle. In addition, their distribution in the human microbiome is presented in both homeostatic and dysbiotic settings. Future efforts should focus on developing cocultures and, if possible, axenic cultures to obtain isolates and therefore genomes that would provide a better understanding of these ultramicrobacteria, the importance of which in the human microbiome is undeniable.

Persistence of salivary antibody responses after COVID-19 vaccination is associated with oral microbiome variation in both healthy and people living with HIV.

Coevolution of microbiome and immunity at mucosal sites is essential for our health. Whether the oral microbiome, the second largest community after the gut, contributes to the immunogenicity of COVID-19 vaccines is not known. We investigated the baseline oral microbiome in individuals in the COVAXID clinical trial receiving the BNT162b2 mRNA vaccine. Participants (n=115) included healthy controls (HC; n=57) and people living with HIV (PLHIV; n=58) who met the study selection criteria. Vaccine-induced Spike antibodies in saliva and serum from 0 to 6 months were assessed and comparative analyses were performed against the individual salivary 16S ASV microbiome diversity. High- versus low vaccine responders were assessed on general, immunological, and oral microbiome features. Our analyses identified oral microbiome features enriched in high- vs. low-responders among healthy and PLHIV participants. In low-responders, an enrichment of Gram-negative, anaerobic species with proteolytic activity were found including Campylobacter, Butyrivibrio, Selenomonas, Lachnoanaerobaculum, Leptotrichia, Megasphaera, Prevotella and Stomatobaculum. In high-responders, enriched species were mainly Gram-positive and saccharolytic facultative anaerobes: Abiotrophia, Corynebacterium, Gemella, Granulicatella, Rothia, and Haemophilus. Combining identified microbial features in a classifier using the area under the receiver operating characteristic curve (ROC AUC) yielded scores of 0.879 (healthy controls) to 0.82 (PLHIV), supporting the oral microbiome contribution in the long-term vaccination outcome. The present study is the first to suggest that the oral microbiome has an impact on the durability of mucosal immunity after Covid-19 vaccination. Microbiome-targeted interventions to enhance long-term duration of mucosal vaccine immunity may be exploited.

Rhizomal Reclassification of Living Organisms.

Living organisms interact with each other during their lifetime, leading to genomes rearrangement and sequences transfer. These well-known phenomena give these organisms mosaic genomes, which challenge their classification. Moreover, many findings occurred between the IXXth and XXIst century, especially the discovery of giant viruses and candidate phyla radiation (CPR). Here, we tried to provide an updated classification, which integrates 216 representative genomes of the current described organisms. The reclassification was expressed through a genetic network based on the total genomic content, not on a single gene to represent the tree of life. This rhizomal exploration represents, more accurately, the evolutionary relationships among the studied species. Our analyses show a separated branch named fifth TRUC (Things Resisting Uncompleted Classifications). This taxon groups CPRs together, independently from Bacteria, Archaea (which regrouped also Nanoarchaeota and Asgard members), Eukarya, and the giant viruses (recognized recently as fourth TRUC). Finally, the broadening of analysis methods will lead to the discovery of new organisms, which justify the importance of updating the classification at every opportunity. In this perspective, our pragmatic representation could be adjusted along with the progress of evolutionary studies.

Proof of Concept of Culturomics Use of Time of Care.

Culturomics, a high throughput culture method with rapid identification of the colonies by Matrix Assisted Laser Desorption Ionization/Time Of Flight Mass Spectrometry (MALDI-TOF MS), has demonstrated its contribution to the exploration of the gut microbiota over the past 10 years. However, the cost, work time and workload, considerably limit its use on a large scale or emergency context. Here, by testing two different stool samples, including a stool sample from a patient requiring rapid immunotherapy treatment, we tested a new fast culturomic protocol using two pre-incubation media, blood culture bottle and YCFA modified medium. Both media were supplemented with 2 ml of rumen fluid filtered at 0.2 µm and 2 ml of defibrinated and sterile sheep blood. Unlike the standard culturomics, subculturing of blood culture bottle were performed at reduced incubation time (3 h, 6 h, 9 h, 24 h) and at a longer incubation time (3 days, 7 days, and 10 days) at 37°C. By testing 5,200 colonies per MALDI-TOF MS and obtaining a comparable number of cultured bacterial species (131 to 143) in a stool sample, this new protocol reduced the number of colonies tested by 57%, working time by 78.6% and cost by 72.2%. In addition, we highlighted that the proportion of strict anaerobic species has increased by 24%, known to be the preferential targets for biotherapy, including Faecalibacterium prausnitzii, Akkermansia muciniphila, Christensenella minuta, and Phascolarctobacterium faecium. Finally, this work showed that some bacterial species grew earlier but disappeared with prolonged incubation times.

State of the Art in the Culture of the Human Microbiota: New Interests and Strategies.

The last 5 years have seen a turning point in the study of the gut microbiota with a rebirth of culture-dependent approaches to study the gut microbiota. High-throughput methods have been developed to study bacterial diversity with culture conditions aimed at mimicking the gut environment by using rich media such as YCFA (yeast extract, casein hydrolysate, fatty acids) and Gifu anaerobic medium in an anaerobic workstation, as well as media enriched with rumen and blood and coculture, to mimic the symbiosis of the gut microbiota. Other culture conditions target phenotypic and metabolic features of bacterial species to facilitate their isolation. Preexisting technologies such as next-generation sequencing and flow cytometry have also been utilized to develop innovative methods to isolate previously uncultured bacteria or explore viability in samples of interest. These techniques have been applied to isolate CPR (Candidate Phyla Radiation) among other, more classic approaches. Methanogenic archaeal and fungal cultures present different challenges than bacterial cultures. Efforts to improve the available systems to grow archaea have been successful through coculture systems. For fungi that are more easily isolated from the human microbiota, the challenge resides in the identification of the isolates, which has been approached by applying matrix-assisted laser desorption ionization-time of flight mass spectrometry technology to fungi. Bacteriotherapy represents a nonnegligible avenue in the future of medicine to correct dysbiosis and improve health or response to therapy. Although great strides have been achieved in the last 5 years, efforts in bacterial culture need to be sustained to continue deciphering the dark matter of metagenomics, particularly CPR, and extend these methods to archaea and fungi.