A High Threshold of Biotherapeutic Aggregate Numbers is Needed to Induce an Immunogenic Response In Vitro, In Vivo, and in the Clinic.

BACKGROUND AND PURPOSE: There is concern that subvisible aggregates in biotherapeutic drug products pose a risk to patient safety. We investigated the threshold of biotherapeutic aggregates needed to induce immunogenic responses. METHODS AND RESULTS: Highly aggregated samples were tested in cell-based assays and induced cellular responses in a manner that depended on the number of particles. The threshold of immune activation varied by disease state (cancer, rheumatoid arthritis, allergy), concomitant therapies, and particle number. Compared to healthy donors, disease state patients showed an equal or lower response at the late phase (7 days), suggesting they may not have a higher risk of responding to aggregates. Xeno-het mice were used to assess the threshold of immune activation in vivo. Although highly aggregated samples (~ 1,600,000 particles/mL) induced a weak and transient immunogenic response in mice, a 100-fold dilution of this sample (~ 16,000 particles/mL) did not induce immunogenicity. To confirm this result, subvisible particles (up to ~ 18,000 particles/mL, containing aggregates and silicone oil droplets) produced under representative administration practices (created upon infusion of a drug product through an IV catheter) did not induce a response in cell-based assays or appear to increase the rate of adverse events or immunogenicity during phase 3 clinical trials. CONCLUSION: The ability of biotherapeutic aggregates to elicit an immune response in vitro, in vivo, and in the clinic depends on high numbers of particles. This suggests that there is a high threshold for aggregates to induce an immunogenic response which is well beyond that seen in standard biotherapeutic drug products.

Evaluation of Various Diagnostic Strategies for Bacterial Vaginosis, Including a New Approach Based on MALDI-TOF Mass Spectrometry.

Bacterial vaginosis (BV) is a common dysbiosis of unclear etiology but with potential consequences representing a public health problem. The diagnostic strategies vary widely. The Amsel criteria and Nugent score have obvious limitations, while molecular biology techniques are expensive and not yet widespread. We set out to evaluate different diagnostic strategies from vaginal samples using (1) a combination of abnormal vaginal discharge and vaginal pH > 4.5; (2) the Amsel-like criteria (replacing the "whiff test" with "malodorous discharge"); (3) the Nugent score; (4) the molecular quantification of Fannyhessea vaginae and Gardnerella vaginalis (qPCR); (5) and MALDI-TOF mass spectrometry (we also refer to it as "VAGI-TOF"). Overall, 54/129 patients (42%) were diagnosed with BV using the combination of vaginal discharge and pH, 46/118 (39%) using the Amsel-like criteria, 31/130 (24%) using qPCR, 32/130 (25%) using "VAGI-TOF", and 23/84 (27%) using the Nugent score (not including the 26 (31%) with intermediate flora). Of the 84 women for whom the five diagnostic strategies were performed, the diagnosis of BV was considered for 38% using the combination of vaginal discharge and pH, 34.5% using the Amsel-like criteria, 27% using the Nugent score, 25% using qPCR, and 25% using "VAGI-TOF". When qPCR was considered as the reference, the sensitivity rate for BV was 76.2% for the combination of vaginal discharge and pH, 90.5% for the Amsel-like criteria, 95.2% for the Nugent score, and 90.5% for "VAGI-TOF", while the specificity rates were 74.6%, 84.1%, 95.3%, and 95.3%, respectively. When the Nugent score was considered as the reference, the sensitivity for BV was 69.6% for the combination of vaginal discharge and pH, 82.6% for the Amsel-like criteria, 87% for qPCR, and 78.7% for "VAGI-TOF", while the specificity rates were 80%, 94.3%, 100%, and 97.1%, respectively. Overall, the use of qPCR and "VAGI-TOF" provided a consistent diagnosis of BV, followed by the Nugent score. If qPCR seems tedious and for some costly, "VAGI-TOF" could be an inexpensive, practical, and less time-consuming alternative.

Culturomics reveals a hidden world of vaginal microbiota with the isolation of 206 bacteria from a single vaginal sample.

The composition of the vaginal microbiota is known to be influenced by various factors and to be associated with several disorders affecting women's health. Although metagenomics is currently a widely used method for studying the human microbiota, it has certain limitations, such as a lack of information on bacterial viability. It is therefore important to use culture-based methods such as culturomics. Here, we used 35 different culture conditions to comprehensively characterize the vaginal bacterial diversity of a single woman's flora. A total of 206 bacterial species, belonging to six phyla (for a little more than half to Firmicutes, followed mainly by Actinobacteria, Bacteroidetes, and Proteobacteria) and 45 families, and 2 fungal species were cultivated. While several species of lactobacilli have been isolated, a wide variety of other bacteria were also separated, including 65 never reported before in vaginal flora, including a new bacterial species, Porphyromonas vaginalis sp. nov. Extensive culture-based methods are essential to establish a comprehensive, evidence-based repertoire of bacterial viability. If combined with molecular methods, they can provide a much more thorough understanding of the vaginal microbiota and fulfil the unknown part of metagenomic studies.

Relationship between Bacterial Vaginosis and Sexually Transmitted Infections: Coincidence, Consequence or Co-Transmission?

Sexually transmitted infections (STIs) are a serious global problem, causing disease, suffering, and death. Although bacterial vaginosis (BV) is not considered to be an STI, it may be associated with an increased risk of contracting a wide range of STIs. We sought to assess the link between the different microorganisms involved in STIs and BV. A total of 290 vaginal swabs from 290 women sent for diagnostic purposes to the clinical microbiology laboratory of the Marseille University Public Hospitals were tested by specific qPCR targeting STI-causing microorganisms and BV. Of these 290 swabs, 15.2% (44/290) were diagnosed with at least one STI-causing microorganism and 17.2% (50/290) with BV. The prevalence of STIs was significantly higher in women with BV (28%, 14/50) than in those without (20.4%, 51/240). The prevalence of co-infections involving two STI-causing microorganisms was significantly more frequent in women with BV than in those without (18% [8/50] vs. 2% [5/250]; p < 0.001). The prevalence of monoinfections and polyinfections with STI-causing microorganisms was lower in women without BV than in those with (8.8% [21/240] vs. 28% [14/50]), p < 0.001 and 2% (5/240) vs. 8% (4/50), p = 0.05, respectively). Our data suggest that a correlation between BV and STI may exist, with a higher prevalence of both monoinfections and polyinfections involving STI-causing microorganisms in women with BV. Further research is needed to better understand BV and its links to STIs.

Anaerococcus ihuae sp. nov. and Mediannikoviicoccus vaginalis gen. nov., sp. nov., two new bacteria isolated from human vaginal samples.

Strains Marseille-Q5893 (= CSUR Q5893 = CECT 30496) and Marseille-Q5883 (= CSUR Q5883 = CECT 30497) were isolated from vaginal samples using the culturomics approach. The 16S rRNA gene sequences of each strain were sequenced and then compared by BLASTn to the NCBI database. Strains Marseille-Q5893 and Marseille-Q5883 were most closely related to Anaerococcus obesiensis and Finegoldia magna, with identities of 98.5% and 90.0%, respectively. Strain Marseille-Q5893 is strictly anaerobic, while strain Marseille-Q5883 is facultative anaerobic. Both strains are Gram-positive, coccus-shaped, oxidase- and catalase-negative. The most abundant fatty acid for both strains is hexadecanoic acid, followed by 9-octadecenoic acid and tetradecanoic acid. Strain Marseille-Q5893 has a genome size of 1,831,271 bp with a G+C content of 29.4 mol%, whereas strain Marseille-Q5883 has a genome of 1,997,945 bp with a 33.6 mol% G+C content. The genomic comparison of closely related species with strains Marseille-Q5893 and Marseille-Q5883 showed that all digital DNA-DNA hybridization (dDDH) and orthologous average nucleotide identity (OrthoANI) values were lower than the published species thresholds (70% and 95-96%, respectively). Based on these data, we conclude that strain Marseille-Q5893 belongs to a new species in the family Peptoniphilaceae and strain Marseille-Q5883 belongs to a new genus in the family Peptostreptococcaceae. For these two new bacterial species, the names Anaerococcus ihuae sp. nov. and Mediannikoviicoccus vaginalis gen. nov., sp. nov., were proposed.