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Monoclonal antibodies: A production boost The use of antibody therapy has been limited in part because fully human monoclonal antibodies (mAbs) targeting specific antigens are difficult to generate. Now by identifying a time window in which the body's immune response to a particular pathogen is at its peak, Wrammert et al . have developed a new technique for the efficient generation of high-affinity human mAbs. In individuals given a booster vaccination against influenza, the number of influenza specific IgG + antibody-secreting cells in the bloodstream peaks a week after vaccination, accounting for about 6% of all peripheral B cells. By harvesting B cells during this time window, it was possible to produce more than 50 human mAbs that bound to the three target influenza strains with high affinity. The whole procedure took less than a month, which is much quicker that than conventional methods of producing mAbs. The technique should be applicable to any infectious disease for which vaccines are available. This paper demonstrates the efficient generation of high-affinity human monoclonal antibodies after booster vaccination with a conventional influenza vaccine. Pre-existing neutralizing antibody provides the first line of defence against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG + antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG + memory B cells that peaked 14–21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B-cell receptor (BCR) repertoire that in some donors was dominated by only a few B-cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over 50 human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high-affinity mAbs from humans within a month after vaccination. The panel of influenza-virus-specific human mAbs allowed us to address the issue of original antigenic sin (OAS): the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared with the virus strain present in the vaccine 1 . However, we found that most of the influenza-virus-specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal, healthy adults receiving influenza vaccination.

Key Clinical Message We report a patient with nonimmune fetal hydrops and multiple pathologic fractures. RNA analysis revealed a novel PIEZO1 variant. This report is the first to elucidate PIEZO1's role as a critical regulator of bone mass and strength. Graphical representation of our patient with a novel PIEZO1 variant, showing an overview of the patient's pedigree, physical exam findings, and radiologic studies.

RNA sequencing (RNA-seq) is a complementary diagnostic tool to exome sequencing (ES), only recently clinically available to undiagnosed patients post-ES, that provides functional information on variants of unknown significance (VUS) by evaluating its effect on RNA transcription. ES became clinically available in the early 2010s and promised an agnostic platform for patients with a neurological disease, especially for those who believed to have a genetic etiology. However, the massive data generated by ES pose challenges in variant interpretation, especially for rare missense, synonymous, and deep intronic variants that may have a splicing effect. Without functional study and/or family segregation analysis, these rare variants would be likely interpreted as VUS which is difficult for clinicians to use in clinical care. Clinicians are able to assess the VUS for phenotypic overlap, but this additional information alone is usually not enough to re-classify a variant. Here, we report a case of a 14-month-old male who presented to clinic with a history of seizures, nystagmus, cerebral palsy, oral aversion, global developmental delay, and poor weight gain requiring gastric tube placement. ES revealed a previously unreported homozygous missense VUS, c.7406A > G p.(Asn2469Ser), in VPS13D . This variant has not been previously reported in genome aggregation database (gnomAD), ClinVar, or in any peer-reviewed published literature. By RNA-seq, we demonstrated that this variant mainly impacts splicing and results in a frameshift and early termination. It is expected to generate either a truncated protein, p.(Val2468fs*19), or no protein from this transcript due to nonsense-mediated mRNA decay leading to VPS13D deficiency. To our knowledge, this is the first case utilizing RNA-seq to further functionally characterize a homozygous novel missense VUS in VPS13D and confirm its impact on splicing. This confirmed pathogenicity gave the diagnosis of VPS13D movement disorder to this patient. Therefore, clinicians should consider utilizing RNA-seq to clarify VUS by evaluating its effect on RNA transcription.

The use of viral vectors as vaccine candidates has shown promise against a number of pathogens. However, preexisting immunity to these vectors is a concern that must be addressed when deciding which viruses are suitable for use. A number of properties, including the existence of antigenically distinct subtypes, make influenza viruses attractive candidates for use as viral vectors. Here, we evaluate the ability of influenza viral vectors containing inserts of foreign pathogens to elicit antibody and CD8 + T cell responses against these foreign antigens in the presence of preexisting immunity to influenza virus in mice. Specifically, responses to an H3N1-based vector expressing a 90 amino acid polypeptide derived from the protective antigen (PA) of Bacillus anthracis or an H1N1-based vector containing a CD8 + T cell epitope from the glycoprotein (GP) of lymphocytic choriomeningitis virus were evaluated following infections with either homosubtypic or heterosubtypic influenza viruses. We found that mice previously infected with influenza viruses, even those expressing HA and NA proteins of completely different subtypes, were severely compromised in their ability to mount an immune response against the inserted epitopes. This inhibition was demonstrated to be mediated by CD8 + T cells, which recognize multiple strains of influenza viruses. These CD8 + T cells were further shown to protect mice from a lethal challenge by a heterologous influenza subtype. The implication of these data for the use of influenza virus vectors and influenza vaccination in general are discussed.

Monoclonal antibodies: A production boost