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Background Influenza vaccination efficacy is reduced after hematopoietic stem cell transplantation (HSCT) and patient factors determining vaccination outcomes are still poorly understood. Methods We investigated the antibody response to seasonal influenza vaccination in 135 HSCT patients and 69 healthy volunteers (HVs) in a prospective observational multicenter cohort study. We identified patient factors associated with hemagglutination inhibition titers against A/California/2009/H1N1, A/Texas/2012/H3N2, and B/Massachusetts/2012 by multivariable regression on the observed titer levels and on seroconversion/seroprotection categories for comparison. Results Both regression approaches yielded consistent results but regression on titers estimated associations with higher precision. HSCT patients required 2 vaccine doses to achieve average responses comparable to a single dose in HVs. Prevaccination titers were positively associated with time after transplantation, confirming that HSCT patients can elicit potent antibody responses. However, an unrelated donor, absolute lymphocyte counts below the normal range, and treatment with calcineurin inhibitors lowered the odds of responding. Conclusions HSCT patients show a highly heterogeneous vaccine response but, overall, patients benefited from the booster shot and can acquire seroprotective antibodies over the years after transplantation. Several common patient factors lower the odds of responding, urging identification of additional preventive strategies in the poorly responding groups. Clinical Trials Registration NCT03467074., The Journal of Infectious Diseases, 225 (8), ISSN:0022-1899, ISSN:1537-6613

Determining antigen specificity is vital for understanding B cell biology and for producing human monoclonal antibodies. We describe here a powerful method for identifying B cells that recognize membrane antigens expressed on cells. The technique depends on two characteristics of the interaction between a B cell and an antigen-expressing cell: antigen-receptor-mediated extraction of antigen from the membrane of the target cell, and B cell activation. We developed the method using influenza hemagglutinin as a model viral membrane antigen, and tested it using acetylcholine receptor (AChR) as a model membrane autoantigen. The technique involves co-culturing B cells with adherent, bioorthogonally labeled cells expressing GFP-tagged antigen, and sorting GFP-capturing, newly activated B cells. Hemagglutinin-specific B cells isolated this way from vaccinated human donors expressed elevated CD20, CD27, CD71, and CD11c, and reduced CD21, and their secreted antibodies blocked hemagglutination and neutralized viral infection. Antibodies cloned from AChR-capturing B cells derived from patients with myasthenia gravis bound specifically to the receptor on cell membrane. The approach is sensitive enough to detect antigen-specific B cells at steady state, and can be adapted for any membrane antigen.

Background Immune checkpoint inhibiting antibodies were introduced into routine clinical practice for cancer patients. Checkpoint blockade has led to durable remissions in some patients, but may also induce immune-related adverse events (irAEs). Lung cancer patients show an increased risk for complications, when infected with influenza viruses. Therefore, vaccination is recommended. However, the efficacy and safety of influenza vaccination during checkpoint blockade and its influence on irAEs is unclear. Similarly, the influence of vaccinations on T cell-mediated immune reactions in patients during PD-1 blockade remains poorly defined. Methods We vaccinated 23 lung cancer patients and 11 age-matched healthy controls using a trivalent inactivated influenza vaccine to investigate vaccine-induced immunity and safety during checkpoint blockade. Results We did not observe significant differences between patients and healthy controls in vaccine-induced antibody titers against all three viral antigens. Influenza vaccination resulted in protective titers in more than 60% of patients/participants. In cancer patients, the post-vaccine frequency of irAEs was 52.2% with a median time to occurrence of 3.2 months after vaccination. Six of 23 patients (26.1%) showed severe grade 3/4 irAEs. This frequency of irAEs might be higher than the rate previously published in the literature and the rate observed in a non-study population at our institution (all grades 25.5%, grade 3/4 9.8%). Conclusions Although this is a non-randomized trial with a limited number of patients, the increased rate of immunological toxicity is concerning. This finding should be studied in a larger patient population.

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay). As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

A luminescence immunoassay (LIA) was developed for the diagnosis of bovine spongiform encephalopathy (BSE) in brain tissue using two different monoclonal antibodies for capture and detection of the protease-resistant fragment of the pathological prion protein (PrP27-30). PrP27-30 currently represents the most reliable marker for the infectious particle (denominated prion) causing transmissible spongiform encephalopathies (TSEs). Internal and official validation studies of this assay are described using brain homogenates from ascertained BSE positive and negative cows. Using more than 300 positive and 1400 negative bovine or ovine samples, an excellent sensitivity and specificity of 100% were demonstrated. More than 1000-fold dilutions of a BSE positive homogenate still resulted in a clear positive signal. In combination with a simple homogenisation procedure for the preparation of the samples, this assay lends itself for large scale screening of cattle and sheep for TSEs using complete automation of the process.

e14523 Background: Cancer patients (pts) are at risk to develop complications when infected with seasonal influenza viruses. Current guidelines propose that pts on PD-1 directed therapies may receive concurrent inactivated influenza immunization if not medically contraindicated. In this observational study, we aimed to assess the vaccine induced immune response and safety of immunotherapy and influenza vaccine. Methods: Metastatic cancer pts treated with at least one dose of either nivolumab or pembrolizumab were vaccinated with a trivalent inactive influenza vaccination between October and November 2015. Partners of the pts were vaccinated and included in our analysis as age-matched controls. Antibody titers against vaccine virus strains were measured by hemagglutination inhibition assay at days 7, 30, 60 and 180. Cytokine/chemokine profile and changes in peripheral immune cells were assessed in cancer pts at the same time points. Immune-related adverse events (irAE) were documented according to CTCAE v4.0. Results: We included 23 pts and 7 age-matched healthy controls. Median time between initiation of PD-1 inhibition and influenza vaccination was 74 days (range, 44-57). 22 pts were treated with nivolumab and 1 pt with pembrolizumab. 16 pts had a diagnosis of non-small cell lung cancer, 3 pts had renal cell carcinoma and 3 pts a malignant melanoma. In total, 12 pts (52.2%) experienced an irAE. 6 pts (26.1%) had grade 3 or 4 (G3/4) irAEs. This frequency seems to be higher than reported in the literature. G3/4 irAEs include colitis (n = 2), encephalitis (n = 1), vasculitis (n = 1), pneumonitis (n = 1), peripheral neuritis (n = 1). There was no major difference over time in the generation of antibody titers against strains in the vaccine. Peripheral leukocyte counts and cytokine/inflammatory chemokine levels were unchanged after vaccination. We did not observe unexpected local toxicities at the injection site. Conclusions: The seasonal influence vaccination reaches a protective range in these pts. Unexpectedly, however, an increased rate of clinically relevant irAEs was observed. Confirmation in a larger population and mechanistic understanding is required.

The maturation of the iliac apophysis (Risser sign) and of the bones of the hand and wrist of 34 girls with scoliosis with 46 pairs of data were analyzed to get information upon their predictive value for further growth and development of the scoliotic curve. A strong correlation was found between the Risser sign and the maturation of the hand and wrist (r = 0.74, p less than 0.005). The prognostic value of the Risser sign (r = -0.57) for further height gain is superior to chronological age only (r = -0.52), the maturation of the hand and wrist (r = -0.62) however is superior to the Risser sign. It can be assumed that this also holds for their prognostic value for the progression of the scoliotic curve. We feel that in the clinician routine for girls with scoliosis the determination of the skeletal maturity from X-rays of the hand is sufficient and with its probably unavoidable irradiation risks to the gonads the determination of the Risser sign should be abandoned.