Immune recognition of putative alien microbial structures: Host-pathogen interactions in the age of space travel.

Human space travel is on the verge of visiting Mars and, in the future, even more distant places in the solar system. These journeys will be also made by terrestrial microorganisms (hitchhiking on the bodies of astronauts or on scientific instruments) that, upon arrival, will come into contact with new planetary environments, despite the best measures to prevent contamination. These microorganisms could potentially adapt and grow in the new environments and subsequently recolonize and infect astronauts. An even more challenging situation would be if truly alien microorganisms will be present on these solar system bodies: What will be their pathogenic potential, and how would our immune host defenses react? It will be crucial to anticipate these situations and investigate how the immune system of humans might cope with modified terrestrial or alien microbes. We propose several scenarios that may be encountered and how to respond to these challenges.

Adaptation of Bordetella pertussis to the Respiratory Tract.

There is a lack of insight into the basic mechanisms by which Bordetella pertussis adapts to the local host environment during infection. We analyzed B. pertussis gene expression in the upper and lower airways of mice and compared this to SO4-induced in vitro Bvg-regulated gene transcription. Approximately 30% of all genes were differentially expressed between in vitro and in vivo conditions. This included several novel potential vaccine antigens that were exclusively expressed in vivo. Significant differences in expression profile and metabolic pathways were identified between the upper versus the lower airways, suggesting distinct antigenic profiles. We found high-level expression of several Bvg-repressed genes during infection, and mouse vaccination experiments using purified protein fractions from both Bvg- and Bvg+ cultures demonstrated protection against intranasal B. pertussis challenge. This study provides novel insights into the in vivo adaptation of B. pertussis and may facilitate the improvement of pertussis vaccines.

Streptococcus pneumoniae PspC Subgroup Prevalence in Invasive Disease and Differences in Contribution to Complement Evasion.

The pneumococcal capsular serotype is an important determinant of complement resistance and invasive disease potential, but other virulence factors have also been found to contribute. Pneumococcal surface protein C (PspC), a highly variable virulence protein that binds complement factor H to evade C3 opsonization, is divided into two subgroups: choline-bound subgroup I and LPxTG-anchored subgroup II. The prevalence of different PspC subgroups in invasive pneumococcal disease (IPD) and functional differences in complement evasion are unknown. The prevalence of PspC subgroups in IPD isolates was determined in a collection of 349 sequenced strains of Streptococcus pneumoniae isolated from adult patients. pspC deletion mutants and isogenic pspC switch mutants were constructed to study differences in factor H binding and complement evasion in relation to capsule thickness. Subgroup I pspC was far more prevalent in IPD isolates than subgroup II pspC The presence of capsule was associated with a greater ability of bound factor H to reduce complement opsonization. Pneumococcal subgroup I PspC bound significantly more factor H and showed more effective complement evasion than subgroup II PspC in isogenic encapsulated pneumococci. We conclude that variation in the PspC subgroups, independent of capsule serotypes, affects pneumococcal factor H binding and its ability to evade complement deposition.

A thioesterase bypasses the requirement for exogenous fatty acids in the plsX deletion of Streptococcus pneumoniae.

PlsX is an acyl-acyl carrier protein (ACP):phosphate transacylase that interconverts the two acyl donors in Gram-positive bacterial phospholipid synthesis. The deletion of plsX in Staphylococcus aureus results in a requirement for both exogenous fatty acids and de novo type II fatty acid biosynthesis. Deletion of plsX (SP0037) in Streptococcus pneumoniae did not result in an auxotrophic phenotype. The ΔplsX S. pneumoniae strain was refractory to myristic acid-dependent growth arrest, and unlike the wild-type strain, was susceptible to fatty acid synthesis inhibitors in the presence of exogenous oleate. The ΔplsX strain contained longer chain saturated fatty acids imparting a distinctly altered phospholipid molecular species profile. An elevated pool of 18- and 20-carbon saturated fatty acids was detected in the ΔplsX strain. A S. pneumoniae thioesterase (TesS, SP1408) hydrolyzed acyl-ACP in vitro, and the ΔtesS ΔplsX double knockout strain was a fatty acid auxotroph. Thus, the TesS thioesterase hydrolyzed the accumulating acyl-ACP in the ΔplsX strain to liberate fatty acids that were activated by fatty acid kinase to bypass a requirement for extracellular fatty acid. This work identifies tesS as the gene responsible for the difference in exogenous fatty acid growth requirement of the ΔplsX strains of S. aureus and S. pneumoniae.

Deciphering the genetic basis of Moraxella catarrhalis complement resistance: a critical role for the disulphide bond formation system.

The complement system is an important innate defence mechanism, and the ability to resist complement-mediated killing is considered a key virulence trait of the respiratory tract pathogen M. catarrhalis. We studied the molecular basis of complement resistance by transcriptional profiling and Tn-seq, a genome-wide negative-selection screenings technology. Exposure of M. catarrhalis to human serum resulted in increased expression of 84 genes and reduced expression of 134 genes, among which genes encoding ABC transporter systems and surface proteins UspA1 and McaP. By subjecting a ∼ 15 800 transposon mutant library to serum, mutants of 53 genes were negatively selected, including the key complement-resistance factor uspA2H. Validation with directed mutants confirmed Tn-seq phenotypes of uspA2H and 11 newly identified genes, with mutants of MCR_0424, olpA, MCR_1483, and dsbB most severely attenuated. Detailed analysis showed that both components of the disulphide bond formation (DSB) system, DsbB and DsbA, were required for complement-resistance in multiple isolates, and fulfil a critical role in evasion of IgG-dependent classical pathway-mediated killing. Lipooligosaccharide (LOS) structure and membrane stability were severely affected in ΔdsbA strains, suggesting a pivotal role for the DSB system in LOS structure safeguarding and membrane stability maintenance.

A predictive signature gene set for discriminating active from latent tuberculosis in Warao Amerindian children.

BACKGROUND: Tuberculosis (TB) continues to cause a high toll of disease and death among children worldwide. The diagnosis of childhood TB is challenged by the paucibacillary nature of the disease and the difficulties in obtaining specimens. Whereas scientific and clinical research efforts to develop novel diagnostic tools have focused on TB in adults, childhood TB has been relatively neglected. Blood transcriptional profiling has improved our understanding of disease pathogenesis of adult TB and may offer future leads for diagnosis and treatment. No studies applying gene expression profiling of children with TB have been published so far. RESULTS: We identified a 116-gene signature set that showed an average prediction error of 11% for TB vs. latent TB infection (LTBI) and for TB vs. LTBI vs. healthy controls (HC) in our dataset. A minimal gene set of only 9 genes showed the same prediction error of 11% for TB vs. LTBI in our dataset. Furthermore, this minimal set showed a significant discriminatory value for TB vs. LTBI for all previously published adult studies using whole blood gene expression, with average prediction errors between 17% and 23%. In order to identify a robust representative gene set that would perform well in populations of different genetic backgrounds, we selected ten genes that were highly discriminative between TB, LTBI and HC in all literature datasets as well as in our dataset. Functional annotation of these genes highlights a possible role for genes involved in calcium signaling and calcium metabolism as biomarkers for active TB. These ten genes were validated by quantitative real-time polymerase chain reaction in an additional cohort of 54 Warao Amerindian children with LTBI, HC and non-TB pneumonia. Decision tree analysis indicated that five of the ten genes were sufficient to classify 78% of the TB cases correctly with no LTBI subjects wrongly classified as TB (100% specificity). CONCLUSIONS: Our data justify the further exploration of our signature set as biomarkers for potential childhood TB diagnosis. We show that, as the identification of different biomarkers in ethnically distinct cohorts is apparent, it is important to cross-validate newly identified markers in all available cohorts.

Tryptophan-like side chain holding aptamers inhibit respiratory syncytial virus infection of lung epithelial cells.

Respiratory syncytial virus (RSV) is a leading cause of serious and even fatal acute lower respiratory tract infections in infants and in the elderly. Potent RSV neutralization has been achieved by antibodies that selectively bind the prefusion form of the viral fusion (F) protein. We hypothesised that similar potent neutralization could be achieved using F protein targeting aptamers. Aptamers have yet to reach their translational potential for therapeutics or diagnostics due to their short half-life and limited range of target-aptamer interactions; these shortcomings can, however, be ameliorated by application of amino acid-like side chain holding nucleotides. In this study, a stabilized version of the prefusion RSV F protein was targeted by aptamer selection using an oligonucleotide library holding a tryptophan-like side chain. This process resulted in aptamers that bound the F protein with high affinity and differentiated between its pre- and postfusion conformation. Identified aptamers inhibited viral infection of lung epithelial cells. Moreover, introduction of modified nucleotides extended aptamer half-lives. Our results suggest that targeting aptamers to the surface of viruses could yield effective drug candidates, which could keep pace with the continuously evolving pathogens.

Differential Pneumococcal Growth Features in Severe Invasive Disease Manifestations.

The nasopharyngeal commensal Streptococcus pneumoniae can become invasive and cause metastatic infection. This requires the pneumococcus to have the ability to adapt, grow, and reside in diverse host environments. Therefore, we studied whether the likelihood of severe disease manifestations was related to pneumococcal growth kinetics. For 383 S. pneumoniae blood isolates and 25 experimental mutants, we observed highly reproducible growth curves in nutrient-rich medium. The derived growth features were lag time, maximum growth rate, maximum density, and stationary-phase time before lysis. First, the pathogenicity of each growth feature was probed by comparing isolates from patients with and without marked preexisting comorbidity. Then, growth features were related to the propensity of causing severe manifestations of invasive pneumococcal disease (IPD). A high maximum bacterial density was the most pronounced pathogenic growth feature, which was also an independent predictor of 30-day mortality (P = 0.03). Serotypes with an epidemiologically higher propensity for causing meningitis displayed a relatively high maximum density (P < 0.005) and a short stationary phase (P < 0.005). Correspondingly, isolates from patients diagnosed with meningitis showed an especially high maximum density and short stationary phase compared to isolates from the same serotype that had caused uncomplicated bacteremic pneumonia. In contrast, empyema-associated strains were characterized by a relatively long lag phase (P < 0.0005), and slower growth (P < 0.005). The course and dissemination of IPD may partly be attributable to the pneumococcal growth features involved. If confirmed, we should tailor the prevention and treatment strategies for the different infection sites that can complicate IPD. IMPORTANCE Streptococcus pneumoniae is a leading infectious cause of deaths worldwide. To understand the course and outcome of pneumococcal infection, most research has focused on the host and its response to contain bacterial growth. However, bacterial epidemiology suggest that certain pneumococcal serotypes are particularly prone to causing complicated infections. Therefore, we took the bacterial point of view, simply examining in vitro growth features for hundreds of pneumococcal blood isolates. Their growth curves were very reproducible. Certain poles of pneumococcal growth features were indeed associated with specific clinical manifestations like meningitis or pleural empyema. This indicates that bacterial growth style potentially affects the progression of infection. Further research on bacterial growth and adaptation to different host environments may therefore provide key insight into pathogenesis of complicated invasive disease. Such knowledge could lead to more tailored vaccine targets or therapeutic approaches to reduce the million deaths that are caused by pneumococcal disease every year.

BCG-induced trained immunity enhances acellular pertussis vaccination responses in an explorative randomized clinical trial.

Acellular pertussis (aP) booster vaccines are central to pertussis immunization programs, although their effectiveness varies. The Bacille Calmette-Guérin (BCG) vaccine is a prototype inducer of trained immunity, which enhances immune responses to subsequent infections or vaccinations. While previous clinical studies have demonstrated that trained immunity can protect against heterologous infections, its effect on aP vaccines in humans is unknown. We conducted a clinical study in order to determine the immunological effects of trained immunity on pertussis vaccination. Healthy female volunteers were randomly assigned to either receive BCG followed by a booster dose of tetanus-diphteria-pertussis inactivated polio vaccine (Tdap-IPV) 3 months later (BCG-trained), BCG + Tdap-IPV concurrently, or Tdap-IPV followed by BCG 3 months later. Primary outcomes were pertussis-specific humoral, T- and B-cell responses and were quantified at baseline of Tdap-IPV vaccination and 2 weeks thereafter. As a secondary outcome in the BCG-trained cohort, ex vivo leukocyte responses were measured in response to unrelated stimuli before and after BCG vaccination. BCG vaccination 3 months prior to, but not concurrent with, Tdap-IPV improves pertussis-specific Th1-cell and humoral responses, and also increases total memory B cell responses. These responses were correlated with enhanced IL-6 and IL-1ß production at the baseline of Tdap-IPV vaccination in the BCG-trained cohort. Our study demonstrates that prior BCG vaccination potentiates immune responses to pertussis vaccines and that biomarkers of trained immunity are the most reliable correlates of those responses.

Lack of Cell Cycle Inhibitor p21 and Low CD4+ T Cell Suppression in Newborns After Exposure to IFN-ß.

Type I IFNs, such as interferon alpha and interferon beta, are key regulators of the adaptive immune response during infectious diseases. Type I IFNs are induced upon infection, bind interferon α/ß receptors on T-cells and activate intracellular pathways. The activating and inhibitory consequences of type I IFN-signaling are determined by cell type and cellular environment. The neonatal immune system is associated with increased vulnerability to infectious diseases which could partly be explained by an immature CD4+ T-cell compartment. Here, we show low IFN-ß-mediated inhibition of CD4+ T-cell proliferation, phosphorylation of retinoblastoma protein and cytokine production in human newborns compared to adults. In addition, both naïve and total newborn CD4+ T-cells are unable to induce the cell-cycle inhibitor p21 upon exposure to IFN-ß in contrast to adults. The distinct IFN-ß-signaling in newborns provides novel insights into T cell functionality and regulation of T cell-dependent inflammation during early life immune responses.

Berberine and Obatoclax Inhibit SARS-Cov-2 Replication in Primary Human Nasal Epithelial Cells In Vitro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a new human pathogen in late 2019 and it has infected over 100 million people in less than a year. There is a clear need for effective antiviral drugs to complement current preventive measures, including vaccines. In this study, we demonstrate that berberine and obatoclax, two broad-spectrum antiviral compounds, are effective against multiple isolates of SARS-CoV-2. Berberine, a plant-derived alkaloid, inhibited SARS-CoV-2 at low micromolar concentrations and obatoclax, which was originally developed as an anti-apoptotic protein antagonist, was effective at sub-micromolar concentrations. Time-of-addition studies indicated that berberine acts on the late stage of the viral life cycle. In agreement, berberine mildly affected viral RNA synthesis, but it strongly reduced infectious viral titers, leading to an increase in the particle-to-pfu ratio. In contrast, obatoclax acted at the early stage of the infection, which is in line with its activity to neutralize the acidic environment in endosomes. We assessed infection of primary human nasal epithelial cells that were cultured on an air-liquid interface and found that SARS-CoV-2 infection induced and repressed expression of specific sets of cytokines and chemokines. Moreover, both obatoclax and berberine inhibited SARS-CoV-2 replication in these primary target cells. We propose berberine and obatoclax as potential antiviral drugs against SARS-CoV-2 that could be considered for further efficacy testing.

Phenotypic and genomic characterization of pneumococcus-like streptococci isolated from HIV-seropositive patients.

Accurate differentiation between pneumococci and other viridans streptococci is essential given their differences in clinical significance. However, classical phenotypic tests are often inconclusive, and many examples of atypical reactions have been reported. In this study, we applied various phenotypic and genotypic methods to discriminate between a collection of 12 streptococci isolated from the upper respiratory tract of HIV-seropositive individuals in 1998 and 1999. Conventional phenotypic characterization initially classified these streptococci as Streptococcus pneumoniae, as they were all sensitive to optochin and were all bile soluble. However, they did not agglutinate with anti-pneumococcal capsular antibodies and were also far more resistant to antimicrobial agents than typeable pneumococci isolated in the same period. Genotypic characterization of these isolates and control isolates by both multilocus sequence analysis (MLSA) and comparative genomic hybridization (CGH) showed that only a single isolate was genetically considered to be a true S. pneumoniae isolate, and that the remaining 11 non-typable isolates were indeed distinct from true pneumococci. Of these, 10 most closely resembled a subgroup of Streptococcus mitis isolates genetically, while one strain was identified as a Streptococcus pseudopneumoniae isolate. CGH also showed that a considerable part of the proposed pneumococcal core genome, including many of the known pneumococcal virulence factors, was conserved in the non-typable isolates. Sequencing of part of the 16S rRNA gene and investigation for the presence of ply by PCR corroborated these results. In conclusion, our findings confirm the close relationship between streptococci of the Mitis group, and show that both MLSA and CGH enable pneumococci to be distinguished from other Mitis group streptococci.

Highly penicillin-resistant multidrug-resistant pneumococcus-like strains colonizing children in Oeiras, Portugal: genomic characteristics and implications for surveillance.

While performing surveillance studies in Oeiras, Portugal, designed to describe the impact of pneumococcal conjugate vaccine on colonization, we observed an increase from 0.7% in 2003 to 5% in 2006 in the prevalence of penicillin resistance (MIC of 2 to 6 mg/liter) among presumptively identified pneumococcal isolates. Although 15 of the 22 penicillin-resistant isolates obtained in 2006 were optochin resistant, they were bile soluble and thus considered to be bona fide pneumococci. This study aimed to clarify the nature of these isolates by using a combination of phenotypic and genotypic approaches that included routine strategies for pneumococcal identification, multilocus sequence analysis (MLSA), and comparative genomic hybridization (CGH). By MLSA, all isolates were classified as "streptococci of the mitis group" that, however, were distinct from typical Streptococcus pneumoniae or Streptococcus mitis. A single isolate was identified as Streptococcus pseudopneumoniae. CGH confirmed these findings and further indicated that a considerable part of the proposed pneumococcal core genome is conserved in these isolates, including several pneumococcal virulence genes (e.g., pavA, spxB, cbpE, and cbpD). These results suggest that among pneumococci and closely related streptococci, universal unique phenotypic and genetic properties that could aid species identification are virtually impossible to define. In pneumococcal colonization studies, when atypical strains are found, MLSA and CGH are informative tools that can be used to complement routine tests. In our study, after correct identification of the penicillin-resistant true pneumococci, we found that penicillin resistance levels among pneumococci remained stable from 2003 to 2006.

Effect of FHA and Prn on Bordetella pertussis colonization of mice is dependent on vaccine type and anatomical site.

Bordetella pertussis vaccine escape mutants that lack expression of the pertussis antigen pertactin (Prn) have emerged in vaccinated populations in the last 10-20 years. Additionally, clinical isolates lacking another acellular pertussis (aP) vaccine component, filamentous hemagglutinin (FHA), have been found sporadically. Here, we show that both whole-cell pertussis (wP) and aP vaccines induced protection in the lungs of mice, but that the wP vaccine was more effective in nasal clearance. Importantly, bacterial populations isolated from the lungs shifted to an FHA-negative phenotype due to frameshift mutations in the fhaB gene. Loss of FHA expression was strongly selected for in Prn-deficient strains in the lungs following aP but not wP vaccination. The combined loss of Prn and FHA led to complete abrogation of bacterial surface binding by aP-induced serum antibodies. This study demonstrates vaccine- and anatomical site-dependent adaptation of B. pertussis and has major implications for the design of improved pertussis vaccines.

Growth on Carbohydrates from Carbonaceous Meteorites Alters the Immunogenicity of Environment-Derived Bacterial Pathogens.

The last decade has witnessed a renewed interest in space exploration. Public and private institutions are investing considerable effort toward the direct exploration of the Moon and Mars, as well as more distant bodies in the solar system. Both automated and human-crewed spacecraft are being considered in these efforts. As inevitable fellow travelers on the bodies of astronauts, spaceships, or equipment, terrestrial microorganisms will undoubtedly come into contact with extraterrestrial environments, despite stringent decontamination. These microorganisms could eventually adapt and grow in their new habitats, where they might potentially recolonize and lead to the infection of the human space travelers. In this article, we demonstrate that clinically relevant bacterial species found in the environment are able to grow in minimal media with sugar compounds identified in extraterrestrial carbon sources. As a surrogate model, we used carbohydrates previously isolated from carbonaceous meteorites. The bacteria underwent an adaptation process that caused structural modifications in the cell envelope that sparked changes in pathogenic potential, both in vitro and in vivo. Understanding the adaptation of microorganisms exposed to extraterrestrial environments, with subsequent changes in their immunogenicity and virulence, requires a comprehensive analysis of such scenarios to ensure the safety of major space expeditions in the decades to come.

A novel 6.14 Mb duplication of chromosome 8p21 in a patient with autism and self mutilation.

Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders with a strong genetic etiology. Cytogenetic abnormalities have been detected in 5-10% of the patients with autism. In this study, we present the clinical, cytogenetic and array-comparative genomic hybridization (array-CGH) evaluation of a 13-year-old male with severe developmental delay, facial dysmorphic features, autism and self mutilation. The patient was found to carry a de novo duplication of chromosome region 8p21 of minimally 6.14 and maximally 6.58 Mb as ascertained by bacterial artificial chromosome (BAC)-based array-CGH. Hitherto, only a few patients with autism with cytogenetically visible duplications involving the chromosome 8p21 region have been described, but the extent of these duplications has not been determined at the molecular level. This represents the smallest rearrangement of chromosomal region 8p21 as yet found in a patient with autism. For 11 of the 36 genes with known functions located within this duplication clear transcription in the brain was found. Of those the STMN4 and DPYSL2 genes are the most likely candidate genes to be involved in neuronal development, and, if altered in gene-dosage, in the autistic phenotype of our patient.

Unmasking of a hemizygous WFS1 gene mutation by a chromosome 4p deletion of 8.3 Mb in a patient with Wolf-Hirschhorn syndrome.

The Wolf-Hirschhorn syndrome (WHS (MIM 194190)), which is characterized by growth delay, mental retardation, epilepsy, facial dysmorphisms, and midline fusion defects, shows extensive phenotypic variability. Several of the proposed mutational and epigenetic mechanisms in this and other chromosomal deletion syndromes fail to explain the observed phenotypic variability. To explain the complex phenotype of a patient with WHS and features reminiscent of Wolfram syndrome (WFS (MIM 222300)), we performed extensive clinical evaluation and classical and molecular cytogenetic (GTG banding, FISH and array-CGH) and WFS1 gene mutation analyses. We detected an 8.3 Mb terminal deletion and an adjacent 2.6 Mb inverted duplication in the short arm of chromosome 4, which encompasses a gene associated with WFS (WFS1). In addition, a nonsense mutation in exon 8 of the WFS1 gene was found on the structurally normal chromosome 4. The combination of the 4p deletion with the WFS1 point mutation explains the complex phenotype presented by our patient. This case further illustrates that unmasking of hemizygous recessive mutations by chromosomal deletions represents an additional explanation for the phenotypic variability observed in chromosomal deletion disorders.

Proportional growth failure and oculocutaneous albinism in a girl with a 6.87 Mb deletion of region 15q26.2-->qter.

We report on an 8(1)/(2)-year-old girl with severe pre- and postnatal growth retardation, congenital heart malformation, facial asymmetry, oculocutaneous albinism without misrouting and subluxation of the radial heads. Her intelligence was in the low normal range. By GTG-banding a deletion of band 15q26 was found. Array-CGH, using a 3783 BAC array, revealed a segmental monosomy of the 15(q26.2-->qter) region, which was narrowed down to a 6.87Mb deletion by using the Illumina Infinium 317 K SNP array system, and subsequently confirmed by fluorescence in situ hybridisation (FISH) analysis. The deletion appeared to have arisen de novo. The IGF1R (insulin-like growth factor 1 receptor) and the NR2F2 genes were situated within, but the OCA2 (oculocutaneous albinism II) gene (formerly called the P gene) was located outside the deleted region. Clinical findings in our patient were compared with previously reported cases carrying terminal deletions of 15q26.2. This allowed us to expand the clinical phenotype of terminal 15q26.2 deletions and to indicate candidate genes for several phenotypic features.

Mapping of constitutional translocation breakpoints in renal cell cancer patients: identification of KCNIP4 as a candidate gene.

Our group and others had previously developed a high throughput procedure to map translocation breakpoints using chromosome flow sorting in conjunction with microarray-based comparative genomic hybridization (arrayCGH). Here we applied both conventional positional cloning and integrated arrayCGH procedures to the mapping of constitutional chromosome anomalies in four patients with renal cell cancer (RCC), three with a chromosome 3 translocation, and one with an insertion involving chromosome 3. In one of these patients, who was carrying a t(3;4)(p13;p15), the KCNIP4 gene was found to be disrupted. KCNIP4 belongs to a family of potassium channel-interacting proteins and is highly expressed in normal kidney cells. In addition, KCNIP4 splice variants have specifically been encountered in RCC.

Monitoring of dynamic changes in Keyhole Limpet Hemocyanin (KLH)-specific B cells in KLH-vaccinated cancer patients.

Keyhole limpet hemocyanin (KLH) is used as an immunogenic neo-antigen for various clinical applications and during vaccine development. For advanced monitoring of KLH-based interventions, we developed a flow cytometry-based assay for the ex vivo detection, phenotyping and isolation of KLH-specific B cells. As proof-of-principle, we analyzed 10 melanoma patients exposed to KLH during anti-cancer dendritic cell vaccination. Our assay demonstrated sensitive and specific detection of KLH-specific B cells in peripheral blood and KLH-specific B cell frequencies strongly correlated with anti-KLH serum antibody titers. Profiling of B cell subsets over the vaccination course revealed that KLH-specific B cells matured from naïve to class-switched memory B cells, confirming the prototypic B cell response to a neo-antigen. We conclude that flow-cytometric detection and in-depth phenotyping of KLH-specific B cells is specific, sensitive, and scalable. Our findings provide novel opportunities to monitor KLH-specific immune responses and serve as a blueprint for the development of new flow-cytometric protocols.