A low level of naturally occurring antibodies associates with functional antibody deficiency.

Functional antibody deficiency is clinically assessed from antibody responses to vaccination. However, diagnostic vaccination is complex and may fail in practice. We hypothesized that the levels of naturally occurring antibodies against galactose-α-1,3-galactose (αGal) may represent alternative markers of functional antibody capacity. We included data from 229 patients with suspected primary immunodeficiency in a retrospective study. Antibody levels against αGal and twelve pneumococcal serotypes were determined with solid-phase immunoassays. Pneumococcal vaccinations and treatment with normal human immunoglobulin were assessed from medical records. Anti-αGal antibody levels correlated positively with anti-pneumococcal antibody levels measured before and after pneumococcal vaccination. Contrary to the anti-pneumococcal antibody levels, the anti-αGal antibody level showed potential for predicting subsequent immunoglobulin treatment - a marker of disease severity. Naturally occurring antibodies may reflect the functional capacity tested by diagnostic vaccination but add more useful clinical data. The clinical utility of this easy test should be evaluated in prospective studies.

Diagnostic Vaccination in Clinical Practice.

Testing the antibody response to vaccination (diagnostic vaccination) is crucial in the clinical evaluation of primary immunodeficiency diseases. Guidelines from the American Academy of Allergy, Asthma & Immunology (AAAAI) provide detailed recommendations for diagnostic vaccination with pure pneumococcal polysaccharide vaccines (PPV). However, the degree of compliance with these guidelines and the utility of the guidelines in actual practice are undescribed. To address this, we systematically evaluated diagnostic vaccination in adult patients with suspected primary immunodeficiency diseases in a single tertiary center from 2011 to 2016 (n = 229). We found that full compliance with the AAAAI guidelines was achieved for only 39 patients (17%), suggesting that the guidelines are not easy to follow. Worse, interpretation according to the guidelines was heavily influenced by which serotype-specific antibodies that were used for the evaluation. We found that the arbitrary choices of serotype-specific antibodies could change the fraction of patients deemed to have 'adequate immunity' by a factor of four, exposing an inherent flaw in the guidelines. The flaw relates to dichotomous principles for data interpretation under the AAAAI guidelines. We therefore propose a revised protocol for diagnostic vaccination limited to PPV vaccination, subsequent antibody measurements, and data interpretation using Z-scores. The Z-score compiles multiple individual antibody levels, adjusted for different weighting, into one single continuous variable for each patient. In contrast to interpretation according to the AAAAI guidelines, the Z-scores were robust to variations in the choice of serotype-specific antibodies used for interpretation. Moreover, Z-scores revealed reduced immunity after vaccination in the patients with recurrent pneumonia (a typical symptom of antibody deficiency) compared with control patients. Assessment according to the AAAAI guidelines failed to detect this difference. We conclude that our simplified protocol and interpretation with Z-scores provides more robust clinical results and may enhance the value of diagnostic vaccination.

The level of naturally occurring anti-αGal antibody predicts antibody response to polysaccharide vaccination in HIV-infected adults.

In clinical practice, the capacity for producing anti-carbohydrate antibodies is regarded as an entity, but supportive evidence is lacking. We hypothesized that the outcome of the gold standard test for clinical assessment of this capacity, antibody response to polysaccharide vaccination, correlated with the level of the abundant naturally occurring anti-carbohydrate antibody, anti-αGal. To perform an exploratory study, 47 HIV-infected adults were recruited from a vaccine trial. Participants received a 23-valent pneumococcal capsular polysaccharide vaccine. Plasma samples obtained just before and median 4 weeks after the vaccination were quantified for IgG anti-αGal antibody and IgG antibodies to polysaccharides present in the vaccine (serotypes 1, 7F and 19A) by solid-phase type immunoassays. The vaccination responses were assessed as a categorical variable (based on criteria defined by The American Academy of Allergy, Asthma & Immunology and the American College of Allergy, Asthma & Immunology) and as three different continuous variables (antibody increment, geometrical average and standard normal deviates of the achieved antibody concentrations). The baseline anti-αGal level predicted the vaccine response as a categorical variable (ROC-curve analysis, AUC = 0.71; 95%CI: 0.55-0.86) and as the three continuous variables (eg slope of linear regression of geometrical average = 0.37; 95%CI: 0.15-0.59). The correlation between the anti-αGal level and antibody responses to polysaccharide vaccination fits with a shared underlying capacity. Thus, the present study supports the notion of a measurable capacity for the production of anti-carbohydrate antibodies in each individual. Firm conclusions on the generalizability and clinical utility require further studies.

The human natural anti-αGal antibody targets common pathogens by broad-spectrum polyreactivity.

Naturally occurring antibodies are abundant in human plasma, but their importance in the defence against bacterial pathogens is unclear. We studied the role of the most abundant of such antibodies, the antibody against terminal galactose-α-1,3-galactose (anti-αGal), in the protection against pneumococcal infections (Streptococcus pneumonia). All known pneumococcal capsular polysaccharides lack terminal galactose-α-1,3-galactose, yet highly purified human anti-αGal antibody of the IgG class reacted with 48 of 91 pneumococcal serotypes. Anti-αGal was found to contain multiple antibody subsets that possess distinct specificities beyond their general reactivity with terminal galactose-α-1,3-galactose. These subsets in concert targeted a wide range of microbial polysaccharides. We found that anti-αGal constituted up to 40% of the total antibody reactivity to pneumococci in normal human plasma, that anti-αGal drives phagocytosis of pneumococci by human neutrophils and that the anti-αGal level was twofold lower in patients prone to pneumococcal infections compared with controls. Moreover, during a 48-year period in Denmark, the 48 anti-αGal-reactive serotypes caused fewer invasive pneumococcal infections (n = 10 927) than the 43 non-reactive serotypes (n = 18 107), supporting protection on the population level. Our findings explain the broad-spectrum pathogen reactivity of anti-αGal and support that these naturally occurring polyreactive antibodies contribute significantly to human protective immunity.

Functional and Structural Characterization of a Potent C1q Inhibitor Targeting the Classical Pathway of the Complement System.

The classical pathway of complement is important for protection against pathogens and in maintaining tissue homeostasis, but excessive or aberrant activation is directly linked to numerous pathologies. We describe the development and in vitro characterization of C1qNb75, a single domain antibody (nanobody) specific for C1q, the pattern recognition molecule of the classical pathway. C1qNb75 binds to the globular head modules of human C1q with sub-nanomolar affinity and impedes classical pathway mediated hemolysis by IgG and IgM. Crystal structure analysis revealed that C1qNb75 recognizes an epitope primarily located in the C1q B-chain that overlaps with the binding sites of IgG and IgM. Thus, C1qNb75 competitively prevents C1q from binding to IgG and IgM causing blockade of complement activation by the classical pathway. Overall, C1qNb75 represents a high-affinity nanobody-based inhibitor of IgG- and IgM-mediated activation of the classical pathway and may serve as a valuable reagent in mechanistic and functional studies of complement, and as an efficient inhibitor of complement under conditions of excessive CP activation.

Complement activation by human IgG antibodies to galactose-α-1,3-galactose.

Some human antibodies may paradoxically inhibit complement activation on bacteria and enhance pathogen survival in humans. This property was also claimed for IgG antibodies reacting with terminal galactose-α-1,3-galactose (Galα3Gal; IgG anti-αGal), a naturally occurring and abundant antibody in human plasma that targets numerous different pathogens. To reinvestigate these effects, we used IgG anti-αGal affinity isolated from a pool of normal human IgG and human hypogammaglobulinaemia serum as a complement source. Flow cytometry was performed to examine antibody binding and complement deposition on pig erythrocytes, Escherichia coli O86 and Streptococcus pneumoniae serotype 9V. Specific nanobodies were used to block the effect of single complement factors and to delineate the complement pathways involved. IgG anti-αGal was capable of activating the classical complement pathway on all the tested target cells. The degree of activation was exponentially related to the density of bound antibody on E. coli O86 and pig erythrocytes, but more linearly on S. pneumoniae 9V. The alternative pathway of complement amplified complement deposition. Deposited C3 fragments covered the activating IgG anti-αGal, obstructing its detection and highlighting this as a likely general caveat in studies of antibody density and complement deposition. The inherent capacity for complement activation by the purified carbohydrate reactive IgG anti-αGal was similar to that of normal human IgG. We propose that the previously reported complement inhibition by IgG anti-αGal relates to suboptimal assay configurations, in contrast to the complement activating property of the antibodies demonstrated in this paper.

Abundant human anti-Galα3Gal antibodies display broad pathogen reactivity.

Antibodies of the IgG class to terminal Galα3Gal (IgG anti-αGal) is abundant in human plasma and are reported to bind most sepsis-causing Gram-negative bacteria. However, these seminal findings, made more than two decades ago, have not been reexamined. Our aim was to assess IgG anti-αGal´s pathogen reactivity. We affinity purified IgG anti-αGal from a therapeutic grade normal human IgG pool applying two rounds of positive selection with Galα3Gal-coupled beads and included removal of column matrix reactive antibodies. The purified antibodies were rigorously characterized in terms of specificity and purity in various solid-phase immunoassays. We used flow cytometry to study reactivity against 100 consecutive clinical isolates diagnosed as cause of sepsis in humans. We found that the purified IgG anti-αGal displays high specificity for Galα3Gal. Also, IgG anti-αGal at 5 mg/L bound 56 out of 100 pathogens with predilection for Gram-positive bacteria binding 39 out of 52 strains. We confirm that although IgG anti-αGal comprise a small fraction of the human antibody pool (~0.1%), these antibodies targets an impressively large part of pathogens causing invasive disease.

The genetic component of preeclampsia: A whole-exome sequencing study.

Preeclampsia is a major cause of maternal and perinatal deaths. The aetiology of preeclampsia is largely unknown but a polygenetic component is assumed. To explore this hypothesis, we performed an in-depth whole-exome sequencing study in women with (cases, N = 50) and without (controls, N = 50) preeclampsia. The women were identified in an unselected cohort of 2,545 pregnant women based on data from the Danish National Patient Registry and the Medical Birth Registry. Matching DNA was obtained from a biobank containing excess blood from routine antenatal care visits. Novogene performed the whole-exome sequencing blinded to preeclampsia status. Variants for comparison between cases and controls were filtered in the Ingenuity Variant Analysis software. We applied two different strategies; a disease association panel approach, which included variants in single genes associated with established clinical risk factors for preeclampsia, and a gene panel approach, which included biological pathways harbouring genes previously reported to be associated with preeclampsia. Variant variability was compared in cases and controls at the level of biological processes, signalling pathways, and in single genes. Regardless of the applied strategy and the level of variability examined, we consistently found positive correlations between variant numbers in cases and controls (all R2s>0.88). Contrary to what was expected, cases carried fewer variants in biological processes and signalling pathways than controls (all p-values ≤0.02). In conclusion, our findings challenge the hypothesis of a polygenetic aetiology for preeclampsia with a common network of susceptibility genes. The greater genetic diversity among controls may suggest a protective role of genetic diversity against the development of preeclampsia.

Polysaccharide responsiveness is not biased by prior pneumococcal-conjugate vaccination.

Polysaccharide responsiveness is tested by measuring antibody responses to polysaccharide vaccines to diagnose for humoral immunodeficiency. A common assumption is that this responsiveness is biased by any previous exposure to the polysaccharides in the form of protein-coupled polysaccharide vaccines, such as those used in many childhood vaccination programmes. To examine this assumption, we investigated the effect of protein-coupled polysaccharide vaccination on subsequent polysaccharide responsiveness. HIV-infected adults (n = 47) were vaccinated twice with protein-coupled polysaccharides and six months later with pure polysaccharides. We measured immunoglobulin G responses against three polysaccharides present in only the polysaccharide vaccine (non-memory polysaccharides) and seven recurring polysaccharides (memory polysaccharides). Responsiveness was evaluated according to the consensus guidelines published by the American immunology societies. Impaired responsiveness to non-memory polysaccharides was more frequent than to memory polysaccharides (51% versus 28%, P = 0.015), but the individual polysaccharides did not differ in triggering sufficient responses (74% versus 77%, P = 0.53). Closer analysis revealed important shortcomings of the current evaluation guidelines. The interpreted responses number and their specificities influenced the likelihood of impaired responsiveness in a complex manor. This influence was propelled by the dichotomous approaches inherent to the American guidelines. We therefore define a novel more robust polysaccharide responsiveness measure, the Z-score, which condenses multiple, uniformly weighted responses into one continuous variable. Using the Z-score, responsiveness to non-memory polysaccharides and memory-polysaccharides were found to correlate (R(2) = 0.59, P<0.0001). We found that polysaccharide responsiveness was not biased by prior protein-coupled polysaccharide vaccination in HIV-infected adults. Studies in additional populations are warranted.

Common variable immunodeficiency unmasked by treatment of immune thrombocytopenic purpura with Rituximab.

BACKGROUND: Hypogammaglobulinemia may be part of several different immunological or malignant conditions, and its origin is not always obvious. Furthermore, although autoimmune cytopenias are known to be associated with common variable immunodeficiency (CVID) and even may precede signs of immunodeficiency, this is not always recognized. Despite novel insight into the molecular immunology of common variable immunodeficiency, several areas of uncertainty remain. In addition, the full spectrum of immunological effects of the B cell depleting anti-CD20 antibody Rituximab has not been fully explored. To our knowledge this is the first report of development of CVID in a patient with normal immunoglobulin prior to Rituximab treatment. CASE PRESENTATION: Here we describe the highly unusual clinical presentation of a 34-year old Caucasian male with treatment refractory immune thrombocytopenic purpura and persistent lymphadenopathy, who was splenectomized and received multiple courses of high-dose corticosteroid before treatment with Rituximab resulted in a sustained response. However, in the setting of severe pneumococcal meningitis, hypogammaglobulinemia was diagnosed. An extensive immunological investigation was performed in order to characterize his immune status, and to distinguish between a primary immunodeficiency and a side effect of Rituximab treatment. We provide an extensive presentation and discussion of the literature on the basic immunology of CVID, the mechanism of action of Rituximab, and the immunopathogenesis of hypogammaglobulinemia observed in this patient. CONCLUSIONS: We suggest that CVID should be ruled out in any patient with immune cytopenias in order to avoid diagnostic delay. Likewise, we stress the importance of monitoring immunoglobulin levels before, during, and after Rituximab therapy to identify patients with hypogammaglobulinemia to ensure initiation of immunoglobulin replacement therapy in order to avoid life-threatening invasive bacterial infections. Recent reports indicate that Rituximab is not contra-indicated for the treatment of CVID-associated thrombocytopenia, however concomitant immunoglobulin substitution therapy is of fundamental importance to minimize the risk of infections. Therefore, lessons can be learned from this case report by clinicians caring for patients with immunodeficiencies, haematological diseases or other autoimmune disorders, particularly, when Rituximab treatment may be considered.

Biological variation of anti-αGal-antibodies studied by a novel Time-Resolved ImmunoFluorometric Assay.

As much as one percent of antibodies in human plasma are reported to be specific for the non-human disaccharide structure αGal. Various microbes express αGal. However, the implications of anti-αGal antibodies for the anti-microbial defenses are poorly established. With the perspective of studying the biological importance of the antibodies, we have established a sensitive Time-Resolved ImmunoFluorometric Assay (TRIFMA) for quantification of such antibodies. Two versions were developed, one for IgM antibodies and one for IgG antibodies. Samples were collected from plasma donations of healthy adults (n=120) of known gender (60+60), AB0-type (0: 15+15, A: 15+15, B: 15+15, and AB: 15+15) and age (19-64 yrs). We subsequently examined the potential association between antibody concentration and AB0-type, gender, age, and titers of antibodies to blood type antigens. We found that IgG and IgM anti-αGal concentrations are, 1) stable over time within the individual, 2) vary more than 400-fold between individuals, 3) negatively correlated with age for IgM but not for IgG antibodies, 4) IgM antibodies are 2-fold higher in females whereas no gender difference was observed for the IgG antibodies, 5) inter-mutual correlated, 6) lowest in individuals expressing B-antigen, and 7) AB0-type A individuals may constitute an intermediate group. Our established method and findings pave the way for further studies of the involvement of anti-αGal antibodies in immunity and may be a method to examine the potential of an individual to mount an anti-carbohydrate response.

Real-time relative qPCR without reference to control samples and estimation of run-specific PCR parameters from run-internal mini-standard curves.

BACKGROUND: Real-Time quantitative PCR is an important tool in research and clinical settings. Here, we describe two new approaches that broaden the scope of real-time quantitative PCR; namely, run-internal mini standard curves (RIMS) and direct real-time relative quantitative PCR (drqPCR). RIMS are an efficient alternative to traditional standard curves and provide both run-specific and target-specific estimates of PCR parameters. The drqPCR enables direct estimation of target ratios without reference to conventional control samples. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we compared RIMS-based drqPCR with classical quantifications based on external standard curves and the "comparative Ct method". Specifically, we used a raw real-time PCR dataset as the basis for more than two-and-a-half million simulated quantifications with various user-defined conditions. Compared with classical approaches, we found that RIMS-based drqPCR provided superior precision and comparable accuracy. CONCLUSIONS/SIGNIFICANCE: The obviation of referencing to control samples is attractive whenever unpaired samples are quantified. This may be in clinical and research settings; for instance, studies on chimerism, TREC quantifications, copy number variations etc. Also, lab-to-lab comparability can be greatly simplified.