Structural Basis of Membrane Protein Chaperoning through the Mitochondrial Intermembrane Space.

The exchange of metabolites between the mitochondrial matrix and the cytosol depends on ß-barrel channels in the outer membrane and α-helical carrier proteins in the inner membrane. The essential translocase of the inner membrane (TIM) chaperones escort these proteins through the intermembrane space, but the structural and mechanistic details remain elusive. We have used an integrated structural biology approach to reveal the functional principle of TIM chaperones. Multiple clamp-like binding sites hold the mitochondrial membrane proteins in a translocation-competent elongated form, thus mimicking characteristics of co-translational membrane insertion. The bound preprotein undergoes conformational dynamics within the chaperone binding clefts, pointing to a multitude of dynamic local binding events. Mutations in these binding sites cause cell death or growth defects associated with impairment of carrier and ß-barrel protein biogenesis. Our work reveals how a single mitochondrial "transfer-chaperone" system is able to guide α-helical and ß-barrel membrane proteins in a "nascent chain-like" conformation through a ribosome-free compartment.

Recruitment of epitope-specific T cell clones with a low-avidity threshold supports efficacy against mutational escape upon re-infection.

Repetitive pathogen exposure leads to the dominant outgrowth of T cell clones with high T cell receptor (TCR) affinity to the relevant pathogen-associated antigens. However, low-affinity clones are also known to expand and form immunological memory. While these low-affinity clones contribute less immunity to the original pathogen, their role in protection against pathogens harboring immune escape mutations remains unclear. Based on identification of the TCR repertoire and functionality landscape of naive epitope-specific CD8+ T cells, we reconstructed defined repertoires that could be followed as polyclonal populations during immune responses in vivo. We found that selective clonal expansion is governed by clear TCR avidity thresholds. Simultaneously, initial recruitment of broad TCR repertoires provided a polyclonal niche from which flexible secondary responses to mutant epitopes could be recalled. Elucidating how T cell responses develop "from scratch" is informative for the development of enhanced immunotherapies and vaccines.

Multivalent antigen display on nanoparticle immunogens increases B cell clonotype diversity and neutralization breadth to pneumoviruses.

Nanoparticles for multivalent display and delivery of vaccine antigens have emerged as a promising avenue for enhancing B cell responses to protein subunit vaccines. Here, we evaluated B cell responses in rhesus macaques immunized with prefusion-stabilized respiratory syncytial virus (RSV) F glycoprotein trimer compared with nanoparticles displaying 10 or 20 copies of the same antigen. We show that multivalent display skews antibody specificities and drives epitope-focusing of responding B cells. Antibody cloning and repertoire sequencing revealed that focusing was driven by the expansion of clonally distinct B cells through recruitment of diverse precursors. We identified two antibody lineages that developed either ultrapotent neutralization or pneumovirus cross-neutralization from precursor B cells with low initial affinity for the RSV-F immunogen. This suggests that increased avidity by multivalent display facilitates the activation and recruitment of these cells. Diversification of the B cell response by multivalent nanoparticle immunogens has broad implications for vaccine design.

A High-Throughput Screen for Transcription Activation Domains Reveals Their Sequence Features and Permits Prediction by Deep Learning.

Acidic transcription activation domains (ADs) are encoded by a wide range of seemingly unrelated amino acid sequences, making it difficult to recognize features that promote their dynamic behavior, "fuzzy" interactions, and target specificity. We screened a large set of random 30-mer peptides for AD function in yeast and trained a deep neural network (ADpred) on the AD-positive and -negative sequences. ADpred identifies known acidic ADs within transcription factors and accurately predicts the consequences of mutations. Our work reveals that strong acidic ADs contain multiple clusters of hydrophobic residues near acidic side chains, explaining why ADs often have a biased amino acid composition. ADs likely use a binding mechanism similar to avidity where a minimum number of weak dynamic interactions are required between activator and target to generate biologically relevant affinity and in vivo function. This mechanism explains the basis for fuzzy binding observed between acidic ADs and targets.

Precursor Frequency and Affinity Determine B Cell Competitive Fitness in Germinal Centers, Tested with Germline-Targeting HIV Vaccine Immunogens.

How precursor frequencies and antigen affinities impact interclonal B cell competition is a particularly relevant issue for candidate germline-targeting HIV vaccine designs because of the in vivo rarity of naive B cells that recognize broadly neutralizing epitopes. Knowing the frequencies and affinities of HIV-specific VRC01-class naive human B cells, we transferred B cells with germline VRC01 B cell receptors into congenic recipients to elucidate the roles of precursor frequency, antigen affinity, and avidity on B cell responses following immunization. All three factors were interdependently limiting for competitive success of VRC01-class B cells. In physiological high-affinity conditions using a multivalent immunogen, rare VRC01-class B cells successfully competed in germinal centers (GC), underwent extensive somatic hypermutation, and differentiated into memory B cells. The data reveal dominant influences of precursor frequency, affinity, and avidity for interclonal GC competition and indicate that germline-targeting immunogens can overcome these challenges with high-affinity multimeric designs.

A Novel Anti-nucleocapsid Antibody Avidity Method for Identifying SARS-CoV-2 Reinfections.

Detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfections is challenging with current serology assays and is further complicated by the marked decrease in routine viral testing practices as viral transmission increased during Omicron. Here, we provide proof-of-principle that high-avidity anti-nucleocapsid (N) antibodies detects reinfections after a single infection with higher specificity (85%; 95% confidence interval [95% CI], 80%-90%) compared to anti-N antibody levels (72%; 95% CI, 66%-79%) in a vaccinated cohort. This method could be used to retroactively investigate the epidemiology and incremental long-term health consequences of SARS-CoV-2 reinfections.

Quantity and Quality of Naturally Acquired Antibody Immunity to the Pneumococcal Proteome Throughout Life.

BACKGROUND: Young children and older adults are susceptible for invasive pneumococcal disease (IPD) caused by Streptococcus pneumoniae. Pneumococcal protein-specific antibodies play a protective role against IPD; however, not much is known about the pace of acquisition, maturation, and maintenance of these antibodies throughout life. METHODS: Immunoglobulin G (IgG) and IgA levels, avidity, and/or specificity to the pneumococcal proteome in serum and saliva from healthy young children, adults, and older adults, with known carriage status, were measured by enzyme-linked immunosorbent assay (ELISA) and 2-dimensional western blotting against ΔcpsTIGR4. RESULTS: Eleven-month-old children, the youngest age group tested, had the lowest pneumococcal proteome-specific IgG and IgA levels and avidity in serum and saliva, followed by 24-month-old children and were further elevated in adult groups. Among adult groups, the parents had the highest serum and saliva IgG and IgA antibody levels. In children, antibody levels and avidity correlated with daycare attendance and presence of siblings, posing as proxy for exposure and immunization. Immunodominance patterns slightly varied throughout life. CONCLUSIONS: Humoral immunity against the pneumococcal proteome is acquired through multiple episodes of pneumococcal exposure. Low-level and low-avidity antiproteome antibody profiles in young children may contribute to their IPD susceptibility, while in overall antiproteome antibody-proficient older adults other factors likely play a role.

trans-Interacting Plasma Membrane Proteins and Binding Partner Identification.

Plasma membrane proteins (PMPs) play critical roles in a myriad of physiological and disease conditions. A unique subset of PMPs functions through interacting with each other in trans at the interface between two contacting cells. These trans-interacting PMPs (tiPMPs) include adhesion molecules and ligands/receptors that facilitate cell-cell contact and direct communication between cells. Among the tiPMPs, a significant number have apparent extracellular binding domains but remain orphans with no known binding partners. Identification of their potential binding partners is therefore important for the understanding of processes such as organismal development and immune cell activation. While a number of methods have been developed for the identification of protein binding partners in general, very few are applicable to tiPMPs, which interact in a two-dimensional fashion with low intrinsic binding affinities. In this review, we present the significance of tiPMP interactions, the challenges of identifying binding partners for tiPMPs, and the landscape of method development. We describe current avidity-based screening approaches for identifying novel tiPMP binding partners and discuss their advantages and limitations. We conclude by highlighting the importance of developing novel methods of identifying new tiPMP interactions for deciphering the complex protein interactome and developing targeted therapeutics for diseases.

Affinity fine-tuning anti-CAIX CAR-T cells mitigate on-target off-tumor side effects.

One of the major hurdles that has hindered the success of chimeric antigen receptor (CAR) T cell therapies against solid tumors is on-target off-tumor (OTOT) toxicity due to sharing of the same epitopes on normal tissues. To elevate the safety profile of CAR-T cells, an affinity/avidity fine-tuned CAR was designed enabling CAR-T cell activation only in the presence of a highly expressed tumor associated antigen (TAA) but not when recognizing the same antigen at a physiological level on healthy cells. Using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution, and flow cytometry, we identified high carbonic anhydrase IX (CAIX) density on clear cell renal cell carcinoma (ccRCC) patient samples and low-density expression on healthy bile duct tissues. A Tet-On doxycycline-inducible CAIX expressing cell line was established to mimic various CAIX densities, providing coverage from CAIX-high skrc-59 tumor cells to CAIX-low MMNK-1 cholangiocytes. Assessing the killing of CAR-T cells, we demonstrated that low-affinity/high-avidity fine-tuned G9 CAR-T has a wider therapeutic window compared to high-affinity/high-avidity G250 that was used in the first anti-CAIX CAR-T clinical trial but displayed serious OTOT effects. To assess the therapeutic effect of G9 on patient samples, we generated ccRCC patient derived organotypic tumor spheroid (PDOTS) ex vivo cultures and demonstrated that G9 CAR-T cells exhibited superior efficacy, migration and cytokine release in these miniature tumors. Moreover, in an RCC orthotopic mouse model, G9 CAR-T cells showed enhanced tumor control compared to G250. In summary, G9 has successfully mitigated OTOT side effects and in doing so has made CAIX a druggable immunotherapeutic target.

Analysis of Suspected Measles Cases with Discrepant Measles-Specific IgM and rRT-PCR Test Results, Japan.

We investigated clinically suspected measles cases that had discrepant real-time reverse transcription PCR (rRT-PCR) and measles-specific IgM test results to determine diagnoses. We performed rRT-PCR and measles-specific IgM testing on samples from 541 suspected measles cases. Of the 24 IgM-positive and rRT-PCR--negative cases, 20 were among children who received a measles-containing vaccine within the previous 6 months; most had low IgG relative avidity indexes (RAIs). The other 4 cases were among adults who had an unknown previous measles history, unknown vaccination status, and high RAIs. We detected viral nucleic acid for viruses other than measles in 15 (62.5%) of the 24 cases with discrepant rRT-PCR and IgM test results. Measles vaccination, measles history, and contact history should be considered in suspected measles cases with discrepant rRT-PCR and IgM test results. If in doubt, measles IgG avidity and PCR testing for other febrile exanthematous viruses can help confirm or refute the diagnosis.

Maternal and congenital human cytomegalovirus infection: laboratory testing for detection and diagnosis.

Human cytomegalovirus (CMV) is the leading cause of congenital infection worldwide and the most common cause of non-genetic sensorineural hearing loss. As there is no vaccine or other specific intervention to prevent congenital CMV infection, there is a need to identify maternal and congenital infections with sensitive and specific testing as early as possible. There is no widely accepted practice for screening during pregnancy or in all newborns for identification of possible cases of congenital CMV. Currently, screening during pregnancy is limited to those identified as at risk followed by fetal and/or neonatal testing when congenital infection is suspected. This review focuses primarily on the current status of laboratory testing for diagnosis of maternal and congenital CMV infections. Primary maternal infection is best diagnosed using serologic testing, including CMV IgM, IgG, and avidity testing, while fetal infection should be assessed by nucleic acid amplification testing (NAAT) of amniotic fluid. Urine and saliva NAATs are the mainstay for diagnosis of congenital CMV in the first 3 weeks of life. Testing of dried blood spots can be useful for diagnosis of congenital CMV outside of the newborn period. The gaps in knowledge such as the prognostic value of viral loads in various sample types are addressed.

Advances in preclinical TCR characterization: leveraging cell avidity to identify functional TCRs.

T-cell therapy has emerged as an effective approach for treating viral infections and cancers. However, a significant challenge is the selection of T-cell receptors (TCRs) that exhibit the desired functionality. Conventionally in vitro techniques, such as peptide sensitivity measurements and cytotoxicity assays, provide valuable insights into TCR potency but are labor-intensive. In contrast, measuring ligand binding properties (z-Movi technology) could provide an accelerated processing while showing robust correlations with T-cell functions. In this study, we assessed whether cell avidity can predict functionality also in the context of TCR-engineered T cells. To this end, we developed a flexible system for TCR re-expression by generating a Jurkat-derived T cell clone lacking TCR and CD3 expression through CRISPR-Cas9-mediated TRBC knockout. The knockin of a transgenic TCR into the TRAC locus restored TCR/CD3 expression, allowing for CD3-based purification of TCR-engineered T cells. Subsequently, we characterized these engineered cell lines by functional readouts, and assessment of binding properties through the z-Movi technology. Our findings revealed a strong correlation between the cell avidities and functional sensitivities of Jurkat TCR-T cells. Altogether, by integrating cell avidity measurements with our versatile T cell engineering platform, we established an accelerated system for enhancing the in vitro selection of clinically relevant TCRs.

Easy assessment of the avidity of polyclonal allergen-specific serum antibodies.

INTRODUCTION: Allergen-specific IgE-blocking IgG antibodies contribute to successful allergen immunotherapy (AIT), however, not much is known about their affinity. Since affinity measurements of polyclonal antibodies in serum are technically challenging we evaluated the applicability of acidic disruption of antibody-allergen complexes by a modified ELISA protocol with monoclonal antibodies (mAbs) specific for the relevant major allergens Betv1 and Mald1. Then, AIT-induced blocking and non-blocking Mald1-specific antibodies in sera from individuals with or without reduced apple allergy were compared. METHODS: After testing their pH stability coated recombinant allergens were incubated with (i) mAbs diluted in PBS or human serum and (ii) sera from individuals after sublingual administration of Mald1 or Betv1 for 16 weeks. Immune complexes were exposed to buffers in the pH range of 6.4-3.4 and residual antibodies were measured. Avidity indexes (AI), defined as the pH removing 50% of antibodies, were compared to the dissociation constants (KD) of mAbs determined by surface plasmon resonance. RESULTS: The selected pH range was applicable to disrupt allergen complexes with highly affine mAbs without compromising allergen integrity. AIs of mAbs accorded with KD values and were unaffected by epitope specificity or the presence of serum proteins. The inter-assay variability was <4% CV. Protective Mald1-specific IgG antibodies from individuals with reduced apple allergy showed a higher collective binding strength than that of the non-protective antibodies of individuals without reduced apple allergy. CONCLUSION: Acidic disruption of allergen-antibody complexes may be used to estimate the net-binding force of polyclonal serum antibodies and eases the investigation of affinity-related research questions in AIT.

Effect of SARS-CoV-2 and Toxoplasma gondii co-infection on IFN-γ and TNF-α expression and its impact on disease severity.

The symptomatology of COVID-19 is dependent on the immune status and the cytokine response of the host. The cytokine level of the host is influenced by the presence of chronic persistent or latent infections with co-pathogens. Parasitic diseases are known to induce host immune-modulation which may impact the response to co-infection. Toxoplasmosis is a widespread protozoal infection that remains quiescent in its latent form to be re-activated during states of immune depression. Clinical data on the relation between toxoplasmosis and COVID-19 cytokine profile and symptomatology are still insufficient. Seventy-nine subjects were included in this study. Patients were diagnosed with COVID-19 by PCR. Serological testing for toxoplasmosis was performed by the detection of anti-Toxoplasma IgG antibodies, in addition to IgG avidity testing. IFN-γ and TNF-α levels were determined by RT-PCR. Among patients diagnosed with COVID-19, 67.1% were seronegative for anti-Toxoplasma IgG, while 32.9% were seropositive. High avidity was found in 10 cases (40% of seropositive cases), 4 of whom required ICU administration, while low avidity was found in 15 cases (60%), 7 of which were administered to the ICU. TNF-α and INF-γ levels were significantly higher in COVID-19 patients than in healthy control subjects. No significant association was found between the seroprevalence of toxoplasmosis and the presence of COVID-19 and its severity. Cytokines were significantly higher in both seropositive and seronegative COVID-19 patients than in their control counterparts. The high prevalence of toxoplasmosis merits further exploration of its relation to COVID-19 by mass studies.

Distinct FDG PET/CT avidity among newly diagnosed intravascular large B-cell lymphoma patients: a descriptive observational study.

Intravascular large B-cell lymphoma (IVLBCL) is a rare type of aggressive B-cell non-Hodgkin lymphoma that poses a great diagnostic challenge due to its highly heterogenous clinical manifestations. Although 18F-fluorodeoxyglucose (FDG) is widely used as a diagnostic tool for patients suspected of having lymphoma, as it reveals FDG-avid lesions, the FDG avidity of IVLBCL has not been extensively characterized. Here, we present a comprehensive report of FDG avidity in IVLBCL and its association with clinicopathological features and survival. This descriptive observational study included consecutive patients aged at least 18 years diagnosed with IVLBCL in Peking Union Medical Hospital across 9 years. Among 50 screened IVLBCL patients, 42 had undergone 18F-FDG PET/CT to detect possible lesions for biopsy before pathological diagnosis; their FDG PET/CT (positron emission computed tomography, PET/CT) reports were retrospectively reviewed. The primary endpoint was the clinical description of FDG avidity of newly diagnosed intravascular large B-cell lymphoma and frequency. A total of 73.8% patients showed FDG-avid lesions, with a median SUVmax of 7.4 (range 1-27.7), which was lower than that for other aggressive lymphomas. Clinicopathological features were the same between the FDG-avid group and the non-FDG-avid group, except that the latter had a higher Ki-67 index (median 90% in the nonavid group vs. 80% in the avid group, P = 0.043). The overall survival rate was not different between the PET/CT groups. Our findings demonstrate that FDG PET/CT is a useful diagnostic tool for detecting FDG-avid lesions in IVLBCL patients. A random skin biopsy is essential for assisting in the diagnosis of IVLBCL, even for those with negative PET/CT.

One-dose intradermal rabies booster enhances rabies antibody production and avidity maturation.

The incidence of rabies in Thailand reached its peak in 2018 with 18 human deaths. Preexposure prophylaxis (PrEP) vaccination is thus recommended for high-risk populations. WHO has recently recommended that patients who are exposed to a suspected rabid animal and have already been immunized against rabies should receive a 1-site intradermal (ID) injection of 0.1 mL on days 0 and 3 as postexposure prophylaxis (PEP). In Thailand, village health and livestock volunteers tasked with annual dog vaccination typically receive only a single lifetime PrEP dose and subsequent boosters solely upon confirmed animal bites. However, the adequacy of a single PrEP dose for priming and maintaining immunity in this high-risk group has not been evaluated. Therefore, our study was designed to address two key questions: (1) sufficiency of single-dose PrEP-to determine whether a single ID PrEP dose provides adequate long-term immune protection for high-risk individuals exposed to numerous dogs during their vaccination duties. (2) Booster efficacy for immune maturation-to investigate whether one or two additional ID booster doses effectively stimulate a mature and sustained antibody response in this population. The level and persistence of the rabies antibody were determined by comparing the immunogenicity and booster efficacy among the vaccination groups. Our study demonstrated that rabies antibodies persisted for more than 180 days after cost-effective ID PrEP or the 1st or the 2nd single ID booster dose, and adequate antibody levels were detected in more than 95% of participants by CEE-cELISA and 100% by indirect ELISA. Moreover, the avidity maturation of rabies-specific antibodies occurred after the 1st single ID booster dose. This smaller ID booster regimen was sufficient for producing a sufficient immune response and enhancing the maturation of anti-rabies antibodies. This safe and effective PrEP regimen and a single visit involving a one-dose ID booster are recommended, and at least one one-dose ID booster regimen could be equitably implemented in at-risk people in Thailand and other developing countries. However, an adequate antibody level should be monitored before the booster is administered.

Linker optimization and activity validation of a cell surface vimentin targeted homo-dimeric peptoid antagonist for lung cancer stem cells.

Epithelial-to-mesenchymal transition (EMT) endows epithelia-derived cancer cells with properties of stem cells that govern cancer invasion and metastasis. Vimentin is one of the best studied EMT markers and recent reports indicate that vimentin interestingly translocated onto cell surface under various tumor conditions. We recently reported a cell surface vimentin (CSV) specific peptoid antagonist named JM3A. We now investigated the selective antagonist activity of the optimized homo-dimeric version of JM3A, JM3A-L2D on stem-like cancer cells or cancer stem cells (CSCs) over normal cells in non-small cell lung cancer (NSCLC). Homo-dimerization of JM3A provided the avidity effect and improved the biological activity compared to the monomeric version. We first optimized the central linker length of the dimer by designing seven JM3A derivatives with varying linker lengths/types and evaluated the anti-cancer activity using the standard MTS cell viability assay. The most optimized derivative contains a central lysine linker and two glycines, named JM3A-L2D, which displayed 100 nM vimentin binding affinity (Kd) with an anti-cancer activity (IC50) of 6.7 µM on H1299 NSCLC cells. This is a 190-fold improvement in binding over the original JM3A. JM3A-L2D exhibited better potency on high vimentin-expressing NSCLC cells (H1299 and H460) compared to low vimentin-expressing NSCLC cells (H2122). No activity was observed on normal bronchial HBEC3-KT cells. The anti-CSC activity of JM3A-L2D was evaluated using the standard colony formation assay and JM3A-L2D disrupted the colony formation with IC50 âˆ¼ 400 nM. In addition, JM3A-L2D inhibited cell migration activity at IC50 âˆ¼ 2 µM, assessed via wound healing assay. The underlying mechanism of action seems to be the induction of apoptosis by JM3A-L2D on high-vimentin expressing H1229 and H460 NSCLC cells. Our optimized highly CSV selective peptoid has the potential to be developed as an anti-cancer drug candidate, especially considering the high serum stability and economical synthesis of peptoids.

Toxoplasma gondii IgG avidity for the diagnosis of primary infection in pregnant women: Comparison between chemiluminescent microparticle immunoassay and enzyme-linked immunosorbent assay.

INTRODUCTION: This study evaluated whether IgG avidity measured by chemiluminescent microparticle immunoassay (CMIA) compared with enzyme-linked immunosorbent assay (ELISA) was useful to detect primary T. gondii infection during pregnancy and to estimate the risk for congenital T. gondii infection. METHODS: One hundred six women with positive tests for T. gondii IgG and T. gondii IgM, comprising 21 women (19.8%) with low (<30%), 6 (5.7%) with borderline (30%-35%), and 79 (74.5%) with high (>35%) IgG avidity measured by ELISA were selected. Their stored sera were used for T. gondii IgG avidity measurements by CMIA. RESULTS: In CMIA, 72 (67.9%) women had low (<50%), 12 (11.3%) had borderline (50%-59.9%), and 22 (20.8%) had high (≥60%) IgG avidity. The ratio of low T. gondii IgG avidity index in CMIA was more than three-fold than that in ELISA. Eighteen (85.7%) of 21 women with ELISA low avidity also had CMIA low avidity, and 26 (96.3%) of 27 women with ELISA low or borderline avidity corresponded to CMIA low or borderline avidity, whereas 21 (26.6%) of 79 women with ELISA high avidity were diagnosed with CMIA low avidity. All three cases with congenital T. gondii infection showed coincidentally low IgG avidity in both methods. A positive correlation in IgG avidity indices was found between of ELISA and CMIA. CONCLUSIONS: CMIA for T. gondii avidity measurements compared with ELISA was clinically useful to detect pregnant women at a high risk of developing congenital T. gondii infection.

Congenital cytomegalovirus infection in newborns suspected of congenital rubella syndrome in Iran: a cross-sectional study.

BACKGROUND: Following rubella virus control, the most important cause of congenital infections is human cytomegalovirus (HCMV). Congenital CMV (cCMV) may happen both in primary and non-primary maternal infections. The present study aimed to screen cCMV in symptomatic newborns suspected of congenital rubella syndrome (CRS) in Iran. METHODS: Out of 1629 collected infants' serum samples suspected of CRS but negative for rubella IgM, 524 samples were selected regarding cCMV complications. These samples were divided into two age groups: 1- one month and younger, 2- older than 1 month up to one year. Anti-HCMV IgM detection was performed on these serums. Then HCMV IgG avidity assay and HCMV DNA detection were carried out on all samples with positive and borderline results in IgM detection. RESULTS: Herein, 3.67% of symptomatic infants aged one month and younger had positive and borderline HCMV IgM, 12.5% of which had a low avidity index (AI). HCMV IgM detection rate among symptomatic infants older than one month to one year was 14.5%. Identified genotypes in this study were gB-1(63.63%), gB2 (18.18%), and gB3 (18.18%), respectively. CONCLUSIONS: This comprehensive study was performed on serum samples of symptomatic infants clinically suspected of cCMV from all over Iran. There was a good correlation between serology findings and PCR.

Functionally impaired antibody response to BNT162b2 booster vaccination in CVID IgG responders.

BACKGROUND: Although previous studies described the production of IgG antibodies in a subgroup of patients with common variable immunodeficiency (CVID) following messenger RNA vaccinations with BNT162b2 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (CVID responders), the functionality of these antibodies in terms of avidity as measured by the dissociation rate constant (kdis) and the antibody response to booster immunization has not been studied. OBJECTIVE: We sought to analyze in CVID responders and healthy individuals, the avidity of anti-SARS-CoV-2 serum antibodies and their neutralization capacity as measured by surrogate virus-neutralizing antibodies in addition to IgG-, IgM-, and IgA-antibody levels and the response of circulating (peripheral blood) follicular T-helper cells after a third vaccination with BNT162b2 SARS-CoV-2 messenger RNA vaccine. METHODS: Binding IgG, IgA, and IgM serum levels were analyzed by ELISA in patients with CVID responding to the primary vaccination (CVID responders, n = 10) and healthy controls (n = 41). The binding avidity of anti-spike antibodies was investigated using biolayer interferometry in combination with biotin-labeled receptor-binding-domain of SARS-CoV-2 spike protein and streptavidin-labeled sensors. Antigen-specific recall T-cell responses were assessed by measuring activation-induced markers by flow cytometry. RESULTS: After the third vaccination with BNT162b2, IgG-, IgM-, and IgA-antibody levels, surrogate virus-neutralizing antibody levels, and antibody avidity were lower in CVID responders than in healthy controls. In contrast, anti-SARS-CoV-2 spike protein avidity was comparable in CVID responders and healthy individuals following primary vaccination. Follicular T-helper cell response to booster vaccination in CVID responders was significantly reduced when compared with that in healthy individuals. CONCLUSIONS: Impaired affinity maturation during booster response provides new insight into CVID pathophysiology.