Deficient Generation of Spike-Specific Long-Lived Plasma Cells in the Bone Marrow After Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Generation of a stable long-lived plasma cell (LLPC) population is the sine qua non of durable antibody responses after vaccination or infection. We studied 20 individuals with a prior coronavirus disease 2019 infection and characterized the antibody response using bone marrow aspiration and plasma samples. We noted deficient generation of spike-specific LLPCs in the bone marrow after severe acute respiratory syndrome coronavirus 2 infection. Furthermore, while the regression model explained 98% of the observed variance in anti-tetanus immunoglobulin G levels based on LLPC enzyme-linked immunospot assay, we were unable to fit the same model with anti-spike antibodies, again pointing to the lack of LLPC contribution to circulating anti-spike antibodies.

Structural, Biochemical, and Computational Characterization of Sulfamides as Bimetallic Peptidase Inhibitors.

The sulfonamide function is used extensively as a general building block in various inhibitory scaffolds and, more specifically, as a zinc-binding group (ZBG) of metalloenzyme inhibitors. Here, we provide biochemical, structural, and computational characterization of a metallopeptidase in complex with inhibitors, where the mono- and bisubstituted sulfamide functions are designed to directly engage zinc ions of a bimetallic enzyme site. Structural data showed that while monosubstituted sulfamides coordinate active-site zinc ions via the free negatively charged amino group in a canonical manner, their bisubstituted counterparts adopt an atypical binding pattern divergent from expected positioning of corresponding tetrahedral reaction intermediates. Accompanying quantum mechanics calculations revealed that electroneutrality of the sulfamide function is a major factor contributing to the markedly lower potency of bisubstituted compounds by considerably lowering their interaction energy with the enzyme. Overall, while bisubstituted uncharged sulfamide functions can bolster favorable pharmacological properties of a given inhibitor, their use as ZBGs in metalloenzyme inhibitors might be less advantageous due to their suboptimal metal-ligand properties.

Aerosol delivery of SARS-CoV-2 human monoclonal antibodies in macaques limits viral replication and lung pathology.

Passively administered monoclonal antibodies (mAbs) given before or after viral infection can prevent or blunt disease. Here, we examine the efficacy of aerosol mAb delivery to prevent infection and disease in rhesus macaques inoculated with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant via intranasal and intratracheal routes. SARS-CoV-2 human mAbs or a human mAb directed to respiratory syncytial virus (RSV) are nebulized and delivered using positive airflow via facemask to sedated macaques pre- and post-infection. Nebulized human mAbs are detectable in nasal, oropharyngeal, and bronchoalveolar lavage (BAL) samples. SARS-CoV-2 mAb treatment significantly reduces levels of SARS-CoV-2 viral RNA and infectious virus in the upper and lower respiratory tracts relative to controls. Reductions in lung and BAL virus levels correspond to reduced BAL inflammatory cytokines and lung pathology. Aerosolized antibody therapy for SARS-CoV-2 could be effective for reducing viral burden and limiting disease severity.

The molecular basis of the neutralization breadth of the RBD-specific antibody CoV11.

SARS-CoV-2, the virus behind the COVID-19 pandemic, has changed over time to the extent that the current virus is substantially different from what originally led to the pandemic in 2019-2020. Viral variants have modified the severity and transmissibility of the disease and continue do so. How much of this change is due to viral fitness versus a response to immune pressure is hard to define. One class of antibodies that continues to afford some level of protection from emerging variants are those that closely overlap the binding site for angiotensin-converting enzyme 2 (ACE2) on the receptor binding domain (RBD). Some members of this class that were identified early in the course of the pandemic arose from the VH 3-53 germline gene (IGHV3-53*01) and had short heavy chain complementarity-determining region 3s (CDR H3s). Here, we describe the molecular basis of the SARS-CoV-2 RBD recognition by the anti-RBD monoclonal antibody CoV11 isolated early in the COVID-19 pandemic and show how its unique mode of binding the RBD determines its neutralization breadth. CoV11 utilizes a heavy chain VH 3-53 and a light chain VK 3-20 germline sequence to bind to the RBD. Two of CoV11's four heavy chain changes from the VH 3-53 germline sequence, ThrFWR H128 to Ile and SerCDR H131 to Arg, and some unique features in its CDR H3 increase its affinity to the RBD, while the four light chain changes from the VK 3-20 germline sequence sit outside of the RBD binding site. Antibodies of this type can retain significant affinity and neutralization potency against variants of concern (VOCs) that have diverged significantly from original virus lineage such as the prevalent omicron variant. We also discuss the mechanism by which VH 3-53 encoded antibodies recognize spike antigen and show how minimal changes to their sequence, their choice of light chain, and their mode of binding influence their affinity and impact their neutralization breadth.

Maternal transfer of IgA and IgG SARS-CoV-2 specific antibodies transplacentally and via breast milk feeding.

BACKGROUND: Although there have been many studies on antibody responses to SARS-CoV-2 in breast milk, very few have looked at the fate of these in the infant, and whether they are delivered to immunologically relevant sites in infants. METHODS: Mother/infant pairs (mothers who breast milk fed and who were SARS-CoV-2 vaccinated before or after delivery) were recruited for this cross-sectional study. Mother blood, mother breast milk, infant blood, infant nasal specimen, and infant stool was tested for IgA and IgG antibodies against SARS-CoV-2 spike trimer. RESULTS: Thirty-one mother/infant pairs were recruited. Breast milk fed infants acquired systemic anti-spike IgG antibodies only if their mothers were vaccinated antepartum (100% Antepartum; 0% Postpartum; P<0.0001). Breast milk fed infants acquired mucosal anti-spike IgG antibodies (in the nose) only if their mothers were vaccinated antepartum (89% Antepartum; 0% Postpartum; P<0.0001). None of the infants in either group had anti-spike IgA in the blood. Surprisingly, 33% of the infants whose mothers were vaccinated antepartum had high titer anti-spike IgA in the nose (33% Antepartum; 0% Postpartum; P = 0.03). Half-life of maternally transferred plasma IgG antibodies in the Antepartum infant cohort was ~70 days. CONCLUSION: Vaccination antepartum followed by breast milk feeding appears to be the best way to provide systemic and local anti-SARS-CoV-2 antibodies for infants. The presence of high titer SARS-CoV-2-specific IgA in the nose of infants points to the potential importance of breast milk feeding early in life for maternal transfer of mucosal IgA antibodies. Expectant mothers should consider becoming vaccinated antepartum and consider breast milk feeding for optimal transfer of systemic and mucosal antibodies to their infants.

Replacement of Antiretroviral Therapy with HIV Broadly Neutralizing Antibodies to Maximize the Effectiveness of Chemotherapy in HIV Patients with Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most fatal non-AIDS defining cancer in people living with HIV (PWH) on antiretroviral therapy (ART). Treatment of malignancies in PWH requires concomitant cancer therapy and ART, which can lead to potential drug-drug interactions (DDIs) and overlapping toxicities. In this study, we hypothesize that replacement of ART with HIV broadly neutralizing antibodies (bNAbs) during cancer chemotherapy (chemo) may maintain HIV suppression and tumor inhibition while minimizing DDIs and overlapping toxicities. We compared HIV suppression, tumor inhibition, and toxicity between conventional treatment (ART plus chemo) and a new modality (bNAbs plus chemo) in humanized mice. Humanized mice infected with HIVYU2 and xenografted with human NSCLC A549 cells were treated with NSCLC chemo (cisplatin and gemcitabine) and first-line ART (dolutegravir, tenofovir disoproxil difumarate, and emtricitabine) or bNAbs (N49P9.6-FR and PGT 121) at human equivalent drug doses. We monitored plasma HIV RNA, tumor volume, and toxicities over five cycles of chemo. We found that chemo plus ART or bNAbs were equally effective at maintaining suppression of HIV viremia and tumor growth. Comparative analysis showed that mice on ART and chemo had significant reductions in body weight and significant increases in plasma creatinine concentrations compared with mice on bNAbs and chemo, which suggests that a combination of bNAbs and chemo produces less renal toxicity than an ART and chemo combination. These data suggest that bNAb therapy during concomitant chemo may be an improved treatment option over ART for PWH and NSCLC, and possibly other cancers, because bNAbs maintain HIV suppression while minimizing DDIs and toxicities.

A practical guide to pulsed laser deposition.

Nanoscale thin films are widely implemented across a plethora of technological and scientific areas, and form the basis for many advancements that have driven human progress, owing to the high degree of functional tunability based on the chemical composition. Pulsed laser deposition is one of the multiple physical vapour deposition routes to fabricate thin films, employing laser energy to eject material from a target in the form of a plasma. A substrate, commonly a single-crystal oxide, is placed in the path of the plume and acts as a template for the arriving species from the target to coalesce and self-assemble into a thin film. This technique is tremendously useful to produce crystalline films, due to the wide range of atmospheric conditions and the extent of possible chemical complexity of the target. However, this flexibility results in a high degree of complexity, oftentimes requiring rigorous optimisation of the growth parameters to achieve high quality crystalline films with desired composition. In this tutorial review, we aim to reduce the complexity and the barrier to entry for the controlled growth of complex oxides by pulsed laser deposition. We present an overview of the fundamental and practical aspects of pulsed laser deposition, discuss the consequences of tailoring the growth parameters on the thin film properties, and describe in situ monitoring techniques that are useful in gaining a deeper understanding of the properties of the resultant films. Particular emphasis is placed on the general relationships between the growth parameters and the consequent structural, chemical and functional properties of the thin films. In the final section, we discuss the open questions within the field and possible directions to further expand the utility of pulsed laser deposition.

Mucosal and Systemic Responses to Severe Acute Respiratory Syndrome Coronavirus 2 Vaccination Determined by Severity of Primary Infection.

With much of the world infected with or vaccinated against severe acute respiratory syndrome coronavirus 2 (commonly abbreviated SARS-CoV-2; abbreviated here SARS2), understanding the immune responses to the SARS2 spike (S) protein in different situations is crucial to controlling the pandemic. We studied the clinical, systemic, mucosal, and cellular responses to two doses of SARS2 mRNA vaccines in 62 individuals with and without prior SARS2 infection that were divided into three groups based on antibody serostatus prior to vaccination and/or degree of disease symptoms among those with prior SARS2 infection: antibody negative (naive), low symptomatic, and symptomatic. Antibody negative were subjects who were antibody negative (i.e., those with no prior infection). Low symptomatic subjects were those who were antibody negative and had minimal or no symptoms at time of SARS2 infection. Symptomatic subjects were those who were antibody positive and symptomatic at time of SARS2 infection. All three groups were then studied when they received their SARS2 mRNA vaccines. In the previously SARS2-infected (based on antibody test) low symptomatic and symptomatic groups, reactogenic symptoms related to a recall response were elicited after the first vaccination. Anti-S trimer IgA and IgG titers, and neutralizing antibody titers, peaked after the 1st vaccination in the previously SARS2-infected groups and were significantly higher than for the SARS2 antibody-negative group in the plasma and nasal samples at most time points. Nasal and plasma IgA antibody responses were significantly higher in the low symptomatic group than in the symptomatic group at most time points. After the first vaccination, differences in cellular immunity were not evident between groups, but the activation-induced cell marker (AIM+) CD4+ cell response correlated with durability of IgG humoral immunity against the SARS2 S protein. In those SARS2-infected subjects, severity of infection dictated plasma and nasal IgA responses in primary infection as well as response to vaccination (peak responses and durability), which could have implications for continued protection against reinfection. Lingering differences between the SARS2-infected and SARS2-naive up to 10 months postvaccination could explain the decreased reinfection rates in the SARS2-infected vaccinees recently reported and suggests that additional strategies (such as boosting of the SARS2-naive vaccinees) are needed to narrow the differences observed between these groups. IMPORTANCE This study on SARS2 vaccination in those with and without previous exposure to the virus demonstrates that severity of infection dictates IgA responses in primary infection as well as response to vaccination (peak responses and durability), which could have implications for continued protection against reinfection.

Structural characterization of protective non-neutralizing antibodies targeting Crimean-Congo hemorrhagic fever virus.

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) causes a life-threatening disease with up to a 40% mortality rate. With no approved medical countermeasures, CCHFV is considered a public health priority agent. The non-neutralizing mouse monoclonal antibody (mAb) 13G8 targets CCHFV glycoprotein GP38 and protects mice from lethal CCHFV challenge when administered prophylactically or therapeutically. Here, we reveal the structures of GP38 bound with a human chimeric 13G8 mAb and a newly isolated CC5-17 mAb from a human survivor. These mAbs bind overlapping epitopes with a shifted angle. The broad-spectrum potential of c13G8 and CC5-17 and the practicality of using them against Aigai virus, a closely related nairovirus were examined. Binding studies demonstrate that the presence of non-conserved amino acids in Aigai virus corresponding region prevent CCHFV mAbs from binding Aigai virus GP38. This information, coupled with in vivo efficacy, paves the way for future mAb therapeutics effective against a wide swath of CCHFV strains.

Molecular dynamics simulations provide structural insight into binding of cyclic dinucleotides to human STING protein.

Human stimulator of interferon genes (hSTING) is a signaling adaptor protein that triggers innate immune system by response to cytosolic DNA and second messenger cyclic dinucleotides (CDNs). Natural CDNs contain purine nucleobase with different phosphodiester linkage types (3'-3', 2'-2' or mixed 2'-3'-linkages) and exhibit different binding affinity towards hSTING, ranging from micromolar to nanomolar. High-affinity CDNs are considered as suitable candidates for treatment of chronic hepatitis B and cancer. We have used molecular dynamics simulations to investigate dynamical aspects of binding of natural CDNs (specifically, 2'-2'-cGAMP, 2'-3'-cGAMP, 3'-3'-cGAMP, 3'-3'-c-di-AMP, and 3'-3'-c-di-GMP) with hSTINGwt protein. Our results revealed that CDN/hSTINGwt interactions are controlled by the balance between fluctuations (conformational changes) in the CDN ligand and the protein dynamics. Binding of different CDNs induces different degrees of conformational/dynamics changes in hSTINGwt ligand binding cavity, especially in α1-helices, the so-called lid region and α2-tails. The ligand residence time in hSTINGwt protein pocket depends on different contribution of R232 and R238 residues interacting with oxygen atoms of phosphodiester groups in ligand, water distribution around interacting charged centers (in protein residues and ligand) and structural stability of closed conformation state of hSTINGwt protein. These findings may perhaps guide design of new compounds modulating hSTING activity.Communicated by Ramaswamy H. Sarma.

Combined cART including Tenofovir Disoproxil, Emtricitabine, and Dolutegravir has potent therapeutic effects in HIV-1 infected humanized mice.

HIV-1 reservoirs persist in the presence of combined antiretroviral therapy (cART). However, cART has transformed HIV-1 infection into a chronic disease marked by control of HIV-1 viral load and mortality reduction. Major challenges remain, including viral resistance upon termination of cART and persistence and identification of tissue distribution of HIV-1 reservoirs. Thus, appropriate animal models that best mimic HIV-1 pathogenesis are important, and the current study complements our previously published validation of the CD34+ hematopoietic humanized mouse model for this purpose. Here we analyze viral suppression using the recently developed combination of antiretrovirals that include Tenofovir Disoproxil (TDF), Emtricitabine (FTC), and Dolutegravir (DTG), a choice based on recent clinical outcomes showing its improved antiretroviral potency, CD4+ T cell preservation, tolerability, and prevention of viral drug resistance compared to that of previous regimens. We used quantitative Airyscan-based super resolution confocal microscopy of selected mouse tissues. Our data allowed us to identify specific solid tissue reservoirs of human T cells expressing the HIV-1 core protein p24. In particular, lymph node, brain, spleen, and liver were visualized as reservoirs for residual infected cells. Marked reduction of viral replication was evident. Considering that detection and visualization of cryptic sites of HIV-1 infection in tissues are clearly crucial steps towards HIV-1 eradication, appropriate animal models with pseudo-human immune systems are needed. In fact, current studies with humans and non-human primates have limited sample availability at multiple stages of infection and cannot easily analyze the effects of differently administered combined antiretroviral treatments on multiple tissues. That is easier to manage when working with humanized mouse models, although we realize the limitations due to low human cell recovery and thus the number of cells available for thorough and comprehensive analyses. Nonetheless, our data further confirm that the CD34+ humanized mouse model is a potentially useful pre-clinical model to study and improve current anti-HIV-1 therapies.

Near-Pan-neutralizing, Plasma Deconvoluted Antibody N49P6 Mimics Host Receptor CD4 in Its Quaternary Interactions with the HIV-1 Envelope Trimer.

The first step in HIV-1 entry is the attachment of the envelope (Env) trimer to target cell CD4. As such, the CD4-binding site (CD4bs) remains one of the few universally accessible sites for antibodies (Abs). We recently described a method of isolating Abs directly from the circulating plasma and described a panel of broadly neutralizing Abs (bnAbs) from an HIV-1 "elite neutralizer" referred to as patient N49 (N49 Ab lineage [M. M. Sajadi, A. Dashti, Z. R. Tehrani, W. D. Tolbert, et al., Cell 173:1783-1795.e14, 2018, https://doi.org/10.1016/j.cell.2018.03.061]). Here, we describe the molecular details of antigen recognition by N49P6, an Ab of the N49 lineage that recapitulates most of the neutralization breadth and potency of the donor's plasma IgG. Our studies done in the context of monomeric and trimeric antigens indicate that N49P6 combines many characteristics of known CD4bs-specific bnAbs with features that are unique to the N49 Ab lineage to achieve its remarkable neutralization breadth. These include the omission of the CD4 Phe43 cavity and dependence instead on interactions with highly conserved gp120 inner domain layer 3. Interestingly, when bound to BG505 SOSIP, N49P6 closely mimics the initial contact of host receptor CD4 to the adjacent promoter of the HIV-1 Env trimer to lock the trimer in the closed conformation. Altogether, N49P6 defines a new class of near-pan-neutralizing, plasma deconvoluted CD4bs Abs that we refer to as the N49P series. The details of the mechanisms of action of this new Ab class pave the way for the next generation of HIV-1 bnAbs that can be used as vaccine components of therapeutics. IMPORTANCE Binding to target cell CD4 is the first crucial step required for HIV-1 infection. Thus, the CD4-binding site (CD4bs) is one of the most accessible sites for antibodies (Abs). However, due to steric constraints, only a few Abs are capable of targeting this site. Here, we show that the exceptional neutralization breadth and potency of N49P6, a near-pan-neutralizing Ab targeting the CD4bs isolated from the plasma of an HIV-1 "elite neutralizer," patient N49, are due to its signature combination of more typical CD4bs Ab-binding characteristics with unique interactions with the highly conserved gp120 inner domain. In addition, we also present a structural analysis of N49P6 in complex with the BG505 SOSIP trimer to show that N49P6 exhibits remarkable breadth in part by mimicking CD4's quaternary interaction with the neighboring gp120 protomer. In its mode of antigen interaction, N49P6 is unique and represents a new class of CD4bs-specific bnAbs.

Rational Engraftment of Quaternary-Interactive Acidic Loops for Anti-HIV-1 Antibody Improvement.

Broadly neutralizing antibodies (bNAbs) are the focus of increasing interest for human immunodeficiency virus type 1 (HIV-1) prevention and treatment. Although several bNAbs are already under clinical evaluation, the development of antibodies with even greater potency and breadth remains a priority. Recently, we reported a novel strategy for improving bNAbs against the CD4-binding site (CD4bs) of gp120 by engraftment of the elongated framework region 3 (FR3) from VRC03, which confers the ability to establish quaternary interactions with a second gp120 protomer. Here, we applied this strategy to a new series of anti-CD4bs bNAbs (N49 lineage) that already possess high potency and breadth. The resultant chimeric antibodies bound the HIV-1 envelope (Env) trimer with a higher affinity than their parental forms. Likewise, their neutralizing capacity against a global panel of HIV-1 Envs was also increased. The introduction of additional modifications further enhanced the neutralization potency. We also tried engrafting the elongated CDR1 of the heavy chain from bNAb 1-18, another highly potent quaternary-binding antibody, onto several VRC01-class bNAbs, but none of them was improved. These findings point to the highly selective requirements for the establishment of quaternary contact with the HIV-1 Env trimer. The improved anti-CD4bs antibodies reported here may provide a helpful complement to current antibody-based protocols for the therapy and prevention of HIV-1 infection.IMPORTANCE Monoclonal antibodies represent one of the most important recent innovations in the fight against infectious diseases. Although potent antibodies can be cloned from infected individuals, various strategies can be employed to improve their activity or pharmacological features. Here, we improved a lineage of very potent antibodies that target the receptor-binding site of HIV-1 by engineering chimeric molecules containing a fragment from a different monoclonal antibody. These engineered antibodies are promising candidates for development of therapeutic or preventive approaches against HIV/AIDS.

Conformational energies and equilibria of cyclic dinucleotides in vacuo and in solution: computational chemistry vs. NMR experiments.

Performance of computational methods in modelling cyclic dinucleotides - an important and challenging class of compounds - has been evaluated by two different benchmarks: (1) gas-phase conformational energies and (2) qualitative agreement with NMR observations of the orientation of the χ-dihedral angle in solvent. In gas-phase benchmarks, where CCSD(T) and DLPNO-CCSD(T) methods have been used as the reference, most of the (dispersion corrected) density functional approximations are accurate enough to justify prioritizing computational cost and compatibility with other modelling options as the criterion of choice. NMR experiments of 3'3'-c-di-AMP, 3'3'-c-GAMP, and 3'3'-c-di-GMP show the overall prevalence of the anti-conformation of purine bases, but some population of syn-conformations is observed for guanines. Implicit solvation models combined with quantum-chemical methods struggle to reproduce this behaviour, probably due to a lack of dynamics and explicitly modelled solvent, leading to structures that are too compact. Molecular dynamics simulations overrepresent the syn-conformation of guanine due to the overestimation of an intramolecular hydrogen bond. Our combination of experimental and computational benchmarks provides "error bars" for modelling cyclic dinucleotides in solvent, where such information is generally difficult to obtain, and should help gauge the interpretability of studies dealing with binding of cyclic dinucleotides to important pharmaceutical targets. At the same time, the presented analysis calls for improvement in both implicit solvation models and force-field parameters.

Ligand Strain and Its Conformational Complexity Is a Major Factor in the Binding of Cyclic Dinucleotides to STING Protein.

STING (stimulator of interferon genes) is a key regulator of innate immunity that has recently been recognized as a promising drug target. STING is activated by cyclic dinucleotides (CDNs) which eventually leads to expression of type I interferons and other cytokines. Factors underlying the affinity of various CDN analogues are poorly understood. Herein, we correlate structural biology, isothermal calorimetry (ITC) and computational modeling to elucidate factors contributing to binding of six CDNs-three pairs of natural (ribo) and fluorinated (2'-fluororibo) 3',3'-CDNs. X-ray structural analyses of six {STING:CDN} complexes did not offer any explanation for the different affinities of the studied ligands. ITC showed entropy/enthalpy compensation up to 25 kcal mol-1 for this set of similar ligands. The higher affinities of fluorinated analogues are explained with help of computational methods by smaller loss of entropy upon binding and by smaller strain (free) energy.

Protein-Ligand Interactions in the STING Binding Site Probed by Rationally Designed Single-Point Mutations: Experiment and Theory.

STING protein (stimulator of interferon genes) plays an important role in the innate immune system. A number of potent compounds regulating its activity have been reported, mostly derivatives of cyclic dinucleotides (CDNs), natural STING agonists. Here, we aim to provide complementary information to large-scale "ligand-profiling" studies by probing the importance of STING-CDN protein-ligand interactions on the protein side. We examined in detail six typical CDNs each in complex with 13 rationally devised mutations in STING: S162A, S162T, Y167F, G230A, R232K, R232H, A233L, A233I, R238K, T263A, T263S, R293Q, and G230A/R293Q. The mutations switch on and off various types of protein-ligand interactions: π-π stacking, hydrogen bonding, ionic pairing, and nonpolar contacts. We correlated experimental data obtained by differential scanning fluorimetry, X-ray crystallography, and isothermal titration calorimetry with theoretical calculations. This enabled us to provide a mechanistic interpretation of the differences in the binding of representative CDNs to STING. We observed that the G230A mutation increased the thermal stability of the protein-ligand complex, indicating an increased level of ligand binding, whereas R238K and Y167F led to a complete loss of stabilization (ligand binding). The effects of the other mutations depended on the type of ligand (CDN) and varied, to some extent. A very good correlation (R2 = 0.6) between the experimental binding affinities and interaction energies computed by quantum chemical methods enabled us to explain the effect of the studied mutations in detail and evaluate specific interactions quantitatively. Our work may inspire development of high-affinity ligands against the common STING haplotypes by targeting the key (sometimes non-intuitive) protein-ligand interactions.

Performance of nucleocapsid and spike-based SARS-CoV-2 serologic assays.

There is an urgent need for an accurate antibody test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have developed 3 ELISA methods, trimer spike IgA, trimer spike IgG, and nucleocapsid IgG, for detecting anti-SARS-CoV-2 antibodies. We evaluated their performance along with four commercial ELISAs, EDI™ Novel Coronavirus COVID-19 ELISA IgG and IgM, Euroimmun Anti-SARS-CoV-2 ELISA IgG and IgA, and one lateral flow assay, DPP® COVID-19 IgM/IgG System (Chembio). Both sensitivity and specificity were evaluated and the probable causes of false-positive reactions were determined. The assays were evaluated using 300 pre-epidemic samples and 100 PCR-confirmed COVID-19 samples. The sensitivities and specificities of the assays were as follows: 90%/100% (in-house trimer spike IgA), 90%/99.3% (in-house trimer spike IgG), 89%/98.3% (in-house nucleocapsid IgG), 73.7%/100% (EDI nucleocapsid IgM), 84.5%/95.1% (EDI nucleocapsid IgG), 95%/93.7% (Euroimmun S1 IgA), 82.8%/99.7% (Euroimmun S1 IgG), 82.0%/91.7% (Chembio nucleocapsid IgM), 92%/93.3% (Chembio nucleocapsid IgG). The presumed causes of false positive results from pre-epidemic samples in commercial and in-house assays were mixed. In some cases, assays lacked reproducibility. In other cases, reactivity was abrogated by competitive inhibition (spiking the sample with the same antigen that was used for coating ELISAs prior to performing the assay), suggesting positive reaction could be attributed to the presence of antibodies against these antigens. In other cases, reactivity was consistently detected but not abrogated by the spiking, suggesting positive reaction was not attributed to the presence of antibodies against these antigens. Overall, there was wide variability in assay performance using our samples, with in-house tests exhibiting the highest combined sensitivity and specificity. The causes of "false positivity" in pre-epidemic samples may be due to plasma antibodies apparently reacting with the corresponding antigen, or spurious reactivity may be directed against non-specific components in the assay system. Identification of these targets will be essential to improving assay performance.

The silent presence of Mycoplasma hominis in patients with prostate cancer.

BACKGROUND: Mycoplasma hominis, an opportunistic pathogen in human genitourinary tract, can cause chronic infection in the prostate. Intracellular survival of M. hominis leads to a prolonged presence in the host cells that can affect the cell's biological cycle. In the present study, we aimed to evaluate the frequency of M. hominis DNA in prostate tissue of Iranian patients with prostate cancer (PCa) in comparison to a control group with benign prostatic hyperplasia (BPH). METHODS: This research was a retrospective case-control study using 61 archived formalin-fixed paraffin-embedded (FFPE) blocks of prostate tissue from patients with PCa and 70 FFPE blocks of patients with BPH. Real-time PCR, targeting two different genes, 16S rRNA and yidC, in the M. hominis genome was performed for all specimens. RESULTS: Out of 61 blocks of prostate biopsy from patients with PCa, eight samples (13%) were positive for M. hominis, while the bacterium was not detected in any of the 70 blocks of patients with BPH (P value, 0.002). CONCLUSIONS: The high frequency of M. hominis in patients with PCa likely shows a hidden role of the organism in prostate cancer during its chronic, apparently silent and asymptomatic colonization in prostate.