Potential Cross-reactivity Research Articles

An isothermal lab-on-phone test for easy molecular diagnosis of SARS-CoV-2 near patients and in less than 1 hour

ObjectivesThe performance of a new point-of-care CE-IVD-marked isothermal lab-on-phone COVID-19 assay was assessed in comparison to a gold standard real-time reverse transcriptase–PCR method. MethodsThe study was conducted following a nonprobability sampling of ≥16-year-old volunteers from three different laboratories, using direct mouthwash (N = 24) or nasopharyngeal (N = 191) clinical samples. ResultsThe assay demonstrated 95.19% sensitivity and 100% specificity for detection of SARS-CoV-2 in direct nasopharyngeal crude samples and 78.95% sensitivity and 100% specificity in direct mouthwash crude samples. It also successfully detected currently predominant SARS-CoV-2 variants of concern (Beta B.1.351, Delta B.1.617.2, and Omicron B.1.1.529) and demonstrated to be inert against potential cross-reactions of other common respiratory pathogens that cause infections that present similar symptoms to COVID-19. ConclusionThis lab-on-phone pocket-sized assay relies on an isothermal amplification of SARS-CoV-2’s N and E genes, taking just 50 minutes from sample to result, with only 2 minutes of hands-on time. It presents good performance when using direct nasopharyngeal crude samples, enabling a low-cost, real-time, rapid, and accurate identification of SARS-CoV-2 infections at the point of care, which is important for both clinical management and population screening, as a tool to break the chain of transmission of COVID-19 pandemic, especially in low-resources environments.

Omicron BA.1 Mutations in SARS-CoV-2 Spike Lead to Reduced T-Cell Response in Vaccinated and Convalescent Individuals.

Omicron BA.1 variant can readily infect people with vaccine-induced or naturally acquired SARS-CoV-2 immunity facilitated by escape from neutralizing antibodies. In contrast, T-cell reactivity against the Omicron BA.1 variant seems relatively well preserved. Here, we studied the preexisting T cells elicited by either vaccination with the mRNA-based BNT162b2 vaccine or by natural infection with ancestral SARS-CoV-2 for their cross-reactive potential to 20 selected CD4+ T-cell epitopes of spike-protein-harboring Omicron BA.1 mutations. Although the overall memory CD4+ T-cell responses primed by the ancestral spike protein was still preserved generally, we show here that there is also a clear loss of memory CD4+ T-cell cross-reactivity to immunodominant epitopes across the spike protein due to Omicron BA.1 mutations. Complete or partial loss of preexisting T-cell responsiveness was observed against 60% of 20 nonconserved CD4+ T-cell epitopes predicted to be presented by a broad set of common HLA class II alleles. Monitoring such mutations in circulating strains helps predict which virus variants may escape previously induced cellular immunity and could be of concern.

SARS-CoV-2-Induced Immunosuppression: A Molecular Mimicry Syndrome.

Background Contrary to immunological expectations, decay of adaptive responses against severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) characterizes recovered patients compared with patients who had a severe disease course or died following SARS-CoV-2 infection. This raises the question of the causes of the virus-induced immune immunosuppression. Searching for molecular link(s) between SARS-CoV-2 immunization and the decay of the adaptive immune responses, SARS-CoV-2 proteome was analyzed for molecular mimicry with human proteins related to immunodeficiency. The aim was to verify the possibility of cross-reactions capable of destroying the adaptive immune response triggered by SARS-CoV-2. Materials and Methods Human immunodeficiency–related proteins were collected from UniProt database and analyzed for sharing of minimal immune determinants with the SARS-CoV-2 proteome. Results Molecular mimicry and consequent potential cross-reactivity exist between SARS-CoV-2 proteome and human immunoregulatory proteins such as nuclear factor kappa B (NFKB), and variable diversity joining V(D)J recombination-activating gene (RAG). Conclusion The data (1) support molecular mimicry and the associated potential cross-reactivity as a mechanism that can underlie self-reactivity against proteins involved in B- and T-cells activation/development, and (2) suggest that the extent of the immunosuppression is dictated by the extent of the immune responses themselves. The higher the titer of the immune responses triggered by SARS-CoV-2 immunization, the more severe can be the cross-reactions against the human immunodeficiency–related proteins, the more severe the immunosuppression. Hence, SARS-CoV-2-induced immunosuppression can be defined as a molecular mimicry syndrome. Clinically, the data imply that booster doses of SARS-CoV-2 vaccines may have opposite results to those expected.

Potential impact of SARS COV-2 infection on the performance of serological assays used to diagnose arboviral diseases

BackgroundThe COVID-19 pandemic caused by SARS-CoV-2 was first described in December 2019, in China. In addition, there has also been an increase in arboviral infections in recent years. As both infections have similar symptoms, misdiagnosis may occur if both outbreaks occur at the same time. ObjectiveOur objective was to assess the potential impact of SARS-CoV-2 infection on diagnostic assays used for arboviral diseases. Materials and methodsWe conducted this study by testing samples obtained during the precovid phase (before November 2019) and during the covid period (after February 2020). Samples were further grouped as those with acute febrile illness (AFI) and those without. All samples were tested for anti SARS-CoV-2 Ab, Chikungunya and Dengue specific IgM antibodies to evaluate potential serological cross-reactions between COVID-19 and Arbovirus specific antibodies. ResultsOne sample from the 62 cases of AFI during the pre-covid phase showed seropositivity for SARS-CoV-2 antibodies. Also, in asymptomatic individuals, arboviral seropositivity was significantly higher in the COVID period samples (22%) compared to pre-COVID samples (3%). ConclusionDue to similar clinical symptoms and cross reactions in both infections, relying solely on serological testing for arboviral diagnosis may be less sensitive; other clinical and laboratory parameters may be required.

Structural insights into engineering a T-cell receptor targeting MAGE-A10 with higher affinity and specificity for cancer immunotherapy

BackgroundT-cell receptor (TCR) immunotherapy is becoming a viable modality in cancer treatment with efficacy in clinical trials. The safety of patients is paramount, so innovative cell engineering methods are being...

Immuno-informatic Prediction of B and T cell Epitopes of Cysteine Protease Allergen from Phaseolus vulgaris with Cross-reactive Potential and Population Coverage.

In-silico mapping of epitopes by immune-informatics has simplified the efforts towards understanding antigen-antibody interactions. The knowledge of allergen epitopes may help in advancing the diagnosis and therapy of allergic diseases. This study was intended to identify B and T cell epitopes of cysteine protease allergen of Phaseolus vulgaris. Modeller 9v20 software was used for the generation of three-dimensional model of cysteine protease and quality assessment was performed using SAVES webserver and other in silico software. Linear and conformational B and T cell epitopes were predicted via immuno-informatics based computational servers. Epitopes were synthesized and their immunoreactivity was analyzed using specific IgE ELISA with food allergy positive patient's sera. Cellular immune response of peptides was determined through basophil activation assay. Consurf and SDAP (property distance) were used to examine the evolutionary conservancy and potential cross-reactivity of predicted epitopes. MSA based positional conservancy between HDM allergen epitopes and predicted peptides was also established using IEDB epitope database. Finally, population coverage for each promiscuous T cell epitope was predicted using IEDB population coverage analysis tool. Cysteine protease structure was derived by homology modeling and combination of bioinformatic tools predicted three B- and three T-cell peptides by consensus method and validated computationally. ELISA with kidney bean sensitive patient's sera showed higher IgE binding of B-cell peptides as compared to T-cell or control peptides. Epitope conservancy revealed B-cell epitopes being upto 95% conserved in comparison to variable T-cell epitopes (upto 69%). B-cell peptides were crossreactive with homologous allergens based on PD values. Structural comparison of cysteine protease with Der p 1 and Der f 1 showed similar epitopic regions, validating the prediction accuracy of epitopes. Promiscuous T-cell epitopes binding to broad-spectrum class-II MHC alleles demonstrated the distribution of T-cell peptides world-wide (30-98%) and in Asian population (99%). The current approach can be applied for identification of epitopes. Analysis of crossreactive and widely-distributed specific epitopes of allergen and knowledge about their interactive surfaces will help in understanding of food allergy and related immune responses.

Abstract 578: A functional approach to identifying and engineering TCRs results in highly-potent and specific TCRs for TCR-T cell therapy

Abstract Peptide-HLA (pHLA)- targeting therapeutics, such as T cell receptor-engineered T cells (TCR-T), have had clinical success in treating solid tumors. However, challenges related to safety exist: major concerns remain surrounding the cross-reactivity of T cell receptors (TCRs) as well as the ability of therapeutics to discriminate between on-target vs off-target pHLAs while maintaining high potency. Therefore, approaches that survey the diversity of the T cell repertoire to discover optimal TCRs, as well as platforms to comprehensively identify potential off-target liabilities, are critical to de-risking and accelerating the development of this promising class of pHLA-targeting therapeutics. We have developed a strategy that (1) queries the TCR repertoire to enrich and identify multiple active, sequence-distinct endogenous TCRs; (2) uses 3T-TRACE, a high-diversity pHLA library, to screen for cross-reactivity; and (3) exploits functional selections to simultaneously optimize for TCR potency and specificity. We applied this approach to identify TCRs of optimal specificity and potency targeting a peptide derived from the cancer-testis antigen NY-ESO-1 (SLLMWITQC) displayed by HLA-A2. We profiled 6 sequence-distinct TCRs using 3T-TRACE and validated potential endogenous off-target cross-reactivities. Many of the identified off-targets exhibited little to no sequence homology to the NY-ESO-1 epitope, highlighting the importance of diverse combinatorial libraries in identifying unexpected cross-reactivities. Leveraging the off-target liabilities identified by 3T-TRACE, we designed a functional library and selection scheme that enabled the identification of TCRs with increased potency and specificity. Optimized TCRs exhibited enhanced killing activity and improved safety against an NY-ESO-1-expressing melanoma cell line compared to benchmark TCRs, indicating that this approach has potential to improve clinical safety and efficacy. Using this multi-faceted and comprehensive approach we rapidly identified highly potent and specific TCRs against NY-ESO-1. Identifying cross-reactivities using 3T-TRACE proved to be critical in selecting TCRs suitable for engineering and functional selections. This approach can be extended to any pHLA target to create safe and effective TCR-Ts for clinical development. Citation Format: John D. Leonard, Alejandro Ramirez, Jason Romero, Jake Kleiner, Joanna Dreux, Bindu Hegde, Bryan Xie, Akshay Sharathchandra, Nathan Katz, Venita I. DeAlmeida, Hans-Peter Gerber, Marvin H. Gee, Leah V. Sibener. A functional approach to identifying and engineering TCRs results in highly-potent and specific TCRs for TCR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 578.

Making a meal out of bugs

Making a meal out of bugs

Simultaneous amplification of multiple immunofluorescence signals via cyclic staining of target molecules using mutually cross-adsorbed antibodies

Amplification of immunofluorescence (IF) signals is becoming increasingly critical in cancer research and neuroscience. Recently, we put forward a new signal amplification technique, which we termed fluorescent signal amplification via cyclic staining of target molecules (FRACTAL). FRACTAL amplifies IF signals by repeatedly labeling target proteins with a pair of secondary antibodies that bind to each other. However, simultaneous amplification of multiple IF signals via FRACTAL has not yet been demonstrated because of cross-reactivity between the secondary antibodies. In this study, we show that mutual cross-adsorption between antibodies can eliminate all forms of cross-reactions between them, enabling simultaneous amplification of multiple IF signals. First, we show that a typical cross-adsorption process—in which an antibody binds to proteins with potential cross-reactivity with the antibody—cannot eliminate cross-reactions between antibodies in FRACTAL. Next, we show that all secondary antibodies used in FRACTAL need to be mutually cross-adsorbed to eliminate all forms of cross-reactivity, and then we demonstrate simultaneous amplification of multiple IF signals using these antibodies. Finally, we show that multiplexed FRACTAL can be applied to expansion microscopy to achieve higher fluorescence intensities after expansion. Multiplexed FRACTAL is a highly versatile tool for standard laboratories, as it amplifies multiple IF signals without the need for custom antibodies.

Characterization of Immunological Responses to Borrelia Immunogenic Protein A (BipA), a Species-Specific Antigen for North American Tick-Borne Relapsing Fever.

ABSTRACTTick-borne relapsing fever (TBRF) is a neglected vector-borne bacterial disease distributed worldwide. Borrelia turicatae, Borrelia parkeri, and Borrelia hermsii are three argasid-borne TBRF species previously implicated in human disease in North America. TBRF is likely underdiagnosed due to its nonspecific symptoms and poorly developed diagnostic tests. Studies suggest that the Borrelia immunogenic protein A (BipA) is specific to TBRF Borrelia but heterogenic between species. In this study, we hypothesized that antibody responses generated to BipA are specific to the North American TBRF species infecting a given animal. To test this, we characterized the expression and localization of native BipA in North American species of TBRF Borrelia. We also infected mice by needle inoculation or tick bite with B. turicatae, B. hermsii, or B. parkeri and evaluated serum sample reactivity to recombinant BipA (rBipA) that was produced from each species. Furthermore, serum samples from nonhuman primates and domestic dogs experimentally infected with B. turicatae were assessed. Lastly, we tested human Lyme disease (LD) serum samples to determine potential cross-reactivity to rBipA generated from B. turicatae, B. parkeri, and B. hermsii. Our findings indicate that rBipA has the potential to distinguish between infections of LD- and TBRF-causing spirochetes and that antibody responses were more robust toward the Borrelia species causing infection. This work further supports that rBipA can likely distinguish between B. turicatae, B. hermsii, and B. parkeri infections in mice, canines, and nonhuman primates.IMPORTANCE Borrelia species transmitted by soft or hard ticks cause tick-borne relapsing fever (TBRF). This is a debilitating disease distributed worldwide but is likely underdiagnosed or misdiagnosed as Lyme disease due to poorly developed diagnostic tests. Borrelia turicatae, Borrelia parkeri, and Borrelia hermsii are three TBRF species previously implicated in human disease in North America. Commonly used diagnostic methods do not identify the species causing infection. In this study, we evaluated the potential of recombinant Borrelia immunogenic protein A (rBipA) as a diagnostic antigen capable of distinguishing between infections of TBRF Borrelia species. We show that serum from mice, canines, and nonhuman primates infected with B. turicatae, B. parkeri, or B. hermsii react more strongly to the rBipA from the species causing infection. Furthermore, sera from Lyme disease patients failed to cross-react with our rBipA proteins, indicating the potential to use rBipA as a species-specific diagnostic antigen for TBRF.

Evaluation of a Human T Cell-Targeted Multi-Epitope Vaccine for Q Fever in Animal Models of Coxiella burnetii Immunity

T cell-mediated immunity plays a central role in the control and clearance of intracellular Coxiella burnetii infection, which can cause Q fever. Therefore, we aimed to develop a novel T cell-targeted vaccine that induces pathogen-specific cell-mediated immunity to protect against Q fever in humans while avoiding the reactogenicity of the current inactivated whole cell vaccine. Human HLA class II T cell epitopes from C. burnetii were previously identified and selected by immunoinformatic predictions of HLA binding, conservation in multiple C. burnetii isolates, and low potential for cross-reactivity with the human proteome or microbiome. Epitopes were selected for vaccine inclusion based on long-lived human T cell recall responses to corresponding peptides in individuals that had been naturally exposed to the bacterium during a 2007-2010 Q fever outbreak in the Netherlands. Multiple viral vector-based candidate vaccines were generated that express concatemers of selected epitope sequences arranged to minimize potential junctional neo-epitopes. The vaccine candidates caused no antigen-specific reactogenicity in a sensitized guinea pig model. A subset of the vaccine epitope peptides elicited antigenic recall responses in splenocytes from C57BL/6 mice previously infected with C. burnetii. However, immunogenicity of the vaccine candidates in C57BL/6 mice was dominated by a single epitope and this was insufficient to confer protection against an infection challenge, highlighting the limitations of assessing human-targeted vaccine candidates in murine models. The viral vector-based vaccine candidates induced antigen-specific T cell responses to a broader array of epitopes in cynomolgus macaques, establishing a foundation for future vaccine efficacy studies in this large animal model of C. burnetii infection.

Antigenic Cross-Reactivity Between SARS-CoV-2 S1-RBD and Its Receptor ACE2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus responsible for the ongoing COVID-19 pandemic. SARS-CoV-2 binds to the human cell receptor angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain in the S1 subunit of the spike protein (S1-RBD). The serum levels of autoantibodies against ACE2 are significantly higher in patients with COVID-19 than in controls and are associated with disease severity. However, the mechanisms through which these anti-ACE2 antibodies are induced during SARS-CoV-2 infection are unclear. In this study, we confirmed the increase in antibodies against ACE2 in patients with COVID-19 and found a positive correlation between the amounts of antibodies against ACE2 and S1-RBD. Moreover, antibody binding to ACE2 was significantly decreased in the sera of some COVID-19 patients after preadsorption of the sera with S1-RBD, which indicated that antibodies against S1-RBD can cross-react with ACE2. To confirm this possibility, two monoclonal antibodies (mAbs 127 and 150) which could bind to both S1-RBD and ACE2 were isolated from S1-RBD-immunized mice. Measurement of the binding affinities by Biacore showed these two mAbs bind to ACE2 much weaker than binding to S1-RBD. Epitope mapping using synthetic overlapping peptides and hydrogen deuterium exchange mass spectrometry (HDX-MS) revealed that the amino acid residues P463, F464, E465, R466, D467 and E471 of S1-RBD are critical for the recognition by mAbs 127 and 150. In addition, Western blotting analysis showed that these mAbs could recognize ACE2 only in native but not denatured form, indicating the ACE2 epitopes recognized by these mAbs were conformation-dependent. The protein–protein interaction between ACE2 and the higher affinity mAb 127 was analyzed by HDX-MS and visualized by negative-stain transmission electron microscopy imaging combined with antigen-antibody docking. Together, our results suggest that ACE2-cross-reactive anti-S1-RBD antibodies can be induced during SARS-CoV-2 infection due to potential antigenic cross-reactivity between S1-RBD and its receptor ACE2.

Serological Follow-Up Study Indicates High Seasonal Coronavirus Infection and Reinfection Rates in Early Childhood.

ABSTRACTSeasonal human coronaviruses (HCoVs) cause respiratory infections, especially in children. Currently, the knowledge on early childhood seasonal coronavirus infections and the duration of antibody levels following the first infections is limited. Here we analyzed serological follow-up samples to estimate the rate of primary infection and reinfection(s) caused by seasonal coronaviruses in early childhood. Serum specimens were collected from 140 children at ages of 13, 24, and 36 months (1, 2, and 3 years), and IgG antibody levels against recombinant HCoV nucleoproteins (N) were measured by enzyme immunoassay (EIA). Altogether, 84% (118/140) of the children were seropositive for at least one seasonal coronavirus N by the age of 3 years. Cumulative seroprevalences for HCoVs 229E, HKU1, NL63, and OC43 increased by age, and they were 45%, 27%, 70%, and 44%, respectively, at the age of 3 years. Increased antibody levels between yearly samples indicated reinfections by 229E, NL63, and OC43 viruses in 20–48% of previously seropositive children by the age of 3 years. Antibody levels declined 54–73% or 31–77% during the year after seropositivity in children initially seropositive at 1 or 2 years of age, respectively, in case there was no reinfection. The correlation of 229E and NL63, and OC43 and HKU1 EIA results, suggested potential cross-reactivity between the N specific antibodies inside the coronavirus genera. The data shows that seasonal coronavirus infections and reinfections are common in early childhood and the antibody levels decline relatively rapidly.IMPORTANCE The rapid spread of COVID-19 requires better knowledge on the rate of coronavirus infections and coronavirus specific antibody responses in different population groups. In this work we analyzed changes in seasonal human coronavirus specific antibodies in young children participating in a prospective 3-year serological follow-up study. We show that based on seropositivity and changes in serum coronavirus antibody levels, coronavirus infections and reinfections are common in early childhood and the antibodies elicited by the infection decline relatively rapidly. These observations provide further information on the characteristics of humoral immune responses of coronavirus infections in children.

Tuning T cell receptor sensitivity through catch bond engineering.

Adoptive cell therapy using engineered T cell receptors (TCRs) is a promising approach for targeting cancer antigens, but tumor-reactive TCRs are often weakly responsive to their target ligands, peptide-major histocompatibility complexes (pMHCs). Affinity-matured TCRs can enhance the efficacy of TCR-T cell therapy but can also cross-react with off-target antigens, resulting in organ immunopathology. We developed an alternative strategy to isolate TCR mutants that exhibited high activation signals coupled with low-affinity pMHC binding through the acquisition of catch bonds. Engineered analogs of a tumor antigen MAGE-A3-specific TCR maintained physiological affinities while exhibiting enhanced target killing potency and undetectable cross-reactivity, compared with a high-affinity clinically tested TCR that exhibited lethal cross-reactivity with a cardiac antigen. Catch bond engineering is a biophysically based strategy to tune high-sensitivity TCRs for T cell therapy with reduced potential for adverse cross-reactivity.

Evaluation of a Cryptococcus capsular polysaccharide detection FungiXpert LFA (lateral flow assay) for the rapid diagnosis of Cryptococcosis.

The FungiXpert LFA was developed to diagnose fungal meningitis caused by Cryptococcus yeasts, by using serum or cerebrospinal fluid. It was compared to an existing lateral flow assay (LFA). The FungiXpert LFA performed well in qualitative and semi-quantitative tests.

CRISPR-Cas12a-mediated label-free electrochemical aptamer-based sensor for SARS-CoV-2 antigen detection

Serological antigen testing has emerged as an important diagnostic paradigm in COVID-19, but often suffers from potential cross-reactivity. To address this limitation, we herein report a label-free electrochemical aptamer-based sensor for the detection of SARS-CoV-2 antigen by integrating aptamer-based specific recognition with CRISPR-Cas12a-mediated signal amplification. The sensing principle is based on the competitive binding of antigen and the preassembled Cas12a-crRNA complex to the antigen-specific aptamer, resulting in a change in the collateral cleavage activity of Cas12a. To further generate an electrochemical signal, a DNA architecture was fabricated by in situ rolling circle amplification on a gold electrode, which serves as a novel substrate for Cas12a. Upon Cas12a-based collateral DNA cleavage, the DNA architecture was degraded, leading to a significant decrease in impedance that can be measured spectroscopically. Using SARS-CoV-2 nucleocapsid antigen as the model, the proposed CRISPR-Cas12a-based electrochemical sensor (CRISPR-E) showed excellent analytical performance for the quantitative detection of nucleocapsid antigen. Since in vitro selection can obtain aptamers selective for many SARS-CoV-2 antigens, the proposed strategy can expand this powerful CRISPR-E system significantly for quantitative monitoring of a wide range of COVID-19 biomarkers.

Evaluation of Serologic Cross-Reactivity Between Dengue Virus and SARS-CoV-2 in Patients with Acute Febrile Illness - United States and Puerto Rico, April 2020-March 2021.

The diagnosis of dengue disease, caused by the dengue virus (DENV) (a flavivirus), often requires serologic testing during acute and early convalescent phases of the disease. Some symptoms of DENV infection, such as nonspecific fever, are similar to those caused by infection with SARS-CoV-2, the virus that causes COVID-19. In studies with few COVID-19 cases, positive DENV immunoglobulin M (IgM) results were reported with various serologic tests, indicating possible cross-reactivity in these tests for DENV and SARS-CoV-2 infections (1,2). DENV antibodies can cross-react with other flaviviruses, including Zika virus. To assess the potential cross-reactivity of SARS-CoV-2, DENV, and Zika virus IgM antibodies, serum specimens from 97 patients from Puerto Rico and 12 U.S.-based patients with confirmed COVID-19 were tested using the DENV Detect IgM Capture enzyme-linked immunosorbent assay (ELISA) (InBios International).* In addition, 122 serum specimens from patients with confirmed dengue and 121 from patients with confirmed Zika virus disease (all from Puerto Rico) were tested using the SARS-CoV-2 pan-Ig Spike Protein ELISA (CDC).† Results obtained for DENV, Zika virus IgM, and SARS-CoV-2 antibodies indicated 98% test specificity and minimal levels of cross-reactivity between the two flaviviruses and SARS-CoV-2. These findings indicate that diagnoses of dengue or Zika virus diseases with the serological assays described in this report are not affected by COVID-19, nor do dengue or Zika virus diseases interfere with the diagnosis of COVID-19.

Identification and characterization of Fe3O4/peroxodisulfate advanced oxidation products from sulfameter

Sulfonamides (SAs) are one of the most widely used antibiotics and their residuals in the environment could cause some negative environmental issues. Advanced oxidation such as Fenton-like reaction has been widely applied in the treatment of SAs polluted water. Degradation rates of 95%-99.7% were achieved in this work for the tested 8 SAs, including sulfisomidine, sulfameter (SME), phthalylsulfathiazole, sulfamethoxypyridazine, sulfamonomethoxine, sulfisoxazole, sulfachloropyridazine, and sulfadimethoxine, in the Fe3O4/peroxodisulfate (PDS) oxidation system after the optimization of PDS concentration and pH. Meanwhile, it was found that a lot of unknown oxidation products were formed, which brought up the uncertainty of health risks to the environment, and the identification of these unknown products was critical. Therefore, SME was selected as the model compound, from which the oxidation products were never elucidated, to identify these intermediates/products. With liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS), 10 new products were identified, in which 2-amino-5-methoxypyrimidine (AMP) was confirmed by its standard. The investigation of the oxidation process of SME indicated that most of the products were not stable and the degradation pathways were very complicated as multiple reactions, such as oxidation of the amino group, SO2 extrusion, and potential cross-reaction occurred simultaneously. Though most of the products were not verified due to the lack of standards, our results could be helpful in the evaluation of the treatment performance of SAs containing wastewater.

Vaccine Candidate Double Mutant Variants of Enterotoxigenic Escherichia coli Heat-Stable Toxin.

Heat-stable enterotoxin (ST) producing enterotoxigenic Escherichia coli (ETEC) strains are among the top four enteropathogens associated with moderate-to-severe diarrhea in children under five years in low-to-middle income countries, thus making ST a target for an ETEC vaccine. However, ST must be mutated to abolish its enterotoxicity and to prevent a potential immunological cross-reaction due to its structural resemblance to the human peptides uroguanylin and guanylin. To reduce the risk of eliciting cross-reacting antibodies with our lead STh-A14T toxoid, L9 was chosen as an additional mutational target. A double mutant vaccine candidate immunogen, STh-L9A/A14T, was constructed by conjugation to the synthetic virus-like mi3 nanoparticle using the SpyTag/SpyCatcher technology. This immunogen elicited STh neutralizing antibodies in mice, but with less consistency than STh-A14T peptide control immunogens. Moreover, individual sera from mice immunized with both single and double mutant variants displayed varying levels of unwanted cross-reacting antibodies. The lowest levels of cross-reacting antibodies were observed with STh-L9K/A14T control immunogens, suggesting that it is indeed possible to reduce the risk of eliciting cross-reacting antibodies by mutation. However, mutant-specific antibodies were observed for most double mutant immunogens, demonstrating the delicate balancing act between disrupting cross-reacting epitopes, keeping protective ones, and avoiding the formation of neoepitopes.

Exploring the role of framework mutations in enabling breadth of a cross-reactive antibody (CR3022) against the SARS-CoV-2 RBD and its variants of concern

Cross-reactive and broadly neutralizing antibodies against surface proteins of diverse strains of rapidly evolving viral pathogens like SARS-CoV-2 can prevent infection and therefore are crucial for the development of effective universal vaccines. While antibodies typically incorporate mutations in their complementarity determining regions during affinity maturation, mutations in the framework regions have been reported as players in determining properties of broadly neutralizing antibodies against HIV and the Influenza virus. We propose an increase in the cross-reactive potential of CR3022 against the emerging SARS- CoV-2 variants of concern through enhanced conformational flexibility. In this study, we use molecular dynamics simulations, in silico mutagenesis, structural modeling, and docking to explore the role of light chain FWR mutations in CR3022, a SARS-CoV anti-spike (S)-protein antibody cross-reactive to the S-protein receptor binding domain of SARS-CoV-2. Our study shows that single substitutions in the light chain framework region of CR3022 with conserved epitopes across SARS-CoV strains allow targeting of diverse antibody epitope footprints that align with the epitopes of recently-categorized neutralizing antibody classes while enabling binding to more than one strain of SARS-CoV-2. Our study has implications for rapid and evolution-based engineering of broadly neutralizing antibodies and reaffirms the role of framework mutations in effective change of antibody orientation and conformation via improved flexibility. Communicated by Ramaswamy H. Sarma