ISOLATION OF PAN-SARBECOVIRUS mABs THAT NEUTRALIZE SARS-CoV-1 AND SARS-CoV-2 VARIANTS

Background: The continued emergence of severe acute respiratory syndrome coronaviruses (SARS-CoVs) and recent explosion of the SARS-CoV-2 pandemic highlights the need for broad and potent antibody recognition and understanding the contexts in which they may develop. Antibodies with cross reactivity across SARS lineages may be of particular value in preparing for future outbreaks of new sarbecoviruses. Method(s): We isolated monoclonal antibodies (mAbs) from an individual 60-days post-vaccination, 30-days post Delta-infection. Reconstructed antibodies were screened for binding to a panel of prefusion-stabilized Spike trimers from SARS-CoV-2 and other beta-coronaviruses using enzyme-linked immunosorbent assay (ELISA). Neutralization potency and breadth was assessed using a spike-pseudotyped lentivirus neutralization assay. Additionally, epitope and escape mutant profiling was conducted by deep mutational scanning (DMS) to identify mutations that affect antibody binding. Lastly, binding breadth was further evaluated using a yeast display library of RBDs from SARS-CoV-2 variants and related sarbecoviruses. Result(s): We identified several SARS-CoV-2-specific mAbs that neutralized SARS-CoV-2 variants of concern (VOCs) and SARS-CoV-1. Notably, two of these mAbs (C68.61 and C68.185) neutralized SARS-CoV-1 with an IC50 = 307 and 139 ng/mL (respectively) that is similar to or better than the potency of S309 (IC50 = 206 ng/mL) and CR3022 (IC50 = 981 ng/mL), which are mAbs isolated from individuals with SARS-CoV-1 infections. C68.61 also neutralized all Omicron VOCs tested and retained neutralization activity against currently circulating variants BQ1.1 (IC50=790 ng/ml) and XBB (IC50=590 ng/ml). Key C68.61 mAbescape mutations identified by DMS in the Omicron BA.2 background yeast display library included sites K462, E465, R466, and I468, which are conserved sites across all VOCs and SARS-CoV-1. The isolated mAbs displayed crossreactive binding to RBDs from diverse SARS-CoV-1-related CoVs and African and European sarbecovirus isolates as well as SARS-CoV-2 VOCs. Conclusion(s): Here we describe mAbs from a SARS-CoV-2-infected individual that bound and neutralized both SARS-CoV-2 and SARS-CoV-1, including one that showed breadth across recent VOCs. Given their breadth, these SARS-CoV-2 cross-reactive mAbs may be robust to viral escape and thus could contribute to therapeutic efforts. In addition, these mAbs displayed broad cross-reactive activity across sarbecoviruses and may be beneficial against future spillover events.

Assembling a structural model of the SARS-CoV-2 replication and transcription complex from 60+ subunits

The SARS-CoV-2 replication and transcription complex (RTC) is made up of nine distinct non-structural viral proteins encoded by the ORF1ab gene. These proteins house seven enzymatic sites that synthesize new viral genomic and subgenomic RNA, proofread and correct errors in the synthesis, add a 5'-cap to the nascent RNA, and truncate the intermediate negative sense 5'-poly-U tail. While x-ray crystallography and cryo-EM have provided high resolution structures of each of the individual proteins of the RTC and have shed light on how subsets of the proteins associate, a full picture of the RTC has remained elusive. Using molecular modeling tools, including protein-protein docking, we have generated a model of the RTC centered around hexameric nsp15, which is capped on two faces by trimers of nsp14/nsp16/(nsp10)2. A conformational change of nsp14, necessary to facilitate binding to nsp15, then recruits six nsp12/nsp7/(nsp8)2 polymerase subunits. To this, six nsp13 subunits are distributed around the complex. The resulting superstructure is composed of 60 subunits total and positions the nsp14 exonuclease and nsp15 endonuclease sites in line with the dsRNA exiting the nsp12 polymerase site. Nsp10 acts to separate the RNA strands, directing the nascent strand to the nsp12 NiRAN site, where a transiently associated nsp9 facilitates the first step in mRNA capping. The RNA is then directed to the nsp14 N7-methyltransferase site and the nsp16 2'O-methyltransferase site to complete the capping. Additionally, template switching during transcription is proposed to be facilitated by positioning of the TRS-L RNA-bound N-protein above the polymerase active site, between two subunits of nsp13. The model, while constructed based on structural considerations, offers a unifying set of hypotheses to explain the diverse set of processes involved in coronavirus genome replication and transcription. All work presented was funded by Gilead Sciences.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

VHH DIMER P559-R45L NEUTRALIZES SARS-CoV-2 BY STABILIZING SPIKE IN UP CONFORMATION

Background: SARS-CoV-2 Omicron subvariants are highly resistant to vaccineinduced immunity and therapeutic monoclonal antibodies. We previously reported anti-SARS-CoV-2 spike alpaca nanobodies (VHHs) P86 and P17 that potently neutralize the wild type and VOCs from Alpha to Omicron BA.1 and BA.2, but not Omicron subvariants after that such as BA.4/5. Thus, we tried to establish a new VHH that can neutralize all the variants including BA.4/5. Method(s): We developed VHH trimers and heterodimers based on the structural and computational analysis of Delta spike-immunized alpaca VHH library. We tested representative VHHs against SARS-CoV-2 spike by pseudovirus assays and generated VHH heterodimers. We further obtained Cryo-EM structure of Spike trimer and VHH monomer or heterodimer. Result(s): First, we generated series of P86 mutants to counteract L452R mutation in Delta or Omicron BA.5 subvariants and found that P86 R45L was most potent against D614G with an IC50 of 0.03 mug/mL. From the Delta spike-immunized VHH library, we also identified that homo-trimer of a new clone P559 neutralized SARS-CoV-2 Delta and Omicron BA.5 variants with an IC50 of 0.077 and 0.54 mug/mL, respectively. We finally generated P559-R45L heterodimer that neutralized all the variants so far including Omicron BA.5 with an IC50 of 0.39 mug/mL. Cryo-EM structure revealed that three molecules of P559- R45L heterodimer bridged two RBD molecules in the spike trimer and stabilized spike timers with RBD in the up conformation. Conclusion(s): We developed VHH P559-R45L heterodimer that potently neutralized all the variants including Omicron subvariants through unique structural interaction.

Development of a spin-label model system for investigation of protein oligomerization states

The transmembrane domains of viral proteins are highly conserved and crucial to normal viral function. Oligomeric transmembrane domains present novel opportunities for drug development, as their disruption can prevent the assembly of the virus. The Reichart lab is particularly interested in developing retro-inverso peptide inhibitors. Retro-inverso peptides are peptides using D-amino acids mirroring a region of target protein, which allows the peptide to inhibit viral assembly, but they are also significantly less likely to be catabolized by natural metabolic or immunologic processes. The efficacy of these inhibitors is governed largely by the extent to which they mirror the target protein, making highly conserved regions, such as transmembrane domains, ideal target regions for these inhibitors. The primary technique in the literature for the investigation of oligomerization states uses fluorescence spectroscopy. We are now working on developing a novel alternative system to evaluate protein oligomerization using spin-labeled peptides that are directly incorporated into the peptide sequence. Direct incorporation of the spin-label into the peptide sequence is a more powerful technique than the standard procedures used in the literature. In particular, the ability to incorporate spin labels in various positions within the protein can give novel insights into the relative depth of the protein within a membrane, which is very difficult to study using other techniques and not possible using the fluorescence technique. The transmembrane domains of proteins with known and well-characterized monomer and trimer standard oligomerization states were synthesized using an Fmoc Solid- Phase Peptide Synthesis (SPPS) procedure incorporating an Fmoc-2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid, (Fmoc-TOAC) instead of an alanine. Direct incorporation of stable N-oxide spin labels, which can be contrasted to labeling cysteine residues after the protein synthesis, has been used for the investigation of the secondary structure of proteins for decades, but the application of this spin labeling technique to study the oligomerization states of transmembrane domains of proteins is an understudied application. The products of SPPS were analyzed using a Liquid Chromatography Mass Spectroscopy instrument and purified using High Performance Liquid Chromatography. The spin-label was then deprotected and evaluated using Electron Spin Resonance (ESR) Spectroscopy. There are two primary future directions following this research project: first, the generation of viral proteins with spin labels incorporated in different positions to determine the relative depth of each position within the membrane;second, the incorporation of spin labels into SARS-CoV- 2 proteins to develop a model for in vitro evaluation of retro-inverso peptide assembly inhibitors. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

A novel computational approach for rapid screening of antibody therapeutics for efficacy against SARS-CoV-2 variants of concern including Omicron variant

Multiple severe acute respiratory syndrome coronavirus 2 (SARSCoV- 2) variants continue to evolve carrying flexible amino acid substitutions in the spike protein's receptor binding domain (RBD). These substitutions modify the binding of the SARS-CoV-2 to human angiotensin-converting enzyme 2 (hACE2) receptor and have been implicated in altered host fitness, transmissibility and efficacy against antibody therapeutics and vaccines. Reliably predicting the binding strength of SARS-CoV-2 variants RBD to hACE2 receptor and neutralizing antibodies (NAbs) can help assessing their fitness, and rapid deployment of effective antibody therapeutics, respectively. Here, we introduced a two-step computational framework with threefold validation that first identified dissociation constant as a reliable predictor of binding affinity in hetero-dimeric and -trimeric protein complexes. The second step implements dissociation constant as descriptor of the binding strengths of SARS-CoV-2 variants RBD to hACE2 and NAbs. Then, we examined several variants of concern (VOCs) such as Alpha, Beta, Gamma, Delta, and Omicron and demonstrated that these VOCs RBD bind to the hACE2 with enhanced affinity. Furthermore, the binding affinity of Omicron variant's RBD was reduced with majority of the RBD-directed NAbs, which is highly consistent with the experimental neutralization data. By studying the atomic contacts between RBD and NAbs, we revealed the molecular footprints of four NAbs (GH-12, P2B-1A1, Asarnow-3D11, and C118)-that may likely neutralize the recently emerged omicron variant-facilitating enhanced binding affinity. Finally, our findings suggest a computational pathway that could aid researchers identify a range of current NAbs that may be effective against emerging SARS-CoV-2 variants.

The Impact of N-butyldeoxynojirimycin and 1-deoxymannojirimycin on SARS-CoV-2 S1 Subunit and Angiotensin-Converting Enzyme 2 Interaction in Intestinal Epithelial Cells

The severe acute respiratory syndrome coronavirus 2 (SARSCoV- 2) targets mainly the respiratory tract. In addition to respiratory symptoms, many extrapulmonary manifestations were observed in the gastrointestinal tract and reported by SARS-CoV-2 patients, including abdominal pain, nausea, and diarrhea. SARS-CoV-2 binds initially to angiotensin-converting enzyme 2 (ACE2) on the host cell surface via its spike (S) protein before it undergoes endocytosis and fusion with the lysosomal membrane. The spike protein of SARS-CoV-2 is a heavily N- and O-glycosylated trimer. Glycosylation is an essential posttranslational modification in the life cycle of membrane and secretory proteins that affects their structural and functional characteristics as well as their trafficking and sorting patterns. This study aimed at elucidating the impact of glycosylation modulation on the trafficking of both S1 subunit and ACE2 as well as their interaction at the cell surface of intestinal epithelial cells. For this purpose, the S1 protein was expressed in COS-1 cells and its glycosylation modified using N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of ER-located alpha-glucosidases I and II, and or 1-deoxymannojirimycin (dMM), an inhibitor of the Golgi-located alpha-mannosidase I. The intracellular and secreted S1 proteins were analyzed by endoglycosidase H treatment. Similarly, ACE2 trafficking to the brush border membrane of intestinal Caco-2 cells was also assessed in the presence or absence of the inhibitors. Finally, the interaction between the S1 protein and ACE2 was investigated at the surface of Caco-2 cells by co-immunoprecipitation. Our data show that NB-DNJ significantly reduced the secretion of S1 proteins in COS-1 cells, while dMM affected S1 secretion to a lesser extent. Moreover, NB-DNJ and dMM differentially affected ACE2 trafficking and sorting to the brush border membrane of intestinal Caco-2 cells. Strikingly, the interaction between S1 and ACE2 was significantly reduced when both proteins were processed by the glycosylation inhibitors, rendering glycosylation and its inhibitors potential candidates for SARS-CoV-2 treatment. This work has been supported by a grant from the German Research Foundation (DFG) grant NA331/15-1 to HYN. M.K. was supported by a scholarship from the Hannover Graduate School for Veterinary Pathobiology, Neuroinfectiology, and Translational Medicine (HGNI) and by the DFG grant NA331/15-1.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Assessing the Cholinergic Anti-Inflammatory Response in Human Macrophages Exposed to the Spike Protein of the SARS-CoV-2 Strain from Wuhan, China

To date, the severe acute respiratory syndrome coronavirus 2 that causes the disease Coronavirus 2019, has infected 601 million people, claiming the lives of 6.4 million people worldwide. Of the patients who survive, 60% suffer from inflammatory problems leading to post-acute sequelae of COVID-19 (PASC). Inflammation in these patients is marked by an increase in pro-inflammatory cytokines which ultimately damage the body's organs, contributing to PASC. Understanding the main mechanism by which this cytokine storm occurs is of utmost importance in order to develop therapeutic strategies for counteracting inflammation in people suffering from COVID-19 and PASC. This project seeks to find out if an innate anti-inflammatory mechanism, the cholinergic anti-inflammatory response (CAR), works properly in patients suffering from COVID-19 and PASC by interrogating its functioning in its cellular substrate, macrophages.We hypothesized that disruption of the CAR in primary human monocytederived macrophages (MDMs) exposed to the SARS-CoV-2 spike protein trimer contributes to the chronic inflammation/ cytokine storm exhibited in these patients. To this end, we exposed MDMs to the SARS-CoV-2 spike protein in order to assess levels of the anti-inflammatory alpha-7 nicotinic acetylcholine receptor (alpha7-nAChR) by means of confocal imaging. Our results demonstrate a statistically significant reduction (P <= 0.01) of alpha7-nAChR expression in MDMs, in a time-dependent manner, after the addition of SARS-CoV-2 spike protein concentrations (30 nM and 100 nM), at different time points. Interestingly, when the receptor employed by the virus to infect, Angiotensin-converting enzyme 2 (ACE-2), was blocked, we detected a significant reduction in the levels of alpha7- nAChRs (P <= 0.001). Collectively, our results support the hypothesis of this work given that the SARS-CoV-2 spike protein is capable of compromising the functioning of the CAR by reducing the levels of alpha7-nAChRs available in macrophages to suppress inflammation. These results could position the alpha7- nAChR as a key target for the development of novel anti-inflammatory therapeutic strategies to counteract the inflammatory problem found in patients suffering from COVID-19 and PASC. We would like to acknowledge Dr. Negin Martin & Dr. Jerrel Yakel, for providing the Purified Spike Protein expressed by SARS-CoV-2 and Pseudotyped Virions in this collaborative study. Also, these experiments are being supported by the University of Puerto Rico - Rio Piedras NIH-RISE program. RISE Grant Number: 5R25GM061151-20.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

BROAD ANTIBODIES TO FUNCTIONALLY CONSTRAINED REGIONS OF SARS-CoV-2 SPIKE

Background: While remarkable and rapid progress was made in fighting the SARS-CoV-2 pandemic with vaccines and therapeutic antibodies, these approaches were quickly compromised by viral evolution. Therapeutic monoclonal antibodies (mAbs) that were once authorized for clinical use, which all target the receptor binding domain (RBD), are no longer effective against current variants of concern (VOCs) due to mutations in this region of Spike. Thus, to achieve durable protection against SARS-CoV-2, novel mAbs need to show breadth and potency across VOCs and target epitopes that are more constrained. Method(s): mAbs from an individual who had a breakthrough Delta VOC infection after vaccination were isolated from Spike-specific memory B cells. mAbs were assessed for binding affinity and neutralization potency using Spike-pseudotyped lentivirus (PSV) and live SARS-CoV-2 virus neutralization assays. Epitopes were mapped using deep mutational sequencing (DMS) and structural-based methods. Result(s): Three novel mAbs (C68.3, C68.13, C68.59) demonstrated binding breadth to Spikes from various VOCs including Omicron VOCs despite that C68 had not yet been exposed to Omicron. These mAbs potently neutralized the Wuhan-Hu-1 vaccine and Delta strains (IC50 = 9-61ng/mL), and early Omicron strains BA.1, BA.2, BA.5 (IC50 = 12-149 ng/mL). C68.3 and C68.59 retained potency against recent VOCs BQ.1.1 and XBB (IC50 = 121-122 ng/mL and 56-82 ng/mL, respectively) in the PSV assay. Similar neutralization activity was observed in the live virus assay. The potency of these mAbs was greater against Omicron VOCs than all but one of the mAbs previously authorized for treatment and they showed greater breadth. The mAbs target distinct epitopes on the Spike glycoprotein, two in the RBD (C68.3, C68.13) and one in an invariant region downstream of RBD in subdomain 1 (SD1) (C68.59). Structural analysis of C68.59 Fab binding to Spike trimer revealed significant allosteric changes to regions of Spike outside of the epitope in the S2 unit. Finally, DMS escape pathways showed these mAbs target regions highly conserved across VOCs that are also functionally constrained, suggesting escape could incur a fitness cost. Conclusion(s): Overall, these mAbs are novel in their breadth across VOCs and include a potent mAb targeting a rare epitope outside of the RBD in SD1. These mAbs focus on diverse, functionally constrained regions in Spike making them candidates for development as combination therapeutics with good durability against future VOCs.

Synthesis of the Transmembrane Domain of the Spike Protein from SARS-CoV-2 Using Solid Phase Peptide Synthesis and Determination of Its Oligomerization State

The spike protein in severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is directly responsible for the binding to ACE2 receptors in host cells. While the spike protein overall is known to form trimers, the oligomerization state of the transmembrane domain of the spike protein in SARS-CoV-2 is unknown. It is believed to be essential for the function of this protein. Since the transmembrane domain of the spike protein is highly conserved in SARS-CoV-2 it is important to investigate its character and determine its relationship to the function of the protein as awhole. The goal of this project was to synthesize, characterize, and analyze the function of the transmembrane domain (TM) of the spike protein in SARS-CoV-2. The most practical method to synthesize the TM domain of the S protein is through solid phase peptide synthesis (SPPS). SPPS is a process in which peptides are made by linking amino acids, the monomers of proteins, one at a time until the full sequence is achieved. These peptide chains will then need to be purified using high-performance liquid chromatography (HPLC). The synthesized peptides will be analyzed using liquid chromatography- mass spectrometry (LCMS) to confirm the identity of the synthesized peptides as well as any potential impurities. The continued investigation of the S protein can lead to the discovery of small peptides capable of inhibiting key processes to the binding mechanism of SARS-CoV-2. The function of the S protein is believed to only present when the transmembrane domain forms a trimer. Therefore, the analysis of their oligomerization states will be investigated by synthesizing versions of the peptide that fluoresce when excited using dyes such as nitrobenzodiazole (NBD) and tetramethylrhodamine (TAMRA) in a fluorescence assay. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Cyclin-dependent kinase 4/6 inhibitor in advanced breast cancer during the COVID pandemic period: efficacy in relation to vaccination for SARS-COV 19

Introduction: More than 2 years after the WHO declaration of a pandemic, SARS-Cov-2 still represents a public health problem The pandemic has increased the complexity of cancer treatments including breast cancer. These difficulties were highlighted in adjuvant treatments but above all in metastatic disease. Vaccination has been one of the most important public health factors that has reduced deaths, hospitalizations and the severity of symptoms related to infection. In metastatic breast cancer hormone receptor positive and HER2/neu negative currently the first line of treatment is given by the association between cyclin 4/6 inhibitors and hormone therapy (aromatase inhibitors or fulvestrant) A well-known and frequent side effect of this therapy is the reduction of white blood cell values and neutrophils. The hypothesis that this study is to evaluate whether treatment with cyclin inhibitors initiated before the period of vaccinations may have influenced, due to the reduction in white blood cell values, an increased risk of infection in these patients. Material(s) and Method(s): In our study, we selected patients who had started treatment with cyclin inhibitors before starting the vaccination cycle (in Italy up to the fourth dose in cancer patients) and continue it without evidence of disease progression. All patients were offered a vaccination cycle with mRNA COVID vaccines and were followed during their cancer treatments. All patients, at least 90 days after the last dose of vaccine, have been tested for antibodies against SARS CoV-2 (trimeric spike protein) with a value expressed in binding antibodies unit (BAU) according to international standard WHO During the observation period (starting from the first dose of vaccine administered) the patients were clinically checked and in case of suspicion of infectious pathology with symptoms suggestive of SARS-COV-19 infection, they were tested with molecular swab Results: We evaluated 52 patients who started cyclin treatment before the vaccination course and who are currently without signs of disease recurrence During the study period we found 14 SARSCOV19 infections (28% of patients) and one patient with two infectious episodes. No patients needed treatment in a hospital or resuscitation setting. All patients have fully recovered from the infection and at most after 21 days have resumed the treatment still in place Statistically, a linear regression calculation was applied to evaluate a functional relationship between variables measured on the basis of sample data. We did not find a relationship between spikes or infections compared to the start date of the vaccination cycle;instead we observed a relationship between the value of the spike and the date of last immunization (considered as an active infection or fourth dose of vaccine) with a reduction in the values the further you go away Conclusion(s): The data of the study show that there is a correlation between the time elapsed between the last vaccination and the risk of getting sick. For this reason, the fourth recall represents a strong help to reduce this risk. We did not find any ranges we could refer to regarding the dosage of trimeric spike protein. Considering the positivity rate of infections that does not exceed the general vaccinated population and the absence of serious infectious symptoms with hospitalization, treatment with cyclin inhibitors appears to be a safe treatment even in a pandemic period. (Table Presented).

Correlation between post-vaccination titres of anti-Spike antibodies and protection against breakthrough SARSCoV-2 infection in 7530 UK adults

Background: A robust correlate of vaccine-induced protection against SARS-CoV-2 infection has yet to be found. Aim(s): To explore whether post-vaccination combined IgG, IgA, and IgM responses to the SARS-CoV-2 trimeric spike glycoprotein (anti-S IgGAM) can predict protection against breakthrough SARS-CoV-2 infection. Method(s): In this prospective population-based study, we used dried blood spots to determine post-vaccination anti-S IgGAM responses in SARS-CoV-2-vaccinated UK adults. Using receiver operating characteristic (ROC) curve analysis, we assessed the ability of anti-S IgGAM titres (adjusted for days since vaccination) to predict postvaccination incident SARS-CoV-2 infection. After adjusting for household and behavioural factors reflecting risk of SARS-CoV-2 exposure, we compared the area under the ROC curve (AUROC) between minimally and fully adjusted models. Finding(s): Between Jan 12, 2021, and Jan 31, 2022, 300 (4.0%) of 7530 participants reported a breakthrough SARS-CoV-2 infection during 18 weeks of follow-up (220 [4.4%] ChAdOx1 nCoV-19 [ChadOx1] recipients and 75 [3.1%] BNT162b2 recipients). Anti-S IgGAM titres were modestly predictive of breakthrough infection (overall: AUROC 0.582 [95% CI 0.550-0.614];ChAdOx1: 0.564 [0.526-0.602];BNT162b2: 0.562 [0.488-0.636]). Adjustment for exposure factors significantly improved discrimination (overall: 0.666 [0.633-0.699], p<0.0001;ChAdOx1: 0.656 [0.617-0.695], p<0.0001;BNT162b1: 0.709 [0.649-0.769], p=0.0012). Conclusion(s): Anti-S IgGAM titres correlate with protection against SARS-CoV-2 infection in vaccinated adults, but exposure factors contribute significantly to risk.

DISTURBED CELLULAR IMMUNITY FOLLOWING MRNA VACCINATION AGAINST COVID-19 in PATIENTS with B-CELL DEPLETING THERAPY

Background: Immunocompromised patients are considered high-risk and prioritized for vaccination against COVID-19 (1). Furthermore, vaccination-induced CD4 and CD8 T-cell responses have been suggested to have a protective role in COVID-19 (2). If T-cell responses are diminished after vaccination in immuno-compromised individuals is not known to date. Objectives: To investigate cellular immunity following mRNA vaccination against COVID-19 in healthy individuals and patients undergoing B-cell depletion therapy. Methods: In this interim analysis of the CoVVac study (NCT04858607), we analyzed T-cell responses in autoimmune patients treated with B-cell depleting therapy (BD, n=41) and age-matched healthy controls (HCs, n=50) 3-4 weeks after the second dose of mRNA vaccination against COVID-19. Therefore, we isolated PBMCs and stimulated them with a peptide pool covering the spike protein in vitro. Reactive CD4 and CD8 T-cells were determined by staining for IFNg, TNFa, IL-2 and GzmB by fow cytometry. Anti-SARS-CoV-2 antibody assays targeting the receptor-binding domain (RBD) or trimeric S protein (TSP) were performed to elucidate concomitant B-cell responses. Results: We observed signifcant alterations in anti-SARS-CoV-2 antibody responses in our cohort, the frequency of IFNg+ and IL-2+ CD4 and CD8 T-cells was similar in BD patients and controls. On the other hand, TNFa+ CD4 T-cells were signifcantly enriched in healthy controls versus BD patients (p=0.017) and correlated signifcantly with antibody titres (p=0.003). Similarly, GzmB+ CD8 T-cells were signifcantly diminished in our patient cohort (p<0.001) and also showed a signifcant correlation with antibody titres (p<0.001). Overall, the frequency of GzmB+ CD8 T-cells correlated very well with reactivity of T-cell subsets for other cytokines. This effect, however, is lost in the BD cohort. No difference was observed in the frequency of TNFa+ CD8 T-cells between the groups. Only 21 (42%) healthy individuals and 14 (34%) patients showed reactive T-cells for all the cytokines tested. This observation is mainly explained by a lack of cytokine production of CD8 T-cells in 26 (52%) HCs and 27 (66%) BD patients. In turn, 22 (44%) HCs and 17 (42%) patients didn't show any IL-2 producing CD8 cells. Of note, only 2 (4%) of HCs showed no GzmB+ CD8 T-cells whereas the number increased to 15 (37%) of BD individuals (p<0.001). In contrast, 42 (84%) HCs as well as 32 (78%) of patients showed production of all IFNg, TNFa and IL-2 in CD4 T-cells. Conclusion: Our data suggest that most patients with B-cell depleting therapy are able to mount T-cell responses similar to those of healthy individuals while a minority of these patients did not show complete immunity against SARS CoV-2. Further analyses are needed to better understand a possible link of B-cell depletion therapy and CD8 T-cell responses.

SEROLOGICAL RESPONSE FOLLOWING THREE DOSES of SARS-COV-2 MRNA VACCINE in PATIENTS with GIANT CELL ARTERITIS: EXPERIENCE of A SINGLE-CENTRE COHORT

Background: The spread of COVID-19 pandemic raised the need to perform an additional vaccine dose to overcome the diffusion of the infection and possible life-threatening disease complications. Certain population subsets seem to be at increased risk of developing such complications, such as elderly and/or immu-nocompromised patients. Objectives: To assess the persistence of immunity following SARS-CoV-2 mRNA vaccine and the magnitude of the humoral response after the booster dose in a cohort of patients affected by giant cell arteritis (GCA). Methods: Patients with GCA regularly followed at the Rheumatology Department of the University of Pavia, Italy, who received a booster dose of SARS-CoV-2 mRNA vaccine (BNT162b2 Pfizer/BioNtech or mRNA-1273 Moderna) between October 1st and December 31st, 2021 were included. Humoral response was assessed by measuring SARS-CoV-2 Trimeric S (TSAbs) and Neutralizing (NAbs) antibodies, with a cut-off of 33.8 Binding Antibody Units (BAU)/mL and 1:10 dilution, respectively. Blood samples from each patient were drawn at least 4 months after the second and three weeks after the third vaccine dose. Results: Forty-two patients who received the booster dose of SARS-CoV-2 mRNA vaccine were enrolled. Thirty (71.4%) were females, mean age 73.2±4.7 years, disease duration 58±38 months, 19 (45.2%) had large-vessel vasculitis. Thirty-two (76.2%) were on glucocorticoids (GCs) at a mean dose of 4.9±7.8 mg/day prednisone equivalent, with 7 (16.7%) receiving ≥7.5 mg/day. Eighteen (42.9%) were on methotrexate (MTX) (mean dose 14.2±3.5 mg/week) and 8 (19.0%) were treated with subcutaneous tocilizumab (TCZ) 162 mg/week. SARS-CoV-2 serology was tested prior to the third vaccine dose at an average of 5.4±0.4 months from the former vaccination scheduled: 37 (88.1%) retained TSAbs and 30 (71.4%) NAbs. The median TSAb titre was 134 BAU/mL (IQR 97-292). Four out of 5 patients (80.0%) without TSAbs and 7 out of 12 (58.3%) without NAbs were on both GCs and MTX. Moreover, those on GCs plus MTX had lower pre-third dose TSAb titres as compared to other treatment subgroups (Figure 1A). GC doses ≥7.5 mg/day prednisone equivalents seemed to blunt NAb levels along time: 28.6% patients on GCs ≥7.5 mg/day prednisone equivalents had negative NAbs before the third dose vs. 80.0% of those taking <7.5 mg/day (p=0.007) as well as lower NAb titres (Figure 1B). Data regarding antibody response after the booster dose were available for 35 patients (83.3%). Blood collection occurred at a median of 25 days (IQR 24-32) after the third vaccine dose. All patients developed TSAbs, even those who did not respond to the previous shots. The median TSAb titre rose to 2080 BAU/mL (IQR 2080-2080) (p<0.001), while the median NAb titre increased from 1:10 to 1:320 (p<0.001). One patient (2.9%) treated with prednisone 8.75 mg/day plus MTX 12.5 mg/week did not develop NAbs. NAb levels were lower in patients taking MTX as compared to those who did not (Figure 1C,D), whereas treatment with TCZ or GCs, along with the GC dose, did not affect the magnitude of the antibody response. There were no serious adverse events from the vaccine. However, 3 patients (8.6%) experienced a disease relapse 24±5 days after the booster dose. Conclusion: In our cohort, most patients who seroconverted after the second dose of vaccine retained the humoral immunity, with excellent serocon-version rates following the booster dose. However, GCs, especially at doses ≥7.5 mg/day prednisone equivalents, may contribute to the waning of NAb titres. On the other hand, immunosuppressants like MTX, especially when combined with GCs, might impair the magnitude of the humoral response to the booster dose.

EVALUATION of the EFFICACY and SAFETY of the SARS-COV-2 VACCINE in PATIENTS with CHRONIC INFLAMMATORY DISEASES TREATED with BIOLOGICAL THERAPY

Background: In the current situation of the SARS-CoV-2 pandemic, the Spanish Society of Rheumatology recommends vaccination of patients with chronic infammatory diseases (CID) under treatment with biological DMARDs (bDMARDs). However, the data regarding the generation of protective antibody titers after mRNA vaccines in patients with CID is limited. Objectives: To determine the seroconversion rate and safety after the SARS-CoV-2 vaccine in patients with CID under treatment with bDMARDs Methods: Cross-sectional observational study of 81 patients with CID from the HURS in Córdoba, who have received full vaccination for SARS-CoV-2 (without having previously suffered from COVID-19 disease) according to national guidelines. A determination of specifc IgG-type antibodies against the trimeric spike protein of SARS-CoV2 was performed on all of them. The chemilumines-cence technique with the kit was used in serum samples taken 4-5 weeks after administration of the second dose of the vaccine. Information about sociode-mographic characteristics, disease, type of bDMARDs, concomitant treatments and adverse effects after the second dose of the vaccine were collected in each patient. Results: 81 patients were included (mean age 59.5, 72.8% females). 50.6% of patients had RA, 17.3% SpA, 11% PsoA and 18.5% other CID. 23.5% were under treatment with Rituximab, 38.8% antiTNF, 13.6% Tocilizumab, 9.9% abatacept, 5% anti-JAK and 14.2% under other treatments. Anti-SARS-CoV-2 antibodies and neutralizing activity were detected in 80% of study participants. Rituximab treatment was signifcantly associated with negative seroconver-sion in comparison with patients under antiTNF treatment (OR 84.0 (95%CI 12.9-1709.2)). No interaction was found between the bDMARDs treatment and the type of vaccine with regard to the seroconversion, nor between bDMARDs and concomitant synthetic DMARD. When we evaluated IgG titers against the spike protein of SARS-CoV2, we found that patients under treatment with Rituximab showed the lowest titers levels in comparison with patients with other treatments (Figure 1, Table 1). In addition, patients who received AstraZeneca vaccine developed lower titers of antibodies in comparison with patients who received Pfzer (Table 1). Interestingly, among patients with antiTNF treatment, AstraZeneca was associated with lower IgG titers in comparison with Pfzer and Moderna [405.9 (553.0) vs. 1084.1 (791.2) vs. 1264.0 (1012.6), p=0.016, respectively]. No differences between vaccines were found in patients with the other type of bDMARDs. Only 18.9% presented mild adverse effects. No serious adverse effects were observed and no patient experienced a disease fare after vaccination. Conclusion: Our results show that SARS-CoV-2 mRNA vaccines produce sero-conversion in most patients with CID, except in the case of patients with rituxi-mab. No severe adverse effects or CID reactivation were found. Despite the small number of patients included, this study suggests the need for revaccination in the group of patients treated with rituximab or vaccinated with Astrazeneca.

ANTI-TNF THERAPY for IMMUNE MEDIATED INFLAMMATORY DISEASES MAY BE ASSOCIATED with LOWER ANTIBODY LEVELS and VIRUS NEUTRALIZATION EFFICACY FOLLOWING SARS-COV-2 MRNA VACCINATION

Background: The impact of immunosuppressants on COVID-19 vaccination response and durability in patients with immune-mediated infammatory diseases (IMID) is yet to be fully characterized. Humoral response may be attenuated in these patients especially those on B cell depleting therapy and higher doses of corticosteroids, but data regarding other immunosuppressants are scarce. Objectives: We aimed to investigate antibody and T cell responses and durability to SARS-CoV-2 mRNA vaccines (BNT162b and/or mRNA 1273) in IMID patients on immunomodulatory maintenance therapy other than B-cell depleting therapy and corticosteroids. Methods: This prospective observational cohort study examined the immuno-genicity of SARS-CoV-2 mRNA vaccines in adult patients with IMIDs (psoriatic arthritis, psoriasis, infammatory bowel disease and rheumatoid arthritis) with or without maintenance immunosuppressive therapies (anti-TNF, methotrexate/azathioprine [MTX/AZA], anti-TNF + MTX/AZA, anti IL12/23, anti-IL-17, anti-IL23) compared to healthy controls. Automated ELISA for IgGs to spike trimer, spike receptor binding domain (RBD) and the nucleocapsid (NP) and T-cell release of 9 cytokines (IFNg, IL2, IL4, IL17A, TNF) and cytotoxic molecules (sFasL, GzmA, GzmB, Perforinin) in cell culture supernatants following stimulation with spike or NP peptide arrays were conducted at 4 time points: T1=pre vaccination, T2=me-dian 26 days after dose 1, T3=median 16 days after dose 2 and T4=median 106 days after dose 2. Neutralization assays against four SARS-CoV-2 variants (wild type, delta, beta and gamma) were conducted at T3. Results: We followed 150 subjects: 26 healthy controls and 124 IMID patients: 9 untreated, 44 on anti-TNF, 16 on anti-TNF with MTX/AZA, 10 on anti-IL23, 28 on anti-IL12/23, 9 on anti-IL17, 8 on MTX/AZA (Table 1). Most patients mounted antibody and T cell responses with increases from dose 1 to dose 2 (100% sero-conversion at T3) and some decline by T4, with variability within groups. Antibody levels and neutralization efficacy was lower in anti-TNFgroups (anti-TNF, anti-TNF + MTX/AZA) compared to controls and waned by T4 (Figure 1). T cell responses were not consistently different between groups. Pooled data showed a higher antibody response to mRNA-1273 compared to BNT162b. Conclusion: Following 2 doses of mRNA vaccination there is 100% seroconver-sion in IMID patients on maintenance therapy. Antibody levels and neutralization efficacy in anti-TNF group are lower than controls, and wane substantially by 3 months after dose 2. These fndings highlight the need for third dose in patients undergoing treatment with anti-TNF therapy and continued monitoring of immunity in these patient groups, taking into consideration newer variants and additional vaccine doses.

SEROLOGICAL RESPONSE to BNT162B2 MRNA ANTI-SARS-COV-2 VACCINATION in PATIENTS with INFLAMMATORY RHEUMATIC DISEASES: RESULTS from the RHEUVAX COHORT

Background: To date, globally considered, the literature suggests that AIRD may be at higher risk of infection and death due to COVID19 compared to the general population. Vaccination against SARS-CoV-2 reduces the risk of hospitalization and mortality. However, immunological alteration associated with Autoimmune Infam-matory Rheumatic Diseases (AIRD) and immunosuppressive medications may impair the response to vaccination. Emerging data suggest that immunosuppres-sive treatment may negatively impact the response to anti-SARS-CoV-2 vaccines in the AIRD population;data are robust for some treatments, more controversial for others. Identifying patients at higher risk of lack of protection is essential for shielding them and for adapting therapeutic protocol and vaccination timing. Objectives: In the light of the current COVID19 epidemic and the availability of effective vaccines, this study aims to identify predictors of non-response to anti-SARS-CoV-2 vaccines in patients affected by AIRD. Methods: An observational cross-sectional study was conducted evaluating the serological response and the persistence of antibodies at eight weeks in IRD patient cohort and non-IRD control. IRD and age and sex-matched controls volunteer among the health professionals (CTRL) who underwent vaccination with two doses of BNT162b2 were recruited for this study. Anti-Trimeric Spike protein antibodies were assayed eight ± one week after the second vaccine dose. Uni-variate and logistic regression analyses were performed to identify predictors of non-response and low antibody titers. Results: Samples were obtained from 237 IRD patients (m/f 73/164, mean age 5 7, CI 95% [56-59]): 4 autoinfammatory diseases (AI), 62 connective tissue diseases (CTD), 86 rheumatoid arthritis (RA), 71 spondylarthritis (SpA) and 14 vasculitis (Vsc). 232 CTRL were recruited (m/f 71/161, mean age 5 7, CI 95% [56-58]). Globally, IRD had a lower seroconversion rate (88.6% vs 99.6%, CI 95% OR [1.61-5.73], p<0.0001) and lower antibody titer compared to controls (median (IQR) 403 (131.5-1012) vs 1160 (702.5-1675), p<0.0001). After logistic regression, age, corticosteroid (CCS), Abatacept (ABA), and Mycophenolate Mofetil (MMF) use were predictors of non-response. The antibody titers eight weeks after the second dose of vaccine were lower in AIRD compared to controls, median (IQR) 403 (131.5-1012) vs 1160 (702.5-1675), p<0.0001 with no difference between sexes and age groups. CTD, RA and SpA had lower antibodies levels. However, the logistic regression model identifed treatment with MMF, ABA, CCS, Methotrexate (MTX), Rituximab (RTX), Janus Kinase inhibitors (JAKi) and TNF inhibitors (TNFi) as independent predictors of serum titer. ABA, RTX, MMF, and MTX had the strongest effect size. Conclusion: The response to anti-SARS-CoV-2 vaccines is often impaired in AIRD patients under treatment and may pose them at higher risk of severe COVID-19. Although this work focused on serological response, most of the treatment the impaired vaccine response are known to act on T cells, possibly also influencing the cellular response. Evidence-based protocols are required to time vaccination and treatment to improve immunization of AIRD patients.

SARS-CoV-2 vaccination in liver transplant recipients: factors affecting immune response and refusal to vaccine

Background and aims: The effectiveness of SARS-CoV-2 vaccination in liver transplant (LT) recipients varies between 47.5% to 81% with majority of reports focusing on the immune response assessed in the first month after the vaccination. Data on LT recipients willingness to receive vaccine is limited to only a few reports. Here, we analysed the immune response to the SARS-CoV-2 vaccination, factors affecting response and reasons for refusal to receive this vaccine. Method: Among 300 consecutive LT recipients, 225 (75%) were vaccinated. Seventy-four (25%) subjects were not vaccinated, including 45 (15%) who refused to be vaccinated and 29 (10%) who did not get the vaccine due to medical reasons. The humoral response was assessed by quantitative determination of anti-trimeric spikeprotein- specific-IgG antibodies to SARS-CoV-2 by LIAISON® SARSCoV- 2 TrimericS IgG assay (Diasorin, Italy), which is a chemiluminescence immunoassay (CLIA). Thirty-four vaccinated patients with prior COVID-19 infection were analysed separately. Results: Among 192 LT recipients vaccinated without prior COVID-19, 69% of them had an immune response (median time of 125 days after the second dose). Older age, worse kidney function and dual immunosuppression negatively affected the humoral response. Mycophenolate mofetil increased the risk of non-response (OR 3.0, 95% CI 1.43–6.25). LT recipients with prior COVID-19 presented with a robust immune response (100%) and with significantly higher IgG antibodies (median 2080 vs 134 BAU/ml;p <0.001). The antibodies concentrationwas higher in the first 90 days fromthe second dose (p = 0.034) and stabile when compared between patients who received the vaccination within 90–150 or more than 150 days (Figure 1). Female gender, living in rural area, lower BMI (all p < 0.05) and younger age (p < 0.001) were associated with refusal of the vaccine due to non-medical reasons. In contrast, liver recipients with diabetes and impaired kidney function (both p <0.01)were more prone to get a vaccine. (Figure Presented) Figure: Median SARS-CoV-2 TrimericS IgG concentration among liver transplant recipients (without prior COVID-19) compared between time from the second dose of the vaccine: <90 days, 90–150 days and >150 days. Conclusion: Lower immune response after the vaccine among LT recipients may support administration of a third dose. Previous COVID-19 infection dramatically improves response to vaccination in these patients. Sociodemographic factors may play a role in refusal of being vaccinated but this finding require further investigations in other cohorts of transplanted patients.

SARS-CoV-2 VARIANTS INCREASE KINETIC STABILITY of OPEN SPIKE CONFORMATIONS

Background: SARS-CoV-2 variants of concern harbor mutations in the Spike (S) glycoprotein that confer more efficient transmission and dampen the efficacy of COVID-19 vaccines and antibody therapies. S mediates virus entry and is the primary target for antibody responses, with structural studies of soluble S variants revealing an increased propensity towards conformations accessible to the human Angiotensin-Converting Enzyme 2 (hACE2) receptor. However, real-time observations of conformational dynamics that govern the structural equilibriums of the S variants have been lacking. Methods: Here, we report single-molecule Förster Resonance Energy Transfer (smFRET) studies of S variants of concern containing critical mutations, including D614G and E484K, in the context of virus particles. Results: Investigated variants were shown by smFRET to predominantly occupy more open hACE2-accessible conformations, agreeing with predictions from structures of soluble trimers. Additionally, S variants exhibited decelerated transitions from hACE2-accessible/bound states. Conclusion: Here, we provide the real-time dimension to distinct structures of Spikes in the context of virus particles and present the first experimental evidence of increased stability of Spike variants. Our finding of increased S kinetic stability in the open conformation provides a new perspective on SARS-CoV-2 adaptation to the human population.

STRUCTURAL CHARACTERIZATION of DNA-ENCODED HIV VACCINES INDUCED NEUTRALIZING ANTIBODY

Background: Rapid and large-scale deployment of COVID-19 mRNA vaccines highlights the potential utility of developing nucleic acid vaccines (such as RNA and DNA vaccines) against infectious diseases, including HIV-1. However, as compared to SARS-CoV-2, HIV-1 pose some unique challenges-induction of neutralizing antibodies (NAbs) against HIV-1 (frequently a correlate of protection) requires presentation of trimeric and highly conformational epitopes to the immune system, and whether nucleic acid vaccines can enable direct in vivo production of antigens that retain critical antigenic profile has not yet been elucidated. Additionally, it was previously reported that Tier 2 NAbs cannot be induced in mice due to a lack of antibody repertoire, and vaccine studies were suggested to be performed in larger mammals such as rabbits/NHPs, inadvertently slowing down and increasing the costs of preclinical HIV-1 vaccine studies. Methods: In our study, we used the Antigen Conformation Tracing In Vivo by ELISA (ACTIVE) assay developed in house to characterize antigenic profiles of vaccines produced in vivo (from transfected muscle tissues). We analyzed induced cellular responses, using stimulation with overlapping peptides followed by intracellular cytokine staining and IFN-g ELIspot assays. We analyzed induced humoral responses by using both binding ELISA assays and TZM-BL based neutralizing assays, and attempted to map induced NAb epitopes by engineering selectively mutated pseudovirus. We performed antigen-specific B-cell sorting, and used the 10x genomics pipeline to characterize antibody sequences of proliferating B-cell clones. Results: We confirmed that in vivo produced vaccines retained key trimeric conformational epitopes and glycan profiles. Compared to protein vaccination, DNA vaccination uniquely and strongly induced both TFH, CD4+, CD8+ T-cell responses, and Tier 2 NAbs mapped to a previously unreported Env C3/V5 epitope. 5 unique NAbs were isolated, and confirmed to bind to the epitope using a Cryo-EM structure of NAb-MD39 complex at 3.8Å resolution. Conclusion: Our study confirmed that with appropriate vaccine delivery technology, murine models can be appropriately used for HIV-1 vaccine studies aimed at generating NAb responses. In addition, beyond potential functional immunity gains, DNA vaccines permit in vivo folding of structured antigens and provide significant cost and speed advantages for enabling rapid evaluation of new HIV vaccines.