Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a worldwide public health concern for which no vaccine is available. Elucidation of the prefusion structure of the RSV F glycoprotein and its identification as the main target of neutralizing antibodies have provided new opportunities for development of an effective vaccine. Here, we describe the structure-based design of a self-assembling protein nanoparticle presenting a prefusion-stabilized variant of the F glycoprotein trimer (DS-Cav1) in a repetitive array on the nanoparticle exterior. The two-component nature of the nanoparticle scaffold enabled the production of highly ordered, monodisperse immunogens that display DS-Cav1 at controllable density. In mice and nonhuman primates, the full-valency nanoparticle immunogen displaying 20 DS-Cav1 trimers induced neutralizing antibody responses ∼10-fold higher than trimeric DS-Cav1. These results motivate continued development of this promising nanoparticle RSV vaccine candidate and establish computationally designed two-component nanoparticles as a robust and customizable platform for structure-based vaccine design.

An Unbiased Screen for Human Cytomegalovirus Identifies Neuropilin-2 as a Central Viral Receptor.

Characterizing cell surface receptors mediating viral infection is critical for understanding viral tropism and developing antiviral therapies. Nevertheless, due to challenges associated with detecting protein interactions on the cell surface, the host receptors of many human pathogens remain unknown. Here, we build a library consisting of most single transmembrane human receptors and implement a workflow for unbiased and high-sensitivity detection of receptor-ligand interactions. We apply this technology to elucidate the long-sought receptor of human cytomegalovirus (HCMV), the leading viral cause of congenital birth defects. We identify neuropilin-2 (Nrp2) as the receptor for HCMV-pentamer infection in epithelial/endothelial cells and uncover additional HCMV interactors. Using a combination of biochemistry, cell-based assays, and electron microscopy, we characterize the pentamer-Nrp2 interaction and determine the architecture of the pentamer-Nrp2 complex. This work represents an important approach to the study of host-pathogen interactions and provides a framework for understanding HCMV infection, neutralization, and the development of novel anti-HCMV therapies.

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

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

Limited access to antigen drives generation of early B cell memory while restraining the plasmablast response.

Cell fate decisions during early B cell activation determine the outcome of responses to pathogens and vaccines. We examined the early B cell response to T-dependent antigen in mice by single-cell RNA sequencing. Early after immunization, a homogeneous population of activated precursors (APs) gave rise to a transient wave of plasmablasts (PBs), followed a day later by the emergence of germinal center B cells (GCBCs). Most APs rapidly exited the cell cycle, giving rise to non-GC-derived early memory B cells (eMBCs) that retained an AP-like transcriptional profile. Rapid decline of antigen availability controlled these events; provision of excess antigen precluded cell cycle exit and induced a new wave of PBs. Fate mapping revealed a prominent contribution of eMBCs to the MBC pool. Quiescent cells with an MBC phenotype dominated the early response to immunization in primates. A reservoir of APs/eMBCs may enable rapid readjustment of the immune response when failure to contain a threat is manifested by increased antigen availability.

Active PD-L1 incorporation within HIV virions functionally impairs T follicular helper cells.

The limited development of broadly neutralizing antibodies (BnAbs) during HIV infection is classically attributed to an inadequate B-cell help brought by functionally impaired T follicular helper (Tfh) cells. However, the determinants of Tfh-cell functional impairment and the signals contributing to this condition remain elusive. In the present study, we showed that PD-L1 is incorporated within HIV virions through an active mechanism involving p17 HIV matrix protein. We subsequently showed that in vitro produced PD-L1high but not PD-L1low HIV virions, significantly reduced Tfh-cell proliferation and IL-21 production, ultimately leading to a decreased of IgG1 secretion from GC B cells. Interestingly, Tfh-cell functions were fully restored in presence of anti-PD-L1/2 blocking mAbs treatment, demonstrating that the incorporated PD-L1 proteins were functionally active. Taken together, the present study unveils an immunovirological mechanism by which HIV specifically exploits the regulatory potential of PD-L1 to suppress the immune system during the course of HIV infection.

Non-uptake of COVID-19 vaccines and reasons for non-uptake among healthcare workers in Uganda: a cross-sectional study.

BACKGROUND: Vaccines play a crucial role in eradicating and containing disease outbreaks. Therefore, understanding the reasons behind vaccine refusal and associated factors is essential for improving vaccine acceptance rates. Our objective was to examine the determinants of COVID-19 vaccine non-uptake and explore the reasons for non-uptake among healthcare workers (HCWs) in Uganda. METHODS: Between July and August 2021, we conducted a cross-sectional study among healthcare workers in primary healthcare facilities (private and government) in Entebbe Municipality, Uganda. Participants were recruited using convenience sampling, and consenting individuals received credentials to access an electronic database and complete a structured questionnaire. There were no established HCWs contact registers in the municipality, and the study was conducted during a national lock down, therefore, the HCWs who were on duty at the time of the study were approached. The survey questions were based on the '3Cs' model of vaccine hesitancy and focused on confidence, convenience, and complacency factors. Non-uptake of vaccines was defined as not having received any of the available vaccines in the country. We employed counts, percentages, and simple logit models to summarize the reasons for non-uptake of COVID-19 vaccines and to identify associated factors. RESULTS: The study recruited 360 HCWs, 61.7% of whom were female, with an average age of 31 years (SD = 7.9). Among them, 124 (34.4%) healthcare workers did not receive any COVID-19 vaccine. Non-uptake of COVID-19 vaccines was independently associated with several factors, including age [35 + years adjusted odds ratio (aOR) = 0.30, 95% CI: 0.13-0.66 compared with 18-24 years], facility ownership [government, aOR = 0.22 (0.10-0.49) compared with private not-for-profit], previous testing for coronavirus [yes, aOR = 0.35 (0.19-0.65)], and previous involvement in COVID-19 vaccine activities [yes, aOR = 0.17 (0.10-0.29)]. The primary reasons cited for non-uptake of COVID-19 vaccines were related to a lack of confidence in the vaccines, such as concerns about side effects (79.8%) and the need for more time to understand the vaccines (89.5%), as well as the importance of weighing benefits and risks (84.7%) before being vaccinated. A smaller proportion, approximately 23%, cited reasons related to complacency and lack of convenience in accessing vaccination services. CONCLUSION: The high proportion of non-uptake of COVID-19 vaccines among this population primarily stems from a lack of confidence and trust in the vaccines, coupled with insufficient time allowed for users to make informed decisions. This underscores the urgent need for ongoing monitoring and trend analysis of vaccine non-uptake to guide the development and implementation of strategies aimed at building and sustaining vaccine confidence. Adequate time should be allowed to explain benefits of vaccination to the population to allay fears that might exist before actual vaccination is rolled out.

Rationally designed Human Cytomegalovirus gB nanoparticle vaccine with improved immunogenicity.

Human cytomegalovirus (HCMV) is the primary viral cause of congenital birth defects and causes significant morbidity and mortality in immune-suppressed transplant recipients. Despite considerable efforts in vaccine development, HCMV infection still represents an unmet clinical need. In recent phase II trials, a MF59-adjuvanted gB vaccine showed only modest efficacy in preventing infection. These findings might be attributed to low level of antibodies (Abs) with a neutralizing activity induced by this vaccine. Here, we analyzed the immunogenicity of each gB antigenic domain (AD) and demonstrated that domain I of gB (AD5) is the main target of HCMV neutralizing antibodies. Furthermore, we designed, characterized and evaluated immunogenic responses to two different nanoparticles displaying a trimeric AD5 antigen. We showed that mice immunization with nanoparticles induces sera neutralization titers up to 100-fold higher compared to those obtained with the gB extracellular domain (gBECD). Collectively, these results illustrate with a medically relevant example the advantages of using a general approach combining antigen discovery, protein engineering and scaffold presentation for modern development of subunit vaccines against complex pathogens.

AncesTree: An interactive immunoglobulin lineage tree visualizer.

High-throughput sequencing of human immunoglobulin genes allows analysis of antibody repertoires and the reconstruction of clonal lineage evolution. The study of antibodies (Abs) affinity maturation is of specific interest to understand the generation of Abs with high affinity or broadly neutralizing activities. Moreover, phylogenic analysis enables the identification of the key somatic mutations required to achieve optimal antigen binding. The Immcantation framework provides a start-to-finish set of analytical methods for high-throughput adaptive immune receptor repertoire sequencing (AIRR-Seq; Rep-Seq) data. Furthermore, Immcantation's Change-O package has developed IgPhyML, an algorithm designed to build specifically immunoglobulin (Ig) phylogenic trees. Meanwhile Phylip, an algorithm that has been originally developed for applications in ecology and macroevolution, can also be used for the phylogenic reconstruction of antibodies maturation pathway. To complement Ig lineages made by IgPhyML or Dnaml (Phylip), we developed AncesTree, a graphic user interface (GUI) that aims to give researchers the opportunity to interactively explore antibodies clonal evolution. AncesTree displays interactive immunoglobulins phylogenic tree, Ig related mutations and sequence alignments using additional information coming from specialized antibody tools. The GUI is a Java standalone application allowing interaction with Ig tree that can run under Windows, Linux and Mac OS.

Mitochondrial dysfunction induced by a SH2 domain-targeting STAT3 inhibitor leads to metabolic synthetic lethality in cancer cells.

In addition to its canonical role in nuclear transcription, signal transducer and activator of transcription 3 (STAT3) is emerging as an important regulator of mitochondrial function. Here, we demonstrate that a novel inhibitor that binds with high affinity to the STAT3 SH2 domain triggers a complex cascade of events initiated by interference with mitochondrial STAT3 (mSTAT3). The mSTAT3-drug interaction leads to mitochondrial dysfunction, accumulation of proteotoxic STAT3 aggregates, and cell death. The cytotoxic effects depend directly on the drug's ability to interfere with mSTAT3 and mitochondrial function, as demonstrated by site-directed mutagenesis and use of STAT3 knockout and mitochondria-depleted cells. Importantly, the lethal consequences of mSTAT3 inhibition are enhanced by glucose starvation and by increased reliance of cancer cells and tumor-initiating cells on mitochondria, resulting in potent activity in cell cultures and tumor xenografts in mice. These findings can be exploited for eliciting synthetic lethality in metabolically stressed cancer cells using high-affinity STAT3 inhibitors. Thus, this study provides insights on the role of mSTAT3 in cancer cells and a conceptual framework for developing more effective cancer therapies.

Cross-neutralization of four paramyxoviruses by a human monoclonal antibody.

Broadly neutralizing antibodies reactive against most and even all variants of the same viral species have been described for influenza and HIV-1 (ref. 1). However, whether a neutralizing antibody could have the breadth of range to target different viral species was unknown. Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are common pathogens that cause severe disease in premature newborns, hospitalized children and immune-compromised patients, and play a role in asthma exacerbations. Although antisera generated against either HRSV or HMPV are not cross-neutralizing, we speculated that, because of the repeated exposure to these viruses, cross-neutralizing antibodies may be selected in some individuals. Here we describe a human monoclonal antibody (MPE8) that potently cross-neutralizes HRSV and HMPV as well as two animal paramyxoviruses: bovine RSV (BRSV) and pneumonia virus of mice (PVM). In its germline configuration, MPE8 is HRSV-specific and its breadth is achieved by somatic mutations in the light chain variable region. MPE8 did not result in the selection of viral escape mutants that evaded antibody targeting and showed potent prophylactic efficacy in animal models of HRSV and HMPV infection, as well as prophylactic and therapeutic efficacy in the more relevant model of lethal PVM infection. The core epitope of MPE8 was mapped on two highly conserved anti-parallel ß-strands on the pre-fusion viral F protein, which are rearranged in the post-fusion F protein conformation. Twenty-six out of the thirty HRSV-specific neutralizing antibodies isolated were also found to be specific for the pre-fusion F protein. Taken together, these results indicate that MPE8 might be used for the prophylaxis and therapy of severe HRSV and HMPV infections and identify the pre-fusion F protein as a candidate HRSV vaccine.

Human cytomegalovirus (HCMV)-specific T cell but not neutralizing or IgG binding antibody responses to glycoprotein complexes gB, gHgLgO, and pUL128L correlate with protection against high HCMV viral load reactivation in solid-organ transplant recipients.

Immune correlates of protection against human cytomegalovirus (HCMV) infection are still debated. This study aimed to investigate which arm of the immune response plays a major role in protection against HCMV infection in kidney transplant recipients (n = 40) and heart transplant recipients (n = 12). Overall, patients were divided into 2 groups: one including 37 patients with low viral load (LVL), and the other including 15 patients with high viral load (HVL). All LVL patients resolved the infection spontaneously, whereas HVL patients were all treated with one or more courses of antivirals. In HVL patients, viral DNAemia, which was more than 100 times higher than LVL, appeared and peaked at significantly earlier times, but disappeared much later than in LVL patients. During a 1-year follow-up, all LVL patients had levels of HCMV-specific CD4+ (and CD8+ ) T cells significantly higher than HVL patients. On the contrary, titers of neutralizing antibodies and enzyme-linked immunosorbent assay-IgG antibodies to gB, gHgLgO, and pentamer gHgLpUL128L were overlapping in the 2 patient groups. In conclusion, while a valid HCMV-specific T-cell response was detected in more than 90% of LVL patients, >90% of HVL patients lacked an adequate T-cell response. Antibody responses did not appear to be associated directly or indirectly with protection.

Monoclonal Antibodies to Different Components of the Human Cytomegalovirus (HCMV) Pentamer gH/gL/pUL128L and Trimer gH/gL/gO as well as Antibodies Elicited during Primary HCMV Infection Prevent Epithelial Cell Syncytium Formation.

UNLABELLED: Human cytomegalovirus (HCMV) may cause disseminated/end-organ disease in congenitally infected newborns and immunosuppressed transplant recipients. Two glycoprotein complexes, gH/gL/gO and gH/gL/pUL128/pUL130/pUL131 (gH/gL/pUL128L; referred to as the pentamer), are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively, in the presence of the viral fusion protein gB. In addition, gH/gL/gO was recently reported to also be required for infection of endothelial/epithelial cells. Virus entry into human fibroblasts involves fusion of the virus envelope with the plasma membrane, whereas entry into endothelial/epithelial cells involves macropinocytosis or endocytosis and low-pH-dependent fusion with endosomes. A large set of neutralizing monoclonal antibodies (MAbs), directed to gH, gB, and multiple components of the pentamer, was developed. In addition, novel anti-gO human monoclonal antibodies were recently isolated. It is known that epithelial cell infection with a wild HCMV strain at a high multiplicity of infection produces a large number of syncytia. Incubation of heavily HCMV VR1814-infected ARPE-19 epithelial cells with neutralizing MAbs to one, two, or three components of the pUL128L portion of the pentamer blocked syncytium formation at an antibody concentration of 10 µg/ml, whereas only a partial inhibitory effect was displayed for MAbs to gO, gH, or gB at the same concentration. A blocking effect was also exhibited by convalescent-phase sera from primary HCMV infections. These findings indicate that the pentamer is required for syncytium formation in epithelial cells. IMPORTANCE: Human cytomegalovirus (HCMV) mostly infects epithelial and endothelial cells in vivo Recently, the pentamer protein complex (gH/gL/pUL128L) was identified as being required for infection of these cells, in association with the other protein complex, gH/gL/gO. In primary infections, HCMV migrates to endothelial cells and then to leukocytes, which disseminate the infection throughout the body. The virus then spreads to organs and tissues, mostly infecting either single cells or multinucleated epithelial giant cells (syncytia), depending on the viral load. Potent neutralizing human MAbs directed to distinct binding sites of the pUL128L portion of the pentamer were shown in the past to block virus dissemination. In the present study, MAbs to pUL128L were shown to block syncytium formation with a higher potency than that of MAbs to gO, gH, or gB, thus suggesting their role in limiting virus dissemination. This finding provides additional information useful for the development of anti-HCMV therapeutic antibodies and subunit vaccines.

Antibody-driven design of a human cytomegalovirus gHgLpUL128L subunit vaccine that selectively elicits potent neutralizing antibodies.

The use of neutralizing antibodies to identify the most effective antigen has been proposed as a strategy to design vaccines capable of eliciting protective B-cell immunity. In this study, we analyzed the human antibody response to cytomegalovirus (human cytomegalovirus, HCMV) infection and found that antibodies to glycoprotein (g)B, a surface glycoprotein that has been developed as a HCMV vaccine, were primarily nonneutralizing. In contrast, most of the antibodies to the complex formed by gH, gL, protein (p)UL128, pUL130, and pUL131 (the gHgLpUL128L pentamer) neutralized HCMV infection with high potency. Based on this analysis, we developed a single polycistronic vector encoding the five pentamer genes separated by "self-cleaving" 2A peptides to generate a stably transfected CHO cell line constitutively secreting high levels of recombinant pentamer that displayed the functional antigenic sites targeted by human neutralizing antibodies. Immunization of mice with the pentamer formulated with different adjuvants elicited HCMV neutralizing antibody titers that persisted to high levels over time and that were a hundred- to thousand-fold higher than those found in individuals that recovered from primary HCMV infection. Sera from mice immunized with the pentamer vaccine neutralized infection of both epithelial cells and fibroblasts and prevented cell-to-cell spread and viral dissemination from endothelial cells to leukocytes. Neutralizing monoclonal antibodies from immunized mice showed the same potency as human antibodies and targeted the same as well as additional sites on the pentamer. These results illustrate with a relevant example a general and practical approach of analytic vaccinology for the development of subunit vaccines against complex pathogens.

The C-terminal domain of TPX2 is made of alpha-helical tandem repeats.

BACKGROUND: TPX2 (Targeting Protein for Xklp2) is essential for spindle assembly, activation of the mitotic kinase Aurora A and for triggering microtubule nucleation. Homologs of TPX2 in Chordata and plants were previously identified. Currently, proteins of the TPX2 family have little structural information and only small parts are covered by defined protein domains. METHODS: We have used computational sequence analyses and structural predictions of proteins of the TPX2 family, supported with Circular Dichroism (CD) measurements. RESULTS: Here, we report our finding that the C-terminal domain of TPX2, which is responsible of its microtubule nucleation capacity and is conserved in all members of the family, is actually formed by tandem repeats, covering well above 2/3 of the protein. We propose that this region forms a flexible solenoid involved in protein-protein interactions. Structural prediction and molecular modeling, combined with Circular Dichroism (CD) measurements reveal a predominant alpha-helical content. Furthermore, we identify full length homologs in fungi and shorter homologs with a different domain organization in diptera (including a paralogous expansion in Drosophila). CONCLUSIONS: Our results, represent the first computational and biophysical analysis of the TPX2 proteins family and help understand the structure and evolution of this conserved protein family to direct future structural studies.

RAIN: a Machine Learning-based identification for HIV-1 bNAbs.

Broadly neutralizing antibodies (bNAbs) are promising candidates for the treatment and prevention of HIV-1 infection. Despite their critical importance, automatic detection of HIV-1 bNAbs from immune repertoire is still lacking. Here, we developed a straightforward computational method for Rapid Automatic Identification of bNAbs (RAIN) based on Machine Learning methods. In contrast to other approaches using one-hot encoding amino acid sequences or structural alignment for prediction, RAIN uses a combination of selected sequence-based features for accurate prediction of HIV-1 bNAbs. We demonstrate the performance of our approach on non-biased, experimentally obtained sequenced BCR repertoires from HIV-1 immune donors. RAIN processing leads to the successful identification of novel HIV-1 bNAbs targeting the CD4-binding site of the envelope glycoprotein. In addition, we validate the identified bNAbs using in vitro neutralization assay and we solve the structure of one of them in complex with the soluble native-like heterotrimeric envelope glycoprotein by single-particle cryo-electron microscopy (cryo-EM). Overall, we propose a method to facilitate and accelerate HIV-1 bNAbs discovery from non-selected immune repertoires.

Therapeutic antibodies and alternative formats against SARS-CoV-2.

The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) heavily burdened the entire world. Despite a prompt generation of vaccines and therapeutics to confront infection, the virus remains a threat. The ancestor viral strain has evolved into several variants of concern, with the Omicron variant now having many distinct sublineages. Consequently, most available antibodies targeting the spike went obsolete and thus new therapies or therapeutic formats are needed. In this review we focus on antibody targets, provide an overview of the therapeutic progress made so far, describe novel formats being explored, and lessons learned from therapeutic antibodies that can enhance pandemic preparedness.

High uptake of COVID-19 vaccines among healthcare workers in urban Uganda.

OBJECTIVES: The aim of the study was to describe the facilitators, barriers to and level of uptake of COVID-19 vaccines among healthcare workers in primary healthcare facilities in an urban setting in Uganda. MATERIALS AND METHODS: We conducted a cross-sectional study among HCWs in private and public health facilities in Entebbe municipality between July 2021 and August 2021. Data was collected using a structured questionnaire that was shared, via an online link, to consented participants. Uptake of the vaccines among healthcare workers was analysed as proportions, and logistic regression was used to analyse barriers and facilitators to uptake of COVID-19 vaccines. RESULTS: The study enrolled 360 participants, with 61.7% (n = 222) females. A total of 236 (65.6%) healthcare workers had received at least one dose of COVID-19 vaccine, with higher uptake among females 64% (n = 151). Age above 40 years (OR 2.16), working in a government healthcare facility (OR 3.12), participating in COVID-19 vaccine related activities (OR 4.62), and having tested for SARS-COV-2 (OR 3.05) increased the odds of having been vaccinated. Working in small roadside clinics reduced the odds of being vaccinated by almost 70%, while HCWs in government health services were 3.1 times more likely to have been vaccinated. History of having cared for a COVID-19 patient and having a positive SARS-COV-2 test result did not influence the uptake of the vaccines in the study population. CONCLUSION: Vaccine uptake among HCWs was close to the World Health Organisation (WHO) recommended uptake of 70% by mid-2022.

RAIN: machine learning-based identification for HIV-1 bNAbs.

Broadly neutralizing antibodies (bNAbs) are promising candidates for the treatment and prevention of HIV-1 infections. Despite their critical importance, automatic detection of HIV-1 bNAbs from immune repertoires is still lacking. Here, we develop a straightforward computational method for the Rapid Automatic Identification of bNAbs (RAIN) based on machine learning methods. In contrast to other approaches, which use one-hot encoding amino acid sequences or structural alignment for prediction, RAIN uses a combination of selected sequence-based features for the accurate prediction of HIV-1 bNAbs. We demonstrate the performance of our approach on non-biased, experimentally obtained and sequenced BCR repertoires from HIV-1 immune donors. RAIN processing leads to the successful identification of distinct HIV-1 bNAbs targeting the CD4-binding site of the envelope glycoprotein. In addition, we validate the identified bNAbs using an in vitro neutralization assay and we solve the structure of one of them in complex with the soluble native-like heterotrimeric envelope glycoprotein by single-particle cryo-electron microscopy (cryo-EM). Overall, we propose a method to facilitate and accelerate HIV-1 bNAbs discovery from non-selected immune repertoires.

Intradermal skin test with mRNA vaccines as a surrogate marker of T cell immunity in immunocompromised patients.

OBJECTIVES: Intradermal skin test (IDT) with mRNA vaccines may represent a simple, reliable, and affordable tool to measure T cell response in immunocompromised patients who failed to mount serological responses following vaccination with mRNA covid-19 vaccines. METHODS: We compared anti-SARS-CoV-2 antibodies and cellular responses in vaccinated immunocompromised patients (n = 58), healthy seronegative naive controls (NC, n = 8), and healthy seropositive vaccinated controls (VC, n = 32) by Luminex, spike-induced IFN-γ Elispot and an IDT. A skin biopsy 24 h after IDT and single-cell RNAseq was performed in three vaccinated volunteers. RESULTS: Twenty-five percent of seronegative NC had a positive Elispot (2/8) and IDT (1/4), compared to 95% (20/21) and 93% (28/30) in seropositive VC, respectively. Single-cell RNAseq data in the skin of VC showed a predominant mixed population of effector helper and cytotoxic T cells. The TCR repertoire revealed 18/1064 clonotypes with known specificities against SARS-CoV-2, among which six were spike-specific. Seronegative immunocompromised patients with positive Elispot and IDT were in 83% (5/6) treated with B cell-depleting reagents, while those with negative IDT were all transplant recipients. CONCLUSIONS: Our results indicate that delayed local reaction to IDT reflects vaccine-induced T-cell immunity opening new perspectives to monitor seronegative patients and elderly populations with waning immunity.

Broadly potent anti-SARS-CoV-2 antibody shares 93% of epitope with ACE2 and provides full protection in monkeys.

OBJECTIVES: Due to the rapid evolution of SARS-CoV-2 to variants with reduced sensitivity to vaccine-induced humoral immunity and the near complete loss of protective efficacy of licensed therapeutic monoclonal antibodies, we isolated a potent, broad-spectrum neutralizing antibody that could potentially provide prophylactic protection to immunocompromised patient populations. METHODS: Spike-specific B-cell clones isolated from a vaccinated post-infected donor were profiled for those producing potent neutralizing antibodies against a panel of SARS-CoV-2 variants. The P4J15 antibody was further characterized to define the structural binding epitope, viral resistance, and in vivo efficacy. RESULTS: The P4J15 mAb shows <20 ng/ml neutralizing activity against all variants including the latest XBB.2.3 and EG.5.1 sub-lineages. Structural studies of P4J15 in complex with Omicron XBB.1 Spike show that the P4J15 epitope shares ∼93% of its buried surface area with the ACE2 contact region, consistent with an ACE2 mimetic antibody. In vitro selection of SARS-CoV-2 mutants escaping P4J15 neutralization showed reduced infectivity, poor ACE2 binding, and mutations are rare in public sequence databases. Using a SARS-CoV-2 XBB.1.5 monkey challenge model, P4J15-LS confers complete prophylactic protection with an exceptionally long in vivo half-life of 43 days. CONCLUSIONS: The P4J15 mAb has potential as a broad-spectrum anti-SARS-CoV-2 drug for prophylactic protection of at-risk patient populations.