DC/L-SIGN recognition of spike glycoprotein promotes SARS-CoV-2 trans-infection and can be inhibited by a glycomimetic antagonist.

The efficient spread of SARS-CoV-2 resulted in a unique pandemic in modern history. Despite early identification of ACE2 as the receptor for viral spike protein, much remains to be understood about the molecular events behind viral dissemination. We evaluated the contribution of C-type lectin receptors (CLRS) of antigen-presenting cells, widely present in respiratory mucosa and lung tissue. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. Using pseudovirus and cells derived from monocytes or T-lymphocytes, we demonstrate that while virus capture by the CLRs examined does not allow direct cell infection, DC/L-SIGN, among these receptors, promote virus transfer to permissive ACE2+ Vero E6 cells. A glycomimetic compound designed against DC-SIGN, enable inhibition of this process. These data have been then confirmed using authentic SARS-CoV-2 virus and human respiratory cell lines. Thus, we described a mechanism potentiating viral spreading of infection.

Superior neutralizing response after first versus second SARS-CoV-2 infection in fully vaccinated individuals.

Currently, the majority of the population has been vaccinated against COVID-19 and/or has experienced SARS-CoV-2 infection either before or after vaccination. The immunological response to repeated episodes of infections is not completely clear. We measured SARS-CoV-2 specific neutralization titers by a pseudovirus assay after BA.1 infection and RBD-specific immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) in a cohort of COVID-19 uninfected and triple vaccinated individuals (breakthrough infection group, BTI) as compared with those previously infected by SARS-CoV-2 (reinfection group, REI) who underwent identical vaccination schedule. SARS-CoV-2 specific neutralizing response after BA.1 infection was significantly higher in the BTI group as compared with the REI. Furthermore, neutralization titers in REI were not significant different from convalescent non reinfected controls. RBD-specific IgG and IgA, but not IgM, were also significantly higher in BTI as compared with REI. Our results show that the first episode of SARS-CoV-2 infection induces a significant increase in neutralizing titers in triple vaccinated individuals and that previous SARS-CoV-2 infection compromise significantly the neutralization response induced by reinfection, even by divergent SARS-CoV-2 variants and at least up to 2 years postinfection, suggesting a fundamental limitation in inducing effective booster through the intranasal route in previously infected individuals.

Cross neutralization of SARS-CoV-2 omicron subvariants after repeated doses of COVID-19 mRNA vaccines.

We have measured the humoral response to messenger RNA (mRNA) vaccines in COVID-19 naïve and convalescent individuals. Third doses of mRNA COVID-19 vaccines induced a significant increase in potency and breadth of neutralization against SARS-CoV-2 variants of concern (VoC) including Omicron subvariants BA.1, BA.2, and BA.2.12.1, that were cross-neutralized at comparable levels and less for BA.4/5. This booster effect was especially important in naïve individuals that only after the third dose achieved a level that was comparable with that of vaccinated COVID-19 convalescents except for BA.4/5. Avidity of RBD-binding antibodies was also significantly increased in naïve individuals after the third dose, indicating an association between affinity maturation and cross neutralization of VoC. These results suggest that at least three antigenic stimuli by infection or vaccination with ancestral SARS-CoV-2 sequences are required to induce high avidity cross-neutralizing antibodies. Nevertheless, the circulation of new subvariants such as BA.4/5 with partial resistance to neutralization will have to be closely monitored and eventually consider for future vaccine developments.

Potent Neutralizing Activity of Polyclonal Equine Antibodies Against Severe Acute Respiratory Syndrome Coronavirus 2 Variants of Concern.

Several anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monoclonal antibodies (mAbs) have received emergency authorization for coronavirus disease 2019 (COVID-19) treatment. However, most of these mAbs are not active against the highly mutated Omicron SARS-CoV-2 subvariants. We have tested a polyclonal approach of equine anti-SARS-CoV-2 F(ab')2 antibodies that achieved a high level of neutralizing potency against all SARS-CoV-2 variants of concern tested including Omicron BA.1, BA.2, BA.2.12 and BA.4/5. A repertoire of antibodies targeting conserved epitopes in different regions of the spike protein could plausibly account for this remarkable breadth of neutralization. These results warrant the clinical investigation of equine polyclonal F(ab')2 antibodies as a novel therapeutic strategy against COVID-19.

Neutralizing Response Against SARS-CoV-2 Variants 8 Months After BNT162b2 Vaccination in Naive and COVID-19-Convalescent Individuals.

We have investigated the evolution of the neutralizing response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants at 8 months after Pfizer-BNT162b2 vaccination in coronavirus disease 2019 (COVID-19)-naive (n = 21) and COVID-19-convalescent (n = 21) individuals. Neutralizing levels declined for all variants (range 2- to 3.7-fold). Eight months after vaccination, a significant proportion (4/21) of naive individuals lacked detectable neutralizing activity against the highly transmissible SARS-CoV-2 delta variant. In the convalescent group, the impressive high initial humoral response resulted in detectable neutralizing antibody levels against all variants throughout this period.

Survey of Crimean-Congo Hemorrhagic Fever Enzootic Focus, Spain, 2011-2015.

During 2011-2015, we conducted a Crimean-Congo hemorrhagic fever virus (CCHFV) survey in captured ticks that were feeding mainly on wild and domestic ungulates in Spain, where presence of this virus had been reported previously. We detected CCHFV RNA in Hyalomma lusitanicum and H. marginatum ticks for 3 of the 5 years. The rate of infected ticks was 2.78% (44/1,579), which was similar to those for other countries in Europe with endemic foci for CCHFV (Kosovo, Bulgaria, and Albania). These data confirm the established spread of CCHFV into western Europe. Phylogenetic study of the small RNA segment showed Africa-3 clade as the only genotype identified, although we observed cocirculation of genetic variants during 2011 and 2015. We could not rule out genetic reassortments because of lack of sequence data for the medium and large RNA segments of the virus genome.

Unprecedented selectivity for homologous lectin targets: differential targeting of the viral receptors L-SIGN and DC-SIGN.

DC-SIGN (CD209) and L-SIGN (CD209L) are two C-type lectin receptors (CLRs) that facilitate SARS-CoV-2 infections as viral co-receptors. SARS-CoV-2 manipulates both DC-SIGN and L-SIGN for enhanced infection, leading to interest in developing receptor antagonists. Despite their structural similarity (82% sequence identity), they function differently. DC-SIGN, found in dendritic cells, shapes the immune response by recognizing pathogen-associated carbohydrate patterns. In contrast, L-SIGN, expressed in airway epithelial endothelial cells, is not directly involved in immunity. COVID-19's primary threat is the hyperactivation of the immune system, potentially reinforced if DC-SIGN engages with exogenous ligands. Therefore, L-SIGN, co-localized with ACE2-expressing cells in the respiratory tract, is a more suitable target for anti-adhesion therapy. However, designing a selective ligand for L-SIGN is challenging due to the high sequence identity of the Carbohydrate Recognition Domains (CRDs) of the two lectins. We here present Man84, a mannose ring modified with a methylene guanidine triazole at position 2. It binds L-SIGN with a K D of 12.7µM ± 1 µM (ITC) and is the first known L-SIGN selective ligand, showing 50-fold selectivity over DC-SIGN (SPR). The X-ray structure of the L-SIGN CRD/Man84 complex reveals the guanidinium group's role in achieving steric and electrostatic complementarity with L-SIGN. This allows us to trace the source of selectivity to a single amino acid difference between the two CRDs. NMR analysis confirms the binding mode in solution, highlighting Man84's conformational selection upon complex formation. Dimeric versions of Man84 achieve additional selectivity and avidity in the low nanomolar range. These compounds selectively inhibit L-SIGN dependent trans-infection by SARS-CoV-2 and Ebola virus. Man84 and its dimeric constructs display the best affinity and avidity reported to date for low-valency glycomimetics targeting CLRs. They are promising tools for competing with SARS-CoV-2 anchoring in the respiratory tract and have potential for other medical applications.

MVA-based vaccine candidates expressing SARS-CoV-2 prefusion-stabilized spike proteins of the Wuhan, Beta or Omicron BA.1 variants protect transgenic K18-hACE2 mice against Omicron infection and elicit robust and broad specific humoral and cellular immune responses.

Despite the decrease in mortality and morbidity due to SARS-CoV-2 infection, the incidence of infections due to Omicron subvariants of SARS-CoV-2 remains high. The mutations acquired by these subvariants, mainly concentrated in the receptor-binding domain (RBD), have caused a shift in infectivity and transmissibility, leading to a loss of effectiveness of the first authorized COVID-19 vaccines, among other reasons, by neutralizing antibody evasion. Hence, the generation of new vaccine candidates adapted to Omicron subvariants is of special interest in an effort to overcome this immune evasion. Here, an optimized COVID-19 vaccine candidate, termed MVA-S(3P_BA.1), was developed using a modified vaccinia virus Ankara (MVA) vector expressing a full-length prefusion-stabilized SARS-CoV-2 spike (S) protein from the Omicron BA.1 variant. The immunogenicity and efficacy induced by MVA-S(3P_BA.1) were evaluated in mice in a head-to-head comparison with the previously generated vaccine candidates MVA-S(3P) and MVA-S(3Pbeta), which express prefusion-stabilized S proteins from Wuhan strain and Beta variant, respectively, and with a bivalent vaccine candidate composed of a combination of MVA-S(3P) and MVA-S(3P_BA.1). The results showed that all four vaccine candidates elicited, after a single intramuscular dose, protection of transgenic K18-hACE2 mice challenged with SARS-CoV-2 Omicron BA.1, reducing viral loads, histopathological lesions, and levels of proinflammatory cytokines in the lungs. They also elicited anti-S IgG and neutralizing antibodies against various Omicron subvariants, with MVA-S(3P_BA.1) and the bivalent vaccine candidate inducing higher titers. Additionally, an intranasal immunization in C57BL/6 mice with all four vaccine candidates induced systemic and mucosal S-specific CD4+ and CD8+ T-cell and humoral immune responses, and the bivalent vaccine candidate induced broader immune responses, eliciting antibodies against the ancestral Wuhan strain and different Omicron subvariants. These results highlight the use of MVA as a potent and adaptable vaccine vector against new emerging SARS-CoV-2 variants, as well as the promising feature of combining multivalent MVA vaccine candidates.

The role of DC-SIGN as a trans-receptor in infection by MERS-CoV.

DC-SIGN is a C-type lectin expressed in myeloid cells such as immature dendritic cells and macrophages. Through glycan recognition in viral envelope glycoproteins, DC-SIGN has been shown to act as a receptor for a number of viral agents such as HIV, Ebola virus, SARS-CoV, and SARS-CoV-2. Using a system of Vesicular Stomatitis Virus pseudotyped with MERS-CoV spike protein, here, we show that DC-SIGN is partially responsible for MERS-CoV infection of dendritic cells and that DC-SIGN efficiently mediates trans-infection of MERS-CoV from dendritic cells to susceptible cells, indicating a potential role of DC-SIGN in MERS-CoV dissemination and pathogenesis.

TLR7 Activation in M-CSF-Dependent Monocyte-Derived Human Macrophages Potentiates Inflammatory Responses and Prompts Neutrophil Recruitment.

Toll-like receptor 7 (TLR7) is an endosomal pathogen-associated molecular pattern (PAMP) receptor that senses single-stranded RNA (ssRNA) and whose engagement results in the production of type I IFN and pro-inflammatory cytokines upon viral exposure. Recent genetic studies have established that a dysfunctional TLR7-initiated signaling is directly linked to the development of inflammatory responses. We present evidence that TLR7 is preferentially expressed by monocyte-derived macrophages generated in the presence of M-CSF (M-MØ). We now show that TLR7 activation in M-MØ triggers a weak MAPK, NFκB, and STAT1 activation and results in low production of type I IFN. Of note, TLR7 engagement reprograms MAFB+ M-MØ towards a pro-inflammatory transcriptional profile characterized by the expression of neutrophil-attracting chemokines (CXCL1-3, CXCL5, CXCL8), whose expression is dependent on the transcription factors MAFB and AhR. Moreover, TLR7-activated M-MØ display enhanced pro-inflammatory responses and a stronger production of neutrophil-attracting chemokines upon secondary stimulation. As aberrant TLR7 signaling and enhanced pulmonary neutrophil/lymphocyte ratio associate with impaired resolution of virus-induced inflammatory responses, these results suggest that targeting macrophage TLR7 might be a therapeutic strategy for viral infections where monocyte-derived macrophages exhibit a pathogenic role.

Optimized vaccine candidate MVA-S(3P) fully protects against SARS-CoV-2 infection in hamsters.

The development of novel optimized vaccines against coronavirus disease 2019 (COVID-19) that are capable of controlling the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and the appearance of different variants of concern (VoC) is needed to fully prevent the transmission of the virus. In the present study, we describe the enhanced immunogenicity and efficacy elicited in hamsters by a modified vaccinia virus Ankara (MVA) vector expressing a full-length prefusion-stabilized SARS-CoV-2 spike (S) protein [termed MVA-S(3P)]. Hamsters vaccinated with one or two doses of MVA-S(3P) developed high titers of S-binding IgG antibodies and neutralizing antibodies against the ancestral Wuhan SARS-CoV-2 virus and VoC beta, gamma, and delta, as well as against omicron, although with a somewhat lower neutralization activity. After SARS-CoV-2 challenge, vaccinated hamsters did not lose body weight as compared to matched placebo (MVA-WT) controls. Consistently, vaccinated hamsters exhibited significantly reduced viral RNA in the lungs and nasal washes, and no infectious virus was detected in the lungs in comparison to controls. Furthermore, almost no lung histopathology was detected in MVA-S(3P)-vaccinated hamsters, which also showed significantly reduced levels of proinflammatory cytokines in the lungs compared to unvaccinated hamsters. These results reinforce the use of MVA-S(3P) as a vaccine candidate against COVID-19 in clinical trials.

MAFB shapes human monocyte-derived macrophage response to SARS-CoV-2 and controls severe COVID-19 biomarker expression.

Monocyte-derived macrophages, the major source of pathogenic macrophages in COVID-19, are oppositely instructed by macrophage CSF (M-CSF) or granulocyte macrophage CSF (GM-CSF), which promote the generation of antiinflammatory/immunosuppressive MAFB+ (M-MØ) or proinflammatory macrophages (GM-MØ), respectively. The transcriptional profile of prevailing macrophage subsets in severe COVID-19 led us to hypothesize that MAFB shapes the transcriptome of pulmonary macrophages driving severe COVID-19 pathogenesis. We have now assessed the role of MAFB in the response of monocyte-derived macrophages to SARS-CoV-2 through genetic and pharmacological approaches, and we demonstrate that MAFB regulated the expression of the genes that define pulmonary pathogenic macrophages in severe COVID-19. Indeed, SARS-CoV-2 potentiated the expression of MAFB and MAFB-regulated genes in M-MØ and GM-MØ, where MAFB upregulated the expression of profibrotic and neutrophil-attracting factors. Thus, MAFB determines the transcriptome and functions of the monocyte-derived macrophage subsets that underlie pulmonary pathogenesis in severe COVID-19 and controls the expression of potentially useful biomarkers for COVID-19 severity.

Dendritic Cell-Mediated Cross-Priming by a Bispecific Neutralizing Antibody Boosts Cytotoxic T Cell Responses and Protects Mice against SARS-CoV-2.

Administration of neutralizing antibodies (nAbs) has proved to be effective by providing immediate protection against SARS-CoV-2. However, dual strategies combining virus neutralization and immune response stimulation to enhance specific cytotoxic T cell responses, such as dendritic cell (DC) cross-priming, represent a promising field but have not yet been explored. Here, a broadly nAb, TNT , are first generated by grafting an anti-RBD biparatopic tandem nanobody onto a trimerbody scaffold. Cryo-EM data show that the TNT structure allows simultaneous binding to all six RBD epitopes, demonstrating a high-avidity neutralizing interaction. Then, by C-terminal fusion of an anti-DNGR-1 scFv to TNT , the bispecific trimerbody TNT DNGR-1 is generated to target neutralized virions to type 1 conventional DCs (cDC1s) and promote T cell cross-priming. Therapeutic administration of TNT DNGR-1, but not TNT , protects K18-hACE2 mice from a lethal SARS-CoV-2 infection, boosting virus-specific humoral responses and CD8+ T cell responses. These results further strengthen the central role of interactions with immune cells in the virus-neutralizing antibody activity and demonstrate the therapeutic potential of the Fc-free strategy that can be used advantageously to provide both immediate and long-term protection against SARS-CoV-2 and other viral infections.

Cellular and humoral immune responses and breakthrough infections after three SARS-CoV-2 mRNA vaccine doses.

Background: SARS-CoV-2 vaccination has proven the most effective measure to control the COVID-19 pandemic. Booster doses are being administered with limited knowledge on their need and effect on immunity. Objective: To determine the duration of specific T cells, antibodies and neutralization after 2-dose vaccination, to assess the effect of a third dose on adaptive immunity and to explore correlates of protection against breakthrough infection. Methods: 12-month longitudinal assessment of SARS-CoV-2-specific T cells, IgG and neutralizing antibodies triggered by 2 BNT162b2 doses followed by a third mRNA-1273 dose in a cohort of 77 healthcare workers: 17 with SARS-CoV-2 infection prior to vaccination (recovered) and 60 naïve. Results: Peak levels of cellular and humoral response were achieved 2 weeks after the second dose. Antibodies declined thereafter while T cells reached a plateau 3 months after vaccination. The decline in neutralization was specially marked in naïve individuals and it was this group who benefited most from the third dose, which resulted in a 20.9-fold increase in neutralization. Overall, recovered individuals maintained higher levels of T cells, antibodies and neutralization 1 to 6 months post-vaccination than naïve. Seventeen asymptomatic or mild SARS-CoV-2 breakthrough infections were reported during follow-up, only in naïve individuals. This viral exposure boosted adaptive immunity. High peak levels of T cells and neutralizing antibodies 15 days post-vaccination associated with protection from breakthrough infections. Conclusion: Booster vaccination in naïve individuals and the inclusion of viral antigens other than spike in future vaccine formulations could be useful strategies to prevent SARS-CoV-2 breakthrough infections.

Prime-Boost Vaccination With BNT162b2 Induces High Neutralizing Activity Against SARS-CoV-2 Variants in Naïve and COVID-19-Convalescent Individuals.

BACKGROUND: The objective of this study was to investigate the neutralizing response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoC) during coronavirus disease 2019 (COVID-19) convalescence and after vaccination. METHODS: COVID-19-convalescent and -naïve individuals were tested for neutralizing activity against SARS-CoV-2 VoC Alpha, Beta, Gamma, and Delta at 1 and 7 months postinfection and 4-6 weeks after BNT162b2 vaccination. RESULTS: Vaccination induced a high neutralizing response in naïve individuals. Interestingly, vaccination of convalescent patients induced a boosted response that was able to neutralize all VoC at high titers. CONCLUSIONS: Vaccination with BNT162b2 induced high levels of neutralization against SARS-CoV-2 VoC in most patients; this is especially beneficial in COVID-19-convalescent individuals.

Identification of potential inhibitors of protein-protein interaction useful to fight against Ebola and other highly pathogenic viruses.

Despite the efforts to develop new treatments against Ebola virus (EBOV) there is currently no antiviral drug licensed to treat patients with Ebola virus disease (EVD). Therefore, there is still an urgent need to find new drugs to fight against EBOV. In order to do this, a virtual screening was done on the druggable interaction between the EBOV glycoprotein (GP) and the host receptor NPC1 with a subsequent selection of compounds for further validation. This screening led to the identification of new small organic molecules with potent inhibitory action against EBOV infection using lentiviral EBOV-GP-pseudotype viruses. Moreover, some of these compounds have shown their ability to interfere with the intracellular cholesterol transport receptor NPC1 using an ELISA-based assay. These preliminary results pave the way to hit to lead optimization programs that lead to successful candidates.

Real-time RT-PCR assay to detect Granada virus and the related Massilia and Arrabida phleboviruses.

BACKGROUND: Granada virus belongs to the genus Phlebovirus within the Naples serocomplex and was detected for the first time in sand flies from Spain in 2003. Seroprevalence studies have revealed that Granada virus may infect humans with most cases being asymptomatic. Moreover, recent studies in vector samples revealed that the related Massilia and Arrabida phleboviruses could be also circulating in Spain. The objective of this study was to develop and assess a new sensitive real-time RT-PCR assay for Granada virus diagnosis able to detect the related phleboviruses Massilia and Arrabida. METHODS: Two specific primers and one unique probe to detect Granada, Massilia and Arrabida viruses, without differentiating between them, were designed targeting the conserved L-segment of their genome. Sensitivity was assessed using 10-fold serial dilutions of quantified in vitro DNA samples. Specificity was evaluated by testing different genomic RNA extracted from other representative phleboviruses. The new assay was used for virus detection in sand flies collected in 2012 from the Balearic Archipelago, a touristic hotspot in the Mediterranean. RESULTS: The real-time RT-PCR assay exhibited a sensitivity per reaction of 19 copies for Granada and Arrabida, and 16 copies for Massilia. No other related phleboviruses were detected. From the 37 pools of sand fly samples studied from four different Balearic Islands, we detected one positive in the island of Cabrera. CONCLUSIONS: To our knowledge, the method described here is the first real-time RT-PCR designed to detect Granada virus and the related Massilia and Arrabida phleboviruses. The study demonstrated that this is a rapid, robust and reliable assay for the accurate diagnosis of human infections as well as for virus surveillance in vectors.