Production and Purification of Hantavirus Glycoproteins in Drosophila melanogaster S2 Cells.

Hantaviruses, are rodent-borne viruses found worldwide that are transmitted to humans through inhalation of contaminated excreta. They can cause a renal or a pulmonary syndrome, depending on the virus, and no effective treatment is currently available for either of these diseases. Hantaviral particles are covered by a protein lattice composed of two glycoproteins (Gn and Gc) that mediate adsorption to target cells and fusion with endosomal membranes, making them prime targets for neutralizing antibodies. Here we present the methodology to produce soluble recombinant glycoproteins in different conformations, either alone or as a stabilized Gn/Gc complex, using stably transfected Drosophila S2 cells.

A synthetic delivery vector for mucosal vaccination.

The success of mRNA-based vaccines during the Covid-19 pandemic has highlighted the value of this new platform for vaccine development against infectious disease. However, the CD8+ T cell response remains modest with mRNA vaccines, and these do not induce mucosal immunity, which would be needed to prevent viral spread in the healthy population. To address this drawback, we developed a dendritic cell targeting mucosal vaccination vector, the homopentameric STxB. Here, we describe the highly efficient chemical synthesis of the protein, and its in vitro folding. This straightforward preparation led to a synthetic delivery tool whose biophysical and intracellular trafficking characteristics were largely indistinguishable from recombinant STxB. The chemical approach allowed for the generation of new variants with bioorthogonal handles. Selected variants were chemically coupled to several types of antigens derived from the mucosal viruses SARS-CoV-2 and type 16 human papillomavirus. Upon intranasal administration in mice, mucosal immunity, including resident memory CD8+ T cells and IgA antibodies was induced against these antigens. Our study thereby identifies a novel synthetic antigen delivery tool for mucosal vaccination with an unmatched potential to respond to an urgent medical need.

Microbial Protein Binding to gC1qR Drives PLA2G1B-Induced CD4 T-Cell Anergy.

The origin of the impaired CD4 T-cell response and immunodeficiency of HIV-infected patients is still only partially understood. We recently demonstrated that PLA2G1B phospholipase synergizes with the HIV gp41 envelope protein in HIV viremic plasma to induce large abnormal membrane microdomains (aMMDs) that trap and inactivate physiological receptors, such as those for IL-7. However, the mechanism of regulation of PLA2G1B activity by the cofactor gp41 is not known. Here, we developed an assay to directly follow PLA2G1B enzymatic activity on CD4 T-cell membranes. We demonstrated that gp41 directly binds to PLA2G1B and increases PLA2G1B enzymatic activity on CD4 membrane. Furthermore, we show that the conserved 3S sequence of gp41, known to bind to the innate sensor gC1qR, increases PLA2G1B activity in a gC1qR-dependent manner using gC1qR KO cells. The critical role of the 3S motif and gC1qR in the inhibition of CD4 T-cell function by the PLA2G1B/cofactor system in HIV-infected patients led us to screen additional microbial proteins for 3S-like motifs and to study other proteins known to bind to the gC1qR to further investigate the role of the PLA2G1B/cofactor system in other infectious diseases and carcinogenesis. We have thus extended the PLA2G1B/cofactor system to HCV and Staphylococcus aureus infections and additional pathologies where microbial proteins with 3S-like motifs also increase PLA2G1B enzymatic activity. Notably, the bacteria Porphyromonas gingivalis, which is associated with pancreatic ductal adenocarcinoma (PDAC), encodes such a cofactor protein and increased PLA2G1B activity in PDAC patient plasma inhibits the CD4 response to IL-7. Our findings identify PLA2G1B/cofactor system as a CD4 T-cell inhibitor. It involves the gC1qR and disease-specific cofactors which are gC1qR-binding proteins that can contain 3S-like motifs. This mechanism involved in HIV-1 immunodeficiency could play a role in pancreatic cancer and several other diseases. These observations suggest that the PLA2G1B/cofactor system is a general CD4 T-cell inhibitor and pave the way for further studies to better understand the role of CD4 T-cell anergy in infectious diseases and tumor escape.

Structural flexibility of a conserved antigenic region in hepatitis C virus glycoprotein E2 recognized by broadly neutralizing antibodies.

UNLABELLED: Neutralizing antibodies (NAbs) targeting glycoprotein E2 are important for the control of hepatitis C virus (HCV) infection. One conserved antigenic site (amino acids 412 to 423) is disordered in the reported E2 structure, but a synthetic peptide mimicking this site forms a ß-hairpin in complex with three independent NAbs. Our structure of the same peptide in complex with NAb 3/11 demonstrates a strikingly different extended conformation. We also show that residues 412 to 423 are essential for virus entry but not for E2 folding. Together with the neutralizing capacity of the 3/11 Fab fragment, this indicates an unexpected structural flexibility within this epitope. NAbs 3/11 and AP33 (recognizing the extended and ß-hairpin conformations, respectively) display similar neutralizing activities despite converse binding kinetics. Our results suggest that HCV utilizes conformational flexibility as an immune evasion strategy, contributing to the limited immunogenicity of this epitope in patients, similar to the conformational flexibility described for other enveloped and nonenveloped viruses. IMPORTANCE: Approximately 180 million people worldwide are infected with hepatitis C virus (HCV), and neutralizing antibodies play an important role in controlling the replication of this major human pathogen. We show here that one of the most conserved antigenic sites within the major glycoprotein E2 (amino acids 412 to 423), which is disordered in the recently reported crystal structure of an E2 core fragment, can adopt different conformations in the context of the infectious virus particle. Recombinant Fab fragments recognizing different conformations of this antigenic site have similar neutralization activities in spite of converse kinetic binding parameters. Of note, an antibody response targeting this antigenic region is less frequent than those targeting other more immunogenic regions in E2. Our results suggest that the observed conformational flexibility in this conserved antigenic region contributes to the evasion of the humoral host immune response, facilitating chronicity and the viral spread of HCV within an infected individual.

Structural basis of HCV neutralization by human monoclonal antibodies resistant to viral neutralization escape.

The high mutation rate of hepatitis C virus allows it to rapidly evade the humoral immune response. However, certain epitopes in the envelope glycoproteins cannot vary without compromising virus viability. Antibodies targeting these epitopes are resistant to viral escape from neutralization and understanding their binding-mode is important for vaccine design. Human monoclonal antibodies HC84-1 and HC84-27 target conformational epitopes overlapping the CD81 receptor-binding site, formed by segments aa434-446 and aa610-619 within the major HCV glycoprotein E2. No neutralization escape was yet observed for these antibodies. We report here the crystal structures of their Fab fragments in complex with a synthetic peptide comprising aa434-446. The structures show that the peptide adopts an α-helical conformation with the main contact residues F44² and Y44³ forming a hydrophobic protrusion. The peptide retained its conformation in both complexes, independently of crystal packing, indicating that it reflects a surface feature of the folded glycoprotein that is exposed similarly on the virion. The same residues of E2 are also involved in interaction with CD81, suggesting that the cellular receptor binds the same surface feature and potential escape mutants critically compromise receptor binding. In summary, our results identify a critical structural motif at the E2 surface, which is essential for virus propagation and therefore represents an ideal candidate for structure-based immunogen design for vaccine development.

An alpaca nanobody inhibits hepatitis C virus entry and cell-to-cell transmission.

UNLABELLED: Severe liver disease caused by chronic hepatitis C virus is the major indication for liver transplantation. Despite recent advances in antiviral therapy, drug toxicity and unwanted side effects render effective treatment in liver-transplanted patients a challenging task. Virus-specific therapeutic antibodies are generally safe and well-tolerated, but their potential in preventing and treating hepatitis C virus (HCV) infection has not yet been realized due to a variety of issues, not least high production costs and virus variability. Heavy-chain antibodies or nanobodies, produced by camelids, represent an exciting antiviral approach; they can target novel highly conserved epitopes that are inaccessible to normal antibodies, and they are also easy to manipulate and produce. We isolated four distinct nanobodies from a phage-display library generated from an alpaca immunized with HCV E2 glycoprotein. One of them, nanobody D03, recognized a novel epitope overlapping with the epitopes of several broadly neutralizing human monoclonal antibodies. Its crystal structure revealed a long complementarity determining region (CD3) folding over part of the framework that, in conventional antibodies, forms the interface between heavy and light chain. D03 neutralized a panel of retroviral particles pseudotyped with HCV glycoproteins from six genotypes and authentic cell culture-derived particles by interfering with the E2-CD81 interaction. In contrast to some of the most broadly neutralizing human anti-E2 monoclonal antibodies, D03 efficiently inhibited HCV cell-to-cell transmission. CONCLUSION: This is the first description of a potent and broadly neutralizing HCV-specific nanobody representing a significant advance that will lead to future development of novel entry inhibitors for the treatment and prevention of HCV infection and help our understanding of HCV cell-to-cell transmission.

Vaccine vectors derived from a large collection of simian adenoviruses induce potent cellular immunity across multiple species.

Replication-defective adenovirus vectors based on human serotype 5 (Ad5) induce protective immune responses against diverse pathogens and cancer in animal models, as well as elicit robust and sustained cellular immunity in humans. However, most humans have neutralizing antibodies to Ad5, which can impair the immunological potency of such vaccines. Here, we show that rare serotypes of human adenoviruses, which should not be neutralized in most humans, are far less potent as vaccine vectors than Ad5 in mice and nonhuman primates, casting doubt on their potential efficacy in humans. To identify novel vaccine carriers suitable for vaccine delivery in humans, we isolated and sequenced more than 1000 adenovirus strains from chimpanzees (ChAd). Replication-defective vectors were generated from a subset of these ChAd serotypes and screened to determine whether they were neutralized by human sera and able to grow in human cell lines. We then ranked these ChAd vectors by immunological potency and found up to a thousandfold variation in potency for CD8+ T cell induction in mice. These ChAd vectors were safe and immunologically potent in phase 1 clinical trials, thereby validating our screening approach. These data suggest that the ChAd vectors developed here represent a large collection of non-cross-reactive, potent vectors that may be exploited for the development of new vaccines.

Isolation of Fully Human Antagonistic RON Antibodies Showing Efficient Block of Downstream Signaling and Cell Migration.

RON belongs to the c-MET family of receptor tyrosine kinases. As its well-known family member MET, RON and its ligand macrophage-stimulating protein have been implicated in the progression and metastasis of tumors and have been shown to be overexpressed in cancer. We generated and tested a large number of human monoclonal antibodies (mAbs) against human RON. Our screening yielded three high-affinity antibodies that efficiently block ligand-dependent intracellular AKT and MAPK signaling. This effect correlates with the strong reduction of ligand-activated migration of T47D breast cancer cell line. By cross-competition experiments, we showed that the antagonistic antibodies fall into three distinct epitope regions of the RON extracellular Sema domain. Notably, no inhibition of tumor growth was observed in different epithelial tumor xenografts in nude mice with any of the antibodies. These results suggest that distinct properties beside ligand antagonism are required for anti-RON mAbs to exert antitumor effects in vivo.

Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer.

The EphA2 receptor tyrosine kinase is overexpressed in a variety of human epithelial cancers and is a determinant of malignant cellular behavior in pancreatic adenocarcinoma cells. Moreover, it is expressed in tumor endothelium and its activation promotes angiogenesis. To better clarify the therapeutic potential of monoclonal antibodies (mAbs) directed to the EphA2 receptor, we generated a large number of mAbs by differential screening of phage-Ab libraries by oligonucleotide microarray technology and implemented a strategy for the rapid identification of antibodies with the desired properties. We selected two high-affinity and highly specific EphA2 monoclonal antibodies with different in vitro properties on the human pancreatic tumor cell line MiaPaCa2. One is a potent EphA2-agonistic antibody, IgG25, that promotes receptor endocytosis and subsequent degradation, and the second is a ligand antagonist, IgG28, that blocks the binding to ephrin A1 and is cross-reactive with the mouse EphA2 receptor. We measured the effect of antibody treatment on the growth of MiaPaCa2 cells orthotopically transplanted in nude mice. Both IgG25 and IgG28 had strong antitumor and antimetastatic efficacy. In vivo treatment with IgG25 determined the reduction of the EphA2 protein levels in the tumor and the phosphorylation of FAK on Tyr576 while administration of IgG28 caused a decrease in tumor vascularization as measured by immunohistochemical analysis of CD31 in tumor sections. These data show that in a pancreatic cancer model comparable therapeutic efficacy is obtained either by promoting receptor degradation or by blocking receptor activation.

Modulation of the immune response induced by gene electrotransfer of a hepatitis C virus DNA vaccine in nonhuman primates.

Induction of multispecific, functional CD4+ and CD8+ T cells is the immunological hallmark of acute self-limiting hepatitis C virus (HCV) infection in humans. In the present study, we showed that gene electrotransfer (GET) of a novel candidate DNA vaccine encoding an optimized version of the nonstructural region of HCV (from NS3 to NS5B) induced substantially more potent, broad, and long-lasting CD4+ and CD8+ cellular immunity than naked DNA injection in mice and in rhesus macaques as measured by a combination of assays, including IFN-gamma ELISPOT, intracellular cytokine staining, and cytotoxic T cell assays. A protocol based on three injections of DNA with GET induced a substantially higher CD4+ T cell response than an adenovirus 6-based viral vector encoding the same Ag. To better evaluate the immunological potency and probability of success of this vaccine, we have immunized two chimpanzees and have compared vaccine-induced cell-mediated immunity to that measured in acute self-limiting infection in humans. GET of the candidate HCV vaccine led to vigorous, multispecific IFN-gamma+CD8+ and CD4+ T lymphocyte responses in chimpanzees, which were comparable to those measured in five individuals that cleared spontaneously HCV infection. These data support the hypothesis that T cell responses elicited by the present strategy could be beneficial in prophylactic vaccine approaches against HCV.

A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees.

Three percent of the world's population is chronically infected with the hepatitis C virus (HCV) and at risk of developing liver cancer. Effective cellular immune responses are deemed essential for spontaneous resolution of acute hepatitis C and long-term protection. Here we describe a new T-cell HCV genetic vaccine capable of protecting chimpanzees from acute hepatitis induced by challenge with heterologous virus. Suppression of acute viremia in vaccinated chimpanzees occurred as a result of massive expansion of peripheral and intrahepatic HCV-specific CD8(+) T lymphocytes that cross-reacted with vaccine and virus epitopes. These findings show that it is possible to elicit effective immunity against heterologous HCV strains by stimulating only the cellular arm of the immune system, and suggest a path for new immunotherapy against highly variable human pathogens like HCV, HIV or malaria, which can evade humoral responses.

A novel adenovirus type 6 (Ad6)-based hepatitis C virus vector that overcomes preexisting anti-ad5 immunity and induces potent and broad cellular immune responses in rhesus macaques.

Success in resolving hepatitis C virus (HCV) infection has been correlated to vigorous, multispecific, and sustained CD8(+) T-cell response in humans and chimpanzees. The efficacy of inducing T-cell-mediated immunity by recombinant serotype 5 adenovirus vector has been proven in many animal models of infectious diseases, but its immunogenicity can be negatively influenced by preexisting immunity against the vector itself. To evaluate the less prevalent adenovirus serotype 6 (Ad6) as an alternative vector for and HCV vaccine development, we have generated serotype 5 and 6 adenoviral vectors directing expression of the nonstructural region of HCV (MRKAd5-NSmut and MRKAd6-NSmut). Immunogenicity studies in mice showed that the two vectors induced comparable T-cell responses but that only MRKAd6-NSmut was not suppressed in the presence of anti-Ad5 immunity. In contrast, preexisting anti-Ad5 immunity dramatically blunted the immunogenicity of the serotype 5-based HCV vector. Furthermore, MRKAd6-NSmut showed equivalent potency, breadth, and longevity of HCV-specific T-cell responses in rhesus macaques as the corresponding Ad5-based vector over a wide range of doses and was capable of boosting DNA-primed animals even if administered at low doses. These data support the use of the MRKAd6-NSmut for anti-HCV immunotherapy and, more generally, for the Ad6 serotype as a better genetic vaccine vehicle than Ad5.

Binding of the hepatitis C virus E2 glycoprotein to CD81 is strain specific and is modulated by a complex interplay between hypervariable regions 1 and 2.

The envelope glycoprotein E2 of hepatitis C virus (HCV) is the target of neutralizing antibodies and is presently being evaluated as an HCV vaccine candidate. HCV binds to human cells through the interaction of E2 with the tetraspanin CD81, a putative viral receptor component. We have analyzed four different E2 proteins from 1a and 1b viral isolates for their ability to bind to recombinant CD81 in vitro and to the native receptor displayed on the surface of Molt-4 cells. A substantial difference in binding efficiency between these E2 variants was observed, with proteins derived from 1b subtypes showing significantly lower binding than the 1a protein. To elucidate the mechanism of E2-CD81 interaction and to identify critical regions responsible for the different binding efficiencies of the E2 variants, several mutants were generated in E2 protein regions predicted by computer modeling to be exposed on the protein surface. Functional analysis of these E2 derivatives revealed that at least two distinct domains are responsible for interaction with CD81. A first segment centered around amino acid residues 613 to 618 is essential for recognition, while a second element including the two hypervariable regions (HVRs) modulates E2 receptor binding. Binding inhibition experiments with anti-HVR monoclonal antibodies confirmed this mapping and supported the hypothesis that a complex interplay between the two HVRs of E2 is responsible for modulating receptor binding, possibly through intramolecular interactions. Finally, E2 proteins from different isolates displayed a profile of binding to human hepatic cells different from that observed on Molt-4 cells or isolated recombinant CD81, indicating that additional factors are involved in viral recognition by target liver cells.