Dissecting the Heterogeneous Glycan Profiles of Recombinant Coronavirus Spike Proteins with Individual Ion Mass Spectrometry.

Surface-embedded glycoproteins, such as the spike protein trimers of coronaviruses MERS, SARS-CoV, and SARS-CoV-2, play a key role in viral function and are the target antigen for many vaccines. However, their significant glycan heterogeneity poses an analytical challenge. Here, we utilized individual ion mass spectrometry (I2MS), a multiplexed charge detection measurement with similarities to charge detection mass spectrometry (CDMS), in which a commercially available Orbitrap analyzer is used to directly produce mass profiles of these heterogeneous coronavirus spike protein trimers under native-like conditions. Analysis by I2MS shows that glycosylation contributes to the molecular mass of each protein trimer more significantly than expected by bottom-up techniques, highlighting the importance of obtaining complementary intact mass information when characterizing glycosylation of such heterogeneous proteins. Enzymatic dissection to remove sialic acid or N-linked glycans demonstrates that I2MS can be used to better understand the glycan profile from a native viewpoint. Deglycosylation of N-glycans followed by I2MS analysis indicates that the SARS-CoV-2 spike protein trimer contains glycans that are more difficult to remove than its MERS and SARS-CoV counterparts, and these differences are correlated with solvent accessibility. I2MS technology enables characterization of protein mass and intact glycan profile and is orthogonal to traditional mass analysis methods such as size exclusion chromatography-multiangle light scattering (SEC-MALS) and field flow fractionation-multiangle light scattering (FFF-MALS). An added advantage of I2MS is low sample use, requiring 100-fold less than other methodologies. This work highlights how I2MS technology can enable efficient development of vaccines and therapeutics for pharmaceutical development.

Discovery and multimerization of cross-reactive single-domain antibodies against SARS-like viruses to enhance potency and address emerging SARS-CoV-2 variants.

Coronaviruses have been the causative agent of three epidemics and pandemics in the past two decades, including the ongoing COVID-19 pandemic. A broadly-neutralizing coronavirus therapeutic is desirable not only to prevent and treat COVID-19, but also to provide protection for high-risk populations against future emergent coronaviruses. As all coronaviruses use spike proteins on the viral surface to enter the host cells, and these spike proteins share sequence and structural homology, we set out to discover cross-reactive biologic agents targeting the spike protein to block viral entry. Through llama immunization campaigns, we have identified single domain antibodies (VHHs) that are cross-reactive against multiple emergent coronaviruses (SARS-CoV, SARS-CoV-2, and MERS). Importantly, a number of these antibodies show sub-nanomolar potency towards all SARS-like viruses including emergent CoV-2 variants. We identified nine distinct epitopes on the spike protein targeted by these VHHs. Further, by engineering VHHs targeting distinct, conserved epitopes into multi-valent formats, we significantly enhanced their neutralization potencies compared to the corresponding VHH cocktails. We believe this approach is ideally suited to address both emerging SARS-CoV-2 variants during the current pandemic as well as potential future pandemics caused by SARS-like coronaviruses.

Profiling of hMPV F-specific antibodies isolated from human memory B cells.

Human metapneumovirus (hMPV) belongs to the Pneumoviridae family and is closely related to respiratory syncytial virus (RSV). The surface fusion (F) glycoprotein mediates viral fusion and is the primary target of neutralizing antibodies against hMPV. Here we report 113 hMPV-F specific monoclonal antibodies (mAbs) isolated from memory B cells of human donors. We characterize the antibodies' germline usage, epitopes, neutralization potencies, and binding specificities. We find that unlike RSV-F specific mAbs, antibody responses to hMPV F are less dominant against the apex of the antigen, and the majority of the potent neutralizing mAbs recognize epitopes on the side of hMPV F. Furthermore, neutralizing epitopes that differ from previously defined antigenic sites on RSV F are identified, and multiple binding modes of site V and II mAbs are discovered. Interestingly, mAbs that bind preferentially to the unprocessed prefusion F show poor neutralization potency. These results elucidate the immune recognition of hMPV infection and provide novel insights for future hMPV antibody and vaccine development.

A novel high throughput assay to quantify Epstein-Barr virus neutralizing antibody activity against B-cell and epithelial cell infections for vaccine and therapeutic developments.

Epstein-Barr Virus (EBV) is the causative agent of infectious mononucleosis and has been associated with a variety of malignancies. In vivo, EBV infects B cells and epithelial cells. However, the current EBV neutralization assays, especially those against B cell infection, are low throughput, laborious and lack of sensitivity. In this study, we optimized the EBV-GFP based micro-neutralization assay by selecting the most susceptible cell substrates, Akata 4E3 for B cell and HEK293T for epithelial cell. The newly developed procedure is high throughput. The cell type specific neutralization was confirmed using monoclonal antibodies specific to gp350 and gH/gL/gp42. A panel of human sera was also tested. Natural human EBV seropositive sera could neutralize EBV in both B cell and epithelial cell assays efficiently with a majority of human sera generating near 100% EBV neutralization. The EBV neutralizing antibody titers were highly correlated with antibodies specific to gp350, gH, EBV total proteins, and to a less degree with antibodies against gp42. Collectively, we demonstrated this improved neutralization assay is suitable to evaluating the humoral responses elicited by EBV vaccine candidates in preclinical animal models or in large-scale human trials.

Evaluation of HSV-2 gE Binding to IgG-Fc and Application for Vaccine Development.

Glycoprotein E (gE) and glycoprotein I (gI) are expressed as a heterodimer on the surface of Herpes simplex virus (HSV). Glycoprotein E binds Fc domain of immunoglobulin G (IgG) and inhibits activities mediated by the IgG Fc domain, contributing to immune evasion by HSV. It has been reported that HSV type 1 gE (gE-1) is capable of binding IgG Fc as a monomer and in a heterodimeric complex with gI, with the heterodimer having 50- to100-fold greater affinity for Fc than gE alone. We report the production of both a soluble form of HSV type 2 gE (gE-2) and a soluble HSV-2 gE/gI heterodimer (gE-2/gI-2). Characterization of soluble gE-2 by surface plasmon resonance (SPR) demonstrates that it is incapable of binding human IgG or the IgG Fc domain. Co-expression with HSV-2 gI (gI-2) and purification of the gE-2/gI-2 heterodimer enable gE-2 to bind human IgG through its Fc domain. We hypothesize that functional epitopes of wildtype gE-2 may be masked by plasma IgG Fc and affect the immunogenicity of the gE-2/gI-2 heterodimer as a vaccine antigen. A series of gE-2 mutations within the surface-exposed Fc:gE-2 interface was designed, and gE-2 mutants were co-expressed with gI-2. Evaluation of twelve gE-2 mutant heterodimers by SPR assay identified nine gE-2 mutations which abrogated or reduced Fc binding while maintaining heterodimer formation with gI. Vaccinating rabbits with the four most Fc-binding deficient gE-2/gI-2 heterodimers elicited comparable anti-heterodimer binding antibody titers and statistically significantly higher serum neutralization antibody levels than wildtype heterodimers. Taken together, these data support the concept of rational antigen design for improved vaccine candidates.

Development of a microneutralization assay for HSV-2.

BACKGROUND: Plaque Reduction Neutralization Test (PRNT) is the standard assay used for measuring neutralizing antibody responses to Herpes simplex virus type-2 (HSV-2). The PRNT is a cumbersome, time-consuming and laborious assay. The development of a faster, high throughput microneutralization assay (MNA) for HSV-2 viruses carried out in a 96-well format will allow for rapid testing of large numbers of samples for drug and vaccine development. METHODS: We describe the generation of a MNA that utilizes a pair of anti-HSV human monoclonal antibodies (mAbs) for virus detection in HSV-2 infected Vero cells. Antibodies were generated by B-cell cloning from PBMC's isolated from HSV-1 negative/HSV-2 positive donors. We describe the selection and characterization of the antibodies used for virus detection by ELISA with purified, recombinant anti-HSV glycoproteins, antibody binding in infected cells, and Western Blot. We determine the anti-HSV-2 neutralizing titers of immune sera from mice by MNA and PRNT and compare these results by linear regression analysis. RESULTS: We show that neutralization titers for HSV-2, determined by the 96-well MNA correlate with titers determined by a PRNT completed in 24-well plates in both the absence (R2 = 0.8250) and presence (R2 = 0.7075) of complement. CONCLUSIONS: We have successfully developed an MNA that can be used in place of the burdensome PRNT to determine anti-HSV-2 neutralizing activity in serum. This MNA has much greater throughput than the PRNT, allowing many more samples to be processed in a shorter time saving ∼90 % of the time required by the laboratory scientist to complete the task as compared to the traditional PRNT.

Modified mRNA/lipid nanoparticle-based vaccines expressing respiratory syncytial virus F protein variants are immunogenic and protective in rodent models of RSV infection.

The RSV Fusion (F) protein is a target for neutralizing antibody responses and is a focus for vaccine discovery; however, the process of RSV entry requires F to adopt a metastable prefusion form and transition to a more stable postfusion form, which displays less potent neutralizing epitopes. mRNA vaccines encode antigens that are translated by host cells following vaccination, which may allow conformational transitions similar to those observed during natural infection to occur. Here we evaluate a panel of chemically modified mRNA vaccines expressing different forms of the RSV F protein, including secreted, membrane associated, prefusion-stabilized, and non-stabilized structures, for conformation, immunogenicity, protection, and safety in rodent models. Vaccination with mRNA encoding native RSV F elicited antibody responses to both prefusion- and postfusion-specific epitopes, suggesting that this antigen may adopt both conformations in vivo. Incorporating prefusion stabilizing mutations further shifts the immune response toward prefusion-specific epitopes, but does not impact neutralizing antibody titer. mRNA vaccine candidates expressing either prefusion stabilized or native forms of RSV F protein elicit robust neutralizing antibody responses in both mice and cotton rats, similar to levels observed with a comparable dose of adjuvanted prefusion stabilized RSV F protein. In contrast to the protein subunit vaccine, mRNA-based vaccines elicited robust CD4+ and CD8+ T-cell responses in mice, highlighting a potential advantage of the technology for vaccines requiring a cellular immune response for efficacy.

A potent broadly neutralizing human RSV antibody targets conserved site IV of the fusion glycoprotein.

Respiratory syncytial virus (RSV) infection is the leading cause of hospitalization and infant mortality under six months of age worldwide; therefore, the prevention of RSV infection in all infants represents a significant unmet medical need. Here we report the isolation of a potent and broadly neutralizing RSV monoclonal antibody derived from a human memory B-cell. This antibody, RB1, is equipotent on RSV A and B subtypes, potently neutralizes a diverse panel of clinical isolates in vitro and demonstrates in vivo protection. It binds to a highly conserved epitope in antigenic site IV of the RSV fusion glycoprotein. RB1 is the parental antibody to MK-1654 which is currently in clinical development for the prevention of RSV infection in infants.

Characterization of potent RSV neutralizing antibodies isolated from human memory B cells and identification of diverse RSV/hMPV cross-neutralizing epitopes.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children and older adults. Currently, no licensed vaccine is available, and therapeutic options are limited. The primary target of neutralizing antibodies to RSV is the surface fusion (F) glycoprotein. Understanding the recognition of antibodies with high neutralization potencies to RSV F antigen will provide critical insights in developing efficacious RSV antibodies and vaccines. In this study, we isolated and characterized a panel of monoclonal antibodies (mAbs) with high binding affinity to RSV prefusion F trimer and neutralization potency to RSV viruses. The mAbs were mapped to previously defined antigenic sites, and some that mapped to the same antigenic sites showed remarkable diversity in specificity, binding, and neutralization potencies. We found that the isolated site III mAbs shared highly conserved germline V-gene usage, but had different cross-reactivities to human metapneumovirus (hMPV), possibly due to the distinct modes/angles of interaction with RSV and hMPV F proteins. Furthermore, we identified a subset of potent RSV/hMPV cross-neutralizing mAbs that target antigenic site IV and the recently defined antigenic site V, while the majority of the mAbs targeting these two sites only neutralize RSV. Additionally, the isolated mAbs targeting site Ø were mono-specific for RSV and showed a wide range of neutralizing potencies on different RSV subtypes. Our data exemplify the diversity of anti-RSV mAbs and provide new insights into the immune recognition of respiratory viruses in the Pneumoviridae family.

Design and characterization of a fusion glycoprotein vaccine for Respiratory Syncytial Virus with improved stability.

Respiratory Syncytial Virus (RSV) infection is the leading cause of lower respiratory tract infection in both young children and older adults. Currently, there is no licensed vaccine available, and therapeutic options are limited. The infectious RSV particle is decorated with a type I viral fusion (F) glycoprotein that structurally rearranges from a metastable prefusion form to a highly stable postfusion form. In people naturally infected with RSV, the neutralizing antibodies primarily recognize the prefusion conformation. Therefore, engineered RSV F protein stabilized in its prefusion conformation has been an attractive strategy for developing RSV F vaccine antigens. Long-term stability at 4 °C or higher is a desirable attribute for a RSV F subunit vaccine antigen. We have previously shown that a prefusion stabilized RSV F construct, DS-Cav1, undergoes conformational changes and forms intermediate structures upon long-term storage at 4 °C. Structure-based design was performed to improve the stability of the RSV F subunit vaccine. We identified additional mutations that further stabilize RSV F protein in its prefusion conformation by using binding to a previously described antigenic site I antibody 4D7 as the screening tool. In addition, we designed and identified variants with increased expression levels, which is another desirable attribute for a subunit vaccine. Our data suggested that an RSV F variant F111 is properly folded, and has improved heat stability as well as stability upon long-term storage at 4 °C. A mouse immunogenicity study demonstrated that no compromise in immunogenicity (both binding and neutralizing antibody levels) was observed with the introduction of these additional mutations.

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation.

Chlamydial major outer membrane protein (MOMP) is the major protein constituent of the bacterial pathogen Chlamydia trachomatis. Chlamydia trachomatis Serovars D-K are the leading cause of genital tract infections which can lead to infertility or ectopic pregnancies. A vaccine against Chlamydia is highly desirable but currently not available. MOMP accounts for ~ 60% of the chlamydial protein mass and is considered to be one of the lead vaccine candidates against C. trachomatis. We report on the spectroscopic analysis of C. trachomatis native MOMP Serovars D, E, F, and J as well as C. muridarum MOMP by size exclusion chromatography multi angle light scattering (SEC MALS), circular dichroism (CD) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). MOMP was purified from the native bacterium grown in either adherent HeLa cells or in different suspension cell lines. Our results confirm that MOMP forms homo-trimers in detergent micelles. The secondary structure composition of C. trachomatis MOMP was conserved across serovars, but different from composition of C. muridarum MOMP with a 13% (CD) to 18% (ATR-FTIR) reduction in ß-sheet conformation for C. trachomatis MOMP. When Serovar E MOMP was isolated from suspension cell lines the α-helix content increased by 7% (CD) to 13% (ATIR-FTIR). Maintenance of a native-like tertiary and quaternary structure in subunit vaccines is important for the generation of protective antibodies. This biophysical characterization of MOMP presented here serves, in the absence of functional assays, as a method for monitoring the structural integrity of MOMP.

Stability Characterization of a Vaccine Antigen Based on the Respiratory Syncytial Virus Fusion Glycoprotein.

Infection with Respiratory Syncytial Virus (RSV) causes both upper and lower respiratory tract disease in humans, leading to significant morbidity and mortality in both young children and older adults. Currently, there is no licensed vaccine available, and therapeutic options are limited. During the infection process, the type I viral fusion (F) glycoprotein on the surface of the RSV particle rearranges from a metastable prefusion conformation to a highly stable postfusion form. In people naturally infected with RSV, most potent neutralizing antibodies are directed to the prefusion form of the F protein. Therefore, an engineered RSV F protein stabilized in the prefusion conformation (DS-Cav1) is an attractive vaccine candidate. Long-term stability at 4°C or higher is a desirable attribute for a commercial subunit vaccine antigen. To assess the stability of DS-Cav1, we developed assays using D25, an antibody which recognizes the prefusion F-specific antigenic site Ø, and a novel antibody 4D7, which was found to bind antigenic site I on the postfusion form of RSV F. Biophysical analysis indicated that, upon long-term storage at 4°C, DS-Cav1 undergoes a conformational change, adopting alternate structures that concomitantly lose the site Ø epitope and gain the ability to bind 4D7.

Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F) glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab) is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs) against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction.

Protection provided by a herpes simplex virus 2 (HSV-2) glycoprotein C and D subunit antigen vaccine against genital HSV-2 infection in HSV-1-seropositive guinea pigs.

A prophylactic vaccine for genital herpes disease remains an elusive goal. We report the results of two studies performed collaboratively in different laboratories that assessed immunogenicity and vaccine efficacy in herpes simplex virus 1 (HSV-1)-seropositive guinea pigs immunized and subsequently challenged intravaginally with HSV-2. In study 1, HSV-2 glycoproteins C (gC2) and D (gD2) were produced in baculovirus and administered intramuscularly as monovalent or bivalent vaccines with CpG and alum. In study 2, gD2 was produced in CHO cells and given intramuscularly with monophosphoryl lipid A (MPL) and alum, or gC2 and gD2 were produced in glycoengineered Pichia pastoris and administered intramuscularly as a bivalent vaccine with Iscomatrix and alum to HSV-1-naive or -seropositive guinea pigs. In both studies, immunization boosted neutralizing antibody responses to HSV-1 and HSV-2. In study 1, immunization with gC2, gD2, or both immunogens significantly reduced the frequency of genital lesions, with the bivalent vaccine showing the greatest protection. In study 2, both vaccines were highly protective against genital disease in naive and HSV-1-seropositive animals. Comparisons between gD2 and gC2/gD2 in study 2 must be interpreted cautiously, because different adjuvants, gD2 doses, and antigen production methods were used; however, significant differences invariably favored the bivalent vaccine. Immunization of naive animals with gC2/gD2 significantly reduced the number of days of vaginal shedding of HSV-2 DNA compared with that for mock-immunized animals. Surprisingly, in both studies, immunization of HSV-1-seropositive animals had little effect on recurrent vaginal shedding of HSV-2 DNA, despite significantly reducing genital disease.

Identification of key sequence determinants for the inhibitory function of the prodomain of TACE.

The TNFalpha converting enzyme (TACE) is a zinc metalloproteinase that mediates shedding of multiple cell surface proteins. Regulation of TACE enzymatic activity is ultimately mediated via proteolytic removal of its inhibitory prodomain. Sequence determinants for TACE prodomain inhibition of the catalytic domain are yet to be identified. Surprisingly, although TACE and ADAM 10 (closest homologue) share only 23% sequence identity at their prodomains, the latter in isolation inhibits TACE with the same potency as TACE own prodomain. In contrast, the prodomain of ADAM 9 inhibited TACE only weakly. Detailed analysis of ADAM prodomains revealed two short regions for which TACE and ADAM 10 depart dramatically from all other family members. We prepared TACE prodomain variants containing full or partial switches to ADAM 9 residues at those two regions and examined their functional properties. Variants containing ADAM 9 substitutions including amino acid residues 72-82 and 126-137 were fully inactive for TACE inhibition. A third variant comprising residues 114-125 was active but at lower potency relative to wild type. All inactive variants appeared to be correctly folded. Finally, the amino acid residue Phe72 and the motif Asp-Asp-Val-Ile137 were identified within those regions as key determinants for TACE prodomain inhibitory function. We conclude that TACE and ADAM 10 prodomains are functionally equivalent in a way that separates them from the rest of the ADAM family.