Structural basis for the broad antigenicity of the computationally optimized influenza hemagglutinin X6.

Influenza causes significant morbidity and mortality. As an alternative approach to current seasonal vaccines, the computationally optimized broadly reactive antigen (COBRA) platform has been previously applied to hemagglutinin (HA). This approach integrates wild-type HA sequences into a single immunogen to expand the breadth of accessible antibody epitopes. Adding to previous studies of H1, H3, and H5 COBRA HAs, we define the structural features of another H1 subtype COBRA, X6, that incorporates HA sequences from before and after the 2009 H1N1 influenza pandemic. We determined structures of this antigen alone and in complex with COBRA-specific as well as broadly reactive and functional antibodies, analyzing its antigenicity. We found that X6 possesses features representing both historic and recent H1 HA strains, enabling binding to both head- and stem-reactive antibodies. Overall, these data confirm the integrity of broadly reactive antibody epitopes of X6 and contribute to design efforts for a next-generation vaccine.

Structural insights into the broad protection against H1 influenza viruses by a computationally optimized hemagglutinin vaccine.

Influenza virus poses an ongoing human health threat with pandemic potential. Due to mutations in circulating strains, formulating effective vaccines remains a challenge. The use of computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) proteins is a promising vaccine strategy to protect against a wide range of current and future influenza viruses. Though effective in preclinical studies, the mechanistic basis driving the broad reactivity of COBRA proteins remains to be elucidated. Here, we report the crystal structure of the COBRA HA termed P1 and identify antigenic and glycosylation properties that contribute to its immunogenicity. We further report the cryo-EM structure of the P1-elicited broadly neutralizing antibody 1F8 bound to COBRA P1, revealing 1F8 to recognize an atypical receptor binding site epitope via an unexpected mode of binding.

Epitope Binning of Monoclonal and Polyclonal Antibodies by Biolayer Interferometry.

Understanding the epitopes of antibodies elicited by infection and vaccination is often useful in immunogen design. In this chapter, we describe biolayer interferometry (BLI)-based methods to evaluate such epitopes and permit simultaneous analysis of antibodies from several sources, including monoclonal antibodies (mAbs) and polyclonal serum antibodies (pAbs). Using previously characterized antibodies with known epitopes as controls, the distribution of epitopes for the influenza hemagglutinin (HA) is shown for isolated human mAbs and pooled serum from HA-immunized mice. This method is versatile, high-throughput, and can be adapted to several antigens.

Adjuvant-Mediated Differences in Antibody Responses to Computationally Optimized Hemagglutinin and Neuraminidase Vaccines.

Computationally optimized broadly reactive antigens (COBRAs) are a next-generation universal influenza vaccine candidate. However, how these COBRAs induce antibody responses when combined with different adjuvants has not previously been well-characterized. Therefore, we performed in vivo studies with an HA-based H1 COBRA, Y2, and an NA-based N1 COBRA, N1-I, to assess this effect for the H1N1 subtype. We tested the adjuvants AddaVax, AddaS03, CpG, and Alhydrogel. AddaS03 performed the best, eliciting high IgG titers and hemagglutination inhibition (HAI) activity for Y2 immunizations. Interestingly, serum antibody epitopes were relatively similar across adjuvant groups. Moreover, following N1-I immunization with these adjuvants, AddaS03 also elicited the highest IgG and neuraminidase inhibition (NAI) titers against the 2009 pandemic virus, A/California/07/2009 (A/CA/09). These results inform adjuvant selection efforts for H1 and N1 COBRA HA and NA antigens in a mouse model.

Differential Recognition of Computationally Optimized H3 Hemagglutinin Influenza Vaccine Candidates by Human Antibodies.

Among circulating influenza viruses in humans, H3N2 viruses typically evolve faster than other subtypes and have caused disease in millions of people since emerging in 1968. Computationally optimized broadly reactive antigen (COBRA) technology is one strategy to broaden vaccine-elicited antibody responses among influenza subtypes. In this study, we determined the structural integrity of an H3N2 COBRA hemagglutinin (HA), TJ5, and we probed the antigenic profile of several H3N2 COBRA HAs by assessing recognition of these immunogens by human B cells from seasonally vaccinated human subjects. Of three recently described COBRA H3 HA antigens (TJ5, NG2, and J4), we determined that TJ5 and J4 HA proteins recognize pre-existing B cells more effectively than NG2 HA and a wild-type Hong Kong/4801/2014 protein. We also isolated a panel of 12 H3 HA-specific human monoclonal antibodies (MAbs) and identified that most MAbs recognize both wild-type and COBRA HA proteins and have functional activity against a broad panel of H3N2 viruses. Most MAbs target the receptor-binding site, and one MAb targets the HA stem. MAb TJ5-5 recognizes TJ5 and J4 COBRA HA proteins but has poor recognition of NG2 HA, similar to the global B-cell analysis. We determined a 3.4 Å structure via cryo-electron microscopy of Fab TJ5-5 complexed with the H3 COBRA TJ5, which revealed residues important to the differential binding. Overall, these studies determined that COBRA H3 HA proteins have correct antigenic and structural features, and the proteins are recognized by B cells and MAbs isolated from seasonally vaccinated humans. IMPORTANCE Vaccine development for circulating influenza viruses, particularly for the H3N2 subtype, remains challenging due to consistent antigenic drift. Computationally optimized broadly reactive antigen (COBRA) technology has proven effective for broadening influenza hemagglutinin (HA)-elicited antibody responses compared to wild-type immunogens. Here, we determined the structural features and antigenic profiles of H3 COBRA HA proteins. Two H3 COBRA HA proteins, TJ5 and J4, are better recognized by pre-existing B cells and monoclonal antibodies from the 2017 to 2018 vaccine season compared to COBRA NG2 and a wild-type A/Hong Kong/2014 HA protein. We determined a cryo-electron microscopy (cryo-EM) structure of one MAb that poorly recognizes NG2, MAb TJ5-5, in complex with the TJ5 COBRA HA protein and identified residues critical to MAb recognition. As NG2 is more effective than TJ5 for the recent Hong Kong/2019 virus, these data provide insights into the diminished effectiveness of influenza vaccines across vaccine seasons.

The Pre-Existing Human Antibody Repertoire to Computationally Optimized Influenza H1 Hemagglutinin Vaccines.

Computationally optimized broadly reactive Ag (COBRA) hemagglutinin (HA) immunogens have previously been generated for several influenza subtypes to improve vaccine-elicited Ab breadth. As nearly all individuals have pre-existing immunity to influenza viruses, influenza-specific memory B cells will likely be recalled upon COBRA HA vaccination. We determined the epitope specificity and repertoire characteristics of pre-existing human B cells to H1 COBRA HA Ags. Cross-reactivity between wild-type HA and H1 COBRA HA proteins P1, X6, and Y2 were observed for isolated mAbs. The mAbs bound five distinct epitopes on the pandemic A/California/04/2009 HA head and stem domains, and most mAbs had hemagglutination inhibition and neutralizing activity against 2009 pandemic H1 strains. Two head-directed mAbs, CA09-26 and CA09-45, had hemagglutination inhibition and neutralizing activity against a prepandemic H1 strain. One mAb, P1-05, targeted the stem region of H1 HA, but did not compete with a known stem-targeting H1 mAb. We determined that mAb P1-05 recognizes a recently discovered HA epitope, the anchor epitope, and we identified similar mAbs using B cell repertoire sequencing. In addition, the trimerization domain distance from HA was critical to recognition of this epitope by mAb P1-05, suggesting the importance of protein design for vaccine formulations. Overall, these data indicate that seasonally vaccinated individuals possess a population of functional H1 COBRA HA-reactive B cells that target head, central stalk, and anchor epitopes, and they demonstrate the importance of structure-based assessment of subunit protein vaccine candidates to ensure accessibility of optimal protein epitopes.

Next-Generation Influenza HA Immunogens and Adjuvants in Pursuit of a Broadly Protective Vaccine.

Influenza virus, a highly mutable respiratory pathogen, causes significant disease nearly every year. Current vaccines are designed to protect against circulating influenza strains of a given season. However, mismatches between vaccine strains and circulating strains, as well as inferior vaccine effectiveness in immunodeficient populations, represent major obstacles. In an effort to expand the breadth of protection elicited by influenza vaccination, one of the major surface glycoproteins, hemagglutinin (HA), has been modified to develop immunogens that display conserved regions from multiple viruses or elicit a highly polyclonal antibody response to broaden protection. These approaches, which target either the head or the stalk domain of HA, or both domains, have shown promise in recent preclinical and clinical studies. Furthermore, the role of adjuvants in bolstering the robustness of the humoral response has been studied, and their effects on the vaccine-elicited antibody repertoire are currently being investigated. This review will discuss the progress made in the universal influenza vaccine field with respect to influenza A viruses from the perspectives of both antigen and adjuvant, with a focus on the elicitation of broadly neutralizing antibodies.

Regulation of Mumps Virus Replication and Transcription by Kinase RPS6KB1.

Mumps virus (MuV) caused the most viral meningitis before mass immunization. Unfortunately, MuV has reemerged in the United States in the past several years. MuV is a member of the genus Rubulavirus, in the family Paramyxoviridae, and has a nonsegmented negative-strand RNA genome. The viral RNA-dependent RNA polymerase (vRdRp) of MuV consists of the large protein (L) and the phosphoprotein (P), while the nucleocapsid protein (NP) encapsulates the viral RNA genome. These proteins make up the replication and transcription machinery of MuV. The P protein is phosphorylated by host kinases, and its phosphorylation is important for its function. In this study, we performed a large-scale small interfering RNA (siRNA) screen targeting host kinases that regulated MuV replication. The human kinase ribosomal protein S6 kinase beta-1 (RPS6KB1) was shown to play a role in MuV replication and transcription. We have validated the role of RPS6KB1 in regulating MuV using siRNA knockdown, an inhibitor, and RPS6KB1 knockout cells. We found that MuV grows better in cells lacking RPS6KB1, indicating that it downregulates viral growth. Furthermore, we detected an interaction between the MuV P protein and RPS6KB1, suggesting that RPS6KB1 directly regulates MuV replication and transcription.IMPORTANCE Mumps virus is an important human pathogen. In recent years, MuV has reemerged in the United State, with outbreaks occurring in young adults who have been vaccinated. Our work provides insight into a previously unknown mumps virus-host interaction. RPS6KB1 negatively regulates MuV replication, likely through its interaction with the P protein. Understanding virus-host interactions can lead to novel antiviral drugs and enhanced vaccine production.